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Sample records for adhesion kinase functions

  1. FAK dimerization controls its kinase-dependent functions at focal adhesions

    KAUST Repository

    Brami-Cherrier, Karen

    2014-01-30

    Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

  2. Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

    KAUST Repository

    Kadaré, Gress

    2015-01-02

    Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr925 facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr925 phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr861, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser910 by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.

  3. Focal Adhesion Kinases in Adhesion Structures and Disease

    Directory of Open Access Journals (Sweden)

    Pierre P. Eleniste

    2012-01-01

    Full Text Available Cell adhesion to the extracellular matrix (ECM is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases.

  4. Focal adhesion kinases in adhesion structures and disease.

    Science.gov (United States)

    Eleniste, Pierre P; Bruzzaniti, Angela

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organization and role of focal adhesions, podosomes, and invadopodia in different cells. In addition, we discuss the role of the tyrosine kinases, FAK, Pyk2, and Src, which are critical for the function of the different adhesion structures. Finally, we discuss the essential role of these tyrosine kinases from the perspective of human diseases. PMID:22888421

  5. Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing.

    Directory of Open Access Journals (Sweden)

    Yuu Horikiri

    Full Text Available Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH expression is positively correlated with phosphorylated focal adhesion kinase (FAK Tyr(397. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr(397 were expressed in bone marrow cells and that pFAK Tyr(397 was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr(397 were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr(397 time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b(+ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

  6. Focal Adhesion Kinases in Adhesion Structures and Disease

    OpenAIRE

    Pierre P. Eleniste; Angela Bruzzaniti

    2012-01-01

    Cell adhesion to the extracellular matrix (ECM) is essential for cell migration, proliferation, and embryonic development. Cells can contact the ECM through a wide range of matrix contact structures such as focal adhesions, podosomes, and invadopodia. Although they are different in structural design and basic function, they share common remodeling proteins such as integrins, talin, paxillin, and the tyrosine kinases FAK, Pyk2, and Src. In this paper, we compare and contrast the basic organiza...

  7. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna W.

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  8. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  9. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    Science.gov (United States)

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  10. Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase

    OpenAIRE

    Jun Feng; Blake Mertz

    2015-01-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through confor...

  11. Functionally Graded Adhesives for Composite Joints

    Science.gov (United States)

    Stapleton, Scott E.; Waas, Anthony M.; Arnold, Steven M.

    2012-01-01

    Adhesives with functionally graded material properties are being considered for use in adhesively bonded joints to reduce the peel stress concentrations located near adherend discontinuities. Several practical concerns impede the actual use of such adhesives. These include increased manufacturing complications, alterations to the grading due to adhesive flow during manufacturing, and whether changing the loading conditions significantly impact the effectiveness of the grading. An analytical study is conducted to address these three concerns. An enhanced joint finite element, which uses an analytical formulation to obtain exact shape functions, is used to model the joint. Furthermore, proof of concept testing is conducted to show the potential advantages of functionally graded adhesives. In this study, grading is achieved by strategically placing glass beads within the adhesive layer at different densities along the joint.

  12. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  13. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components......Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration...... in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...

  14. Mitogen-activated protein kinase (MAPK pathway regulates branching by remodeling epithelial cell adhesion.

    Directory of Open Access Journals (Sweden)

    Anneliis Ihermann-Hella

    2014-03-01

    Full Text Available Although the growth factor (GF signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

  15. IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components.

    Science.gov (United States)

    Ariel, Amiram; Novick, Daniela; Rubinstein, Menachem; Dinarello, Charles A; Lider, Ofer; Hershkoviz, Rami

    2002-07-01

    Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, beta(1) integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-gamma (INF-gamma) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-gamma production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites. PMID:12101280

  16. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

    Directory of Open Access Journals (Sweden)

    Yun-Hee Choi

    2015-11-01

    Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

  17. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    Science.gov (United States)

    Seong, Jihye; Tajik, Arash; Sun, Jie; Guan, Jun-Lin; Humphries, Martin J; Craig, Susan E; Shekaran, Asha; García, Andrés J; Lu, Shaoying; Lin, Michael Z; Wang, Ning; Wang, Yingxiao

    2013-11-26

    Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

  18. Focal Adhesion Kinase Is Required for Intestinal Regeneration and Tumorigenesis Downstream of Wnt/c-Myc Signaling

    OpenAIRE

    Ashton, Gabrielle H.; Morton, Jennifer P; Myant, Kevin; Phesse, Toby J.; Ridgway, Rachel A.; Marsh, Victoria; Wilkins, Julie A.; Athineos, Dimitris; Muncan, Vanesa; Kemp, Richard; Neufeld, Kristi; Clevers, Hans; Brunton, Valerie; Winton, Douglas J.; Wang, Xiaoyan

    2010-01-01

    The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein withi...

  19. Diacylglycerol Kinase Inhibition and Vascular Function

    OpenAIRE

    Choi, Hyehun; Allahdadi, Kyan J.; Tostes, Rita C A; Webb, R. Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structu...

  20. The structural features of Trask that mediate its anti-adhesive functions.

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    Danislav S Spassov

    Full Text Available Trask/CDCP1 is a transmembrane protein with a large extracellular and small intracellular domains. The intracellular domain (ICD undergoes tyrosine phosphorylation by Src kinases during anchorage loss and, when phosphorylated, Trask functions to inhibit cell adhesion. The extracellular domain (ECD undergoes proteolytic cleavage by serine proteases, although the functional significance of this remains unknown. There is conflicting evidence regarding whether it functions to signal the phosphorylation of the ICD. To better define the structural determinants that mediate the anti-adhesive functions of Trask, we generated a series of deletion mutants of Trask and expressed them in tet-inducible cell models to define the structural elements involved in cell adhesion signaling. We find that the ECD is dispensable for the phosphorylation of the ICD or for the inhibition of cell adhesion. The anti-adhesive functions of Trask are entirely embodied within its ICD and are specifically due to tyrosine phosphorylation of the ICD as this function is completely lost in a phosphorylation-defective tyrosine-phenylalanine mutant. Both full length and cleaved ECDs are fully capable of phosphorylation and undergo phosphorylation during anchorage loss and cleavage is not an upstream signal for ICD phosphorylation. These data establish that the anti-adhesive functions of Trask are mediated entirely through its tyrosine phosphorylation. It remains to be defined what role, if any, the Trask ECD plays in its adhesion functions.

  1. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    Energy Technology Data Exchange (ETDEWEB)

    Dowling, Catríona M., E-mail: Catriona.Dowling@ul.ie; Kiely, Patrick A., E-mail: Catriona.Dowling@ul.ie [Department of Life Sciences, Materials and Surface Science Institute and Stokes Institute, University of Limerick, Limerick 78666 (Ireland); Health Research Institute (HRI), University of Limerick, Limerick 78666 (Ireland)

    2015-07-15

    The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

  2. Novel Phosphotidylinositol 4,5-Bisphosphate Binding Sites on Focal Adhesion Kinase.

    Directory of Open Access Journals (Sweden)

    Jun Feng

    Full Text Available Focal adhesion kinase (FAK is a protein tyrosine kinase that is ubiquitously expressed, recruited to focal adhesions, and engages in a variety of cellular signaling pathways. Diverse cellular responses, such as cell migration, proliferation, and survival, are regulated by FAK. Prior to activation, FAK adopts an autoinhibited conformation in which the FERM domain binds the kinase domain, blocking access to the activation loop and substrate binding site. Activation of FAK occurs through conformational change, and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2 are known to facilitate this process. PIP2 binding alters the autoinhibited conformation of the FERM and kinase domains and subsequently exposes the activation loop to phosphorylation. However, the detailed molecular mechanism of PIP2 binding and its role in FAK activation remain unclear. In this study, we conducted coarse-grained molecular dynamics simulations to investigate the binding of FAK to PIP2. Our simulations identified novel areas of basic residues in the kinase domain of FAK that potentially undergo transient binding to PIP2 through electrostatic attractions. Our investigation provides a molecular picture of PIP2-initiated FAK activation and introduces promising new pathways for future studies of FAK regulation.

  3. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    International Nuclear Information System (INIS)

    Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects. Treatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs. These results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis

  4. Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase

    OpenAIRE

    WANG, CHUNHUAI; Xiang, Ru; ZHANG, XIANGZHONG; CHEN, YUNXIAN

    2015-01-01

    Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were...

  5. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    Science.gov (United States)

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

  6. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    International Nuclear Information System (INIS)

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  7. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Karin [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden); Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Grawé, Jan [Department of Genetics and Pathology, Uppsala University, Uppsala 75185 (Sweden); McKinney-Freeman, Shannon L. [Department of Hematology, St. Jude Children' s Research Hospital, Memphis, TN 38105 (United States); Daley, George Q. [HHMI, Children' s Hospital Boston, Harvard Medical School, Boston, 02115 MA (United States); Welsh, Michael, E-mail: michael.welsh@mcb.uu.se [Department of Medical Cell Biology, Uppsala University, Uppsala 751 23 (Sweden)

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via

  8. Organization and post-transcriptional processing of focal adhesion kinase gene

    Directory of Open Access Journals (Sweden)

    Enslen Hervé

    2006-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase critical for processes ranging from embryo development to cancer progression. Although isoforms with specific molecular and functional properties have been characterized in rodents and chicken, the organization of FAK gene throughout phylogeny and its potential to generate multiple isoforms are not well understood. Here, we study the phylogeny of FAK, the organization of its gene, and its post-transcriptional processing in rodents and human. Results A single orthologue of FAK and the related PYK2 was found in non-vertebrate species. Gene duplication probably occurred in deuterostomes after the echinoderma embranchment, leading to the evolution of PYK2 with distinct properties. The amino acid sequence of FAK and PYK2 is conserved in their functional domains but not in their linker regions, with the absence of autophosphorylation site in C. elegans. Comparison of mouse and human FAK genes revealed the existence of multiple combinations of conserved and non-conserved 5'-untranslated exons in FAK transcripts suggesting a complex regulation of their expression. Four alternatively spliced coding exons (13, 14, 16, and 31, previously described in rodents, are highly conserved in vertebrates. Cis-regulatory elements known to regulate alternative splicing were found in conserved alternative exons of FAK or in the flanking introns. In contrast, other reported human variant exons were restricted to Homo sapiens, and, in some cases, other primates. Several of these non-conserved exons may correspond to transposable elements. The inclusion of conserved alternative exons was examined by RT-PCR in mouse and human brain during development. Inclusion of exons 14 and 16 peaked at the end of embryonic life, whereas inclusion of exon 13 increased steadily until adulthood. Study of various tissues showed that inclusion of these exons also occurred, independently from each other, in a

  9. Syndecan-4 and focal adhesion function

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    2001-01-01

    Two groups have now reported the viability of mice that lack syndecan-4. These mice have wound healing/angiogenesis problems, and fibroblasts from these animals differ in adhesion and migration from normal. This is consistent with recent in vitro data indicating a need for signaling via syndecan-4...... for focal adhesion formation, and reports that overexpression of proteins that bind syndecan-4 can modify cell adhesion and migration....

  10. Switchable adhesion by chemical functionality and topography

    NARCIS (Netherlands)

    Kamperman, M.M.G.; Synytska, A.

    2012-01-01

    Progress in adhesion technology over the last few decades has led to widespread replacement of mechanical fasteners with adhesive bonds. Despite the advances, it remains challenging to produce materials that are sticky on demand. In this feature article we highlight recent efforts to develop reversi

  11. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    International Nuclear Information System (INIS)

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

  12. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  13. Diacylglycerol Kinase Inhibition and Vascular Function.

    Science.gov (United States)

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction. PMID:21547002

  14. Role of Titanium Surface Topography and Surface Wettability on Focal Adhesion Kinase Mediated Signaling in Fibroblasts

    Directory of Open Access Journals (Sweden)

    Douglas W. Hamilton

    2011-05-01

    Full Text Available Changes of titanium surface roughness and surface free energy may influence protein absorption that increases cell differentiation through activation of focal adhesion kinase related pathways. However, the influence of titanium surface roughness and hydrophilicity on fibroblast behavior is not well understood. The aim of this study was to investigate the influence of topography and hydrophilicity on fibroblast attachment, spreading, morphology, intracellular signaling, proliferation, and collagen I mRNA levels. Using a cellular FAK knockout (FAK−/− model and wild-type (WT controls, we also investigated the contribution of adhesion in fibroblasts cultured on smooth (PT, sand-blasted, large grit, acid-etched (SLA and hydrophilic SLA topographies. Loss of FAK did not significantly affect fibroblast attachment to any surface, but SLA and hydrophilic SLA surface attenuated spreading of WT cells significantly more than FAK−/− fibroblasts. Both FAK−/− and WT cells formed numerous focal adhesions on PT surfaces, but significantly less on SLA and hydrophilic SLA surfaces. In WT cells, phosphorylation levels of FAK were lower on SLA and hydrophilic SLA in comparison with PT 24 h post seeding. Labeling of cells with antibodies to cortactin showed that FAK−/−cells contained significantly more cortactin-rich focal adhesion in comparison with WT cells on PT surfaces, but not on SLA or hydrophilic SLA. ERK 1/2 phosphorylation was highest in WT cells on all surfaces which correlated with collagen I expression levels. We conclude that fibroblasts are sensitive to changes in surface roughness and hydrophilicity, with adhesive interactions mediated through FAK, an important modulator of fibroblast response.

  15. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  16. Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase

    Institute of Scientific and Technical Information of China (English)

    Lun Suo; Huinan Lu; Guoxin Ying; Mario R. Capecchi; Qiang Wu

    2012-01-01

    Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments.The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton.Here,we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration.The knockout of the entire Pcdhα cluster results in the dendritic simplification and spine loss in CA1 pyramidal neurons in vivo and in cultured primary hippocampal neurons in vitro.The knockdown of the whole Pcdhγ cluster or in combination with the Pcdhα knockout results in similar dendritic and spine defects in vitro.The overexpression of proline-rich tyrosine kinase 2 (Pyk2,also known as CAKβ,RAFTK,FAK2,and CADTK) recapitulates these defects and its knockdown rescues the phenotype.Moreover,the genetic deletion of the Pcdhα cluster results in phosphorylation and activation of Pyk2 and focal adhesion kinase (Fak) and the inhibition of Rho GTPases in vivo.Finally,the overexpression of PyK2 leads to inactivation of Rac1 and,conversely,the constitutive active Rac1 rescues the dendritic and spine morphogenesis defects caused by the knockout of the Pcdhα cluster and the knockdown of the Pcdhγ cluster.Thus,the involvement of the Pcdh-CAK-Rho GTPase pathway in the dendritic development and spine morphogenesis has interesting implications for proper assembly of neuronal connections in the brain.

  17. Engineering bio-adhesive functions in an antimicrobial polymer multilayer

    International Nuclear Information System (INIS)

    Functionalization of a biomaterial surface with adhesive ligands is an effective way to promote specific cell adhesion. Ideally, biomaterial for applications in biomedical implants should simultaneously promote host cell adhesion and inhibit bacterial adhesion. Currently, little attention has been paid to the design of antimicrobial biomaterial with selective adhesiveness towards only targeted cells or tissues. In this study, the role of two typical adhesive ligands on the bioadhesion functions of a model antimicrobial film was elucidated. First, an adhesive ligand including an RGD peptide or collagen (CL) was chemically coupled to an antimicrobial polymeric multilayer composed of dextran sulfate (DS) and chitosan (CS). It was demonstrated that the density of RGD and CL immobilized on the DS/CS multilayer ranges between 4 to 137 ng cm−2 and 100 to 1000 ng cm−2, respectively. Then the effect of immobilized RGD or CL on both bacterial and fibroblast adhesion was investigated. By determining the density and morphology of adherent fibroblast on a DS/CS multilayer with or without an adhesive ligand, it was shown that RGD or CL effectively promoted fibroblast adhesion and proliferation in a concentration-dependent manner. Interestingly, the type of adhesive ligands imposed distinct effects in bacterial adhesion. Immobilized RGD did not enhance Staphylococcus aureus and Escherichia coli adhesion on DS/CS multilayers under all concentrations. In contrast, CL triggered significant S. aureus adhesion on DS/CS multilayers even at low surface concentration and when fibroblast adhesion was absent. Moreover, the detachment forces of individual S. aureus on CL coated DS/CS multilayers probed by atomic force microscopy (AFM) was 3 times and 20 times higher than that on the control substrate and on unmodified DS/CS multilayers, respectively. Interestingly, the lowest detachment force of E. coli was found on the CL coated DS/CS multilayers. This study demonstrated the

  18. Mnk kinase pathway: Cellular functions and biological outcomes

    Institute of Scientific and Technical Information of China (English)

    Sonali; Joshi; Leonidas; C; Platanias

    2014-01-01

    The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.

  19. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  20. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    Science.gov (United States)

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  1. The SYK tyrosine kinase: a crucial player in diverse biological functions

    OpenAIRE

    Mócsai, Attila; Ruland, Jürgen; Tybulewicz, Victor L.J.

    2010-01-01

    Spleen tyrosine kinase (SYK) has been known to relay adaptive immune receptor signalling. However, recent reports indicate that SYK also mediates other, unexpectedly diverse biological functions including cellular adhesion, innate immune recognition, osteoclast maturation, platelet activation and vascular development. SYK is activated by C-type lectins and integrins, and activates novel targets including the CARD9/CARMA1–BCL10–MALT1 pathway and the NLRP3 inflammasome. Drosophila studies indic...

  2. Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

    Directory of Open Access Journals (Sweden)

    Ohannessian Arthur

    2004-05-01

    Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

  3. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  4. Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation.

    NARCIS (Netherlands)

    Miltenburg, van M.H.; Nimwegen, van M.J.; Tijdens, R.B.; Lalai, R.A.; Kuiper, R.; Klarenbeek, S.; Schouten, P.C.; Vries, de A.; Jonkers, J.M.M..; Water, van de B.

    2014-01-01

    BACKGROUND Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneo

  5. Focal adhesion kinase regulation in stem cell alignment and spreading on nanofibers.

    Science.gov (United States)

    Andalib, Mohammad Nahid; Lee, Jeong Soon; Ha, Ligyeom; Dzenis, Yuris; Lim, Jung Yul

    2016-05-13

    While electrospun nanofibers have demonstrated the potential for novel tissue engineering scaffolds, very little is known about the molecular mechanism of how cells sense and adapt to nanofibers. Here, we revealed the role of focal adhesion kinase (FAK), one of the key molecular sensors in the focal adhesion complex, in regulating mesenchymal stem cell (MSC) shaping on nanofibers. We produced uniaxially aligned and randomly distributed nanofibers from poly(l-lactic acid) to have the same diameters (about 130 nm) and evaluated MSC behavior on these nanofibers comparing with that on flat PLLA control. C3H10T1/2 murine MSCs exhibited upregulations in FAK expression and phosphorylation (pY397) on nanofibrous cultures as assessed by immunoblotting, and this trend was even greater on aligned nanofibers. MSCs showed significantly elongated and well-spread morphologies on aligned and random nanofibers, respectively. In the presence of FAK silencing via small hairpin RNA (shRNA), cell elongation length in the aligned nanofiber direction (cell major axis length) was significantly decreased, while cells still showed preferred orientation along the aligned nanofibers. On random nanofibers, MSCs with FAK-shRNA showed impaired cell spreading resulting in smaller cell area and higher circularity. Our study provides new data on how MSCs shape their morphologies on aligned and random nanofibrous cultures potentially via FAK-mediated mechanism. PMID:27040763

  6. The secret life of kinases: functions beyond catalysis

    Directory of Open Access Journals (Sweden)

    Romano David

    2011-10-01

    Full Text Available Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  7. The secret life of kinases: functions beyond catalysis.

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  8. Amine-functionalized polypyrrole: Inherently cell adhesive conducting polymer.

    Science.gov (United States)

    Lee, Jae Y; Schmidt, Christine E

    2015-06-01

    Electrically conducting polymers (CPs) have been recognized as novel biomaterials that can electrically communicate with biological systems. For their tissue engineering applications, CPs have been modified to promote cell adhesion for improved interactions between biomaterials and cells/tissues. Conventional approaches to improve cell adhesion involve the surface modification of CPs with biomolecules, such as physical adsorption of cell adhesive proteins and polycationic polymers, or their chemical immobilization; however, these approaches require additional multiple modification steps with expensive biomolecules. In this study, as a simple and effective alternative to such additional biomolecule treatment, we synthesized amine-functionalized polypyrrole (APPy) that inherently presents cell adhesion-supporting positive charges under physiological conditions. The synthesized APPy provides electrical activity in a moderate range and a hydrophilic surface compared to regular polypyrrole (PPy) homopolymers. Under both serum and serum-free conditions, APPy exhibited superior attachment of human dermal fibroblasts and Schwann cells compared to PPy homopolymer controls. Moreover, Schwann cell adhesion onto the APPy copolymer was at least similar to that on poly-l-lysine treated PPy controls. Our results indicate that amine-functionalized CP substrates will be useful to achieve good cell adhesion and potentially electrically stimulate various cells. In addition, amine functionality present on CPs can further serve as a novel and flexible platform to chemically tether various bioactive molecules, such as growth factors, antibodies, and chemical drugs. PMID:25294089

  9. Cell Adhesion on Surface-Functionalized Magnesium.

    Science.gov (United States)

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance. PMID:27089250

  10. Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

    Directory of Open Access Journals (Sweden)

    Duane Delimont

    Full Text Available It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

  11. Focal adhesion kinase as a mechanotransducer during rapid brain growth of the chick embryo.

    Science.gov (United States)

    Desmond, Mary E; Knepper, Janice E; DiBenedetto, Angela J; Malaugh, Elizabeth; Callejo, Sagrario; Carretero, Raquel; Alonso, Maria-Isabel; Gato, Angel

    2014-01-01

    Expansion of the hollow fluid-filled embryonic brain occurs by an increase in intraluminal pressure created by accumulation of cerebrospinal fluid (CSF). Experiments have shown a direct correlation between cavity pressure and cell proliferation within the neuroepithelium. These findings lead us to ask how mechanistically this might come about. Are there perhaps molecules on the luminal surface of the embryonic neuroepithelium, such as focal adhesion kinases (FAKs) known to respond to tension in other epithelial cells? Immunodetection using antibodies to total FAK and p-FAK was performed with subsequent confocal analysis of the pattern of their activation under normal intraluminal pressure and induced chronic pressure. Western analysis was also done to look at the amount of FAK expression, as well as its activation under these same conditions. Using immunolocalization, we have shown that FAK is present and activated on both apical and basolateral surfaces and within the cytoplasm of the neuroepithelial cells. This pattern changed profoundly when the neuroepithelium was under pressure. By Western blot, we have shown that FAK was upregulated and activated in the neuroepithelium of the embryos just after the neural tube becomes a closed pressurized system, with phosphorylation detected on the luminal instead of the basal surface, along with an increase in cell proliferation. Chronic hyper-pressure does not induce an increase in phosphorylation of FAK. In conclusion, here we show that neuroepithelial cells respond to intraluminal pressure via FAK phosphorylation on the luminal surface. PMID:24860993

  12. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    Science.gov (United States)

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  13. Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation.

    Directory of Open Access Journals (Sweden)

    Shiro Mizuno

    Full Text Available BACKGROUND: Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α dependent vascular endothelial growth factor (VEGF expression, and is also required for the activity of lysyl oxidase (LOX to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis via inactivation of focal adhesion kinase (FAK. METHODOLOGY: To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT or FAK small interfering RNA (siRNA. PRINCIPAL FINDINGS: Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim. CONCLUSIONS: These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.

  14. Adhesion

    Science.gov (United States)

    As the body moves, tissues or organs inside are normally able to shift around each other. This is because these tissues have ... occur if the adhesions cause an organ or body part to: Twist Pull ... unable to move normally The risk of forming adhesions is high ...

  15. Interfacial adhesion of laser clad functionally graded materials

    NARCIS (Netherlands)

    De Hosson, JTM; Pei, YT; Ocelik, [No Value; Sudarshan, TS; Stiglich, JJ; Jeandin, M

    2002-01-01

    Specially designed samples of laser clad AlSi40 functionally graded materials (FGM) are made for evaluating the interfacial adhesion. To obtain the interfacial bond strength notches are made right at the interface of the FGMs. In-sitit microstructural observations during straining in an FEG-ESEM (fi

  16. Interfacial adhesion of laser clad functionally graded materials

    NARCIS (Netherlands)

    Pei, Y. T.; Ocelik, V.; De Hosson, J. T. M.

    2003-01-01

    Specially designed samples of laser clad AlSi40 functionally graded materials (FGM) are made for evaluating the interfacial adhesion. To obtain the interfacial bond strength notches are made right at the interface of the FGMs. In-situ microstructural observations during straining in a field-emission

  17. Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules

    International Nuclear Information System (INIS)

    Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient

  18. Down-regulation of integrin β1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

    Directory of Open Access Journals (Sweden)

    Xiaoli Yuan

    Full Text Available Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.

  19. Protein kinase substrate identification on functional protein arrays

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    2008-02-01

    Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

  20. Molecular properties,functions,and potential applications of NAD kinases

    Institute of Scientific and Technical Information of China (English)

    Feng Shi; Yongfu Li; Ye Li; Xiaoyuan Wang

    2009-01-01

    NAD kinase catalyzes the phosphorylation of NAD(H)to form NADP(H),using ATP as phosphoryl donor.It is the only key enzyme leading to the de novo NADP+/NADPH biosynthesis.Coenzymes such as NAD(H)and NADP(H)are known for their important functions.Recent studies have partially demonstrated that NAD kinase plays a crucial role in the regulation of NAD(H)/NADP(H)conversion.Here,the molecular properties,physiologic functions,and potential applications of NAD kinase are discussed.

  1. Relationship between focal adhesion kinase and hepatic stellate cell proliferation during rat hepatic fibrogenesis

    Institute of Scientific and Technical Information of China (English)

    Hui-Qing Jiang; Xiao-Lan Zhang; Li Liu; Chang-Chun Yang

    2004-01-01

    AIM: To investigate the dynamic expression of focal adhesion kinase (FAK) protein and FAK mRNA in fibrotic rat liver tissue,and the relationship between FAK and hepatic stellate cell (HSC) proliferation.METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson′s trichrome method. FAK mRNA in the rat livers was determined by reverse transcription-polymerase chain reaction (RT-PCR), and the distributions of FAK were assessed immunohistochemistrically.The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining.RESULTS: With the development of hepatic fibrosis, the positively stained cells of α-SMA increased obviously, which were mainly resided in the portal ducts, fiber septa and perisinuses accompanied with proliferating bile ducts. The positively stained areas of the rat livers in model groups 1 to 4 wk after ligation of common bile duct (12.88±2.63%,22.65±2.16%, 27.45±1.86%, 35.25±2.34%, respectively)were significantly larger than those in the control group (5.88±1.46%) (P<0.01). The positive staining for FAK significantly increased, which was mainly situated in portal ducts, fiber septa and around the bile ducts, vascular endothelial cells and perisinusoidal cells. The expression of FAK was positively correlated with α-SMA expression (r = 0.963, P<0.05). FAK mRNA expression was obviously up-regulated in the model groups compared to the control group.CONCLUSION: These data suggest that expressions of FAK protein and mRNA are greatly increased in fibrotic rat livers,which may play an important role in HSC proliferation and hepatic fibrogenesis.

  2. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

    Science.gov (United States)

    Nielsen-Preiss, Sheila M; Allen, Melissa P; Xu, Mei; Linseman, Daniel A; Pawlowski, John E; Bouchard, R J; Varnum, Brian C; Heidenreich, Kim A; Wierman, Margaret E

    2007-06-01

    GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration. PMID:17332061

  3. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    OpenAIRE

    Yang, Chen-Chieh; Chang, Shun-Fu; Chao, Jian-Kang; Lai, Yi-Liang; Chang, Wei-En; Hsu, Wen-Hsiu; Kuo, Wu-Hsien

    2014-01-01

    Background Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Methods Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to hu...

  4. Electrostatic adhesion for added functionality of composite structures

    Science.gov (United States)

    Heath, Callum J. C.; Bond, Ian P.; Potter, Kevin D.

    2016-02-01

    Electrostatic adhesion can be used as a means of reversible attachment. The incorporation of electrostatic adhesion into fibre reinforced polymer (FRP) composite structures could provide significant value added functionality. Imparting large potential differences (∼2 kV) across electrodes generates an attractive force, thus providing a means of attachment. This could be used as a reversible latching mechanism or as a means of controllable internal connectivity. Varying the connectivity for discrete elements of a substructure of a given design allows for control of internal load paths and moment of area of the cross section. This could facilitate variable stiffness (both in bending and torsion). Using a combination of existing fabrication techniques, functional electrodes have been integrated within a FRP. Copper polyimide thin film laminate material has been both co-cured with carbon fibre reinforced epoxy and bonded to PVC closed cell foam core material to provide a range of structural configurations with integrated electrodes. The ability of such integrated devices to confer variations in global bending stiffness of basic beam structures is investigated. Through the application of 4 kV across integrated electrostatic adhesive devices, a 112% increase in flexural stiffness has been demonstrated for a composite sandwich structure.

  5. Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer.

    Science.gov (United States)

    Gómez Del Pulgar, Teresa; Cebrián, Arancha; Fernández-Aceñero, Maria Jesús; Borrero-Palacios, Aurea; Del Puerto-Nevado, Laura; Martínez-Useros, Javier; Marín-Arango, Juan Pablo; Caramés, Cristina; Vega-Bravo, Ricardo; Rodríguez-Remírez, María; Cruz-Ramos, Marlid; Manzarbeitia, Félix; García-Foncillas, Jesús

    2016-09-01

    Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio- and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre-treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease-free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence. PMID:27171907

  6. Assembly and mechanosensory function of focal adhesions: experiments and models.

    Science.gov (United States)

    Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

    2006-04-01

    Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex

  7. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1992-01-01

    Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form...... of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and......-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta...

  8. Is kinase activity essential for biological functions of BRI1?

    Institute of Scientific and Technical Information of China (English)

    Weihui Xu; Juan Huang; Baohua Li; Jiayang Li; Yonghong Wang

    2008-01-01

    Brassinosteroids (BRs) are a major group of plant hormones that regulate plant growth and development. BRI1, a protein localized to the plasma membrane, functions as a BR receptor and it has been proposed that its kinase activity has an essential role in BR-regulated plant growth and development. Here we report the isolation and molecular characterization of a new allele of bril, bril-301, which shows moderate morphological phenotypes and a reduced response to BRs under normal growth conditions. Sequence analysis identified a two-base alteration from GG to AT, resulting in a conversion of 989G to 9891 in the BRI1 kinase domain. An in vitro assay of kinase activity showed that bril-301 has no detectable autophosphorylation activity or phosphorylation activity towards the BRI1 substrates TTL and BAK1. Furthermore, our results suggest that bril-301, even with extremely impaired kinase activity, still retains partial function in regulating plant growth and development, which raises the question of whether BRI1 kinase activity is essential for BR-mediated growth and development in higher plants.

  9. Therapeutic effects of tyroservatide on metastasis of lung cancer and its mechanism affecting integrin–focal adhesion kinase signal transduction

    Science.gov (United States)

    Huang, Yu-ting; Zhao, Lan; Fu, Zheng; Zhao, Meng; Song, Xiao-meng; Jia, Jing; Wang, Song; Li, Jin-ping; Zhu, Zhi-feng; Lin, Gang; Lu, Rong; Yao, Zhi

    2016-01-01

    Tyroservatide (YSV) can inhibit the growth and metastasis of mouse lung cancer significantly. This study investigated the therapeutic effects of tripeptide YSV on metastasis of human lung cancer cells and explored its possible mechanism that affects integrin–focal adhesion kinase (FAK) signal transduction in tumor cells. YSV significantly inhibited the adhesion and the invasion of highly metastatic human lung cancer cell lines 95D, A549, and NCI-H1299. In addition, YSV significantly inhibited phosphorylation of FAK Tyr397 and FAK Tyr576/577 in the 95D, A549, and NCI-H1299 human lung cancer cells in vitro. And the mRNA level and protein expression of FAK in these human lung cancer cells decreased at the same time. YSV also significantly inhibited mRNA and protein levels of integrin β1 and integrin β3 in the 95D, A549, and NCI-H1299 human lung cancer cells. Our research showed that YSV inhibited adhesion and invasion of human lung cancer cells and exhibited therapeutic effects on metastasis of lung cancer. PMID:27041993

  10. PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

    OpenAIRE

    Madaro, Luca; Marrocco, Valeria; Fiore, Piera; Aulino, Paola; Smeriglio, Piera; Adamo, Sergio; Molinaro, Mario; Bouché, Marina

    2011-01-01

    Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the...

  11. New adhesive systems based on functionalized block copolymers

    Energy Technology Data Exchange (ETDEWEB)

    Kent, M.; Saunders, R.; Hurst, M.; Small, J.; Emerson, J.; Zamora, D.

    1997-05-01

    The goal of this work was to evaluate chemically-functionalized block copolymers as adhesion promoters for metal/thermoset resin interfaces. Novel block copolymers were synthesized which contain pendant functional groups reactive toward copper and epoxy resins. In particular, imidazole and triazole functionalities that chelate with copper were incorporated onto one block, while secondary amines were incorporated onto the second block. These copolymers were found to self-assemble from solution onto copper surfaces to form monolayers. The structure of the adsorbed monolayers were studied in detail by neutron reflection and time-of-flight secondary ion mass spectrometry. The monolayer structure was found to vary markedly with the solution conditions and adsorption protocol. Appropriate conditions were found for which the two blocks form separate layers on the surface with the amine functionalized block exposed at the air surface. Adhesion testing of block copolymer-coated copper with epoxy resins was performed in both lap shear and peel modes. Modest enhancements in bond strengths were observed with the block copolymer applied to the native oxide. However, it was discovered that the native oxide is the weak link, and that by simply removing the native oxide, and then applying an epoxy resin before the native oxide can reform, excellent bond strength in the as-prepared state as well as excellent retention of bond strength after exposure to solder in ambient conditions are obtained. It is recommended that long term aging studies be performed with and without the block copolymer. In addition, the functionalized block copolymer method should be evaluated for another system that has inherently poor bonding, such as the nickel/silicone interface, and for systems involving metals and alloys which form oxides very rapidly, such as aluminum and stainless steel, where bonding strategies involve stabilizing the native oxide.

  12. The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells

    OpenAIRE

    Salem, Israa; Alsalahi, Manal; Chervoneva, Inna; Aburto, Lucy D.; Addya, Sankar; Ott, Gregory R.; Ruggeri, Bruce A.; Cristofanilli, Massimo; Fernandez, Sandra V.

    2016-01-01

    Background Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC. Methods Cell proliferation assays were performed in the presence of several concentrations of CEP-37...

  13. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    Science.gov (United States)

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  14. Caveolin-1 Up-regulation during Epithelial to Mesenchymal Transition Is Mediated by Focal Adhesion Kinase*

    OpenAIRE

    Bailey, Kelly M.; Liu, Jun

    2008-01-01

    Emerging evidence has shown that caveolin-1 is up-regulated in a number of metastatic cancers and can influence various aspects of cell migration. However, in general, the role of caveolin-1 in cancer progression is poorly understood. In the present study, we examined alterations in caveolin-1 expression during epithelial-to-mesenchymal transition (EMT) and the ability of caveolin-1 to alter cancer cell adhesion, an aspect of cell motility. We employed two EMT cell models, the human embryonic...

  15. Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

    Full Text Available Abstract Background Genetically manipulated embryonic stem (ES cell derived neurons (ESNs provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK, which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. Results Mouse ES cells carrying homozygous null mutations (FAK-/- were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. Conclusion FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

  16. Functional nanoparticles translocation into cell and adhesion force curve analysis.

    Science.gov (United States)

    Lee, Haisung; Veerapandian, Murugan; Kim, Byung Tae; Yun, Kyusik; Seo, Soo-Won

    2012-10-01

    The aim of this research is to investigate the cell translocation of two functional nanoparticles (barium sulfate (BaSO4NPs), europium (III) doped gadolinium oxide nanoparticles (Gd2O3@EuNPs)) into A549 cells by Bio-Atomic Force Microscopy (Bio-AFM). Successful cell translocation of these two nanoparticles are ensured from the measurement of changes in the cell surface roughness and interaction (extension), retraction forces from the vertical deflection of tip towards substrate surfaces through force-distance curve slope analysis. Measurement of typical adhesion forces (i.e., extension and retraction) between the tip-substrate (0.0963 and 1.155 nN), tip-A549 cell substrate (0.1177 and 2.468 nN), tip-Gd2O3@EuNPs/A549 substrate (0.0785 and 0.4276 nN) and tip-BaSO4NPs/A549 substrate (0.518 and 6.838 nN) confirms the successful cell translocation of functional nanoparticles into A549 cells. Further the nanoscale resolution of topographical height and 3D images evinces the surface characteristics of normal A549 cells and nanoparticles translocated A549 cells. PMID:23421137

  17. BRET biosensor analysis of receptor tyrosine kinase functionality

    Directory of Open Access Journals (Sweden)

    Sana eSiddiqui

    2013-04-01

    Full Text Available Bioluminescence resonance energy transfer (BRET is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein-protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the receptor tyrosine kinase (RTK super family involving: neurotrophic receptors (e.g. tropomyosin-related kinase (Trk and p75 neurotrophin receptor (p75NTR; insulinotropic receptors (e.g. insulin receptor (IR and insulin-like growth factor receptor (IGFR and growth factor receptors (e.g. ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR, the vascular endothelial growth factor receptor (VEGFR and the c-kit and platelet-derived growth factor receptor (PDGFR. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e. leptin receptor (OB-R and the growth hormone receptor (GHR. It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.

  18. Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

    Science.gov (United States)

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

    Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field. PMID:23745726

  19. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    Science.gov (United States)

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  20. Quantitative changes in focal adhesion kinase and its inhibitor, FRNK, drive load-dependent expression of costamere components.

    Science.gov (United States)

    Klossner, Stephan; Li, Ruowei; Ruoss, Severin; Durieux, Anne-Cécile; Flück, Martin

    2013-09-15

    Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in antigravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at autoregulatory tyrosine 397 (FAK-pY397) was increased after 20 s of stretch. FAK-pY397 content remained elevated after 24 h of stretch-overload due to upregulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electrotransfer increased the β1-integrin (+56%) and meta-vinculin (+88%) content. α7-Integrin (P = 0.46) and γ-vinculin (P = 0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FAK-related nonkinase (FRNK) reduced FAK-pY397 content by 33% and increased the percentage of fast-type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. Seven days of unloading blunted the elevation of FAK-pY397, β1-integrin, and meta-vinculin content with FAK overexpression, and this was reversed by 1 day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK.

  1. Protein kinase CK2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1994-01-01

    Protein kinase CK2 subunits alpha and beta were expressed either separately or together in a bacterial expression system (pT7-7/BL21(DE3)) and purified to homogeneity. After mixing the subunits, a CK2 holoenzyme (alpha 2 beta 2) was spontaneously reconstituted, which displays identical features...... conditions, (b) it protects the alpha subunit against denaturing agents or conditions, and (c) it alters the substrate specificity of the alpha subunit. By site-directed mutagenesis, certain functions of the beta subunit could be assigned to specific amino acids or domains. Twenty one mutants of the beta...

  2. Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

    Science.gov (United States)

    Wangpu, Xiongzhi; Lu, Jiaoyang; Xi, Ruxing; Yue, Fei; Sahni, Sumit; Park, Kyung Chan; Menezes, Sharleen; Huang, Michael L H; Zheng, Minhua; Kovacevic, Zaklina; Richardson, Des R

    2016-05-01

    Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway. PMID:26895766

  3. Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth

    OpenAIRE

    Golubovskaya, Vita M.; Ho, Baotran; Zheng, Min; Magis, Andrew; Ostrov, David; Morrison, Carl; Cance, William G.

    2013-01-01

    Background Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis. Methods We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compoun...

  4. Reduced Mechanical Stretch Induces Enhanced Endothelin B Receptor-mediated Contractility via Activation of Focal Adhesion Kinase and Extra Cellular-regulated Kinase 1/2 in Cerebral Arteries from Rat

    DEFF Research Database (Denmark)

    Rasmussen, Marianne N P; Spray, Stine; Skovsted, Gry F;

    2016-01-01

    Cerebral ischaemia results in enhanced endothelin B (ETB ) receptor-mediated contraction and receptor protein expression in the affected cerebrovascular smooth muscle cells (SMC). Organ culture of cerebral arteries is a method to induce similar alterations in ETB receptor expression. We hypothesize...... ETB receptor agonist sarafotoxin 6c. The involvement of extracellular regulated kinase (ERK) 1/2 and focal adhesion kinase (FAK) were studied by their specific inhibitors U0126 and PF-228, respectively. Compared to their stretched counterparts, un-stretched MCA segments showed a significantly...

  5. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

    Science.gov (United States)

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M; Bergmeier, Wolfgang; Wagner, Denisa D

    2010-03-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

  6. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China)

    2014-08-01

    Focal adhesion kinase (FAK) acts as a regulator of cellular signaling and may promote cell spreading, motility, invasion and survival in malignancy. Elevated expression and activity of FAK frequently correlate with tumor cell metastasis and poor prognosis in breast cancer. However, the mechanisms by which the turnover of FAK is regulated remain elusive. Here we report that heat shock protein 90β (HSP90β) interacts with FAK and the middle domain (amino acids 233–620) of HSP90β is mainly responsible for this interaction. Furthermore, we found that HSP90β regulates FAK stability since HSP90β inhibitor 17-AAG triggers FAK ubiquitylation and subsequent proteasome-dependent degradation. Moreover, disrupted FAK-HSP90β interaction induced by 17-AAG contributes to attenuation of tumor cell growth, migration, and invasion. Together, our results reveal how HSP90β regulates FAK stability and identifies a potential therapeutic strategy to breast cancer. - Highlights: • HSP90β protects FAK from degradation by the ubiquitin-proteasome pathway. • Inhibition of HSP90β or FAK attenuates tumorigenesis of breast cancer cells. • Genetic repression of HSP90β or FAK inhibits tumor cell migration and proliferation. • Inhibition of HSP90β or FAK interferes cell invasion and cytoskeleton.

  7. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion.

    Science.gov (United States)

    Heuslein, Joshua L; Murrell, Kelsey P; Leiphart, Ryan J; Llewellyn, Ryan A; Meisner, Joshua K; Price, Richard J

    2016-01-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase's (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6C(hi) and Ly6C(lo) blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK. PMID:27244251

  8. Soluble fms-like tyrosine kinase-1 and endothelial adhesion molecules (intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1) as predictive markers for blood pressure reduction after renal sympathetic denervation.

    Science.gov (United States)

    Dörr, Oliver; Liebetrau, Christoph; Möllmann, Helge; Gaede, Luise; Troidl, Christian; Rixe, Johannes; Hamm, Christian; Nef, Holger

    2014-05-01

    Renal sympathetic denervation (RSD) is a treatment option for patients with resistant arterial hypertension, but in some patients it is not successful. Predictive parameters on the success of RSD remain unknown. The angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are known to be associated with endothelial dysfunction, vascular remodeling, and hypertension. We evaluated whether sFLT-1, ICAM-1, and VCAM-1 are predictive markers for blood pressure reduction after RSD. Consecutive patients (n=55) undergoing renal denervation were included. Venous serum samples for measurement of sFlt-1, ICAM-1, and VCAM-1 were collected before and 6 months after RSD. A therapeutic response was defined as an office systolic blood pressure reduction of >10 mm Hg 6 months after RSD. A significant mean office systolic blood pressure reduction of 31.2 mm Hg was observed in 46 patients 6 months after RSD. Nine patients were classified as nonresponders, with a mean systolic blood pressure reduction of 4.6 mm Hg. At baseline, sFLT-1 levels were significantly higher in responders than in nonresponders (P<0.001) as were ICAM-1 (P<0.001) and VCAM-1 levels (P<0.01). The areas under the curve for sFLT-1, ICAM-1, and VCAM-1 were 0.82 (interquartile range, 0.718-0.921; P<0.001), 0.754 (0.654-0.854; P<0.001), and 0.684 (0.564-804; P=0.01), respectively, demonstrating prediction of an RSD response. Responders showed significantly higher serum levels of sFLT-1, ICAM-1, and VCAM-1 at baseline compared with nonresponders. Thus, this study identified for the first time potential biomarkers with a predictive value indicating a responder or nonresponder before renal denervation. PMID:24470464

  9. Heterologous desensitization of T cell functions by CCR5 and CXCR4 ligands: inhibition of cellular signaling, adhesion and chemotaxis.

    Science.gov (United States)

    Hecht, Iris; Cahalon, Liora; Hershkoviz, Rami; Lahat, Adi; Franitza, Suzanne; Lider, Ofer

    2003-01-01

    T cells migrate into inflamed sites through the extracellular matrix (ECM) in response to chemotactic areas and are then simultaneously or sequentially exposed to multiple chemotactic ligands. We examined the responses of human peripheral blood T cells, present in an ECM-like context, to combinatorial signaling transduced by SDF-1alpha (CXCL12), and two CCR5 ligands, RANTES (CCL5) and MIP-1beta (CCL4). Separately, these chemokines, at G protein-coupled receptor (GPCR)-stimulating concentrations, induced T cell adhesion to fibronectin (FN) and T cell chemotaxis. However, the pro-adhesive and pro-migratory capacities of SDF-1alpha and RANTES or MIP-1beta were mutually suppressed by the simultaneous or sequential exposure of the cells to these CCR5 or CXCR4 ligands. This cross-talk did not involve the internalization of the SDF-1alpha receptor, CXCR4, but rather, a decrease in phosphorylation of ERK and Pyk-2, as well as inhibition of Ca(2+) mobilization. Strikingly, early CXCR4 signaling of phosphatidylinositol-3-kinase, detected by SDF-1alpha-induced AKT phosphorylation, was insensitive to RANTES-CCR5 signals. Accordingly, early chemotaxis to SDF-1alpha was not susceptible to CCR5 occupancy, whereas late stages of T cell chemotaxis were markedly down-regulated. This is an example of a specialized functional desensitization of heterologous chemokine receptors that induces GPCR interference with T cell adhesion to ECM ligands and chemotaxis within chemokine-rich extravascular contexts. PMID:12502723

  10. Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

    OpenAIRE

    Yun-Hee Choi; Dong Joo Yang; Atul Kulkarni; Sang Hyun Moh; Ki Woo Kim

    2015-01-01

    Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles ...

  11. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M;

    1998-01-01

    experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB......-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB....... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...

  12. Oxidative and adhesive responses of human neutrophils to nitrovasodilators in vitro: the role of protein kinases

    Directory of Open Access Journals (Sweden)

    Magdalena Klink

    2003-01-01

    Full Text Available Background: Sodium nitroprusside (SNP and molsidomine are used in the treatment of coronary heart disease. Since the neutrophils play a pathological role in ischaemic heart disease, it is important to understand the direct action of nitrovasodilators on their function.

  13. Calsyntenins Function as Synaptogenic Adhesion Molecules in Concert with Neurexins

    OpenAIRE

    Ji Won Um; Gopal Pramanik; Ji Seung Ko; Min-Young Song; Dongmin Lee; Hyun Kim; Kang-Sik Park; Thomas C. Südhof; Katsuhiko Tabuchi; Jaewon Ko

    2014-01-01

    Multiple synaptic adhesion molecules govern synapse formation. Here, we propose calsyntenin-3/alcadein-β as a synapse organizer that specifically induces presynaptic differentiation in heterologous synapse-formation assays. Calsyntenin-3 (CST-3) was highly expressed during various postnatal periods of mouse brain development. The simultaneous knockdown of all three CSTs, but not CST-3 alone, decreased inhibitory, but not excitatory, synapse densities in cultured hippocampal neurons. Moreover,...

  14. Adhesion of DOPA-Functionalized Model Membranes to Hard and Soft Surfaces.

    Science.gov (United States)

    Guvendiren, Murat; Brass, David A; Messersmith, Phillip B; Shull, Kenneth R

    2009-01-01

    The adhesive proteins secreted by marine mussels form a natural glue that cures rapidly to form strong and durable bonds in aqueous environments. These mussel adhesive proteins contain an unusual amino acid, 3,4-dihydroxy-L-phenylalanine (DOPA), which is largely responsible for their cohesive and adhesive strengths. In this study, we incorporated DOPA into diblock and triblock polymers and developed a membrane contact experiment to assess the adhesive interactions of these materials with TiO(2) and tissue surfaces. In a typical experiment a micrometer-thick DOPA-functionalized elastomeric membrane is attached to the end of a cylindrical glass tube. Application of a positive pressure to the tube brings the membrane into contact with the surface of interest. The negative pressure needed to separate the membrane from the substrate is a measure of the strength of the adhesive interaction. The test confirms previous results obtained with TiO(2) substrates. Because the membrane geometry is well suited for rough or chemically heterogeneous surfaces, it is ideal for studies of tissue adhesion. DOPA was found to give strong adhesion to tissue surfaces, with the strongest adhesion obtained when the DOPA groups were oxidized while in contact with the tissue surface. PMID:21461121

  15. Progress of the mechanisms of focal adhesion kinase induced angiogenesis%黏着斑激酶促进血管生成机制的进展

    Institute of Scientific and Technical Information of China (English)

    李越; 林春龙

    2013-01-01

    黏着斑激酶(focal adhesion kinase,FAK)是一种胞质非受体蛋白酪氨酸激酶,在人、鸟、果蝇、鼠、非洲蟾蜍中均有表达并呈高度同源性.FAK在多器官和组织中均有表达,尤其是在肿瘤细胞中高水平表达.FAK介导的信号通路是细胞内各信号通路的交汇点,对正常细胞及肿瘤细胞黏附、迁移、增殖均起调控作用.在血管形成过程中,FAK可以通过FAK-Rho GTPases通路、FAK-PI3K通路等信号通路调节血管内皮细胞的黏附及迁移,调节细胞骨架重建.%Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase and has its expression in humans,birds,fruit flies,mice and xenopus with a high degree of homology.FAK has its expression as well in multiple organs and tissues,especially high expression in tumor cell.FAKmediated signal pathway is the intersection of all signal pathways,playing a regulatory role in the adhesion,migration and proliferation of both normal cells and the tumor cell.FAK can also through such signal pathways as FAK Rho GTPases pathway and FAK-PI3K pathway and etc.Regulate both the reconstruction of cytoskeleton and the adhesion and migration of vascular endothelial cells that is of great significance in that process.

  16. Big Roles of Small Kinases:The Complex Functions of Receptor-Like Cytoplasmic Kinases in Plant Immunity and Development

    Institute of Scientific and Technical Information of China (English)

    Wenwei Lin; Xiyu Ma; Libo Shan; Ping He

    2013-01-01

    Plants have evolved a large number of receptor-like cytoplasmic kinases (RLCKs) that often functionally and physically associate with receptor-like kinases (RLKs) to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.

  17. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis.

    Science.gov (United States)

    Faltermeier, Claire M; Drake, Justin M; Clark, Peter M; Smith, Bryan A; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N

    2016-01-12

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention. PMID:26621741

  18. Augmentation of RANTES-induced extracellular signal-regulated kinase mediated signaling and T cell adhesion by elastase-treated fibronectin.

    Science.gov (United States)

    Brill, A; Hershkoviz, R; Vaday, G G; Chowers, Y; Lider, O

    2001-06-15

    T cells migrating across extracellular matrix (ECM) barriers toward their target, the inflammatory site, should respond to chemoattractant cytokines and to the degradation of ECM by specific enzymes. In this study, we examined the effects of RANTES and ECM proteins treated with human leukocyte elastase on T cell activation and adhesion to the ECM. We found that human peripheral blood T cells briefly suspended with RANTES (0.1-100 ng/ml) had increased phosphorylation of their intracellular extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase involved in the activation of several intracellular downstream effector molecules implicated in cell adhesion and migration. Consequently, a small portion (12-20%) of the responding cells adhered to fibronectin (FN). However, when the T cells were exposed to RANTES in the presence of native immobilized FN, laminin, or collagen type I, ERK phosphorylation was partially inhibited, suggesting that this form of the ECM proteins can down-regulate RANTES-induced intracellular signaling. In contrast, when the T cells were exposed to RANTES in the presence of elastase-treated immobilized FN, but not to elastase-treated laminin, ERK phosphorylation was markedly increased. Furthermore, a large percentage (30%) of RANTES-activated T cells adhered to the enzymatically treated FN in a beta1 integrin-dependent fashion. Thus, while migrating along chemotactic gradients within the ECM, T cells can adapt their adhesive performance according to the level of cleavage induced by enzymes to the matrix. PMID:11390457

  19. Functions of the Lyn tyrosine kinase in health and disease

    Directory of Open Access Journals (Sweden)

    Ingley Evan

    2012-07-01

    Full Text Available Abstract Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML, chronic myeloid leukaemia (CML and B-cell lymphocytic leukaemia (BCLL. Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. Lay Abstract To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information

  20. Interconversion of functional motions between mesophilic and thermophilic adenylate kinases.

    Directory of Open Access Journals (Sweden)

    Michael D Daily

    2011-07-01

    Full Text Available Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK, for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō simulation of mesophilic AK from E. coli (AKmeso to simulations of thermophilic AK from Aquifex aeolicus (AKthermo. In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A "heated" simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the "corresponding states" hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.

  1. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    Science.gov (United States)

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  2. Aplysia cell adhesion molecule and a novel protein kinase C activity in the postsynaptic neuron are required for presynaptic growth and initial formation of specific synapses

    OpenAIRE

    Hu, Jiang-Yuan; Chen, Yang; Bougie, Joanna K; Sossin, Wayne S.; Schacher, Samuel

    2010-01-01

    To explore the role of both Aplysia cell adhesion molecule (ApCAM) and activity of specific protein kinase C (PKC) isoforms in the initial formation of sensory neuron synapses with specific postsynaptic targets (L7 but not L11), we examined presynaptic growth, initial synapse formation, and the expression of the presynaptic neuropeptide sensorin following cell-specific reduction of ApCAM or of a novel PKC activity. Synapse formation between sensory neurons and L7 begins by 3 h after plating a...

  3. STUDY ON FUNCTION OF FOCAL ADHENSIVE KINASE AND INTEGRIN α1 IN HYPERTROPHIC SCAR FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    FU Min-gang; PING Ping; FAN Zhi-hong

    2008-01-01

    Objective To study the function of focal adhesion kinase (FAK) in the formation of hyper-trophic scar and its interrelationship with integrin α1. Methods Original fibroblasts from human hypertrophic scar and human normal dermis were cultured, and immanocytochemistry was applied to detect localization of expres-sion of FAK and integrin α1 in hypertrophic scar and human normal skin fibroblasts. The expression of integrin α1 was detected before and after FAK antibody blocking hypertrophic scar fibroblasts (HSFB) 48 h later. Meanwhile the collagen synthesis was evaluated by [3H] -proline incorporation and HSFB cell proliferation was measured by MTT method. Results The expression of FAK and integrin α1 of hypertrophic scar fibroblasts was higher than that of the normal skin fibroblasts significantly (P <0. 01). The expression of integrinct, was reduced after FAK be-ing blocked (P<0.01). Meanwhile the collagen synthesis of human scar-derived fibroblasts by [3H] -proline incor-poration was depressed respectively (P<0.01). The cell proliferation was inhibited by using 1:100 and 1:200 FAK antibody with MTT method (P<0.01). Conclusion FAK is the key point of signal transmission pathway medi-ated by integrin α1, which regulates protein synthesis of integrin α1, it may play an important role in the prolifera-tion and constriction of hypertrophic scar. FAK antibody can inhibit the collagen synthesis and cell proliferation of hypertrophic scar fibroblasts.

  4. The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

    DEFF Research Database (Denmark)

    Ditlevsen, Dorte K; Køhler, Lene B; Pedersen, Martin Volmer;

    2003-01-01

    kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating...... that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data...

  5. The role(s) of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function.

    Science.gov (United States)

    Horne, William C; Sanjay, Archana; Bruzzaniti, Angela; Baron, Roland

    2005-12-01

    The osteoclast resorbs mineralized bone during bone development, homeostasis, and repair. The deletion of the gene encoding the nonreceptor tyrosine kinase c-Src produces an osteopetrotic skeletal phenotype that is the consequence of the inability of the mature osteoclast to efficiently resorb bone. Src-/- osteoclasts exhibit reduced motility and abnormal organization of the apical secretory domain (the ruffled border) and attachment-related cytoskeletal elements that are necessary for bone resorption. A key function of Src in osteoclasts is to promote the rapid assembly and disassembly of the podosomes, the specialized integrin-based attachment structures of osteoclasts and other highly motile cells. Once recruited to the activated integrins, especially alphavbeta3), by the adhesion tyrosine kinase Pyk2, Src binds and phosphorylates Cbl and Cbl-b, homologous multisite adapter proteins with ubiquitin ligase activity. The Cbl proteins in turn recruit and activate additional signaling effectors, including phosphatidylinositol 3-kinase and dynamin, which play key roles in the development of cell polarity and the regulation of cell attachment and motility. In addition, Src and the Cbl proteins contribute to signaling cascades that are activated by several important receptors, including receptor activator of nuclear factor kappaB and the macrophage colony-stimulating factor receptor, and also downregulate the signaling from many of these receptors. PMID:16313344

  6. Catechol-Functionalized Synthetic Polymer as a Dental Adhesive to Contaminated Dentin Surface for a Composite Restoration

    OpenAIRE

    Lee, Sang-Bae; González-Cabezas, Carlos; Kim, Kwang-mahn; Kim, Kyoung-Nam; Kuroda, Kenichi

    2015-01-01

    This study reports a synthetic polymer functionalized with catechol groups as dental adhesives. We hypothesize that a catechol-functionalized polymer functions as a dental adhesive for wet dentin surfaces, potentially eliminating the complications associated with saliva contamination. We prepared a random copolymer containing catechol and methoxyethyl groups in the side chains. The mechanical and adhesive properties of the polymer to dentin surface in the presence of water and salivary compon...

  7. Glycan-functionalized diamond nanoparticles as potent E. coli anti-adhesives.

    Science.gov (United States)

    Barras, Alexandre; Martin, Fernando Ariel; Bande, Omprakash; Baumann, Jean-Sébastien; Ghigo, Jean-Marc; Boukherroub, Rabah; Beloin, Christophe; Siriwardena, Aloysius; Szunerits, Sabine

    2013-03-21

    Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with mannose moieties by a "click" chemistry approach, are able to efficiently inhibit E. coli type 1 fimbriae-mediated adhesion to eukaryotic cells with relative inhibitory potency (RIP) of as high as 9259 (bladder cell adhesion assay), which is unprecedented when compared with RIP values previously reported for alternate multivalent mannose-functionalized nanostructures designed to inhibit E. coli adhesion. Also remarkable is that these novel mannose-modified NDs reduce E. coli biofilm formation, a property previously not observed for multivalent glyco-nanoparticles and rarely demonstrated for other multivalent or monovalent mannose glycans. This work sets the stage for the further evaluation of these novel NDs as an anti-adhesive therapeutic strategy against E. coli-derived infections.

  8. Adaptive Shape Functions and Internal Mesh Adaptation for Modelling Progressive Failure in Adhesively Bonded Joints

    Science.gov (United States)

    Stapleton, Scott; Gries, Thomas; Waas, Anthony M.; Pineda, Evan J.

    2014-01-01

    Enhanced finite elements are elements with an embedded analytical solution that can capture detailed local fields, enabling more efficient, mesh independent finite element analysis. The shape functions are determined based on the analytical model rather than prescribed. This method was applied to adhesively bonded joints to model joint behavior with one element through the thickness. This study demonstrates two methods of maintaining the fidelity of such elements during adhesive non-linearity and cracking without increasing the mesh needed for an accurate solution. The first method uses adaptive shape functions, where the shape functions are recalculated at each load step based on the softening of the adhesive. The second method is internal mesh adaption, where cracking of the adhesive within an element is captured by further discretizing the element internally to represent the partially cracked geometry. By keeping mesh adaptations within an element, a finer mesh can be used during the analysis without affecting the global finite element model mesh. Examples are shown which highlight when each method is most effective in reducing the number of elements needed to capture adhesive nonlinearity and cracking. These methods are validated against analogous finite element models utilizing cohesive zone elements.

  9. Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence.

    Science.gov (United States)

    Smith, M Shane; Bivins-Smith, Elizabeth R; Tilley, A Michael; Bentz, Gretchen L; Chan, Gary; Minard, Jessica; Yurochko, Andrew D

    2007-07-01

    Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.

  10. Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

    Science.gov (United States)

    Mohanty, Smita; Oruganty, Krishnadev; Kwon, Annie; Byrne, Dominic P; Ferries, Samantha; Ruan, Zheng; Hanold, Laura E; Katiyar, Samiksha; Kennedy, Eileen J; Eyers, Patrick A; Kannan, Natarajan

    2016-02-01

    Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they

  11. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    Energy Technology Data Exchange (ETDEWEB)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A. [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States); Nusrat, Asma, E-mail: anusrat@emory.edu [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States)

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  12. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    International Nuclear Information System (INIS)

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion

  13. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Directory of Open Access Journals (Sweden)

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  14. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    Directory of Open Access Journals (Sweden)

    Wang HY

    2016-05-01

    Full Text Available Hsian-Yu Wang,1,2 Min-Kung Hsu,3,4 Kai-Hsuan Wang,1 Ching-Ping Tseng,2,4 Feng-Chi Chen,3,4 John T-A Hsu1,4 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 2Institute of Molecular Medicine and Bioengineering, National Chiao Tung University (NCTU, Hsinchu, 3Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 4Department of Biological Science and Technology, National Chiao Tung University (NCTU, Hsinchu, Taiwan, Republic of China Background: Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.Results: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits

  15. Cooperation between c-Met and focal adhesion kinase family members in medulloblastoma and implications for therapy

    OpenAIRE

    Guessous, Fadila; Yang, Yanzhi; Johnson, Elizabeth; Marcinkiewicz, Lukasz; Smith, Matthew; Zhang, Ying; Kofman, Alexander; Schiff, David; Christensen, James; Abounader, Roger

    2011-01-01

    We previously demonstrated the involvement of the tyrosine kinase receptor c-Met in medulloblastoma malignancy. The nonreceptor tyrosine kinases FAK and Pyk2, are key players in the progression of different cancers. However, their role in medulloblastoma malignancy is not well understood. In this study, using a protein array approach, we found that c-Met phosphorylates FAK and Pyk2 in medulloblastoma cells. We therefore studied the interactions between c-Met and FAK/Pyk2 and their implication...

  16. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    Science.gov (United States)

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  17. Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

    Directory of Open Access Journals (Sweden)

    Anthony John Walker

    2014-07-01

    Full Text Available Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavour, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behaviour, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis.

  18. Molecular cloning and chemical synthesis of a region of platelet glycoprotein IIb involved in adhesive function.

    OpenAIRE

    Loftus, J C; Plow, E F; Frelinger, A.L.; D'Souza, S E; Dixon, D; Lacy, J.; Sorge, J; Ginsberg, M H

    1987-01-01

    Membrane glycoprotein (GP) IIb-IIIa is a component of a platelet adhesive protein receptor. A region of the heavy chain of GPIIb, defined by the monoclonal antibody PMI-1, is involved in adhesion receptor function. We have localized and chemically synthesized this region of GPIIb. A cDNA clone that directs the synthesis of a fusion protein reactive with the PMI-1 antibody was isolated from a phage lambda gt11 expression library constructed with mRNA from an erythroleukemia (HEL) cell line. Th...

  19. Cell Adhesion Selectivity of Stent Material to improve Bio-functionality by Ion Beam Modification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jaesang; Park, JUngchan; Jung, Myunghwan; Kim, Yongki [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Park, Junkyu [Bio alpha., Co. Ltd., Gimhae (Korea, Republic of)

    2014-05-15

    In this study, ion implantation into collagen coated Co-Cr alloy, which is a cheaper material of the artificial stent product comparing with Ti alloy, has been studied to develop small diameter artificial stent by the cell adhesion control. The size of stent was 1.6mm of the diameter and 18mm of the length. The life-time of artificial stent depends on adhesion property of endothelial-cells. We successfully controlled cell adhesion selectivity between endothelial cell and muscle cell by using collagen coated and He{sup +} ion beam irradiated Co-Cr-alloy to apply to artificial stent. But, we did not achieve the inhibition of platelet adhesion, yet by using collagen coating and He{sup +} ion beam irradiation. Based on this study, we have plan to research about separation between collagen coating effect and ion beam effect. Also, we will have more detail analysis of the mechanism of cell attachment. In recent years, ion implantation has been applied to the surface modification of prosthesis to improve blood compatibility and tissue compatibility in field of biomedical application. As well known, bio compatibility was concerned with the cell adhesion selectivity for bio-functionality. The biomedical application of ion beam technology would be used more widely in the future such as catheter and artificial graft.

  20. Cyclin-Dependent Kinase-Like Function Is Shared by the Beta- and Gamma- Subset of the Conserved Herpesvirus Protein Kinases

    OpenAIRE

    Kuny, Chad V.; Karen Chinchilla; Culbertson, Michael R.; Kalejta, Robert F.

    2010-01-01

    The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been...

  1. Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    Directory of Open Access Journals (Sweden)

    Ren-You Gan

    2014-09-01

    Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

  2. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

    Institute of Scientific and Technical Information of China (English)

    LIN Chun-long; ZHANG Zhen-xiang; XU Yong-jian; NI Wang; CHEN Shi-xin

    2005-01-01

    Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group

  3. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N;

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  4. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

    Directory of Open Access Journals (Sweden)

    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  5. MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.

    Science.gov (United States)

    Madhani, H D; Styles, C A; Fink, G R

    1997-11-28

    Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK. PMID:9393860

  6. A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei.

    Science.gov (United States)

    Hu, Huiqing; Zhou, Qing; Li, Ziyin

    2015-10-01

    The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.

  7. Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

    Institute of Scientific and Technical Information of China (English)

    Urai Pongchairerk; Jun-Lin Guan; Vijittra Leardkamolkarn

    2005-01-01

    AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

  8. STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF MUCUS ADHESION PROTEINS OF LACTOBACILLUS REUTERI

    OpenAIRE

    Etzold, Sabrina

    2013-01-01

    Mucus is the first point of contact between the gut microbiota and the host. Mucus adhesins are thought to be key mediators in the mucus adhesion of commensal Lactobacillus species. However, knowledge on the structural or functional basis of adhesin interaction with mucin glycoproteins, the main component of mucus, is limited. This work describes the biochemical and structural properties of two cell-surface proteins from Lactobacillus reuteri, the mucus-binding protein (MUB) and the Lar0958 p...

  9. Synthesis and Cell Adhesive Properties of Linear and Cyclic RGD Functionalized Polynorbornene Thin Films

    OpenAIRE

    Patel, Paresma R.; Kiser, Rosemary Conrad; Lu, Ying Y.; Fong, Eileen; Ho, Wilson C.; Tirrell, David A.; Grubbs, Robert H.

    2012-01-01

    Described herein is the efficient synthesis and evaluation of bioactive arginine-glycine-aspartic acid (RGD) functionalized polynorbornene-based materials for cell adhesion and spreading. Polynorbornenes containing either linear or cyclic RGD peptides were synthesized by ring-opening metathesis polymerization (ROMP) using the well-defined ruthenium initiator [(H_(2)IMes)(pyr)_(2)(Cl)_(2)Ru═CHPh]. The random copolymerization of three separate norbornene monomers allowed for the incorporation o...

  10. The direct effect of Focal Adhesion Kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    International Nuclear Information System (INIS)

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA. Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal specific expression of genes affected by

  11. The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhang Li

    2009-08-01

    Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

  12. Selective disruption of aurora C kinase reveals distinct functions from aurora B kinase during meiosis in mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Ahmed Z Balboula

    2014-02-01

    Full Text Available Aurora B kinase (AURKB is the catalytic subunit of the chromosomal passenger complex (CPC, an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I. We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.

  13. The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

    DEFF Research Database (Denmark)

    Ditlevsen, Dorte K; Køhler, Lene B; Pedersen, Martin V;

    2003-01-01

    kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that...... indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be...

  14. A Site-Specific Phosphorylation of the Focal Adhesion Kinase Controls the Formation of Spheroid Cell Clusters

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Gosau, Martin; Kristensen, Lars Peter;

    2014-01-01

    Human dental follicle cells (DFCs) are ectomesenchymal multipotent stem cells that form spheroid cell clusters (SCCs) under serum free medium cell culture conditions (SFM). Until today, molecular mechanisms for the formation of SCCs are unknown. In this study a quantitative phosphoproteomics...... kinase (FAK) signaling pathway. In addition to the phosphoproteomics approach we showed that a specific phosphorylation of FAK (Y397) was required for the formation of SCCs. In conclusion, this study disclosed the phosphoproteome of SCCs for the first time and showed that the FAK signaling pathway...

  15. In Vitro Functional Study of Rice Adenosine 5'-Phosphosulfate Kinase

    Institute of Scientific and Technical Information of China (English)

    WANG De-zhen; CHEN Guo-guo; LU Lu-jia; JIANG Zhao-jun; RAO Yu-chun; SUN Mei-hao

    2016-01-01

    Sulfate can be activated by ATP sulfurylase and adenosine 5'-phosphosulfate kinase (APSK)in vivo. Recent studies suggested that APSK inArabidopsis thaliana regulated the partition between APS reduction and phosphorylation and its activity can be modulated by cellular redox status. In order to study regulation of APSK in rice (OsAPSK),OsAPSK1 gene was cloned and its activity was analyzed. OsAPSK1 C36 and C69 were found to be the conserved counterparts of C86 and C119, which involved in disulfide formation in AtAPSK.C36A/C69A OsAPSK1 double mutation was made by site directed mutagenesis. OsAPSK1 and its mutant were prokaryotically over-expressed and purified, and then assayed for APS phosphorylation activity. OsAPSK1 activity was depressed by oxidized glutathione, while the activity of its mutantwas not. Further studies in the case that oxidative stress will fluctuatein vivo3'-phosphoadenosine-5'-phosphosulfate content, and all APSK isoenzymes have similar regulation patterns are necessary to be performed.

  16. Substrate-specific reorganization of the conformational ensemble of CSK implicates novel modes of kinase function.

    Directory of Open Access Journals (Sweden)

    Michael A Jamros

    Full Text Available Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.

  17. Abl family kinases regulate endothelial barrier function in vitro and in mice.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Chislock

    Full Text Available The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg, as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR, Kit, colony stimulating factor 1 receptor (CSF1R, and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use

  18. Functional morphology and adhesive performance of the stick-capture apparatus of the rove beetles Stenus spp. (Coleoptera, Staphylinidae).

    Science.gov (United States)

    Koerner, Lars; Gorb, Stanislav N; Betz, Oliver

    2012-04-01

    The adhesive prey-capture apparatus of the representatives of the rove beetle genus Stenus (Coleoptera, Staphylinidae) is an outstanding example of biological adhesive systems. This unique prey-capture device is used for catching elusive prey by combining (i) hierarchically structured adhesive outgrowths, (ii) an adhesive secretion, and (iii) a network of cuticular fibres within the pad. The outgrowths arise from a pad-like cuticle and are completely immersed within the secretion. To date, the forces generated during the predatory strike of these beetles have only been estimated theoretically. In the present study, we used force transducers to measure both the compressive and adhesive forces during the predatory strike of two Stenus species. The experiments revealed that the compressive forces are low, ranging from 0.10 mN (Stenus bimaculatus) to 0.18 mN (Stenus juno), whereas the corresponding adhesive forces attain up to 1.0 mN in S. juno and 1.08 mN in S. bimaculatus. The tenacity or adhesive strength (adhesive force per apparent unit area) amounts to 51.9 kPa (S. bimaculatus) and 69.7 kPa (S. juno). S. juno beetles possess significantly smaller pad surface areas than S. bimaculatus but seem to compensate for this disadvantage by generating higher compressive forces. Consequently, S. juno beetles reach almost identical adhesive properties and an equal prey-capture success in attacks on larger prey. The possible functions of the various parts of the adhesive system during the adhesive prey-capture process are discussed in detail.

  19. Chemical functionalization of ceramic tile surfaces by silane coupling agents: polymer modified mortar adhesion mechanism implications

    Directory of Open Access Journals (Sweden)

    Alexandra Ancelmo Piscitelli Mansur

    2008-09-01

    Full Text Available Adhesion between tiles and mortars are crucial to the stability of ceramic tile systems. From the chemical point of view, weak forces such as van der Waals forces and hydrophilic interactions are expected to be developed preferably at the tiles and polymer modified Portland cement mortar interface. The main goal of this paper was to use organosilanes as primers to modify ceramic tile hydrophilic properties to improve adhesion between ceramic tiles and polymer modified mortars. Glass tile surfaces were treated with several silane derivatives bearing specific functionalities. Contact angle measurements and Fourier Transform Infrared Spectroscopy (FTIR were used for evaluating the chemical changes on the tile surface. In addition, pull-off tests were conducted to assess the effect on adhesion properties between tile and poly(ethylene-co-vinyl acetate, EVA, modified mortar. The bond strength results have clearly shown the improvement of adherence at the tile-polymer modified mortar interface, reflecting the overall balance of silane, cement and polymer interactions.

  20. Silencing HIF-1α reduces the adhesion and secretion functions of acute leukemia hBMSCs

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Dong-Feng [Department of Hematology, XinQiao Hospital, Third Military Medical University, ChongQing (China); Liu, Ting [Department of Ophthalmology, DaPing Hospital, Third Military Medical University, ChongQing (China); Chang, Cheng; Zhang, Xi; Liang, Xue; Chen, Xing-Hua; Kong, Pei-Yan [Department of Hematology, XinQiao Hospital, Third Military Medical University, ChongQing (China)

    2012-06-29

    Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenvironment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1α secreted by stromal cells was decreased. When HIF-1α was blocked, the co-cultured Jurkat cell's adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

  1. Protein kinase inhibitors CK59 and CID755673 alter primary human NK cell effector functions

    Directory of Open Access Journals (Sweden)

    Maxi eScheiter

    2013-03-01

    Full Text Available Natural killer (NK cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory theraphies. In this study, we have tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against CaMKII (CK59 and PKD family kinases (CID755673 that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Dose dependent treatment with CK59 and CID755673 indeed results in a significant reduction of NK cell degranulation markers and cytokine release in freshly isolated PBMC populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest PKD2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.

  2. Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

    Directory of Open Access Journals (Sweden)

    Naadiya Carrim

    Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

  3. PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

    Science.gov (United States)

    Madaro, Luca; Marrocco, Valeria; Fiore, Piera; Aulino, Paola; Smeriglio, Piera; Adamo, Sergio; Molinaro, Mario; Bouché, Marina

    2011-01-01

    Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKC

  4. Human cell adhesion molecules: annotated functional subtypes and overrepresentation of addiction-associated genes.

    Science.gov (United States)

    Zhong, Xiaoming; Drgonova, Jana; Li, Chuan-Yun; Uhl, George R

    2015-09-01

    Human cell adhesion molecules (CAMs) are essential for proper development, modulation, and maintenance of interactions between cells and cell-to-cell (and matrix-to-cell) communication about these interactions. Despite the differential functional significance of these roles, there have been surprisingly few systematic studies to enumerate the universe of CAMs and identify specific CAMs in distinct functions. In this paper, we update and review the set of human genes likely to encode CAMs with searches of databases, literature reviews, and annotations. We describe likely CAMs and functional subclasses, including CAMs that have a primary function in information exchange (iCAMs), CAMs involved in focal adhesions, CAM gene products that are preferentially involved with stereotyped and morphologically identifiable connections between cells (e.g., adherens junctions, gap junctions), and smaller numbers of CAM genes in other classes. We discuss a novel proposed mechanism involving selective anchoring of the constituents of iCAM-containing lipid rafts in zones of close neuronal apposition to membranes expressing iCAM binding partners. We also discuss data from genetic and genomic studies of addiction in humans and mouse models to highlight the ways in which CAM variation may contribute to a specific brain-based disorder such as addiction. Specific examples include changes in CAM mRNA splicing mediated by differences in the addiction-associated splicing regulator RBFOX1/A2BP1 and CAM expression in dopamine neurons. PMID:25988664

  5. Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase.

    Science.gov (United States)

    Ng, Ho Yin; Oliver, Brian Gregory George; Burgess, Janette Kay; Krymskaya, Vera P; Black, Judith Lee; Moir, Lyn M

    2015-11-01

    Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction. PMID:26282580

  6. Adhesion of alkane as a functional group on muscovite and quartz

    DEFF Research Database (Denmark)

    Juhl, Klaus; Pedersen, Christian Schack; Bovet, Nicolas Emile;

    2014-01-01

    The interactions between mineral surfaces and organic molecules in water control many processes in nature and in the production of modern materials. To improve the understanding of fluid-surface interactions, we investigated the interface behavior of quartz and muscovite, a model for clay minerals...... experiments and DLVO calculations, we were able to determine the surface charge density. We observed increased adhesion between the mineral surface and the hydrophobic tips as the contact time increased from 7 ms to ?2 s. The di ffusion of dissolved ions takes time, and density functional theory (DFT...

  7. The FGGY carbohydrate kinase family: insights into the evolution of functional specificities.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2011-12-01

    Full Text Available Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose. Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families.

  8. GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass

    OpenAIRE

    Menon, Prashanthi; Yin, Guoyong; Smolock, Elaine M.; Zuscik, Michael J.; Yan, Chen; Berk, Bradford C.

    2010-01-01

    G-protein coupled receptor (GPCR) kinase 2 interacting protein-1 (GIT1) is a scaffold protein expressed in various cell types including neurons, endothelial and vascular smooth muscle cells. The GIT1 knockout (KO) mouse has a pulmonary phenotype due to impaired endothelial function. Because GIT1 is tyrosine phosphorylated by Src kinase, we anticipated that GIT1 KO should have a bone phenotype similar to Src KO. Microcomputed tomography of the long bones revealed that GIT1 KO mice have a 2.3-f...

  9. Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion

    Science.gov (United States)

    Heuslein, Joshua L.; Murrell, Kelsey P.; Leiphart, Ryan J.; Llewellyn, Ryan A.; Meisner, Joshua K.; Price, Richard J.

    2016-05-01

    Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase’s (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6Chi and Ly6Clo blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

  10. Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-07-01

    The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion.

  11. Function and regulation of Aurora/Ipl1p kinase family in cell division

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p fanily,a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer.

  12. Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

    International Nuclear Information System (INIS)

    Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells

  13. Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Boulatnikov, Igor; Popov, Kirill M

    2003-02-21

    Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general. In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4). These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated. Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell. The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography. The purified kinase was stable and was not subjected to reassortment of the subunits. The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation. These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity. PMID:12573248

  14. Expression of focal adhesion kinase and α5 and β1 integrins in carcinomas and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Su; Lu Gui; Yi-Ping Zhou; Xi-Liang Zha

    2002-01-01

    AIM: To detect the expression pattern of FAK (focaladhesion kinase) and integrin α5 and β1 subunits indifferent kinds of cancerous tissues and to study theircorrelation with clinicopathological data includingtumor type, grade and lymph node status. METHODS:Using an immunohistochemical technique, weexamined the expression of FAK and integrin andsubunits in cancerous and noncancerous tissuesobtained from 75 patients with gastric carcinomas, 21colorectal carcinomas, 16 hepatocellular carcinomas,20 uterocervical carcinomas, and 20 breast carcinomas.RESULTS: The staining of FAK was stronger in cancerousthan in noncancerous areas. Enhanced expression ofFAKwas detected in poor-differentiated carcinoma ofthe stomach and colorectum. Tumors with lymph nodemetastases had more FAK protein than those withoutmetastases. In addition, the deeper the extent of tumorinfiltration, the higher the FAK expression. Theexpression of integrin α5 and β1 subunits was lower incancerous areas than in noncancerous areas, but it washigher in well-differentiated cancerous tissues than inpoor differentiated tissues. The relationship betweenthe expression of integrin α5 and β1 subunits andinfiltration or metastasis was not significant. Cancerous tissues with stronger FAK expression (++ or +++) alsohad a higher expression of integrin α5 and β1 subunitsin the tumor and its unaffected margins.CONCLUSION: FAK is a better marker for carcinogenesisand the progression of cancer than integrin α5 or β1subunit, and it may be not only a transformation-linkedenzyme but also a progression-linked enzyme.

  15. Identification of interphase functions for the NIMA kinase involving microtubules and the ESCRT pathway.

    Directory of Open Access Journals (Sweden)

    Meera Govindaraghavan

    2014-03-01

    Full Text Available The Never in Mitosis A (NIMA kinase (the founding member of the Nek family of kinases has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell

  16. A small physiological electric field mediated responses of extravillous trophoblasts derived from HTR8/SVneo cells: involvement of activation of focal adhesion kinase signaling.

    Directory of Open Access Journals (Sweden)

    Juan Zhang

    Full Text Available Moderate invasion of trophoblast cells into endometrium is essential for the placental development and normal pregnancy. Electric field (EF-induced effects on cellular behaviors have been observed in many cell types. This study was to investigate the effect of physiological direct current EF (dc EF on cellular responses such as elongation, orientation and motility of trophoblast cells. Immortalized first trimester extravillous trophoblast cells (HTR-8/SVneo were exposed to the dc EF at physiological magnitude. Cell images were recorded and analyzed by image analyzer. Cell lysates were used to detect protein expression by Western blot. Cultured in the dc EFs the cells showed elongation, orientation and enhanced migration rate compared with non-EF stimulated cells at field strengths of 100 mV/mm to 200 mV/mm. EF exposure increased focal adhesion kinase (FAK phosphorylation in a time-dependent manner and increased expression levels of MMP-2. Pharmacological inhibition of FAK impaired the EF-induced responses including motility and abrogated the elevation of MMP-2 expression. However, the expression levels of integrins like integrin α1, α5, αV and β1 were not affected by EF stimulation. Our results demonstrate the importance of FAK activation in migration/motility of trophobalst cells driven by EFs. In addition, it raises the feasibility of using applied EFs to promote placentation through effects on trophoblast cells.

  17. Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Bin [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@hotmail.com [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China); Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Lu, Jian; Zhang, Li; Zhao, Miao; Shi, Changcan; Khan, Musammir [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Guo, Jintang [School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China)

    2013-07-01

    In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices. - Highlights: • MPC was successfully grafted onto polycarbonate urethane surface via Michael reaction. • High concentration of PC functional groups on the surface via TAEA molecule • Biomimetic surface modification • The modified surface showed high hydrophilicity and anti-platelet adhesion.

  18. KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

    Energy Technology Data Exchange (ETDEWEB)

    Goettel, Jeremy A. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Liang, Dongchun [Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Gould, Kathleen L. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Hanks, Steven K. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Polk, D. Brent, E-mail: dbpolk@chla.usc.edu [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)

    2011-02-15

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1{sup -/-} colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

  19. Selective adhesion of intestinal epithelial cells on patterned films with amine functionalities formed by plasma enhanced chemical vapor deposition

    International Nuclear Information System (INIS)

    Control of cell adhesion to surfaces is important to develop analytical tools in the areas of biomedical engineering. To control cell adhesiveness of the surface, we constructed a variety of plasma polymerized hexamethyldisiloxane (PPHMDSO) thin films deposited at the plasma power range of 10-100 W by plasma enhanced chemical vapor deposition (PECVD). The PPHMDSO film that was formed at 10 W was revealed to be resistant to cell adhesion. The resistance to cell adhesion is closely related to physicochemical properties of the film. Atomic force microscopic data show an increase in surface roughness from 0.52 nm to 0.74 nm with increasing plasma power. From Fourier transform infrared (FT-IR) absorption spectroscopy data, it was also determined that the methyl (-CH3) peak intensity increases with increasing plasma power, whereas the hydroxyl (-OH) peak decreases. X-ray photoelectron spectroscopy data reveal an increase in C-O bonding with increasing plasma power. These results suggest that C-O bonding and hydroxyl (-OH) and methyl (-CH3) functional groups play a critical part in cell adhesion. Furthermore, to enhance a diversity of film surface, we accumulated the patterned plasma polymerized ethylenediamine (PPEDA) thin film on the top of the PPHMDSO thin film. The PPEDA film is established to be strongly cell-adherent. This patterned two-layer film stacking method can be used to form the selectively limited cell-adhesive PPEDA spots over the adhesion-resistant surface.

  20. Selective adhesion of intestinal epithelial cells on patterned films with amine functionalities formed by plasma enhanced chemical vapor deposition

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung Seop; Choi, Changrok; Kim, Soo Heon; Choi, Kun oh [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Kim, Jeong Min [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Kim, Hong Ja [Department of Internal Medicine, Dankook University College of Medicine, Cheonan 330-715 (Korea, Republic of); Yeo, Sanghak [R and D Center, ELBIO Incorporation, 426-5 Gasan-dong Geumchun-gu, Seoul (Korea, Republic of); Park, Heonyong [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Jung, Donggeun, E-mail: djung@skku.ac.kr [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2010-11-01

    Control of cell adhesion to surfaces is important to develop analytical tools in the areas of biomedical engineering. To control cell adhesiveness of the surface, we constructed a variety of plasma polymerized hexamethyldisiloxane (PPHMDSO) thin films deposited at the plasma power range of 10-100 W by plasma enhanced chemical vapor deposition (PECVD). The PPHMDSO film that was formed at 10 W was revealed to be resistant to cell adhesion. The resistance to cell adhesion is closely related to physicochemical properties of the film. Atomic force microscopic data show an increase in surface roughness from 0.52 nm to 0.74 nm with increasing plasma power. From Fourier transform infrared (FT-IR) absorption spectroscopy data, it was also determined that the methyl (-CH{sub 3}) peak intensity increases with increasing plasma power, whereas the hydroxyl (-OH) peak decreases. X-ray photoelectron spectroscopy data reveal an increase in C-O bonding with increasing plasma power. These results suggest that C-O bonding and hydroxyl (-OH) and methyl (-CH{sub 3}) functional groups play a critical part in cell adhesion. Furthermore, to enhance a diversity of film surface, we accumulated the patterned plasma polymerized ethylenediamine (PPEDA) thin film on the top of the PPHMDSO thin film. The PPEDA film is established to be strongly cell-adherent. This patterned two-layer film stacking method can be used to form the selectively limited cell-adhesive PPEDA spots over the adhesion-resistant surface.

  1. Nonmetabolic functions of pyruvate kinase isoform M2 in controlling cell cycle progression and tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Zhimin Lu

    2012-01-01

    Pyruvate kinase catalyzes the rate-limiting final step of glycolysis,generating adenosine triphosphate (ATP) and pyruvate.The M2 tumor-specific isoform of pyruvate kinase (PKM2) promotes glucose uptake and lactate production in the presence of oxygen,known as aerobic glycolysis or the Warburg effect.As recently reported in Nature,PKM2,besides its metabolic function,has a nonmetabolic function in the direct control of cell cycle progression by activating β-catenin and inducing expression of the β-catenin downstream gene CCND1 (encoding for cyclin D1).This nonmetabolic function of PKM2 is essential for epidermal growth factor receptor (EGFR) activation-induced tumorigenesis.

  2. Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Sandra Franz; Britta Ehlert; Anja Liese; Joachim Kurth; Anne-Claire Cazalé; Tina Romeis

    2011-01-01

    Calcium-dependent protein kinases(CDPKs)comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule.So far,a biological function in abiotic stress signaling has only been reported for few CDPK isoforms,whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown.Here,we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress.Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation.In transgenic Arabidopsis complementation lines in the cpk21 mutant background,in which either CPK21 wildtype,or a full-length enzyme variant carrying an amino-acid substitution were stably expressed,stress responsitivity was restored by CPK21 but not with the kinase inactive variant.The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain,N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity,suggesting a crucial role for the N-terminal EF-hand pair.Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

  3. Functional expression of adhesive peptides as fusions to Escherichia coli flagellin.

    Science.gov (United States)

    Westerlund-Wikström, B; Tanskanen, J; Virkola, R; Hacker, J; Lindberg, M; Skurnik, M; Korhonen, T K

    1997-11-01

    An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins. PMID:9514121

  4. Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

    Science.gov (United States)

    Dormond, Olivier; Ponsonnet, Lionel; Hasmim, Meriem; Foletti, Alessandro; Rüegg, Curzio

    2004-07-01

    Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

  5. Reversing adhesion with light: a general method for functionalized bead release from cells.

    Science.gov (United States)

    Goulet-Hanssens, Alexis; Magdesian, Margaret H; Lopez-Ayon, G Monserratt; Grutter, Peter; Barrett, Christopher J

    2016-07-19

    Coated beads retain great importance in the study of cell adhesion and intracellular communication; we present a generally applicable method permitting spatiotemporal control of bead adhesion from cells. Herein we demonstrate in vitro release of a poly-d-lysine (PDL) layer from anionic polystyrene beads, allowing complete bead release from rat cortical neurons post-adhesion. PMID:27165466

  6. Advanced adhesives in electronics

    CERN Document Server

    Bailey, C

    2011-01-01

    Adhesives are widely used in the manufacture of electronic devices to act as passive and active components. Recently there has been considerable interest in the use of conductive adhesives. This book reviews key types of conductive adhesives, processing methods, properties and the way they can be modelled as well as potential applications.$bAdhesives for electronic applications serve important functional and structural purposes in electronic components and packaging, and have developed significantly over the last few decades. Advanced adhesives in electronics reviews recent developments in adhesive joining technology, processing and properties. The book opens with an introduction to adhesive joining technology for electronics. Part one goes on to cover different types of adhesive used in electronic systems, including thermally conductive adhesives, isotropic and anisotropic conductive adhesives and underfill adhesives for flip-chip applications. Part two focuses on the properties and processing of electronic ...

  7. Changes in focal adhesion kinase expression in rats with collagen-induced arthritis and efficacy of intervention with disease modifying anti-rheumatic drugs alone or in combination.

    Science.gov (United States)

    Gao, Hui-Ying; Luo, Jing; Li, Xiao-Feng; Lv, Qian; Wen, Hong-Yan; Song, Qing-Zhen; Zhao, Wen-Peng; Zhao, Xiang-Cong; Zhang, Ting-Ting; Zhang, Si-Yu; Zhi, Jian-Ming

    2015-01-01

    Focal adhesion kinase (FAK) is known to promote the proliferation, migration and survival of synovial cells and plays an important role in the occurrence, development and pathological process of rheumatoid arthritis (RA). The aim of the present study was to observe FAK changes in synovial cells of rats with collagen-induced arthritis (CIA) and after intervention with disease modifying anti-rheumatic drugs (DMARDs) alone or in combination in a CIA female SD rat model induced by collagen type II. The rats were randomized to 8 groups: normal control group, CIA model control group, methotrexate (MTX, 0.9 mg/kg/w) group, cyclophosphamide (CTX, 24 mg/kg/3 w) group, leflunomide (LEF, 1.2 mg/kg/d) group, MTX + CTX group, LEF + CTX group, and MTX + LEF group. They were intervened with DMARDs alone or in combination for six weeks. The experiment lasted a total of 9 weeks in vivo. Articular inflammation was measured during the process of drug intervention in terms of the degree of swelling degree in the right hind foot using a venire caliper. All animals were sacrificed by breaking the neck after 9 weeks. Then, the ankle was fixed, decalcified, embedded, and HE stained, and prepared into slices to observe pathological changes in the synovial tissue. FAK expression in synovial cells was assayed by immunohistochemistry and the mean optical density (OD) value was measured using the HPIAS-2000 image analysis system. It was found that FAK expression was negative in normal control group, positive in CIA model control group, and decreased in the three DMARD combination treatment groups significantly as compared with that in the three single-drug groups (P < 0.05). FAK expression in LEF + CTX group or MTX + CTX group decreased more significantly than that in MTX + LEF group (P < 0.05), and there was no statistically significant difference between LEF + CTX and MTX + CTX groups. The arthritis index and pathological change in the synovial tissue in LEF + CTX group or MTX + CTX group

  8. Small Molecule Agonists of Cell Adhesion Molecule L1 Mimic L1 Functions In Vivo.

    Science.gov (United States)

    Kataria, Hardeep; Lutz, David; Chaudhary, Harshita; Schachner, Melitta; Loers, Gabriele

    2016-09-01

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery after injury, leading to severe disabilities in motor functions and pain. Peripheral nerve injury impairs motor, sensory, and autonomic functions, particularly in cases where nerve gaps are large and chronic nerve injury ensues. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration after acute injury. We screened libraries of known drugs for small molecule agonists of L1 and evaluated the effect of hit compounds in cell-based assays in vitro and in mice after femoral nerve and spinal cord injuries in vivo. We identified eight small molecule L1 agonists and showed in cell-based assays that they stimulate neuronal survival, neuronal migration, and neurite outgrowth and enhance Schwann cell proliferation and migration and myelination of neurons in an L1-dependent manner. In a femoral nerve injury mouse model, enhanced functional regeneration and remyelination after application of the L1 agonists were observed. In a spinal cord injury mouse model, L1 agonists improved recovery of motor functions, being paralleled by enhanced remyelination, neuronal survival, and monoaminergic innervation, reduced astrogliosis, and activation of microglia. Together, these findings suggest that application of small organic compounds that bind to L1 and stimulate the beneficial homophilic L1 functions may prove to be a valuable addition to treatments of nervous system injuries. PMID:26253722

  9. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Grant A Howe

    Full Text Available Blockade of epidermal growth factor receptor (EGFR activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC. As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs, there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975 were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271 both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be

  10. Catechol-Functionalized Synthetic Polymer as a Dental Adhesive to Contaminated Dentin Surface for a Composite Restoration.

    Science.gov (United States)

    Lee, Sang-Bae; González-Cabezas, Carlos; Kim, Kwang-Mahn; Kim, Kyoung-Nam; Kuroda, Kenichi

    2015-08-10

    This study reports a synthetic polymer functionalized with catechol groups as dental adhesives. We hypothesize that a catechol-functionalized polymer functions as a dental adhesive for wet dentin surfaces, potentially eliminating the complications associated with saliva contamination. We prepared a random copolymer containing catechol and methoxyethyl groups in the side chains. The mechanical and adhesive properties of the polymer to dentin surface in the presence of water and salivary components were determined. It was found that the new polymer combined with an Fe(3+) additive improved bond strength of a commercial dental adhesive to artificial saliva contaminated dentin surface as compared to a control sample without the polymer. Histological analysis of the bonding structures showed no leakage pattern, probably due to the formation of Fe-catechol complexes, which reinforce the bonding structures. Cytotoxicity test showed that the polymers did not inhibit human gingival fibroblast cells proliferation. Results from this study suggest a potential to reduce failure of dental restorations due to saliva contamination using catechol-functionalized polymers as dental adhesives.

  11. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Directory of Open Access Journals (Sweden)

    Lorna S Kategaya

    Full Text Available Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite with external cues (e.g., daylight and food. In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2 as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

  12. Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

    Science.gov (United States)

    Wagner, Florence F; Bishop, Joshua A; Gale, Jennifer P; Shi, Xi; Walk, Michelle; Ketterman, Joshua; Patnaik, Debasis; Barker, Doug; Walpita, Deepika; Campbell, Arthur J; Nguyen, Shannon; Lewis, Michael; Ross, Linda; Weïwer, Michel; An, W Frank; Germain, Andrew R; Nag, Partha P; Metkar, Shailesh; Kaya, Taner; Dandapani, Sivaraman; Olson, David E; Barbe, Anne-Laure; Lazzaro, Fanny; Sacher, Joshua R; Cheah, Jaime H; Fei, David; Perez, Jose; Munoz, Benito; Palmer, Michelle; Stegmaier, Kimberly; Schreiber, Stuart L; Scolnick, Edward; Zhang, Yan-Ling; Haggarty, Stephen J; Holson, Edward B; Pan, Jen Q

    2016-07-15

    The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders. PMID:27128528

  13. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Science.gov (United States)

    Kategaya, Lorna S; Hilliard, Aisha; Zhang, Louying; Asara, John M; Ptáček, Louis J; Fu, Ying-Hui

    2012-01-01

    Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

  14. Roles of LIM kinases in central nervous system function and dysfunction.

    Science.gov (United States)

    Cuberos, H; Vallée, B; Vourc'h, P; Tastet, J; Andres, C R; Bénédetti, H

    2015-12-21

    LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin. In this review, we outline studies that have shown an involvement of LIMKs in neuronal function and we detail some of the pathways and molecular mechanisms involving LIMKs in neurodevelopment and synaptic plasticity. We also review the involvement of LIMKs in neuronal diseases and emphasize the differences in the regulation of LIMKs expression and mode of action. We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

  15. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

    OpenAIRE

    Angelopoulos, Nicos; Stebbing, Justin; Xu, Yichen; Giamas, Georgios; Hua ZHANG

    2016-01-01

    Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling netw...

  16. Mechanisms of regulation and function of G-protein-coupled receptor kinases

    Institute of Scientific and Technical Information of China (English)

    Wen Yang; Shi-Hai Xia

    2006-01-01

    G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptor (GPCR) to affect receptor phosphorylation and to initiate profound impairment of receptor signaling,or desensitization. GPCR forms the largest family of cell surface receptors, and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations.

  17. Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity.

    Science.gov (United States)

    Soe, Katherine C; Devaiah, Ballachanda N; Singer, Dinah S

    2013-11-01

    The Major Histocompatibility Complex (MHC) class II transactivator (CIITA) mediates activated immune responses and its deficiency results in the Type II Bare Lymphocyte Syndrome. CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of MHC class I and class II genes. It is also a functional homolog of TAF1, a component of the general transcription factor complex TFIID. TAF1 and CIITA both possess intrinsic acetyltransferase (AT) activity that is required for transcription initiation. In response to induction by γ-interferon, CIITA and it's AT activity bypass the requirement for TAF1 AT activity. TAF1 also has kinase activity that is essential for its function. However, no similar activity has been identified for CIITA thus far. Here we report that CIITA, like TAF1, is a serine-threonine kinase. Its substrate specificity parallels, but does not duplicate, that of TAF1 in phosphorylating the TFIID component TAF7, the RAP74 subunit of the general transcription factor TFIIF and histone H2B. Like TAF1, CIITA autophosphorylates, affecting its interaction with TAF7. Additionally, CIITA phosphorylates histone H2B at Ser36, a target of TAF1 that is required for transcription during cell cycle progression and stress response. However, unlike TAF1, CIITA also phosphorylates all the other histones. The identification of this novel kinase activity of CIITA further clarifies its role as a functional homolog of TAF1 which may operate during stress and γ-IFN activated MHC gene transcription.

  18. Tumor Suppressive Function of p21-activated Kinase 6 in Hepatocellular Carcinoma.

    Science.gov (United States)

    Liu, Weisi; Liu, Yidong; Liu, Haiou; Zhang, Weijuan; Fu, Qiang; Xu, Jiejie; Gu, Jianxin

    2015-11-20

    Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict a favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with tumor node metastasis stage progression and unfavorable overall survival in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal deletion mutants of PAK6 experiments indicated the tumor suppressive function of PAK6 was partially dependent on its kinase activity and nuclear translocation. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components, including EZH2, SUZ12, and EED, elucidated epigenetic control of H3K27me3-arbitrated PAK6 down-regulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.

  19. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet;

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region....... In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF...... recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs....

  20. PROTEIN ADSORPTION AND CELL ADHESION ON RGD-FUNCTIONALIZED SILICON SUBSTRATE SURFACES

    Institute of Scientific and Technical Information of China (English)

    Wei-fang Tong; Xiao-li Liu; Fei Pan; Zhao-qiang Wu; Wen-wen Jiang

    2013-01-01

    A method was developed to modify silicon surfaces with good protein resistance and specific cell attachment.A silicon surface was initially deposited using a block copolymer of N-vinylpyrrolidone (NVP) and 2-hydroxyethyl methacrylate (HEMA) (PVP-b-PHEMA) film through surface-initiated atom transfer radical polymerization and then further immobilized using a short arginine-glycine-aspartate (RGD) peptide.Our results demonstrate that the RGD-modified surfaces (Si-RGD) can suppress non-specific adsorption of proteins and induce the adhesion of L929 cells.The Si-RGD surface exhibited higher cell proliferation rates than the unmodified silicon surface.This research established a simple method for the fabrication of dual-functional silicon surface that combines antifouling and cell attachment promotion.

  1. Functionalization of a Rigid Divalent Ligand for LecA, a Bacterial Adhesion Lectin**

    Science.gov (United States)

    Fu, Ou; Pukin, Aliaksei V; Quarles van Ufford, H C; Kemmink, Johan; de Mol, Nico J; Pieters, Roland J

    2015-01-01

    The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P. aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer–protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC). PMID:26478841

  2. HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Wilfried Jonkers

    2014-11-01

    Full Text Available Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this

  3. Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

    Science.gov (United States)

    Kessler, Sharon A; Lindner, Heike; Jones, Daniel S; Grossniklaus, Ueli

    2015-01-01

    The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception. PMID:25490905

  4. Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases.

    Directory of Open Access Journals (Sweden)

    Chad V Kuny

    Full Text Available The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5, is a kinase that phosphorylates the cellular retinoblastoma (Rb tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks. A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs and disruption of cytoplasmic or nuclear aggresomes showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs

  5. An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin.

    Science.gov (United States)

    Lo Sardo, Valentina; Zuccato, Chiara; Gaudenzi, Germano; Vitali, Barbara; Ramos, Catarina; Tartari, Marzia; Myre, Michael A; Walker, James A; Pistocchi, Anna; Conti, Luciano; Valenza, Marta; Drung, Binia; Schmidt, Boris; Gusella, James; Zeitlin, Scott; Cotelli, Franco; Cattaneo, Elena

    2012-05-01

    The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt’s ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development. PMID:22466506

  6. An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin.

    Science.gov (United States)

    Lo Sardo, Valentina; Zuccato, Chiara; Gaudenzi, Germano; Vitali, Barbara; Ramos, Catarina; Tartari, Marzia; Myre, Michael A; Walker, James A; Pistocchi, Anna; Conti, Luciano; Valenza, Marta; Drung, Binia; Schmidt, Boris; Gusella, James; Zeitlin, Scott; Cotelli, Franco; Cattaneo, Elena

    2012-05-01

    The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt’s ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

  7. Adjustment of surface chemical and physical properties with functionalized polymers to control cell adhesion

    Science.gov (United States)

    Zhou, Zhaoli

    Cell-surface interaction is crucial in many cellular functions such as movement, growth, differentiation, proliferation and survival. In the present work, we have developed several strategies to design and prepare synthetic polymeric materials with selected cues to control cell attachment. To promote neuronal cell adhesion on the surfaces, biocompatible, non-adhesive PEG-based materials were modified with neurotransmitter acetylcholine functionalities to produce hydrogels with a range of porous structures, swollen states, and mechanical strengths. Mice hippocampal cells cultured on the hydrogels showed differences in number, length of processes and exhibited different survival rates, thereby highlighting the importance of chemical composition and structure in biomaterials. Similar strategies were used to prepare polymer brushes to assess how topographical cues influence neuronal cell behaviors. The brushes were prepared using the "grown from" method through surface-initiated atom transfer radical polymerization (SI-ATRP) reactions and further patterned via UV photolithography. Protein absorption tests and hippocampal neuronal cell culture of the brush patterns showed that both protein and neuronal cells can adhere to the patterns and therefore can be guided by the patterns at certain length scales. We also prepared functional polymers to discourage attachment of undesirable cells on the surfaces. For example, we synthesized PEG-perfluorinated alkyl amphiphilic surfactants to modify polystyrene-block-poly(ethylene-ran-butylene)- block-polyisoprene (SEBI or K3) triblock copolymers for marine antifouling/fouling release surface coatings. Initial results showed that the polymer coated surfaces can facilitate removal of Ulva sporelings on the surfaces. In addition, we prepared both bioactive and dual functional biopassive/bioactive antimicrobial coatings based on SEBI polymers. Incubating the polymer coated surfaces with gram-positive bacteria (S. aureus), gram

  8. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  9. Functions of Polo-Like Kinases: A Journey From Yeast To Humans.

    Science.gov (United States)

    Vaid, Rajni; Sharma, Nitin; Chauhan, Sujata; Deshta, Ankita; Dev, Kamal; Sourirajan, Anuradha

    2016-01-01

    Polo-like kinases (PLKs) belong to the serine/threonine kinase subfamily, characterized by the presence of the signature motif called Polo-box domain. PLK members studied so far have emerged as the conserved regulator of cell cycle and cell division in eukaryotes. The Polo-box domain adds diversity to PLK functions by targeting the enzyme to an array of substrates found at different sub-cellular structures for exquisite regulation of cell cycle. More than a dozen members of PLK subfamily have been identified in the eukaryotic world except in the higher plants. Despite the similarities in governing cell division, PLKs have diverse and unique functions in different organisms. This review summarizes the plethora of functions of PLK in yeast to humans. Along with its classical functions, this review also emphasizes on the role of PLKs in regulating DNA replication, repair, genome integrity, development, and morphogenesis pathways. Perturbations in PLK functions have disease implications, such as cancer in humans, and thus human PLK1 is targeted for cancer therapeutics. PLKs also play a vital role in regulating several stages of meiotic cell division. Thus, PLKs are emerging as a unique class of proteins with multiple and diverse functions in different organisms. PMID:26786872

  10. Use of integrin-linked kinase to extend function of encapsulated pancreatic tissue

    Energy Technology Data Exchange (ETDEWEB)

    Blanchette, James O [Department of Chemical Engineering, University of South Carolina, Columbia, SC (United States); Langer, Steven J; Leinwand, Leslie L [Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO (United States); Sahai, Suchit; Topiwala, Pritesh S [Biomedical Engineering Program, University of South Carolina, Columbia, SC (United States); Anseth, Kristi S, E-mail: blanchej@cec.sc.ed [Howard Hughes Medical Institute, Boulder, CO (United States)

    2010-12-15

    We have studied the impact of overexpression of an intracellular signaling protein, integrin-linked kinase (ILK), on the survival and function of encapsulated islet tissue used for the treatment of type 1 diabetes. The dimensions of the encapsulated tissue can impact the stresses placed on the tissue and ILK overexpression shows the ability to extend function of dissociated cells as well as intact islets. These results suggest that lost cell-extracellular matrix interactions in cell encapsulation systems can lead to decreased insulin secretion and ILK signaling is a target to overcome this phenomenon. (communication)

  11. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  12. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

    Science.gov (United States)

    Espinosa-Urgel, M; Salido, A; Ramos, J L

    2000-05-01

    Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

  13. Functional mapping of the cytoplasmic region of intercellular adhesion molecule-3 reveals important roles for serine residues

    NARCIS (Netherlands)

    Hayflick, J S; Stine, J; Fox, R; Hoekstra, D; Gallatin, W M

    1997-01-01

    Intercellular adhesion molecule-3 (ICAM-3), a ligand for beta2 integrins, elicits a variety of activation responses in lymphocytes. We describe a functional mapping study that focuses on the 37-residue cytoplasmic region of ICAM-3. Carboxyl-terminal truncations delineated portions involved in T cell

  14. Functional adhesive surfaces with “gecko” effect: the concept of contact splitting

    NARCIS (Netherlands)

    Kamperman, M.M.G.; Kroner, E.; Campo, del A.; McMeeking, R.M.; Arzt, E.

    2010-01-01

    Nature has developed reversibly adhesive surfaces whose stickiness has attracted much research attention over the last decade. The central lesson from nature is that “patterned” or “fibrillar” surfaces can produce higher adhesion forces to flat and rough substrates than smooth surfaces. This paper c

  15. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

    Science.gov (United States)

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-01-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases.

  16. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    Directory of Open Access Journals (Sweden)

    Raouf A. Khalil

    2013-03-01

    Full Text Available Blood pressure (BP is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN. In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i, which forms a complex with calmodulin, activates myosin light chain (MLC kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC. PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK and MAPK kinase (MEK, a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in

  17. Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea.

    Science.gov (United States)

    Li, Lei; Xue, Chaoyang; Bruno, Kenneth; Nishimura, Marie; Xu, Jin-Rong

    2004-05-01

    In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea. PMID:15141959

  18. Raf Kinase Inhibitory Protein (RKIP): Functional Pleiotropy in the Mammalian Brain

    Science.gov (United States)

    Ling, Harrod H.; Mendoza-Viveros, Lucia; Mehta, Neel; Cheng, Hai-Ying M.

    2016-01-01

    In 1984, a cytosolic protein was isolated from bovine brain and coined phosphatidylethanolamine binding protein (PEBP) to describe its phospholipid-binding potential. Its cellular function remained elusive for more than a decade until it was discovered that PEBP had the ability to suppress the Raf1-mitogen activated protein kinase (MAPK) pathway, earning it the new name of Raf1 kinase inhibitory protein (RKIP). This milestone discovery has paved the way for numerous studies that have now extended the reach of RKIP’s function to other signaling cascades, within the context of various physiological and pathophysiological systems. This review will summarize our current knowledge of the neurophysiological roles of RKIP in the mammalian brain, including its function in the circadian clock and synaptic plasticity. It will also discuss evidence for an involvement of RKIP and its derived neuropeptide, hippocampal cholinergic neurostimulating peptide (HCNP), in neural development and differentiation. Implications in certain pathologies such as Alzheimer’s disease and brain cancer will be highlighted. By chronicling the diverse functions of RKIP in the brain, we hope that this review will serve as a timely resource that ignites future studies on this versatile, multifaceted protein in the nervous system. PMID:25597360

  19. Rate-dependence of 'wet' biological adhesives and the function of the pad secretion in insects.

    Science.gov (United States)

    Labonte, David; Federle, Walter

    2015-11-28

    Many insects use soft adhesive footpads for climbing. The surface contact of these organs is mediated by small volumes of a liquid secretion, which forms thin films in the contact zone. Here, we investigate the role of viscous dissipation by this secretion and the 'bulk' pad cuticle by quantifying the rate-dependence of the adhesive force of individual pads. Adhesion increased with retraction speed, but this effect was independent of the amount of pad secretion present in the contact zone, suggesting that the secretion's viscosity did not play a significant role. Instead, the rate-dependence can be explained by relating the strain energy release rate to the speed of crack propagation, using an established empirical power law. The 'wet' pads' behaviour was akin to that of 'dry' elastomers, with an equilibrium energy release rate close to that of dry van-der-Waals contacts. We suggest that the secretion mainly serves as a 'release layer', minimising viscous dissipation and thereby reducing the time- and 'loading-history'-dependence of the adhesive pads. In contrast to many commercial adhesives which derive much of their strength from viscous dissipation, we show that the major modulator of adhesive strength in 'wet' biological adhesive pads is friction, exhibiting a much larger effect than retraction speed. A comparison between 'wet' and 'dry' biological adhesives, using both results from this study and the literature, revealed a striking lack of differences in attachment performance under varying experimental conditions. Together, these results suggest that 'wet' and 'dry' biological adhesives may be more similar than previously thought.

  20. Rate-dependence of 'wet' biological adhesives and the function of the pad secretion in insects.

    Science.gov (United States)

    Labonte, David; Federle, Walter

    2015-11-28

    Many insects use soft adhesive footpads for climbing. The surface contact of these organs is mediated by small volumes of a liquid secretion, which forms thin films in the contact zone. Here, we investigate the role of viscous dissipation by this secretion and the 'bulk' pad cuticle by quantifying the rate-dependence of the adhesive force of individual pads. Adhesion increased with retraction speed, but this effect was independent of the amount of pad secretion present in the contact zone, suggesting that the secretion's viscosity did not play a significant role. Instead, the rate-dependence can be explained by relating the strain energy release rate to the speed of crack propagation, using an established empirical power law. The 'wet' pads' behaviour was akin to that of 'dry' elastomers, with an equilibrium energy release rate close to that of dry van-der-Waals contacts. We suggest that the secretion mainly serves as a 'release layer', minimising viscous dissipation and thereby reducing the time- and 'loading-history'-dependence of the adhesive pads. In contrast to many commercial adhesives which derive much of their strength from viscous dissipation, we show that the major modulator of adhesive strength in 'wet' biological adhesive pads is friction, exhibiting a much larger effect than retraction speed. A comparison between 'wet' and 'dry' biological adhesives, using both results from this study and the literature, revealed a striking lack of differences in attachment performance under varying experimental conditions. Together, these results suggest that 'wet' and 'dry' biological adhesives may be more similar than previously thought. PMID:26376599

  1. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    Science.gov (United States)

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  2. Src Family Kinases Modulate the Loss of Endothelial Barrier Function in Response to TNF-α: Crosstalk with p38 Signaling.

    Science.gov (United States)

    Adam, Alejandro P; Lowery, Anthony M; Martino, Nina; Alsaffar, Hiba; Vincent, Peter A

    2016-01-01

    Activation of Src Family Kinase (SFK) signaling is required for the increase in endothelial permeability induced by a variety of cytokines and growth factors. However, we previously demonstrated that activation of endogenous SFKs by expression of dominant negative C-terminal Src Kinase (DN-Csk) is not sufficient to decrease endothelial adherens junction integrity. Basal SFK activity has been observed in normal venular endothelia and was not associated with increased basal permeability. The basal SFK activity however was found to contribute to increased sensitivity of the venular endothelium to inflammatory mediator-induced leakage. How SFK activation achieves this is still not well understood. Here, we show that SFK activation renders human dermal microvascular endothelial cells susceptible to low doses of TNF-α. Treatment of DN-Csk-expressing cells with 50 pg/ml TNF-α induced a loss of TEER as well as drastic changes in the actin cytoskeleton and focal adhesion proteins. This synergistic effect was independent of ROCK or NF-κB activity. TNF-α-induced p38 signaling was required for the synergistic effect on barrier function, and activation of the p38 MAPK alone was also able to induce changes in permeability only in monolayers with active SFKs. These results suggest that the activation of endogenous levels of SFK renders the endothelial barrier more susceptible to low, physiologic doses of TNF-α through activation of p38 which leads to a loss of endothelial tight junctions. PMID:27603666

  3. Diacylglycerol Kinases: Regulated Controllers of T Cell Activation, Function, and Development

    Directory of Open Access Journals (Sweden)

    Gary A. Koretzky

    2013-03-01

    Full Text Available Diacylglycerol kinases (DGKs are a diverse family of enzymes that catalyze the conversion of diacylglycerol (DAG, a crucial second messenger of receptor-mediated signaling, to phosphatidic acid (PA. Both DAG and PA are bioactive molecules that regulate a wide set of intracellular signaling proteins involved in innate and adaptive immunity. Clear evidence points to a critical role for DGKs in modulating T cell activation, function, and development. More recently, studies have elucidated factors that control DGK function, suggesting an added complexity to how DGKs act during signaling. This review summarizes the available knowledge of the function and regulation of DGK isoforms in signal transduction with a particular focus on T lymphocytes.

  4. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Hudmon, Andy; Schulman, Howard

    2002-06-15

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase. PMID:11931644

  5. Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

    Science.gov (United States)

    Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

    2016-09-15

    Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. PMID:27402795

  6. Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

    Science.gov (United States)

    Huang, Yafan; Li, Hui; Hutchison, Claire E.; Laskey, James; Kieber, Joseph J.

    2003-01-01

    CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

  7. Expression and function of neural cell adhesion molecule during limb regeneration.

    OpenAIRE

    Maier, C E; Watanabe, M.(Niigata University, 950-2181, Niigata, Japan); Singer, M.; McQuarrie, I G; Sunshine, J.; Rutishauser, U.

    1986-01-01

    The neural cell adhesion molecule (NCAM) has been detected in regenerating limb bud of adult newts in addition to brain and peripheral nerves. In the regenerating tissue, NCAM was found primarily on mesenchymal cells and also in wound epidermis. Infusion of Fab fragments of antibodies to NCAM into limb buds at the early blastema stage delayed the regenerative process. Previous studies have indicated that NCAM serves as a homophilic ligand for adhesion among cells that express this molecule an...

  8. Insights into the structure-function relationship of Brugia malayi thymidylate kinase (BmTMK).

    Science.gov (United States)

    Doharey, Pawan Kumar; Singh, Sudhir Kumar; Verma, Pravesh; Verma, Anita; Rathaur, Sushma; Saxena, Jitendra Kumar

    2016-07-01

    Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of B. malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, B. malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. The study delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme. PMID:27044348

  9. Functional Redundancy of ERK1 and ERK2 MAP Kinases during Development

    Directory of Open Access Journals (Sweden)

    Christophe Frémin

    2015-08-01

    Full Text Available ERK1 and ERK2 are the effector kinases of the ERK1/2 MAP-kinase signaling pathway, which plays a central role in transducing signals controlling cell proliferation, differentiation, and survival. Deregulated activity of the ERK1/2 pathway is linked to a group of developmental syndromes and contributes to the pathogenesis of various human diseases. One fundamental question that remains unaddressed is whether ERK1 and ERK2 have evolved unique physiological functions or whether they are used redundantly to reach a threshold of global ERK activity. Here, we show that the extent of development of the mouse placenta and embryo bearing different combinations of Erk1 and Erk2 alleles is strictly correlated with total ERK1/2 activity. We further demonstrate that transgenic expression of ERK1 fully rescues the embryonic and placental developmental defects associated with the loss of ERK2. We conclude that ERK1 and ERK2 exert redundant functions in mouse development.

  10. Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations

    Institute of Scientific and Technical Information of China (English)

    Hui Jun XIA; Guang YANG

    2005-01-01

    Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) plays an important role in signal transduction in animal cells by phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4). Both IP3 and IP4 are critical second messengers which regulate calcium (Ca2+) homeostasis. Mammalian IP3Ks are involved in many biological processes, including brain development, memory, learning and so on. It is widely reported that Ca2+ is a canonical second messenger in higher plants. Therefore, plant IP3K should also play a crucial role in plant development. Recently,we reported the identification of plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis thaliana and its characterization.Here, we summarize the molecular cloning, biochemical properties and biological functions of IP3Ks from animal, yeast and plant. This review also discusses potential functions of IP3Ks in signaling crosstalk, inositol phosphate metabolism,gene transcriptional control and so on.

  11. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis

    Science.gov (United States)

    Kollmann, Karoline; Heller, Gerwin; Schneckenleithner, Christine; Warsch, Wolfgang; Scheicher, Ruth; Ott, Rene G.; Schäfer, Markus; Fajmann, Sabine; Schlederer, Michaela; Schiefer, Ana-Iris; Reichart, Ursula; Mayerhofer, Matthias; Hoeller, Christoph; Zöchbauer-Müller, Sabine; Kerjaschki, Dontscho; Bock, Christoph; Kenner, Lukas; Hoefler, Gerald; Freissmuth, Michael; Green, Anthony R.; Moriggl, Richard; Busslinger, Meinrad; Malumbres, Marcos; Sexl, Veronika

    2013-01-01

    Summary In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a. PMID:23948297

  12. The adhesion molecule PECAM-1 enhances the TGFβ-mediated inhibition of T cell function

    Science.gov (United States)

    Newman, Debra K.; Fu, Guoping; Adams, Tamara; Cui, Weiguo; Arumugam, Vidhyalakshmi; Bluemn, Theresa; Riese, Matthew J.

    2016-01-01

    Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine that inhibits the pro-inflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical signaling results in the activation of Smad proteins, transcription factors that regulate target gene expression. Here, we found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitates non-canonical (Smad-independent) TGF-β signaling in T cells. Subcutaneously injected tumor cells dependent on TGF-β-mediated suppression of immunity grew more slowly in PECAM-1−/− mice than in their wild type counterparts. T cells isolated from PECAM-1−/− mice demonstrated relative insensitivity to the TGF-β-dependent inhibition of interferon- γ (IFN-γ) production, granzyme B synthesis and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-β in a manner that was partially restored by re-expression of PECAM-1. Co-incubation of T cells with TGF-β and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2). Such stimulatory conditions also induced the co-localization of PECAM-1 with the TGF-β receptor complex as identified by co-immunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-β in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-β inhibitory axis represents a means to overcome TGF-β-dependent immunosuppression within the tumor microenvironment. PMID:26956486

  13. Green Tea Epigallocatechin Gallate Exhibits Anticancer Effect in Human Pancreatic Carcinoma Cells via the Inhibition of Both Focal Adhesion Kinase and Insulin-Like Growth Factor-I Receptor

    Directory of Open Access Journals (Sweden)

    Hoang Anh Vu

    2010-01-01

    Full Text Available The exact molecular mechanism by which epigallocatechin gallate (EGCG suppresses human pancreatic cancer cell proliferation is unclear. We show here that EGCG-treated pancreatic cancer cells AsPC-1 and BxPC-3 decrease cell adhesion ability on micro-pattern dots, accompanied by dephosphorylations of both focal adhesion kinase (FAK and insulin-like growth factor-1 receptor (IGF-1R whereas retained the activations of mitogen-activated protein kinase and mammalian target of rapamycin. The growth of AsPC-1 and BxPC-3 cells can be significantly suppressed by EGCG treatment alone in a dose-dependent manner. At a dose of 100 μM which completely abolishes activations of FAK and IGF-1R, EGCG suppresses more than 50% of cell proliferation without evidence of apoptosis analyzed by PARP cleavage. Finally, the MEK1/2 inhibitor U0126 enhances growth-suppressive effect of EGCG. Our data suggests that blocking FAK and IGF-1R by EGCG could prove valuable for targeted therapy, which can be used in combination with other therapies, for pancreatic cancer.

  14. Functional groups grafted nonwoven fabrics for blood filtration-The effects of functional groups and wettability on the adhesion of leukocyte and platelet

    Energy Technology Data Exchange (ETDEWEB)

    Yang Chao [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Cao Ye [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China); Sun Kang, E-mail: ksun@sjtu.edu.cn [State Key Lab of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Liu Jiaxin; Wang Hong [Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu 610081 (China)

    2011-01-15

    In this work, the effects of grafted functional groups and surface wettability on the adhesion of leukocyte and platelet were investigated by the method of blood filtration. The filter materials, poly(butylene terephthalate) nonwoven fabrics bearing different functional groups including hydroxyl (OH), carboxyl (COOH), sulfonic acid group (SO{sub 3}H) and zwitterionic sulfobetaine group ({sup +}N((CH{sub 3}){sub 2})(CH{sub 2}){sub 3}SO{sub 3}{sup Circled-Minus }) with controllable wettability were prepared by UV radiation grafting vinyl monomers with these functional groups. Our results emphasized that both surface functional groups and surface wettability had significant effects on the adhesion of leukocyte and platelet. In the case of filter materials with the same wettability, leukocytes adhering to filter materials decreased in the order: the surface bearing OH only > the surface bearing both OH and COOH > the surface bearing sulfobetaine group > the surface bearing SO{sub 3}H, while platelets adhering to filter materials decreased as the following order: the surface bearing SO{sub 3}H > the surface bearing both OH and COOH > the surface bearing OH only > the surface bearing sulfobetaine group. As the wettability of filter materials increased, both leukocyte and platelet adhesion to filter materials declined, except that leukocyte adhesion to the surface bearing OH only remained unchanged.

  15. Fabrication of Functional Wrinkled Interfaces from Polymer Blends: Role of the Surface Functionality on the Bacterial Adhesion

    Directory of Open Access Journals (Sweden)

    Marta Palacios-Cuesta

    2014-11-01

    Full Text Available The generation of nano-microstructured surfaces is a current challenge in polymer science. The fabrication of such surfaces has been accomplished mainly following two different alternatives i.e., by adapting techniques, such as molding (embossing or nano/microimprinting, or by developing novel techniques including laser ablation, soft lithography or laser scanning. Surface instabilities have been recently highlighted as a promising alternative to induce surface features. In particular, wrinkles have been extensively explored for this purpose. Herein, we describe the preparation of wrinkled interfaces by confining a photosensitive monomeric mixture composed of monofunctional monomer and a crosslinking agent within a substrate and a cover. The wrinkle characteristics can be controlled by the monomer mixture and the experimental conditions employed for the photopolymerization. More interestingly, incorporation within the material of a functional copolymer allowed us to vary the surface chemical composition while maintaining the surface structure. For that purpose we incorporated either a fluorinated copolymer that enhanced the surface hydrophobicity of the wrinkled interface or an acrylic acid containing copolymer that increased the hydrophilicity of the wrinkled surface. Finally, the role of the hydrophobicity on the bacterial surface adhesion will be tested by using Staphylococcus aureus.

  16. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

    Directory of Open Access Journals (Sweden)

    Nicos Angelopoulos

    2016-06-01

    Full Text Available Tyrosine kinases (TKs play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015 [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015 [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

  17. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

    Science.gov (United States)

    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert

    2011-10-25

    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  18. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

  19. Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-β signaling and metastasis

    OpenAIRE

    Wendt, Michael K.; William P. Schiemann

    2009-01-01

    Introduction Mammary tumorigenesis is associated with the increased expression of several proteins in the focal adhesion complex, including focal adhesion kinase (FAK) and various integrins. Aberrant expression of these molecules occurs concomitant with the conversion of TGF-β function from a tumor suppressor to a tumor promoter. We previously showed that interaction between β3 integrin and TβR-II facilitates TGF-β-mediated oncogenic signaling, epithelial-mesenchymal transition (EMT), and met...

  20. Snail1 controls epithelial–mesenchymal lineage commitment in focal adhesion kinase–null embryonic cells

    OpenAIRE

    Li, Xiao-Yan; Zhou, Xiaoming; Rowe, R. Grant; Hu, Yuexian; Schlaepfer, David D.; Ilić, Dusko; Dressler, Gregory; Park, Ann; Guan, Jun-Lin; Weiss, Stephen J.

    2011-01-01

    Mouse embryonic cells isolated from focal adhesion kinase (FAK)–null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epi...

  1. Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in others functions.

    Science.gov (United States)

    Labbé, Marie; Péroval, Marylène; Bourdieu, Christiane; Girard-Misguich, Fabienne; Péry, Pierre

    2006-12-01

    Two cDNA codings for glycolytic enzymes were cloned from a cDNA library constructed from the schizont stage of the avian parasite Eimeria tenella. Enolase and pyruvate kinase cDNA were fully sequenced and compared with sequences of enzymes from other organisms. Although these enzymes were already detected in the sporozoite stage, their expression was enhanced during the first schizogony in accordance with the anaerobic conditions of this part of the life cycle of the parasite. Under activating conditions, microscopic observations suggest that these glycolytic enzymes were relocalised inside sporozoites and moreover were in part secreted. The enzymes were also localised at the apex of the first generation of merozoites. Enolase was partly observed inside the nucleus of sporozoites and schizonts. Taken together, these results suggest that glycolytic enzymes not only have a function in glycolysis during anaerobic intracellular stages but may also participate in the invasion process and, for enolase, in the control of gene regulation.

  2. Identification, Expression and Functional Analysis of a Receptor-like Cytoplasmic Kinase, OsRLCK1, in Rice

    Institute of Scientific and Technical Information of China (English)

    Zhaosheng Kong; Wenying Xu; Qun Li; Yongbiao Xue

    2007-01-01

    Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCKI, which belongs to the receptor-like cytoplasmic kinase VIM subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCKI is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCKI recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCKI is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.

  3. Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Huiquan Liu

    2015-06-01

    Full Text Available Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data

  4. Functional Role and Therapeutic Potential of the Pim-1 Kinase in Colon Carcinoma

    Directory of Open Access Journals (Sweden)

    Ulrike Weirauch

    2013-07-01

    Full Text Available PURPOSE: The provirus integration site for Moloney murine leukemia virus 1 (Pim-1 kinase is overexpressed in various tumors and has been linked to poor prognosis. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes. Here, we explore the functional relevance of Pim-1 in colon carcinoma. EXPERIMENTAL DESIGN: RNAi-based knockdown approaches, as well as a specific small molecule inhibitor, were used to inhibit Pim-1 in colon carcinoma cells. The effects were analyzed regarding proliferation, apoptosis, sensitization toward cytostatic treatment, and overall antitumor effect in vitro and in mouse tumor models in vivo. RESULTS: We demonstrate antiproliferative, proapoptotic, and overall antitumor effects of Pim-1 inhibition. The sensitization to 5-fluorouracil (5-FU treatment upon Pim-1 knockdown offers new possibilities for combinatorial treatment approaches. Importantly, this also antagonizes a 5-FU-triggered Pim-1 up-regulation, which is mediated by decreased levels of miR-15b, a microRNA we newly identify to regulate Pim-1. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21Cip1/WAF1, STAT3, c-jun-N-terminal kinase (JNK, c-Myc, and survivin and in the levels of apoptosis-related proteins Puma, Bax, and Bcl-xL. CONCLUSIONS: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signaling pathways and establish the functional relevance of Pim-1 in colon carcinoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric polyethylenimine/ small interfering RNA nanoparticles as a promising therapeutic approach.

  5. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide;

    2012-01-01

    at the Institute of Physiology of the University of Würzburg on September 6-8, 2012, assembled a majority of the investigators currently actively pursuing research on adhesion-GPCRs, including scientists from laboratories in Europe, the United States, and Asia. The meeting featured the nascent mechanistic...

  6. Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men

    NARCIS (Netherlands)

    Esser, D.; Mars, M.; Oosterink, E.; Stalmach, A.; Müller, M.R.; Afman, L.A.

    2014-01-01

    Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We invest

  7. Spatiotemporal distribution and function of N-cadherin in postnatal Schwann cells: A matter of adhesion?

    DEFF Research Database (Denmark)

    Corell, Mikael; Wicher, Grzegorz; Limbach, Christoph;

    2010-01-01

    During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells. In this ...

  8. Gold nanoparticles functionalized with a fragment of the neural cell adhesion molecule L1 stimulate L1-mediated functions

    Science.gov (United States)

    Schulz, Florian; Lutz, David; Rusche, Norman; Bastús, Neus G.; Stieben, Martin; Höltig, Michael; Grüner, Florian; Weller, Horst; Schachner, Melitta; Vossmeyer, Tobias; Loers, Gabriele

    2013-10-01

    The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1 sequence of the third fibronectin type III domain of murine L1 was identified and conjugated to gold nanoparticles (AuNPs) to obtain constructs that interact homophilically with the extracellular domain of L1 and trigger the cognate beneficial L1-mediated functions. Covalent conjugation was achieved by reacting mixtures of two cysteine-terminated forms of this L1 peptide and thiolated poly(ethylene) glycol (PEG) ligands (~2.1 kDa) with citrate stabilized AuNPs of two different sizes (~14 and 40 nm in diameter). By varying the ratio of the L1 peptide-PEG mixtures, an optimized layer composition was achieved that resulted in the expected homophilic interaction of the AuNPs. These AuNPs were stable as tested over a time period of 30 days in artificial cerebrospinal fluid and interacted with the extracellular domain of L1 on neurons and Schwann cells, as could be shown by using cells from wild-type and L1-deficient mice. In vitro, the L1-derivatized particles promoted neurite outgrowth and survival of neurons from the central and peripheral nervous system and stimulated Schwann cell process formation and proliferation. These observations raise the hope that, in combination with other therapeutic approaches, L1 peptide-functionalized AuNPs may become a useful tool to ameliorate the deficits resulting from acute and chronic injuries of the mammalian nervous system.The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1

  9. P-Selectin Cross-Links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent, but GPCR-Independent Mechanism

    OpenAIRE

    Xu, Tao; Zhang, Lei; Geng, Zhen H; Wang, Hai-Bo; Wang, Jin-Tao; Chen, Ming; Geng, Jian-Guo

    2007-01-01

    Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeri...

  10. Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line

    DEFF Research Database (Denmark)

    Holland, J; Owens, T

    1997-01-01

    Intercellular adhesion molecule 1 (ICAM-1) (CD54) is an adhesion molecule of the immunoglobulin superfamily. The interaction between ICAM-1 on B lymphocytes and leukocyte function-associated antigen 1 on T cells plays a major role in several aspects of the immune response, including T-dependent B...... cell activation. While it was originally believed that ICAM-1 played a purely adhesive role, recent evidence suggests that it can itself transduce biochemical signals. We demonstrate that cross-linking of ICAM-1 results in the up-regulation of class II major histocompatibility complex, and we...... investigate the biochemical mechanism for the signaling role of ICAM-1. We show that cross-linking of ICAM-1 on the B lymphoma line A20 induces an increase in tyrosine phosphorylation of several cellular proteins, including the Src family kinase p53/p56(lyn). In vitro kinase assays showed that Lyn kinase...

  11. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Directory of Open Access Journals (Sweden)

    Evangelos Dioletis

    Full Text Available Death-associated protein kinase (DAPk is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD, NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2 and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural

  12. Multiple faces of protein interacting with C kinase 1 (PICK1): Structure, function, and diseases.

    Science.gov (United States)

    Li, Yun-Hong; Zhang, Nan; Wang, Ya-Nan; Shen, Ying; Wang, Yin

    2016-09-01

    Protein interacting with C-kinase 1 (PICK1) has received considerable attention because it is the only protein that contains both PSD-95/DlgA/ZO-1 (PDZ) domain and Bin-Amphiphysin-Rvs (BAR) domain. Through PDZ and BAR domains, PICK1 binds to a large number of membrane proteins and lipid molecules, and is thereby of multiple functions. PICK1 is widely expressed in various tissues, particularly abundant in the brain and testis. In the central nervous system (CNS), PICK1 interacts with numerous neurotransmitters receptors, transporters, ion channels, and enzymes, and controls their trafficking. The best characterized function of PICK1 is that it regulates trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit GluA2 during long-term depression and long-term potentiation. Recent evidence shows that PICK1 participates in various diseases including neurobiological disorders, such as chronic pain, epilepsy, oxidative stress, stroke, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and non-neurological disorders, such as globozoospermia, breast cancer, and heart failure. In this review, we will summarize recent advances focusing on the structure and regulation of PICK1 and its functions in protein trafficking, neurological and non-neurological diseases. PMID:26970394

  13. Abdominal Adhesions

    Science.gov (United States)

    ... adhesions? Abdominal adhesions can cause intestinal obstruction and female infertility—the inability to become pregnant after a year of trying. Abdominal adhesions can lead to female infertility by preventing fertilized eggs from reaching the uterus, ...

  14. Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance.

    Science.gov (United States)

    Wang, Ji-Peng; Xu, You-Ping; Munyampundu, Jean-Pierre; Liu, Tian-Yu; Cai, Xin-Zhong

    2016-04-01

    Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato (Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca(2+) binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance. PMID:26520101

  15. Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate.

    Science.gov (United States)

    Saouaf, S J; Wolven, A; Resh, M D; Bolen, J B

    1997-05-19

    Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases. PMID:9177269

  16. Expression, purification and functional characterization of IkappaB kinase-2 (IKK-2) mutants.

    Science.gov (United States)

    Mathialagan, Sumathy; Poda, Gennadiy I; Kurumbail, Ravi G; Selness, Shaun R; Hall, Troii; Reitz, Beverly A; Weinberg, Robin A; Kishore, Nandini; Mbalaviele, Gabriel

    2010-08-01

    NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.

  17. Surface Treatments and Functional Coatings for Biocompatibility Improvement and Bacterial Adhesion Reduction in Dental Implantology

    Directory of Open Access Journals (Sweden)

    Pietro Mandracci

    2016-01-01

    Full Text Available Surface modification of dental implants is a key process in the production of these medical devices, and especially titanium implants used in the dental practice are commonly subjected to surface modification processes before their clinical use. A wide range of treatments, such as sand blasting, acid etching, plasma etching, plasma spray deposition, sputtering deposition and cathodic arc deposition, have been studied over the years in order to improve the performance of dental implants. Improving or accelerating the osseointegration process is usually the main goal of these surface processes, but the improvement of biocompatibility and the prevention of bacterial adhesion are also of considerable importance. In this review, we report on the research of the recent years in the field of surface treatments and coatings deposition for the improvement of dental implants performance, with a main focus on the osseointegration acceleration, the reduction of bacterial adhesion and the improvement of biocompatibility.

  18. Similitude law for the creep-adhesion function in dry contact

    Energy Technology Data Exchange (ETDEWEB)

    Sciammarella, C.A.; Kumar, S.; Nailescu, L.; Seth, B.B.

    1979-08-01

    An experimental study is described on the dry friction-creep phenomenon. The study was carried out on a test-rig. The simulated wheel has an elliptical area of contact similar to the actual locomotive wheel and is made of a 1070 plain carbon steel. The testing was conducted with variable contact stresses up to approximately 200,000 psi and equivalent speeds of approximately 5 miles per hour. The results of the test show that all the different creep-adhesion curves obtained by changing the normal loads can be reduced to one single dimesionless curve. This can be achieved if the actual nominal area of contact experimentally measured, is introduced in the corresponding expressions. A law of constancy of the product of the creep times the area of contact for dry, clean contact surfaces and a constant value of adhesion and normal load is presented.

  19. Fracture R-curve of a toughened epoxy adhesive as a function of irreversible degradation

    Energy Technology Data Exchange (ETDEWEB)

    Ameli, A. [Department of Mechanical and Industrial Engineering, University of Toronto, 5 King' s College Road, Toronto, Ontario, M5S 3G8 (Canada); Papini, M. [Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, Ontario, M5B 2K3 (Canada); Spelt, J.K., E-mail: spelt@mie.utoronto.ca [Department of Mechanical and Industrial Engineering, University of Toronto, 5 King' s College Road, Toronto, Ontario, M5S 3G8 (Canada)

    2010-07-25

    Open-faced double cantilever beam (DCB) specimens of a toughened epoxy-aluminum adhesive system were degraded over a relatively wide range of temperature, relative humidity (RH) and exposure time, dried and tested to characterize the irreversible evolution of the mixed-mode fracture resistance curves (R-curves). The water diffusion properties of the bulk adhesive were modeled using an earlier sequential dual Fickian (SDF) model for the same adhesive in order to predict the adhesive water content. Three temporal stages of degradation possessing different R-curve and fracture surface characteristics were observed. In general, the steady-state critical strain energy release rate (G{sub cs}), the rate of toughening (dG{sub cr}/da) and the length of the rising part of the R-curve decreased with increasing exposure temperature, RH and water concentration, while the initiation G{sub c} (G{sub ci}) remained unchanged. It is hypothesized that crack initiation is governed by the properties of the epoxy matrix and that the toughening action of rubber particles does not become appreciable until after a certain amount of crack extension (more than about 50 {mu}m in the present case). The irreversible degradation of fracture toughness was found to be insensitive to the phase angle, which simplifies the construction of the fracture toughness envelope for a given level of degradation. These effects were incorporated into a new R-curve degradation model which has an application in the R-curve prediction for closed joints having nonuniform degradation.

  20. Functional polyaniline nanofibre mats for human adipose-derived stem cell proliferation and adhesion

    International Nuclear Information System (INIS)

    Conductive polymer poly(aniline-co-m-aminobenzoic acid) (P(ANI-co-m-ABA)) and polyaniline (PANI) were blended with a biodegradable, biocompatible polymer, poly(L-lactic acid) and were electrospun into nanofibres to investigate their potential application as a scaffold for human adipose-derived stem cells (hASCs). These polymers, in both conductive and non-conductive form, were electrospun with average fibre diameters of less than 400 nm. Novel nanoindentation results obtained on the individual nanofibres revealed that the elastic moduli of the nanofibres are much higher at the surface (4–10 GPa, hmax max >75 nm). The composite nanofibres showed great promise as a scaffold for hASCs as they supported the cell adhesion and proliferation. After 1 week of cell culture hASCs were well spread on the substrates with abundant focal adhesions. The electrospun mats provide the cells with comparably stiff, sub-micron sized fibres as anchoring points on a substrate of high porosity. The conductive nature of these composite nanofibres offers exciting opportunities for electrical stimulation of the cells. - Highlights: ► Polyaniline and its copolymer's nanofibres were prepared by electrospinning. ► The elastic modulus of a single polyaniline composite nanofibres were determined. ► Elastic moduli of the nanofibres are much higher at the surface than the inner core. ► The electrospun mats supported the cell adhesion and proliferation. ► The nanofibres show great promise as a scaffold for adipose derived stem cells

  1. Adhesion Strength of Cellulosic Varnish Coated Wood Species as Function of Their Surface Roughness

    Directory of Open Access Journals (Sweden)

    Turgay Ozdemir

    2015-01-01

    Full Text Available The objective of this study was to evaluate adhesion strength of four wood species, namely, beech (Fagus orientalis Lipsky, alder (Alnus glutinosa subsp. barbata Yalt., spruce (Picea orientalis L. Link, and fir (Abies nordmanniana subsp. coated with cellulosic varnish. Samples were prepared in tangential and radial grain orientations from the above species. Surface quality of the specimens was also measured employing stylus type equipment after samples of all four types of species were sanded with 80- and 180-grit sandpaper prior to coating process. Surface roughness of the specimens sanded with 80-grit sandpaper resulted in significantly higher mean peak-to-valley height (Rz values based on the measurement employing stylus type profilometer. The highest adhesion strength values of 2.39 N/mm2 and 2.03 N/mm2 were found for beech and alder samples, respectively. It appears that overall higher roughness characteristics of the specimens exhibited enhanced adherence between substrate and varnish resulting in higher adhesion strength values.

  2. Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site

    Directory of Open Access Journals (Sweden)

    Man-Ho eOh

    2015-07-01

    Full Text Available BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T cytoplasmic domain protein (the S891T protein was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein

  3. “Stop Ne(c)king around”: How interactomics contributes to functionally characterize Nek family kinases

    Institute of Scientific and Technical Information of China (English)

    Gabriela; Vaz; Meirelles; Arina; Marina; Perez; Edmárcia; Elisa; de; Souza; Ferna; Luisa; Basei; Priscila; Ferreira; Papa; Talita; Diniz; Melo; Hanchuk; Vanessa; Bomfim; Cardoso; Jrg; Kobarg

    2014-01-01

    Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A(NIMA)-related kinases(Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals:(1) centrioles/mitosis;(2) primary ciliary function/ciliopathies; and(3) DNA damage response(DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.

  4. Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells.

    Science.gov (United States)

    Resnick, M S; Kang, B S; Luu, D; Wickham, J T; Sando, J J; Hahn, C S

    1998-10-16

    Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation. PMID:9765301

  5. Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    Amy; K; Erbe; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    The Ikaros gene encodes a zinc finger,DNA-binding protein that regulates gene transcription and chromatin remodeling.Ikaros is a master regulator of hematopoiesis and an established tumor suppressor.Moderate alteration of Ikaros activity (e.g.haploinsufficiency) appears to be sufficient to promote malignant transformation in human hematopoietic cells.This raises questions about the mechanisms that normally regulate Ikaros function and the potential of these mechanisms to contribute to the development of leukemia.The focus of this review is the regulation of Ikaros function by phosphorylation/dephosphorylation.Site-specific phosphorylation of Ikaros by casein kinase 2 (CK2) controls Ikaros DNA-binding ability and subcellular localization.As a consequence,the ability of Ikaros to regulate cell cycle progression,chromatin remodeling,target gene expression,and thymocyte differentiation are controlled by CK2.In addition,hyperphosphorylation of Ikaros by CK2 leads to decreased Ikaros levels due to ubiquitinmediated degradation.Dephosphorylation of Ikaros by protein phosphatase 1 (PP1) acts in opposition to CK2 to increase Ikaros stability and restore Ikaros DNA binding ability and pericentromeric localization.Thus,the CK2 and PP1 pathways act in concert to regulate Ikaros activity in hematopoiesis and as a tumor suppressor.This highlights the importance of these signal transduction pathways as potential mediators of leukemogenesis via their role in regulating the activities of Ikaros.

  6. Identification and functional analysis of tomato BRI1 and BAK1 receptor kinase phosphorylation sites

    Science.gov (United States)

    Brassinosteroids (BRs) are essential plant hormones that are perceived at the cell surface by a membrane bound receptor kinase, BRASSINOSTEROID INSENSITIVE 1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation an...

  7. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  8. Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

    DEFF Research Database (Denmark)

    Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim;

    2005-01-01

    The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional...... for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent...

  9. Dry adhesives with sensing features

    Science.gov (United States)

    Krahn, J.; Menon, C.

    2013-08-01

    Geckos are capable of detecting detachment of their feet. Inspired by this basic observation, a novel functional dry adhesive is proposed, which can be used to measure the instantaneous forces and torques acting on an adhesive pad. Such a novel sensing dry adhesive could potentially be used by climbing robots to quickly realize and respond appropriately to catastrophic detachment conditions. The proposed torque and force sensing dry adhesive was fabricated by mixing Carbon Black (CB) and Polydimethylsiloxane (PDMS) to form a functionalized adhesive with mushroom caps. The addition of CB to PDMS resulted in conductive PDMS which, when under compression, tension or torque, resulted in a change in the resistance across the adhesive patch terminals. The proposed design of the functionalized dry adhesive enables distinguishing an applied torque from a compressive force in a single adhesive pad. A model based on beam theory was used to predict the change in resistance across the terminals as either a torque or compressive force was applied to the adhesive patch. Under a compressive force, the sensing dry adhesive was capable of measuring compression stresses from 0.11 Pa to 20.9 kPa. The torque measured by the adhesive patch ranged from 2.6 to 10 mN m, at which point the dry adhesives became detached. The adhesive strength was 1.75 kPa under an applied preload of 1.65 kPa for an adhesive patch with an adhesive contact area of 7.07 cm2.

  10. Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions.

    Directory of Open Access Journals (Sweden)

    Eli Zamir

    Full Text Available BACKGROUND: Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: We present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition. CONCLUSIONS/SIGNIFICANCE: Multicolor compositional imaging resolves "molecular signatures" characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional "contents-resolved" dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.

  11. Functional polyaniline nanofibre mats for human adipose-derived stem cell proliferation and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Abdul Rahman, Norizah, E-mail: norizah@science.putra.edu.my [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Department of Chemistry, University of Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan (Malaysia); Feisst, Vaughan [School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Dickinson, Michelle E. [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Malmström, Jenny [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Dunbar, P. Rod [School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); Maurice Wilkins Centre, Private Bag 92019, Auckland (New Zealand); Travas-Sejdic, Jadranka, E-mail: j.travas-sejdic@auckland.ac.nz [Polymer Electronics Research Centre, School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland (New Zealand); MacDiarmid Institute for Advanced Materials and Nanotechnology, P.O. Box 600, Wellington 6140 (New Zealand)

    2013-02-15

    Conductive polymer poly(aniline-co-m-aminobenzoic acid) (P(ANI-co-m-ABA)) and polyaniline (PANI) were blended with a biodegradable, biocompatible polymer, poly(L-lactic acid) and were electrospun into nanofibres to investigate their potential application as a scaffold for human adipose-derived stem cells (hASCs). These polymers, in both conductive and non-conductive form, were electrospun with average fibre diameters of less than 400 nm. Novel nanoindentation results obtained on the individual nanofibres revealed that the elastic moduli of the nanofibres are much higher at the surface (4–10 GPa, h{sub max} <75 nm) than in the inner fibre core (2–4 GPa, h{sub max} >75 nm). The composite nanofibres showed great promise as a scaffold for hASCs as they supported the cell adhesion and proliferation. After 1 week of cell culture hASCs were well spread on the substrates with abundant focal adhesions. The electrospun mats provide the cells with comparably stiff, sub-micron sized fibres as anchoring points on a substrate of high porosity. The conductive nature of these composite nanofibres offers exciting opportunities for electrical stimulation of the cells. - Highlights: ► Polyaniline and its copolymer's nanofibres were prepared by electrospinning. ► The elastic modulus of a single polyaniline composite nanofibres were determined. ► Elastic moduli of the nanofibres are much higher at the surface than the inner core. ► The electrospun mats supported the cell adhesion and proliferation. ► The nanofibres show great promise as a scaffold for adipose derived stem cells.

  12. Tumour necrosis factor-alpha interacts with laminin and functions as a pro-adhesive cytokine.

    Science.gov (United States)

    Hershkoviz, R; Goldkorn, I; Lider, O

    1995-05-01

    Certain cytokines, chemokines and growth factors interact with components of the extracellular matrix (ECM) and, in particular, sulphated polysaccharides and proteoglycans. Recently, we demonstrated that tumour necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, can bind fibronectin (FN), a cell-adhesive glycoprotein of the ECM, and that TNF-alpha bound to FN enhances the binding of T cells to the glycoprotein. In the present study, we studied the interactions of TNF-alpha and laminin (LN), another glycoprotein present in basement membranes and extracellular matrices. 125I-labelled TNF-alpha was found to bind to immobilized LN, and more avidly to the E1 and P1 fragments of LN, which contain its integrin- and non-integrin-dependent cell-adhesive sites, suggesting that cryptic TNF-alpha-binding sites are exposed upon proteolytic fragmentation of LN by enzymes such as elastase or pepsin. The bound cytokine did not dissociate from the LN and its fragments during a 24-hr period, indicating that in vivo LN can serve to restrict TNF-alpha adjacent to inflammatory sites. The LN-associated TNF-alpha retained at least some of its biological activities, since both diffusible and, to a greater extent, LN-bound TNF-alpha elevated the beta 1-integrin-dependent adhesion to LN of phorbol ester-activated human CD4+ T cells. Thus, LN and TNF-alpha may act in concert to transmit synergistic activating signals to infiltrating leucocytes, and thereby regulate immune cell reactions in extravascular inflammatory tissue. PMID:7635514

  13. Extracellular Microvesicles from Astrocytes Contain Functional Glutamate Transporters: Regulation by Protein Kinase C and Cell Activation

    Directory of Open Access Journals (Sweden)

    Romain-Daniel eGosselin

    2013-12-01

    Full Text Available Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes and because the Protein Kinase C (PKC family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

  14. A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

    Directory of Open Access Journals (Sweden)

    Elena Nekhoroshkova

    Full Text Available BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149 that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.

  15. Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

    Directory of Open Access Journals (Sweden)

    Uta Gey

    Full Text Available The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p. The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p, Lys114 (Kgd2p and Lys102 (Gcv3p, respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

  16. The rice diacylglycerol kinase family: functional analysis using transient RNA interference

    Directory of Open Access Journals (Sweden)

    Hongliang eGe

    2012-03-01

    Full Text Available Diacylglycerol kinase (DGK is a pivotal enzyme that phosphorylates diacylglycerol (DAG to form phosphatidic acid (PA. The production of PA from phospholipase D (PLD and the coupled phospholipase C (PLC/DGK route is an important signaling process in animal and plant cells. In this study, we report a genomic analysis of eight putative rice DGKs encoded by a gene family (OsDGKs grouped into three clusters. To further investigate the functions of the OsDGKs, a double-stranded RNA (dsRNA-induced RNA silencing method was established. Introduction of in vitro-synthesized dsRNAs corresponding to a unique or conserved region of OsDGKs into rice protoplasts abolished or diminished the expression of individual or multiple OsDGK genes. Suppressing the expression of OsDGKs resulted in a distinct depletion of the transcripts of the defense gene OsNPR1 and the salt-responsive gene OsCIPK15. Our primary results suggest that OsDGKs are involved in the signaling of stress responses.

  17. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  18. Functional analysis of Casein Kinase 1 in a minimal circadian system.

    Directory of Open Access Journals (Sweden)

    Gerben van Ooijen

    Full Text Available The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1 is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.

  19. Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    李登举; 张瑶珍; 曹文静; 孙岚; 徐慧珍; 路武

    2002-01-01

    Summary: In order to investigate the regulative function of telomerase and phosphorylated (acti-vated) extracelluar regulated protein kinase (ERK) i and 2 in the leukemic cell lines HL-60 andK562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT),Vincristine(VCR)and Etoposide(Vp16)were selected as inducers. The proliferation inhibition ratewas detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometryand the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP)assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression wasdetected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cellproliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expres-sion of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involvedin the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treat-ed by HRT, VCR and Vp16.

  20. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    Science.gov (United States)

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  1. The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

    Science.gov (United States)

    Miguel-Rojas, Cristina; Hera, Concepcion

    2016-01-01

    F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.

  2. Regulation of embryonic cell adhesion by the prion protein.

    Directory of Open Access Journals (Sweden)

    Edward Málaga-Trillo

    2009-03-01

    Full Text Available Prion proteins (PrPs are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1 mediates Ca(+2-independent homophilic cell adhesion and signaling; and (2 modulates Ca(+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin-based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

  3. Dietary carbohydrate restriction improves insulin sensitivity, blood pressure, microvascular function, and cellular adhesion markers in individuals taking statins.

    Science.gov (United States)

    Ballard, Kevin D; Quann, Erin E; Kupchak, Brian R; Volk, Brittanie M; Kawiecki, Diana M; Fernandez, Maria Luz; Seip, Richard L; Maresh, Carl M; Kraemer, William J; Volek, Jeff S

    2013-11-01

    Statins positively impact plasma low-density lipoprotein cholesterol, inflammation and vascular endothelial function (VEF). Carbohydrate restricted diets (CRD) improve atherogenic dyslipidemia, and similar to statins, have been shown to favorably affect markers of inflammation and VEF. No studies have examined whether a CRD provides additional benefit beyond that achieved by habitual statin use. We hypothesized that a CRD (carb/fat/pro) and averaged across the intervention (11/58/28% carb/fat/pro) demonstrated dietary compliance, with carbohydrate intake at baseline nearly 5-fold greater than during the intervention (P < .001). Compared to baseline, both systolic and diastolic blood pressure decreased after 3 and 6 weeks (P < .01). Peak forearm blood flow, but not flow-mediated dilation, increased at week 6 compared to baseline and week 3 (P ≤ .03). Serum triglyceride, insulin, soluble E-Selectin and intracellular adhesion molecule-1 decreased (P < .01) from baseline at week 3, and this effect was maintained at week 6. In conclusion, these findings demonstrate that individuals undergoing statin therapy experience additional improvements in metabolic and vascular health from a 6 weeks CRD as evidenced by increased insulin sensitivity and resistance vessel endothelial function, and decreased blood pressure, triglycerides, and adhesion molecules. PMID:24176230

  4. The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation.

    Science.gov (United States)

    Lasek, Amber L; McPherson, Brittany M; Trueman, Natalie G; Burkard, Mark E

    2016-01-01

    Mitosis is coordinated by carefully controlled phosphorylation and ubiquitin-mediated proteolysis. Polo-like kinase 1 (Plk1) plays a central role in regulating mitosis and cytokinesis by phosphorylating target proteins. Yet, Plk1 is itself a target for posttranslational modification by phosphorylation and ubiquitination. We developed a chemical-genetic complementation assay to evaluate the functional significance of 34 posttranslational modifications (PTMs) on human Plk1. To do this, we used human cells that solely express a modified analog-sensitive Plk1 (Plk1AS) and complemented with wildtype Plk1. The wildtype Plk1 provides cells with a functional Plk1 allele in the presence of 3-MB-PP1, a bulky ATP-analog inhibitor that specifically inhibits Plk1AS. Using this approach, we evaluated the ability of 34 singly non-modifiable Plk1 mutants to complement Plk1AS in the presence of 3-MB-PP1. Mutation of the T-loop activating residue T210 and adjacent T214 are lethal, but surprisingly individual mutation of the remaining 32 posttranslational modification sites did not disrupt the essential functions of Plk1. To evaluate redundancy, we simultaneously mutated all phosphorylation sites in the kinase domain except for T210 and T214 or all sites in the C-terminal polo-box domain (PBD). We discovered that redundant phosphorylation events within the kinase domain are required for accurate chromosome segregation in anaphase but those in the PBD are dispensable. We conclude that PTMs within the T-loop of Plk1 are essential and nonredundant, additional modifications in the kinase domain provide redundant control of Plk1 function, and those in the PBD are dispensable for essential mitotic functions of Plk1. This comprehensive evaluation of Plk1 modifications demonstrates that although phosphorylation and ubiquitination are important for mitotic progression, many individual PTMs detected in human tissue may have redundant, subtle, or dispensable roles in gene function. PMID

  5. Serine34 phosphorylation of RHO guanine dissociation inhibitor (RHOGDI{alpha}) links signaling from conventional protein kinase C to RHO GTPase in cell adhesion

    DEFF Research Database (Denmark)

    Dovas, Athanassios; Choi, Youngsil; Yoneda, Atsuko;

    2010-01-01

    . Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with FRET microscopy sensing GTP-RhoA levels, the data reveal a common pathway......, resulting in a specific decrease in affinity for RhoA, but not Rac1 or cdc42. The mechanism of RhoGDIalpha phosphorylation is distinct, requiring PKCalpha and phosphatidylinositol 4,5 bisphosphate, consistent with recent evidence that the inositide can activate, localize and orient PKCalpha in membranes...

  6. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    OpenAIRE

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways...

  7. Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases.

    OpenAIRE

    Z.Zhou; Gartner, A...; Cade, R.; Ammerer, G; Errede, B

    1993-01-01

    Protein phosphorylation plays an important role in pheromone-induced differentiation processes of haploid yeast cells. Among the components necessary for signal transduction are the STE7 and STE11 kinases and either one of the redundant FUS3 and KSS1 kinases. FUS3 and presumably KSS1 are phosphorylated and activated during pheromone induction by a STE7-dependent mechanism. Pheromone also induces the accumulation of STE7 in a hyperphosphorylated form. This modification of STE7 requires the STE...

  8. Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte

    Institute of Scientific and Technical Information of China (English)

    HUO Lijun; FAN Hengyu; CHEN Dayuan; SUN Qingyuan

    2004-01-01

    Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.

  9. Electron work functions of ferrite and austenite phases in a duplex stainless steel and their adhesive forces with AFM silicon probe.

    Science.gov (United States)

    Guo, Liqiu; Hua, Guomin; Yang, Binjie; Lu, Hao; Qiao, Lijie; Yan, Xianguo; Li, Dongyang

    2016-01-01

    Local electron work function, adhesive force, modulus and deformation of ferrite and austenite phases in a duplex stainless steel were analyzed by scanning force microscopy. It is demonstrated that the austenite has a higher electron work function than the ferrite, corresponding to higher modulus, smaller deformation and larger adhesive force. Relevant first-principles calculations were conducted to elucidate the mechanism behind. It is demonstrated that the difference in the properties between austenite and ferrite is intrinsically related to their electron work functions. PMID:26868719

  10. Identification of a BET family Bromodomain / Casein Kinase II / TAF-containing complex as a regulator of mitotic condensin function

    OpenAIRE

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B.; Silva, Andrea C.; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J.; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G.; Greenblatt, Jack F.; Krogan, Nevan J.; Fillingham, Jeffrey S.

    2014-01-01

    Condensin is a central regulator of mitotic genome structure, with mutants showing poorly condensed chromosomes and profound segregation defects. Here we identify NCT complex, comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), Casein Kinase II (CKII) and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions, but only briefly co-localize during the periods of chromosome condensation and decondensation. This pattern ...

  11. A novel dry chemical path way for diene and dienophile surface functionalization toward thermally responsive metal-polymer adhesion.

    Science.gov (United States)

    Moreno-Couranjou, Maryline; Manakhov, Anton; Boscher, Nicolas D; Pireaux, Jean-Jacques; Choquet, Patrick

    2013-09-11

    In this paper, we report a new and easily up-scalable dry chemical method to functionalize with diene and dienophile groups a large range of surfaces, such as metal, polymer, or glass, and we demonstrate the potentiality of this technique to realize thermally responsive adhesion between these materials. A complete and extensive surface chemistry analysis of the grafted surfaces, based on the deposition of an anhydride-rich thin plasma polymer layer by using an atmospheric pressure dielectric barrier discharge (DBD) plasma process, and its subsequent gas phase aminolysis reaction with specific diene or dienophile compound is discussed. The optimization of the assembling condition for these tailored surfaces has led to achieve a Diels-Alder adhesion force up to 0.6 N/mm at ambient temperature, which can be reduced by a factor of 50 when the retro Diels-Alder is ignited at a heating temperature around 200 °C. The study of the failure interface produced after peeling tests is presented and a mechanism of failure is proposed, based on forensic analyses involving surface analytical techniques such as XPS, ToF-SIMS, and SEM combined to AFM analyses for the retrieving of chemical and morphological information.

  12. Tyrosine kinase inhibitor NVP-BGJ398 functionally improves FGFR3-related dwarfism in mouse model.

    Science.gov (United States)

    Komla-Ebri, Davide; Dambroise, Emilie; Kramer, Ina; Benoist-Lasselin, Catherine; Kaci, Nabil; Le Gall, Cindy; Martin, Ludovic; Busca, Patricia; Barbault, Florent; Graus-Porta, Diana; Munnich, Arnold; Kneissel, Michaela; Di Rocco, Federico; Biosse-Duplan, Martin; Legeai-Mallet, Laurence

    2016-05-01

    Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the fibroblast growth factor receptor 3-encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral growth plate and calvaria in organ cultures from embryos of the Fgfr3Y367C/+ mouse model of ACH. Moreover, we demonstrated that a low dose of NVP-BGJ398, injected subcutaneously, was able to penetrate into the growth plate of Fgfr3Y367C/+ mice and modify its organization. Improvements to the axial and appendicular skeletons were noticeable after 10 days of treatment and were more extensive after 15 days of treatment that started from postnatal day 1. Low-dose NVP-BGJ398 treatment reduced intervertebral disc defects of lumbar vertebrae, loss of synchondroses, and foramen-magnum shape anomalies. NVP-BGJ398 inhibited FGFR3 downstream signaling pathways, including MAPK, SOX9, STAT1, and PLCγ, in the growth plates of Fgfr3Y367C/+ mice and in cultured chondrocyte models of ACH. Together, our data demonstrate that NVP-BGJ398 corrects pathological hallmarks of ACH and support TKIs as a potential therapeutic approach for ACH. PMID:27064282

  13. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

  14. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  15. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Science.gov (United States)

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  16. Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis.

    Directory of Open Access Journals (Sweden)

    Prasad Vaddepalli

    Full Text Available Tissue morphogenesis in plants requires the coordination of cellular behavior across clonally distinct histogenic layers. The underlying signaling mechanisms are presently being unraveled and are known to include the cell surface leucine-rich repeat receptor-like kinase STRUBBELIG in Arabidopsis. To understand better its mode of action an extensive structure-function analysis of STRUBBELIG was performed. The phenotypes of 20 EMS and T-DNA-induced strubbelig alleles were assessed and homology modeling was applied to rationalize their possible effects on STRUBBELIG protein structure. The analysis was complemented by phenotypic, cell biological, and pharmacological investigations of a strubbelig null allele carrying genomic rescue constructs encoding fusions between various mutated STRUBBELIG proteins and GFP. The results indicate that STRUBBELIG accepts quite some sequence variation, reveal the biological importance for the STRUBBELIG N-capping domain, and reinforce the notion that kinase activity is not essential for its function in vivo. Furthermore, individual protein domains of STRUBBELIG cannot be related to specific STRUBBELIG-dependent biological processes suggesting that process specificity is mediated by factors acting together with or downstream of STRUBBELIG. In addition, the evidence indicates that biogenesis of a functional STRUBBELIG receptor is subject to endoplasmic reticulum-mediated quality control, and that an MG132-sensitive process regulates its stability. Finally, STRUBBELIG and the receptor-like kinase gene ERECTA interact synergistically in the control of internode length. The data provide genetic and molecular insight into how STRUBBELIG regulates intercellular communication in tissue morphogenesis.

  17. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  18. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer

  19. A novel dual kinase function of the RET proto-oncogene negatively regulates activating transcription factor 4-mediated apoptosis.

    Science.gov (United States)

    Bagheri-Yarmand, Rozita; Sinha, Krishna M; Gururaj, Anupama E; Ahmed, Zamal; Rizvi, Yasmeen Q; Huang, Su-Chen; Ladbury, John E; Bogler, Oliver; Williams, Michelle D; Cote, Gilbert J; Gagel, Robert F

    2015-05-01

    The RET proto-oncogene, a tyrosine kinase receptor, is widely known for its essential role in cell survival. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2, a dominant inherited cancer syndrome that affects neuroendocrine organs. However, the mechanisms by which RET promotes cell survival and prevents cell death remain elusive. We demonstrate that in addition to cytoplasmic localization, RET is localized in the nucleus and functions as a tyrosine-threonine dual specificity kinase. Knockdown of RET by shRNA in medullary thyroid cancer-derived cells stimulated expression of activating transcription factor 4 (ATF4), a master transcription factor for stress-induced apoptosis, through activation of its target proapoptotic genes NOXA and PUMA. RET knockdown also increased sensitivity to cisplatin-induced apoptosis. We observed that RET physically interacted with and phosphorylated ATF4 at tyrosine and threonine residues. Indeed, RET kinase activity was required to inhibit the ATF4-dependent activation of the NOXA gene because the site-specific substitution mutations that block threonine phosphorylation increased ATF4 stability and activated its targets NOXA and PUMA. Moreover, chromatin immunoprecipitation assays revealed that ATF4 occupancy increased at the NOXA promoter in TT cells treated with tyrosine kinase inhibitors or the ATF4 inducer eeyarestatin as well as in RET-depleted TT cells. Together these findings reveal RET as a novel dual kinase with nuclear localization and provide mechanisms by which RET represses the proapoptotic genes through direct interaction with and phosphorylation-dependent inactivation of ATF4 during the pathogenesis of medullary thyroid cancer.

  20. Mechanism of activation and functional role of protein kinase Ceta in human platelets.

    Science.gov (United States)

    Bynagari, Yamini S; Nagy, Bela; Tuluc, Florin; Bhavaraju, Kamala; Kim, Soochong; Vijayan, K Vinod; Kunapuli, Satya P

    2009-05-15

    The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation. PMID:19286657

  1. Comparison of a new single-donor human fibrin adhesive with suture for posterior tibial nerve repair in rat: biomechanical resistance and functional analysis

    Institute of Scientific and Technical Information of China (English)

    Reza Erfanian; Masoumeh Firouzi; Mohammad Hossein Nabian; Masoud Darvishzadeh; Leila Oryadi Zanjani; Shayan Abdollah Zadegan; Reza Shahryar Kamrani

    2014-01-01

    Objective:The use of fibrin adhesives has a broad background in nerve repair.Currently the suboptimal physical properties of singledonor fibrin adhesives have restricted their usage.The present experiment studies the performance and physical characteristics of a modified fibrin glue prepared from single-donor human plasma in the repair of posterior tibial nerve of rat.Methods:Forty Wistar rats were divided into 5 groups; in the control group,tibial nerve was completely transected and no treatment was done,while in the four experimental groups the nerve stumps were reconnected by one suture,three sutures,one suture with fibrin glue and fibrin glue alone respectively.During 8 weeks of follow-up,Tibial Function Index was measured weekly and adhesive strength,inflammation and scar formation were assessed at the end of the study.Results:Nerve stumps dehiscence rate and adhesive strength were similar in all experimental groups and significantly differed from control group (P<0.05).By the end of the eighth follow-up week,functional recovery of one and three sutures groups were significantly higher than groups in which fibrin glue was used for repair (P<0.05).The amount of inflammation and scar tissue formation was similar among all groups.Conclusion:The study results show that the prepared single-donor fibrin adhesive has acceptable mechanical properties which could provide required adhesiveness and hold nerve stumps in the long term; yet,we acknowledge that more studies are needed to improve functional outcome of single donor fibrin adhesive repair.

  2. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2003-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well...... to rewriting on arbitrary adhesive categories....

  3. Adhesive Categories

    DEFF Research Database (Denmark)

    Lack, Stephen; Sobocinski, Pawel

    2004-01-01

    We introduce adhesive categories, which are categories with structure ensuring that pushouts along monomorphisms are well-behaved. Many types of graphical structures used in computer science are shown to be examples of adhesive categories. Double-pushout graph rewriting generalises well...... to rewriting on arbitrary adhesive categories....

  4. Aldosterone stimulates nuclear factor-kappa B activity and transcription of intercellular adhesion molecule-1 and connective tissue growth factor in rat mesangial cells via serum- and glucocorticoid-inducible protein kinase-1.

    Science.gov (United States)

    Terada, Yoshio; Ueda, Satoko; Hamada, Kazu; Shimamura, Yoshiko; Ogata, Koji; Inoue, Kosuke; Taniguchi, Yoshinori; Kagawa, Toru; Horino, Taro; Takao, Toshihiro

    2012-02-01

    Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. To determine the signaling pathway of aldosterone in relation to fibrosis and inflammation in mesangial cells, we investigated the effects of aldosterone on expression and activation of serum- and glucocorticoid-inducible protein kinase-1 (SGK1), the activation of nuclear factor-kappa B (NF-κB activation, and the expressions of intercellular adhesion molecule-1 (ICAM-1) and connective tissue growth factor (CTGF). Aldosterone stimulated SGK1 expression, phosphorylation (Ser-256), and kinase activity. The increments of phosphorylation and expression of SGK1 induced by aldosterone were inhibited by mineralocorticoid receptor (MR) inhibitor (eplerenone). Aldosterone stimulated NF-κB activity measured by NF-κB responsive elements, luciferase assay, and the levels of inhibitor of kappa B (IκB) phosphorylation. This aldosterone-induced activation of NF-κB was inhibited by the transfection of dominant-negative SGK1. Furthermore, aldosterone augmented the promoter activities and protein expressions of ICAM-1 and CTGF. The effects of aldosterone on ICAM-1 and CTGF promoter activities and protein expressions were inhibited by the transfection of dominant-negative SGK1 and dominant-negative IκBα. We also found that the MR antagonist significantly ameliorated the glomerular injury and enhancements in SGK1, ICAM-1, and CTGF expressions induced by 1% sodium chloride and aldosterone in vivo. In conclusion, our findings suggest that aldosterone stimulates ICAM-1 and CTGF transcription via activation of SGK1 and NF-κB, which may be involved in the progression of aldosterone-induced mesangial fibrosis and inflammation. MR antagonists may serve as useful therapeutic targets for the treatment of glomerular inflammatory disease.

  5. Diacylglycerol kinase theta and zeta isoforms : regulation of activity, protein binding partners and physiological functions

    NARCIS (Netherlands)

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblasto

  6. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

    Science.gov (United States)

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans

    2007-03-01

    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  7. Terminal regions of β-catenin are critical for regulating its adhesion and transcription functions.

    Science.gov (United States)

    Dar, Mohd Saleem; Singh, Paramjeet; Singh, Gurjinder; Jamwal, Gayatri; Hussain, Syed Sajad; Rana, Aarti; Akhter, Yusuf; Monga, Satdarshan P; Dar, Mohd Jamal

    2016-09-01

    β-Catenin, the central molecule of canonical Wnt signaling pathway, has multiple binding partners and performs many roles in the cell. Apart from being a transcriptional activator, β-catenin acts as a crucial effector component of cadherin/catenin complex to physically interact with actin cytoskeleton along with α-catenin and E-cadherin for regulating cell-cell adhesion. Here, we have generated a library of β-catenin point and deletion mutants to delineate regions within β-catenin that are important for α-catenin-β-catenin interaction, nuclear localization, and transcriptional activity of β-catenin. We observed a unique mechanism for nuclear localization of β-catenin and its mutants and show that N-terminal exon-3 region and C-terminal domain of β-catenin are critical for this activity of β-catenin. Furthermore, we show HepG2 cells have high β-catenin mediated transcriptional activity due to the presence of an interstitial deletion at the N-terminal region of β-catenin. Due to this deletion mutant (hereupon called TM), GSK3β and HDAC inhibitors failed to show any impact whereas curcumin significantly inhibited β-catenin mediated transcriptional activity reiterating that TM is primarily responsible for the high transcriptional activity of HepG2 cells. Moreover, we show the recombinant TM does not physically interact with α-catenin, localizes predominantly in the nucleus, and has nearly two-fold higher transcriptional activity than the wildtype β-catenin. PMID:27368802

  8. Induction of Macrophage Function in Human THP-1 Cells is Associated with MAPK Signaling and Activation of MAP3K7 (TAK1 Protein Kinase

    Directory of Open Access Journals (Sweden)

    Erik eRichter

    2016-03-01

    Full Text Available Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cycline-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1 acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

  9. Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

    Science.gov (United States)

    Min, Xiaoshan; Ungureanu, Daniela; Maxwell, Sarah; Hammarén, Henrik; Thibault, Steve; Hillert, Ellin-Kristina; Ayres, Merrill; Greenfield, Brad; Eksterowicz, John; Gabel, Chris; Walker, Nigel; Silvennoinen, Olli; Wang, Zhulun

    2015-11-01

    JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. PMID:26359499

  10. Adhesion of ZAP-70+ chronic lymphocytic leukemia cells to stromal cells is enhanced by cytokines and blocked by inhibitors of the PI3-kinase pathway.

    Science.gov (United States)

    Lafarge, Sandrine T; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2014-01-01

    CLL cell survival and proliferation is enhanced through direct contact with supporting cells present in lymphoid tissues. PI3Ks are critical signal transduction enzymes controlling B cell survival and activation. PI3K inhibitors have entered clinical trials and show promising therapeutic activity; however, it is unclear whether PI3K inhibitor drugs differentially affect ZAP-70 positive versus negative CLL cells or target specific microenvironmental interactions. Here we provide evidence that CD40L+IL-4, IL-8 or IL-6 enhance adhesion to stromal cells, with IL-6 showing a selective effect on ZAP-70 positive cells. Stimulatory effects of IL-8 or IL-6 are fully reversed by PI3K inhibition, while the effects of CD40L+IL-4 are partially reversed. While CD40L+IL-4 is the only stimulation increasing CLL cell survival for all patient groups, IL-6 protects ZAP-70 positive cells from cell death induced by PI3K inhibition. Altogether, our results indicate that targeting the PI3K pathway can reverse protective CLL-microenvironment interactions in both ZAP-70 positive and negative CLL despite their differences in cytokine responsiveness.

  11. Protein kinase G1 α overexpression increases stem cell survival and cardiac function after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Linlin Wang

    Full Text Available BACKGROUND: We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs contributing to regeneration of the ischemic heart. METHODS AND RESULTS: MSCs from male rats were transduced with adenoviral vector encoding for PKG1α ((PKG1αMSCs.Controls included native MSCs ((NatMSCs and MSCs transduced with an empty vector ((NullMSCs. PKG1α activity was increased approximately 20, 5 and 16 fold respectively in (PKG1αMSCs. (PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in (PKG1αMSCs compared to (NatMSCs and (NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in (PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, (PKG1αMSCs group showed higher survival compared with (NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of (PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and (NullMSCs group, (PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05 and attenuated infarct size (27.2±2.5%, p<0.01. Heart function indices including ejection fraction (52.1±2.2%, p<0.01 and fractional shortening (24.8%±1.3%, p<0.01 were improved significantly in (PKG1αMSCs group. CONCLUSION: Overexpression of PKG1α transgene could be a powerful approach to improve MSCs

  12. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  13. Biomimetic Mussel Adhesive Inspired Clickable Anchors Applied to the Functionalization of Fe3O4 Nanoparticles

    NARCIS (Netherlands)

    Goldmann, Anja S.; Schoedel, Christine; Walther, Andreas; Yuan, Jiayin; Loos, Katja; Mueller, Axel H. E.; Müller, Axel H.E.

    2010-01-01

    The functionalization of magnetite (Fe3O4) nanoparticles with dopamine-derived clickable biomimetic anchors is reported. Herein, an alkyne-modified catechol-derivative is employed as the anchor, as i) the catechol-functional anchor groups possess irreversible covalent binding affinity to Fe3O4 nanop

  14. Recent progress on type II diacylglycerol kinases: the physiological functions of diacylglycerol kinase δ, η and κ and their involvement in disease.

    Science.gov (United States)

    Sakai, Hiromichi; Sakane, Fumio

    2012-11-01

    Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to produce phosphatidic acid (PA) and plays an important role in signal transduction by modulating the balance between these signalling lipids. To date, 10 mammalian DGK isozymes have been identified, and these isozymes are subdivided into five groups according to their structural features. The type II DGKs, consisting of δ1, δ2, η1, η2 and κ isoforms, possess a pleckstrin homology (PH) domain at their N-termini in addition to the separate catalytic region. Moreover, DGKs δ1, δ2 and η2 have a sterile α motif domain at their C-termini. Recent studies have revealed that type II DGKs play pivotal roles in a wide variety of mammalian signal transduction pathways for cell proliferation and differentiation and glucose metabolism and that the DGKs are involved in cancer, type II diabetes, seizures, hypospadias and bipolar disorder. This review summarizes the current knowledge on the properties and physiological functions of type II DGKs and their involvement in disease. PMID:22984004

  15. Facile fabrication of functional PDMS surfaces with tunable wettablity and high adhesive force via femtosecond laser textured templating

    Directory of Open Access Journals (Sweden)

    Yanlei Hu

    2014-12-01

    Full Text Available Femtosecond laser processing is emerged as a promising tool to functionalize surfaces of various materials, including metals, semiconductors, and polymers. However, the productivity of this technique is limited by the low efficiency of laser raster scanning. Here we report a facile approach for efficiently producing large-area functional polymer surfaces, by which metal is firstly textured by a femtosecond laser, and the as-prepared hierarchical structures are subsequently transferred onto polydimethylsiloxane (PDMS surfaces. Aluminum pieces covered by laser induced micro/nano-structures act as template masters and their performance of displaying diverse colors are investigated. Polymer replicas are endowed with tunable wetting properties, which are mainly attributed to the multi-scale surface structures. Furthermore, the surfaces are found to have extremely high adhesive force for water drops because of the high water penetration depth and the resultant high contact angle hysteresis. This characteristic facilitates many potential applications like loss-free tiny water droplets transportation. The reusability of metal master and easiness of soft lithography make it to be a very simple, fast and cost-efficient way for mass production of functional polymeric surfaces.

  16. Effects of Estrogen Level on the Function of Vascular Endothelial Cells and Expression of Vascular Cell Adhesion Molecule - 1φ

    Institute of Scientific and Technical Information of China (English)

    WU Saizhu(吴赛珠); LIU Jiangguo(刘建国); TAN Jiayu(谭家余); ZHoU Kexiang(周可祥); Gorge D Webb; WEI Heming(隗和明); GUO Zhiguang(郭志刚)

    2002-01-01

    Objectives To ob- serve the effect of different estrogen levels on the se- cretory function of vascular endothelial cells of female rats, and study the effect of modulation of estrogen level on the expression of vascular cell adhesion molecule - 1 and the concentration of estrogen receptorin vascular endothelial cells. Methods Radioim-munology was used to measure the serum concentrationof endothelin and PGI2, and copper-cadmium re-duction was employed to measure the serum content ofnitrogen monoxide. Radioligand binding and flowcy-tometry were used to measure the expression of estrogenreceptor and vascular cell adhesion molecule (VCAM-1 ) of vascular endothelial cells respectively. Re-sults 1. The serum concentration of nitric oxide andPGI2 decreased when the ovaries of female rats wereremoved. In ovariectomized rats, given estrogen, theconcentration rose ( P < 0.05), but the plasma con-centration of endothelin was adverse to it. 2. Theconcentration of estrogen receptor of vascular endothe-lial cells decreased remarkably when the ovaries of fe-male rats were removed. When given estrogen, it in-creased. 3. The percent of expressed VCAM - 1 in-creased siguificantly after interleukin- lβoperated onthe cells, but 17 - βestradiol at 3 × 10-8 ~ 10-6 mol/lall decreased the percent. Conclusions Estrogenlevel can influence the secretion of nitrogen monoxide,PGI2 and endothlin of vascular endothelial cells, andalso influence the concentration of estrogen receptor ofvascular endothelial cells. 17 -β Estradiol at 3 × 10-8~ 10-6 M can decrease the elevation of VCAM - 1 ofvascular endothelial cells induced by interleukin - 1 β.

  17. RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

    Directory of Open Access Journals (Sweden)

    Frank Stenner

    Full Text Available RP1 (synonym: MAPRE2, EB2 is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

  18. Functional Characterization of AtSP1, A Sucrose-Responsive Receptor-Like Kinase

    Institute of Scientific and Technical Information of China (English)

    Xuna Wu; Kerstin Zander; Eugenia Maximova; Gerhard Obermeyer; Waltraud Schulze

    2012-01-01

    SP1-dependent sucrose response or resupply.Among 42 phosphoproteins with lower phosphorylation in the sp1 mutant,three aquaporins were inactivated in sp1 mutant based on phosphoproteomics results.Swelling assay of protoplasts indicated that AtSP1 was involved in modulation water transport ability by phosphorylating aquaporins in sucrose-induced osmolarity.In addition,aquaporin PIP3,v-type ATPase VHA-D,ribosomal protein RPP1C and RPS2C,a putative translational activator (AT1G64790),and a putative LRR-receptor like kinase (AT3G02880) were found that phosphorylation of these proteins was furthermore dependent on Ser-744 of AtSP1.In future,interaction network of AtSP1 need to be investigated based on the transgenic lines with GFP tag.Targeted phosphorylation studies using the identified potential targets of AtSP1 on protein arrays could elucidate if the functional interactions discovered in this work are direct or indirect.

  19. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  20. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    Science.gov (United States)

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs.

  1. Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

    Science.gov (United States)

    Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

    2015-12-01

    Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development.

  2. Functional Role of Glycogen synthase Kinase-3β on Glucocorticoid-mediated signaling

    OpenAIRE

    Rubio Patiño, Camila

    2012-01-01

    Glucocorticoids (GC) induce cell cycle arrest and apoptosis in different cell types and therefore are widely used to treat a variety of diseases including autoimmune disorders and cancer. This effect is mediated by the GC receptor (GR), a ligandactivated transcription factor that translocates into the nucleus where it modulates transcription of target genes in a promoter-specific manner. Glycogen synthase kinase-3 (GSK3) regulates GR response by genomic and nongenomic mechanisms, although the...

  3. Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

    OpenAIRE

    Los, Alrik Pieter

    2007-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the second messenger diacylglycerol (DAG) yielding phosphatidic acid (PA). In this thesis, we investigated which structural domains of DGKtheta are required for DGK activity. Furthermore, we showed that DGKzeta binds to and is activated by the Retinoblastoma tumour suppressor protein (pRB) and the pRB-related proteins p107 and p130, key regulators of the cell-cycle, differentiation and apoptosis. The interaction between pRB and DGKzeta is regulated ...

  4. Solvent-free functionalization of silicone rubber and efficacy of PAAm brushes grafted from an amino-PPX layer against bacterial adhesion

    NARCIS (Netherlands)

    Fundeanu, Irina; Klee, Doris; Schouten, Arend J.; Busscher, Henk J.; van der Mei, Henny C.

    2010-01-01

    Silicone rubber is a frequently employed biomaterial that is prone to bacterial adhesion and biofilm formation. In this study, the surface of silicone rubber was solvent-free functionalized by chemical vapor deposition (CVD) of poly(o-amino-p-xylylene-co-p-xylylene (amino-PPX). Subsequently, the ami

  5. Guanine Nucleotide-Binding Proteins of the G(12) Family Shape Immune Functions by Controlling CD4(+) T Cell Adhesiveness and Motility

    NARCIS (Netherlands)

    S. Herroeder; P. Reichardt; A. Sassmann; B. Zimmermann; D. Jaeneke; J. Hoeckner; M.W. Hollmann; K.D. Fischer; S. Vogt; R. Grosse; N. Hogg; M. Gunzer; S. Offermanns; N. Wettschureck

    2009-01-01

    Integrin-mediated adhesion plays a central role in T cell trafficking and activation. Genetic inactivation of the guanine nucleotide-binding (G) protein alpha-subunits G alpha(12) and G alpha(13) resulted in an increased activity of integrin leukocyte-function-antigen-1 in murine CD4(+) T cells. The

  6. Effect of various surface conditioning methods on the adhesion of dual-cure resin cement with MDP functional monomer to zirconia after thermal aging

    NARCIS (Netherlands)

    Ozcan, Mutlu; Nijhuis, Henk; Felipe Valandro, Luiz

    2008-01-01

    This study evaluated the effect of chairside and laboratory types of surface conditioning methods on the adhesion of dual-cure resin cement with MDP functional monomer to zirconia ceramic after thermocycling. Disk-shaped (diameter: 10 mm, thickness: 2 mm) Y-TZP ceramics (Lava(TM), 3M ESPE) were used

  7. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L.; Spiller, Benjamin W. (Vanderbilt)

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

  8. Lactobacillus Adhesion to Mucus

    Directory of Open Access Journals (Sweden)

    Maxwell L. Van Tassell

    2011-05-01

    Full Text Available Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host.

  9. Isolation and functional characterization of the C-terminus of rice phosphatidylinositol 4-kinase in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A partial rice (Oryza sativa L.) cDNA clone, OsPI4K1c, was isolated through screening of a cDNA library constructed from tillering materials. OsPI4K1c encoded a peptide of 608 amino acids with a calculated molecular mass of 68.4 kDa. The OsPI4K1c peptide shared high homology and possessed the highly conserved domains present in most isolated cloned PI4-kinases, i.e. a lipid kinase unique (LKU) domain and a catalytic (CAT) domain. A region with similarity to pleckstrin homology (PH) domain was present in OsPI4K1c as well. Further comparison with genomic sequences in databases revealed that OsPI4K1c is located at the 3'-end of a putative rice PI 4-kinase coding gene OsPI4K1, and its coding region corresponded to the C-terminal half of OsPI4K1 protein. Twelve exons (49-562 bp in size) and 11 introns (77-974 bp in size) were identified in OsPI4K1c. The recombinant protein expressed in Escherichia coli phosphorylates phosphatidylinositol at the D4 position of the inositol ring. OsPI4K1 transcript levels were detected in a low but constitutive manner in shoot, stem, leaf, spike and root tissues and did not change upon treatment with different hormones, calcium and jasmonic acid (JA). However, treatment with salicylic acid (SA) elevated the mRNA level of the OsPI4K1 gene, which suggested the involvement of OsPI4K1 in wounding responses.

  10. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  11. Stereoselectivity of isoflurane in adhesion molecule leukocyte function-associated antigen-1.

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    Weiming Bu

    Full Text Available BACKGROUND: Isoflurane in clinical use is a racemate of S- and R-isoflurane. Previous studies have demonstrated that the effects of S-isoflurane on relevant anesthetic targets might be modestly stronger (less than 2-fold than R-isoflurane. The X-ray crystallographic structure of the immunological target, leukocyte function-associated antigen-1 (LFA-1 with racemic isoflurane suggested that only S-isoflurane bound specifically to this protein. If so, the use of specific isoflurane enantiomers may have advantage in the surgical settings where a wide range of inflammatory responses is expected to occur. Here, we have further tested the hypothesis that isoflurane enantioselectivity is apparent in solution binding and functional studies. METHODS: First, binding of isoflurane enantiomers to LFA-1 was studied using 1-aminoanthracene (1-AMA displacement assays. The binding site of each enantiomer on LFA-1 was studied using the docking program GLIDE. Functional studies employed the flow-cytometry based ICAM binding assay. RESULTS: Both enantiomers decreased 1-AMA fluorescence signal (at 520 nm, indicating that both competed with 1-AMA and bound to the αL I domain. The docking simulation demonstrated that both enantiomers bound to the LFA-1 "lovastatin site." ICAM binding assays showed that S-isoflurane inhibited more potently than R-isoflurane, consistent with the result of 1-AMA competition assay. CONCLUSIONS: In contrast with the x-ray crystallography, both enantiomers bound to and inhibited LFA-1. S-isoflurane showed slight preference over R-isoflurane.

  12. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

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    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  13. Protein Kinase G1 α Overexpression Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction

    OpenAIRE

    Linlin Wang; Zeeshan Pasha; Shuyun Wang; Ning Li; Yuliang Feng; Gang Lu; Millard, Ronald W.; Muhammad Ashraf

    2013-01-01

    BACKGROUND: We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart. METHODS AND RESULTS: MSCs from male rats were transduced with adenoviral vector encoding for PKG1α ((PKG1α)MSCs).Controls included native MSCs ((Nat)MSCs) and MSCs transduced with an empty vector ((Null)MSCs). PKG1α activity was increased appr...

  14. Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2.

    Science.gov (United States)

    Li, Hanjun; Spagnol, Gaelle; Zheng, Li; Stauch, Kelly L; Sorgen, Paul L

    2016-07-22

    Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43. PMID:27235399

  15. Pro-B-cell-specific transcription and proapoptotic function of protein kinase Ceta.

    Science.gov (United States)

    Morrow, T A; Muljo, S A; Zhang, J; Hardwick, J M; Schlissel, M S

    1999-08-01

    Using a subtractive cloning scheme on cDNA prepared from primary pro-B and pre-B cells, we identified several genes whose products regulate apoptosis. We further characterized one of these genes, encoding protein kinase Ceta (PKCeta). PKCeta transcripts were readily detected in pro-B cells but were absent in pre-B cells. Although both a full-length and a truncated form of PKCeta were detectable in bone marrow pro-B cells, transition to the pre-B-cell stage was associated with increased relative levels of truncated PKCeta. We found that PKCeta is proteolyzed in apoptotic lymphocytes, generating a kinase-active fragment identical to the truncated form which is capable of inducing apoptosis when expressed in a pro-B cell line. Caspase-3 can generate an identical PKCeta cleavage product in vitro, and caspase inhibitors prevent the generation of this product during apoptosis in transfected cell lines. Inducible overexpression of either the full-length or truncated form of PKCeta results in cell cycle arrest at the G(1)/S transition. These results suggest that the expression and proteolytic activation of PKCeta play an important role in the regulation of cell division and cell death during early B-cell development. PMID:10409750

  16. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    Science.gov (United States)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  17. Molecular mechanism underlying the functional loss of cyclindependent kinase inhibitors pl6 and p27 in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Yasunobu Matsuda

    2008-01-01

    Hepatocellular carcinoma (HCC) is one of the most common human cancers, and its incidence is still increasing in many countries. The prognosis of HCC patients remains poor, and identification of useful molecular prognostic markers is required. Many recent studies have shown that functional alterations of cell-cycle regulators can be observed in HCC. Among the various types of cell-cycle regulators, pl6 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors. pl6, a Gl-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase (CDK) 4 and CDK6 with cyclin Dl, is frequently inactivated in HCC via CpG methylation of its promoter region. pl6 may be involved in the early steps of hepatocarcinogenesis, since pl6 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients. p27, a negative regulator of the Gl-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes, is now considered to be an adverse prognostic factor in HCC. In some cases of HCC with increased cell proliferation, p27 is overexpressed but inactivated by sequestration into cyclin Dl-CDK4-containing complexes. Since loss of pl6 is closely related to functional inactivation of p27 in HCC, investigating both pl6 and p27 may be useful for precise prognostic predictions in individuals with HCC.

  18. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    Science.gov (United States)

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  19. Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Vasculogenic mimicry (VM is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2/focal adhesion kinase (FAK/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR, respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.

  20. Identification of novel gene targets and functions of p21-activated kinase 1 during DNA damage by gene expression profiling.

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    Mona Motwani

    Full Text Available P21-activated kinase 1 (PAK1, a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR, and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG, Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new

  1. Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1

    Directory of Open Access Journals (Sweden)

    Jose-Antonio Rodriguez-Rodriguez

    2016-05-01

    Full Text Available To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release and MEN (mitotic exit network. Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions.

  2. Structure-function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell.

    Science.gov (United States)

    Ziercher, Léa; Filhol, Odile; Laudet, Béatrice; Prudent, Renaud; Cochet, Claude; Buchou, Thierry

    2011-10-01

    Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2β) revealed a viability defect. However, analysis of CK2β functions along the neural lineage established CK2β as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2β involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis. PMID:21861102

  3. Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects.

    Science.gov (United States)

    Levine, K; Oehlen, L J; Cross, F R

    1998-01-01

    The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits. PMID:9418876

  4. Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

    Science.gov (United States)

    Ramakrishnan, N.; Tourdot, Richard W.; Eckmann, David M.; Ayyaswamy, Portonovo S.; Muzykantov, Vladimir R.; Radhakrishnan, Ravi

    2016-01-01

    In order to achieve selective targeting of affinity–ligand coated nanoparticles to the target tissue, it is essential to understand the key mechanisms that govern their capture by the target cell. Next-generation pharmacokinetic (PK) models that systematically account for proteomic and mechanical factors can accelerate the design, validation and translation of targeted nanocarriers (NCs) in the clinic. Towards this objective, we have developed a computational model to delineate the roles played by target protein expression and mechanical factors of the target cell membrane in determining the avidity of functionalized NCs to live cells. Model results show quantitative agreement with in vivo experiments when specific and non-specific contributions to NC binding are taken into account. The specific contributions are accounted for through extensive simulations of multivalent receptor–ligand interactions, membrane mechanics and entropic factors such as membrane undulations and receptor translation. The computed NC avidity is strongly dependent on ligand density, receptor expression, bending mechanics of the target cell membrane, as well as entropic factors associated with the membrane and the receptor motion. Our computational model can predict the in vivo targeting levels of the intracellular adhesion molecule-1 (ICAM1)-coated NCs targeted to the lung, heart, kidney, liver and spleen of mouse, when the contributions due to endothelial capture are accounted for. The effect of other cells (such as monocytes, etc.) do not improve the model predictions at steady state. We demonstrate the predictive utility of our model by predicting partitioning coefficients of functionalized NCs in mice and human tissues and report the statistical accuracy of our model predictions under different scenarios. PMID:27429783

  5. Biophysically inspired model for functionalized nanocarrier adhesion to cell surface: roles of protein expression and mechanical factors

    Science.gov (United States)

    Ramakrishnan, N.; Tourdot, Richard W.; Eckmann, David M.; Ayyaswamy, Portonovo S.; Muzykantov, Vladimir R.; Radhakrishnan, Ravi

    2016-06-01

    In order to achieve selective targeting of affinity-ligand coated nanoparticles to the target tissue, it is essential to understand the key mechanisms that govern their capture by the target cell. Next-generation pharmacokinetic (PK) models that systematically account for proteomic and mechanical factors can accelerate the design, validation and translation of targeted nanocarriers (NCs) in the clinic. Towards this objective, we have developed a computational model to delineate the roles played by target protein expression and mechanical factors of the target cell membrane in determining the avidity of functionalized NCs to live cells. Model results show quantitative agreement with in vivo experiments when specific and non-specific contributions to NC binding are taken into account. The specific contributions are accounted for through extensive simulations of multivalent receptor-ligand interactions, membrane mechanics and entropic factors such as membrane undulations and receptor translation. The computed NC avidity is strongly dependent on ligand density, receptor expression, bending mechanics of the target cell membrane, as well as entropic factors associated with the membrane and the receptor motion. Our computational model can predict the in vivo targeting levels of the intracellular adhesion molecule-1 (ICAM1)-coated NCs targeted to the lung, heart, kidney, liver and spleen of mouse, when the contributions due to endothelial capture are accounted for. The effect of other cells (such as monocytes, etc.) do not improve the model predictions at steady state. We demonstrate the predictive utility of our model by predicting partitioning coefficients of functionalized NCs in mice and human tissues and report the statistical accuracy of our model predictions under different scenarios.

  6. T cell-mediated modulation of mast cell function: heterotypic adhesion-induced stimulatory or inhibitory effects.

    Science.gov (United States)

    Mekori, Yoseph A; Hershko, Alon Y

    2012-01-01

    Close physical proximity between mast cells and T cells has been demonstrated in several T cell mediated inflammatory processes such as rheumatoid arthritis and sarcoidosis. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We have identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells, and showed that this pathway is associated with degranulation and cytokine release. The signaling events associated with this pathway of mast cell activation have also been elucidated confirming the activation of the Ras mitogen-activated protein kinase systems. More recently, we hypothesized and demonstrated that mast cells may also be activated by microparticles released from activated T cells that are considered as miniature version of a cell. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site. Recent works have also focused on the effects of regulatory T cells (Treg) on mast cells. These reports highlighted the importance of the cytokines IL-2 and IL-9, produced by mast cells and T cells, respectively, in obtaining optimal immune suppression. Finally, physical contact, associated by OX40-OX40L engagement has been found to underlie the down-regulatory effects exerted by Treg on mast cell function. PMID:22566892

  7. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  8. Glycosynapses: microdomains controlling carbohydrate-dependent cell adhesion and signaling

    Directory of Open Access Journals (Sweden)

    Hakomori Senitiroh

    2004-01-01

    Full Text Available The concept of microdomains in plasma membranes was developed over two decades, following observation of polarity of membrane based on clustering of specific membrane components. Microdomains involved in carbohydrate-dependent cell adhesion with concurrent signal transduction that affect cellular phenotype are termed "glycosynapse". Three types of glycosynapse have been distinguished: "type 1" having glycosphingolipid associated with signal transducers (small G-proteins, cSrc, Src family kinases and proteolipids; "type 2" having O-linked mucin-type glycoprotein associated with Src family kinases; and "type 3" having N-linked integrin receptor complexed with tetraspanin and ganglioside. Different cell types are characterized by presence of specific types of glycosynapse or their combinations, whose adhesion induces signal transduction to either facilitate or inhibit signaling. E.g., signaling through type 3 glycosynapse inhibits cell motility and differentiation. Glycosynapses are distinct from classically-known microdomains termed "caveolae", "caveolar membrane", or more recently "lipid raft", which are not involved in carbohydrate-dependent cell adhesion. Type 1 and type 3 glycosynapses are resistant to cholesterol-binding reagents, whereas structure and function of "caveolar membrane" or "lipid raft" are disrupted by these reagents. Various data indicate a functional role of glycosynapses during differentiation, development, and oncogenic transformation.

  9. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael;

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea...... that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell....

  10. Identification of Differentially Expressed Kinase and Screening Potential Anticancer Drugs in Papillary Thyroid Carcinoma

    Science.gov (United States)

    Zhang, Huairong

    2016-01-01

    Aim. We aim to identify protein kinases involved in the pathophysiology of papillary thyroid carcinoma (PTC) in order to provide potential therapeutic targets for kinase inhibitors and unfold possible molecular mechanisms. Materials and Methods. The gene expression profile of GSE27155 was analyzed to identify differentially expressed genes and mapped onto human protein kinases database. Correlation of kinases with PTC was addressed by systematic literature search, GO and KEGG pathway analysis. Results. The functional enrichment analysis indicated that “mitogen-activated protein kinases pathway” expression was extremely enriched, followed by “neurotrophin signaling pathway,” “focal adhesion,” and “GnRH signaling pathway.” MAPK, SRC, PDGFRa, ErbB, and EGFR were significantly regulated to correct these pathways. Kinases investigated by the literature on carcinoma were considered to be potential novel molecular therapeutic target in PTC and application of corresponding kinase inhibitors could be possible therapeutic tool. Conclusion. SRC, MAPK, and EGFR were the most important differentially expressed kinases in PTC. Combined inhibitors may have high efficacy in PTC treatment by targeting these kinases. PMID:27703281

  11. Molecular behavior adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH.

    Science.gov (United States)

    Gilat, D; Hershkoviz, R; Goldkorn, I; Cahalon, L; Korner, G; Vlodavsky, I; Lider, O

    1995-05-01

    Migration of lymphocytes into inflammatory sites requires their adhesion to the vascular endothelium and subendothelial extracellular matrix (ECM). The ensuing penetration of the ECM is associated with the expression of ECM-degrading enzymes, such as endo-beta-D glucuronidase (heparanase), which cleaves heparan sulfate (HS) proteoglycans. We now report that, depending on the local pH, a mammalian heparanase can function either as an enzyme or as an adhesion molecule. At relatively acidified pH conditions, heparanase performs as an enzyme, degrading HS. In contrast, at the hydrogen ion concentration of a quiescent tissue, heparanase binds specifically to HS molecules without degrading them, and thereby anchors CD4+ human T lymphocytes. Thus, the local state of a tissue can regulate the activities of heparanase and can determine whether the molecule will function as an enzyme or as a proadhesive molecule. PMID:7722469

  12. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Science.gov (United States)

    Colabardini, Ana Cristina; Brown, Neil Andrew; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2013-01-01

    The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the

  13. Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK.

    Directory of Open Access Journals (Sweden)

    Ana Cristina Colabardini

    Full Text Available The serum- and glucocorticoid-regulated protein kinase (SGK is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene and niiA (from the nitrate reductase gene. Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets

  14. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    Science.gov (United States)

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P

    2013-03-01

    phosphorylation levels of protein kinase A, protein kinase B, and CREB. Similarly, CREB inhibition reduced CREB activation and PGC-1α protein levels in selenoprotein H transfected cells. Moreover, selenoprotein H transfection increased the activity of mitochondrial complexes and prevented the ultraviolet B induced fall of mitochondrial membrane potential. We conclude that the effects of selenoprotein H on mitochondrial biogenesis and mitochondrial function are probably mediated through protein kinase A-CREB-PGC-1α and Akt/protein kinase B-CREB-PGC-1α pathways.

  15. Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart.

    Science.gov (United States)

    Jiang, Mao; Xu, Ande; Narayanan, Njanoor

    2006-09-01

    Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (approximately 40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment. PMID:16617128

  16. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    Science.gov (United States)

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  17. Cloning and functions analysis of a pyruvate dehydrogenase kinase in Brassica napus.

    Science.gov (United States)

    Li, Rong-Jun; Hu, Zhi-Yong; Zhang, Hua-Shan; Zhan, Gao-Miao; Wang, Han-Zhong; Hua, Wei

    2011-08-01

    Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), which plays a key role in intermediary metabolism. In this study, a 1,490-bp PDK in Brassica napus (BnPDK1) was isolated and cloned from Brassica cDNA library. BnPDK1 has an 1,104 open reading frame encoding 367 amino acids. Genomic DNA gel blot analysis result indicated that BnPDK1 is a multi-copy gene. RNA gel blot analysis and RNA in situ hybridization were used to determine the expression of BnPDK1 in different organs. BnPDK1 gene was ubiquitously expressed in almost all the tissues tested, having the highest expression in the stamen and the young silique. Over-expression of BnPDK1 in transgenic Arabidopsis lines would repress the PDC activity, and resulted in the decrease of seed oil content and leaf photosynthesis. These results implied that BnPDK1 was involved in the regulation of fatty acid biosynthesis in developing seeds.

  18. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  19. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    Science.gov (United States)

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  20. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation

    DEFF Research Database (Denmark)

    Steffen, Anika; Ladwein, Markus; Dimchev, Georgi A;

    2013-01-01

    Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation...... restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed...... deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but...

  1. Genomic Dissection and Expression Profiling Revealed Functional Divergence in Triticum aestivum Leucine Rich Repeat Receptor Like Kinases (TaLRRKs)

    Science.gov (United States)

    Shumayla; Sharma, Shailesh; Kumar, Rohit; Mendu, Venugopal; Singh, Kashmir; Upadhyay, Santosh K.

    2016-01-01

    The leucine rich repeat receptor like kinases (LRRK) constitute the largest subfamily of receptor like kinases (RLK), which play critical roles in plant development and stress responses. Herein, we identified 531 TaLRRK genes in Triticum aestivum (bread wheat), which were distributed throughout the A, B, and D sub-genomes and chromosomes. These were clustered into 233 homologous groups, which were mostly located on either homeologous chromosomes from various sub-genomes or in proximity on the same chromosome. A total of 255 paralogous genes were predicted which depicted the role of duplication events in expansion of this gene family. Majority of TaLRRKs consisted of trans-membrane region and localized on plasma-membrane. The TaLRRKs were further categorized into eight phylogenetic groups with numerous subgroups on the basis of sequence homology. The gene and protein structure in terms of exon/intron ratio, domains, and motifs organization were found to be variably conserved across the different phylogenetic groups/subgroups, which indicated a potential divergence and neofunctionalization during evolution. High-throughput transcriptome data and quantitative real time PCR analyses in various developmental stages, and biotic and abiotic (heat, drought, and salt) stresses provided insight into modus operandi of TaLRRKs during these conditions. Distinct expression of majority of stress responsive TaLRRKs homologous genes suggested their specified role in a particular condition. These results provided a comprehensive analysis of various characteristic features including functional divergence, which may provide the way for future functional characterization of this important gene family in bread wheat.

  2. Signaling transduction pathways involved in basophil adhesion and histamine release

    DEFF Research Database (Denmark)

    Sha, Quan; Poulsen, Lars K.; Gerwien, Jens;

    2006-01-01

    Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles ...... of beta1 and beta2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase (PI3K), src-kinases and extracellular signal regulated kinase (ERK) 1/2 in basophil adhesion and histamine release (HR)....

  3. Functional expression of double-stranded RNA-dependent protein kinase in rat intestinal epithelial cells.

    Science.gov (United States)

    Sato, Nagahiro; Morimoto, Hiroyuki; Baba, Ryoko; Nakamata, Junichi; Doi, Yoshiaki; Yamaguchi, Koji

    2010-05-01

    Intestinal epithelial cells (IECs) are exposed to external environment, microbial and viral products, and serve as essential barriers to antigens. Recent studies have shown that IECs express Toll-like receptors (TLRs) and respond to microbial components. The antimicrobial and antiviral barriers consist of many molecules including TLRs. To investigate the further component of this barrier in intestine, we examined the expression of double-stranded RNA-dependent protein kinase (PKR). PKR is a player in the cellular antiviral response and phosphorylates alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) to block protein synthesis and induces apoptosis. In this study, we showed that the expression of PKR was restricted to the cytoplasm of absorptive epithelial cells in the intestine of adult rat. We also demonstrated that PKR was expressed in the cultured rat intestinal epithelial cells (IEC-6). The level of PKR protein expression and the activity of alkaline phosphatase (ALP) increased in the cultured IEC-6 cells in a time-dependent manner. Inhibition of PKR by the 2-aminopurine treatment decreased ALP activity in the IEC-6 cells. Treatment of IEC-6 cells with synthetic double-stranded RNA (dsRNA) induced cell death in a dose-dependent manner. The addition of hydrocortisone also provoked suppression of PKR expression and ALP activity. This modulation might be mediated by signal transducers and activators of transcription-1 (STAT-1) protein. We concluded that PKR is expressed in IECs as potent barriers to antigens and is a possible modulator of the differentiation of rat IECs. PMID:20213745

  4. Targeting polo-like kinase 1 suppresses essential functions of alloreactive T cells.

    Science.gov (United States)

    Berges, Carsten; Chatterjee, Manik; Topp, Max S; Einsele, Hermann

    2016-06-01

    Acute graft-versus-host disease (aGvHD) is still a major cause of transplant-related mortality after allogeneic stem cell transplantation (ASCT). It requires immunosuppressive treatments that broadly abrogate T cell responses including beneficial ones directed against tumor cells or infective pathogens. Polo-like kinase 1 (PLK1) is overexpressed in many cancer types including leukemia, and clinical studies demonstrated that targeting PLK1 using selective PLK1 inhibitors resulted in inhibition of proliferation and induction of apoptosis predominantly in tumor cells, supporting the feasibility of PLK1 as target for anticancer therapy. Here, we show that activation of alloreactive T cells (Tallo) up-regulate expression of PLK1, suggesting that PLK1 is a potential new candidate for dual therapy of aGvHD and leukemia after ASCT. Inhibition of PLK1, using PLK1-specific inhibitor GSK461364A selectively depletes Tallo by preventing activation and by inducing apoptosis in already activated Tallo, while memory T cells are preserved. Activated Tallo cells which survive exposure to PLK1 undergo inhibition of proliferation by induction of G2/M cell cycle arrest, which is accompanied by accumulation of cell cycle regulator proteins p21(WAF/CIP1), p27(Kip1), p53 and cyclin B1, whereas abundance of CDK4 decreased. We also show that suppressive effects of PLK1 inhibition on Tallo were synergistically enhanced by concomitant inhibition of molecular chaperone Hsp90. Taken together, our data suggest that PLK1 inhibition represents a reasonable dual strategy to suppress residual tumor growth and efficiently deplete Tallo, and thus provide a rationale to selectively prevent and treat aGvHD. PMID:26724940

  5. The phosphoinositide 3-kinase signalling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions

    Directory of Open Access Journals (Sweden)

    Samantha D Pauls

    2012-08-01

    Full Text Available The phosphoinositide 3-kinase (PI3K pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunogloblulin isotype switch, germinal center responses and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia.

  6. The phosphoinositide 3-kinase signaling pathway in normal and malignant B cells: activation mechanisms, regulation and impact on cellular functions.

    Science.gov (United States)

    Pauls, Samantha D; Lafarge, Sandrine T; Landego, Ivan; Zhang, Tingting; Marshall, Aaron J

    2012-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is a central signal transduction axis controlling normal B cell homeostasis and activation in humoral immunity. The p110δ PI3K catalytic subunit has emerged as a critical mediator of multiple B cell functions. The activity of this pathway is regulated at multiple levels, with inositol phosphatases PTEN and SHIP both playing critical roles. When deregulated, the PI3K pathway can contribute to B cell malignancies and autoantibody production. This review summarizes current knowledge on key mechanisms that activate and regulate the PI3K pathway and influence normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell anergy. We also discuss PI3K pathway alterations reported in select B cell malignancies and highlight studies indicating the functional significance of this pathway in malignant B cell survival and growth within tissue microenvironments. Finally, we comment on early clinical trial results, which support PI3K inhibition as a promising treatment of chronic lymphocytic leukemia.

  7. Natural variation in the thermotolerance of neural function and behavior due to a cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    Ken Dawson-Scully

    Full Text Available Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG as well as to its downstream target, protein phosphatase 2A (PP2A. Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations.

  8. Natural Variation in the Thermotolerance of Neural Function and Behavior due to a cGMP-Dependent Protein Kinase

    Science.gov (United States)

    Dawson-Scully, Ken; Armstrong, Gary A.B.; Kent, Clement; Robertson, R. Meldrum; Sokolowski, Marla B.

    2007-01-01

    Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations. PMID:17712421

  9. Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-cyclin B kinases to regulate lipid synthesis in yeast.

    Science.gov (United States)

    Su, Wen-Min; Han, Gil-Soo; Casciano, Jessica; Carman, George M

    2012-09-28

    Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.

  10. The neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Berezin, V; Bock, E; Poulsen, F M

    2000-01-01

    During the past year, the understanding of the structure and function of neural cell adhesion has advanced considerably. The three-dimensional structures of several of the individual modules of the neural cell adhesion molecule (NCAM) have been determined, as well as the structure of the complex...... between two identical fragments of the NCAM. Also during the past year, a link between homophilic cell adhesion and several signal transduction pathways has been proposed, connecting the event of cell surface adhesion to cellular responses such as neurite outgrowth. Finally, the stimulation of neurite...

  11. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  12. Cyclin H binding to the RARα activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7

    OpenAIRE

    Bour, Gaétan; Gaillard, Emilie; Bruck, Nathalie; Lalevée, Sébastien; Plassat, Jean-Luc; Busso, Didier; Samama, Jean-Pierre; Rochette-Egly, Cécile

    2005-01-01

    The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARα is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show tha...

  13. Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

    OpenAIRE

    Levine, Kristi; Oehlen, L. J. W. M.; Cross, Frederick R.

    1998-01-01

    The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 w...

  14. Separation of the gluconeogenic and mitochondrial functions of pgc-1α through s6 kinase

    DEFF Research Database (Denmark)

    Lustig, Y.; Ruas, J.L.; Estall, J.L.;

    2011-01-01

    PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle...

  15. Protective coupling of mitochondrial function and protein synthesis via the eIF2α kinase GCN-2.

    Directory of Open Access Journals (Sweden)

    Brooke M Baker

    Full Text Available Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2-dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans. Reactive oxygen species (ROS generated from dysfunctional mitochondria are required for GCN-2-dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPR(mt.

  16. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    Science.gov (United States)

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  17. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan;

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation......-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...

  18. Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

    Directory of Open Access Journals (Sweden)

    I. O. Zhaleyko

    2014-07-01

    Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

  19. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  20. Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133)

    OpenAIRE

    Fischer, Liane; Wilde, Caroline; Schöneberg, Torsten; Liebscher, Ines

    2016-01-01

    Background: A large number of human inherited and acquired diseases and phenotypes are caused by mutations in G protein-coupled receptors (GPCR). Genome-wide association studies (GWAS) have shown that variations in the ADGRD1 (GPR133) locus are linked with differences in metabolism, human height and heart frequency. ADGRD1 is a Gs protein-coupled receptor belonging to the class of adhesion GPCRs. Results: Analysis of more than 1000 sequenced human genomes revealed approximately 9000 single nu...

  1. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Takayuki [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Murata, Ryo [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Sun, Shu-lan [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Saito, Yutaro; Yamaga, Shuhei [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Tanaka, Nobuyuki; Tamai, Keiichi [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Moriya, Kunihiko [Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kasai, Noriyuki [Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Ishii, Naoto [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  2. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    Science.gov (United States)

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development. PMID:24585212

  3. Crystal structure of LpxK, the 4'-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface.

    Science.gov (United States)

    Emptage, Ryan P; Daughtry, Kelly D; Pemble, Charles W; Raetz, Christian R H

    2012-08-01

    In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4'-kinase (LpxK) of the diverse P-loop-containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg(2+)-bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg(2+) cation using a 25° hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg(2+) binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design. PMID:22826246

  4. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: structural and functional studies.

    Science.gov (United States)

    Drakou, Christina E; Malekkou, Anna; Hayes, Joseph M; Lederer, Carsten W; Leonidas, Demetres D; Oikonomakos, Nikos G; Lamond, Angus I; Santama, Niovi; Zographos, Spyros E

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells. PMID:22038794

  5. [FTIR spectroscopic studies of facial prosthetic adhesives].

    Science.gov (United States)

    Kang, Biao; Yang, Qing-fang; Liang, Jian-feng; Zhao, Yi-min

    2008-10-01

    According to the composition of the traditional facial prosthetic adhesives, most of adhesives can be classified into two categories: acrylic polymer-based adhesive and silicone-based adhesive. In previous studies, measurements of various mechanical bond strengths were carried out, whereas the functional groups of the adhesives were evaluated seldom during the adhesion. In the present study the analysis of two facial prosthetic adhesives (Epithane and Secure Adhesive) was carried out by using infrared spectroscopy. Two adhesives in the form of fluid or semisolid were submitted to FTIR spectroscopy, respectively. The results showed that water and ammonia residue volatilized during the solidification of Epithane, and absorption peak reduction of carbonyl was due to the volatilization of acetate vinyl from Secure Adhesive. Similar silicone functional groups both in the silicone-based adhesive and in silicone elastomer could be the key to higher bond strength between silicone elastomer and skin with silicone-based adhesive. The position, shape of main absorption peaks of three adhesives didn't change, which showing that their main chemicals and basic structures didn't change during solidification. PMID:19123392

  6. Yeast GSK-3 kinase regulates astral microtubule function through phosphorylation of the microtubule-stabilizing kinesin Kip2.

    Science.gov (United States)

    Drechsler, Hauke; Tan, Ann Na; Liakopoulos, Dimitris

    2015-11-01

    The S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs. PMID:26395399

  7. E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

    Directory of Open Access Journals (Sweden)

    Bill B. Chen

    2014-04-01

    Full Text Available Acute lung injury (ALI is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1. Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN-induced putative kinase 1 (PINK1, which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

  8. p38 signaling and receptor recycling events in a microfluidic endothelial cell adhesion assay.

    Directory of Open Access Journals (Sweden)

    Dwayne A L Vickers

    Full Text Available Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome.

  9. The PlA2 polymorphism of integrin β3 enhances outside-in signaling and adhesive functions

    OpenAIRE

    Vijayan, K. Vinod; Pascal J. Goldschmidt-Clermont; Roos, Christine; Bray, Paul F.

    2000-01-01

    Genetic factors are believed to influence the development of arterial thromboses. Because integrin αIIbβ3 plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the PlA1 or PlA2 polymorphic forms of αIIbβ3. Soluble fibrinogen binding was no different between PlA1 and PlA2 cells, either in a resting state or when αIIbβ3 was activated with anti-LIBS6. PlA1 and PlA2 cells bound equivalen...

  10. Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >or=1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

  11. LIM kinase1 modulates function of membrane type matrix metalloproteinase 1: implication in invasion of prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Chakrabarti Ratna

    2011-01-01

    Full Text Available Abstract Background LIM kinase 1 (LIMK1 is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP is a critical modulator of extracellular matrix (ECM turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion. Results Our results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues. Conclusion Our results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.

  12. Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

    Institute of Scientific and Technical Information of China (English)

    Liping Yang; Yongming Yao; Zhiyong Sheng; Xiaomei Zhu; Yong Jiang

    2009-01-01

    Objective p38 Mitogen-activated protein kinase (MAPK) is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus (HIV)-1 transactivator of transcription (TAT), which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor (ATF) 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

  13. Integrin-linked kinase regulates interphase and mitotic microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Simin Lim

    Full Text Available Integrin-linked kinase (ILK localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

  14. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation.

    Science.gov (United States)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M; Gil, Francisco J; Rodriguez, Daniel

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria-cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties.

  15. Adhesion versus coreceptor function of CD4 and CD8: role of the cytoplasmic tail in coreceptor activity.

    OpenAIRE

    Miceli, M C; Von Hoegen, P.; Parnes, J R

    1991-01-01

    CD4 and CD8 play an important role in T-cell recognition and activation; however, their mechanisms of action are not well understood. We compare the effects of expressing CD4 and CD8 alpha either individually or together in a class II-restricted T-cell hybridoma. We also compare the effects of expressing truncated forms of CD4 or CD8 alpha that do not have a cytoplasmic tail and thus do not associate with the T-cell-specific tyrosine kinase p56lck, which has been implicated in T-cell activati...

  16. Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase1[C][W][OA

    Science.gov (United States)

    Bi, Dongling; Cheng, Yu Ti; Li, Xin; Zhang, Yuelin

    2010-01-01

    Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases. PMID:20508139

  17. Synthesis and application of glycoconjugate-functionalized magnetic nanoparticles as potent anti-adhesion agents for reducing enterotoxigenic Escherichia coli infections

    Science.gov (United States)

    Raval, Yash S.; Stone, Roland; Fellows, Benjamin; Qi, Bin; Huang, Guohui; Mefford, O. Thompson; Tzeng, Tzuen-Rong J.

    2015-04-01

    Polyethylene oxide stabilized magnetic nanoparticles (PEO-MNPs) bio-functionalized with glycoconjugate (Neu5Ac(α2-3)Gal(β1-4)Glcβ-sp) (GM3-MNPs) are synthesized using click chemistry. Interaction of GM3-MNPs with Enterotoxigenic Escherichia coli (ETEC) strain K99 (EC K99) is investigated using different microscopic techniques. Our results suggest that GM3-MNPs can effectively act as non-antibiotic anti-adhesion agents for treating ETEC infections.Polyethylene oxide stabilized magnetic nanoparticles (PEO-MNPs) bio-functionalized with glycoconjugate (Neu5Ac(α2-3)Gal(β1-4)Glcβ-sp) (GM3-MNPs) are synthesized using click chemistry. Interaction of GM3-MNPs with Enterotoxigenic Escherichia coli (ETEC) strain K99 (EC K99) is investigated using different microscopic techniques. Our results suggest that GM3-MNPs can effectively act as non-antibiotic anti-adhesion agents for treating ETEC infections. Electronic supplementary information (ESI) available: Materials and methods used in the synthesis and characterization of the polymer and particles described in this manuscript. See DOI: 10.1039/c5nr00511f

  18. GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover

    OpenAIRE

    Frank, Scott R.; Adelstein, Molly R; Hansen, Steen H.

    2006-01-01

    G protein-coupled receptor kinase interactors (GITs) regulate focal adhesion (FA) turnover, cell spreading, and motility through direct interaction with paxillin and the Rac-exchange factor Pak-interacting exchange factor β (βPIX). However, it is not clear whether GITs function to activate or repress motility or if the predominant GIT forms, GIT1 and GIT2, serve distinct or redundant roles. Here we demonstrate an obligatory role for endogenous GIT2 in repression of lamellipodial extension and...

  19. Therapeutic targeting of Janus kinases

    OpenAIRE

    Pesu, Marko; Laurence, Arian; Kishore, Nandini; Zwillich, Sam; Chan, Gary; O’Shea, John J.

    2008-01-01

    Cytokines play pivotal roles in immunity and inflammation, and targeting cytokines and their receptors is an effective means of treating such disorders. Type I and II cytokine receptors associate with Janus family kinases (JAKs) to effect intracellular signaling. These structurally unique protein kinases play essential and specific roles in immune cell development and function. One JAK, JAK3, has particularly selective functions. Mutations of this kinase underlie severe combined immunodeficie...

  20. FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase.

    Science.gov (United States)

    Imeri, Faik; Blanchard, Olivier; Jenni, Aurelio; Schwalm, Stephanie; Wünsche, Christin; Zivkovic, Aleksandra; Stark, Holger; Pfeilschifter, Josef; Huwiler, Andrea

    2015-12-01

    Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation. PMID:26267293

  1. Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

    OpenAIRE

    Hyun-Soo Kim; Rituparna Mukhopadhyay; Scott B. Rothbart; Andrea C. Silva; Vincent Vanoosthuyse; Ernest Radovani; Thomas Kislinger; Assen Roguev; Colm J. Ryan; Jiewei Xu; Harlizawati Jahari; Kevin G. Hardwick; Jack F. Greenblatt; Nevan J. Krogan; Jeffrey S. Fillingham

    2014-01-01

    Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern...

  2. CHARACTERIZATION AND FUNCTIONAL STUDY OF A PUTATIVE JUVENILE HORMONE DIOL KINASE IN THE COLORADO POTATO BEETLE Leptinotarsa decemlineata (Say).

    Science.gov (United States)

    Fu, Kai-Yun; Lü, Feng-Gong; Guo, Wen-Chao; Li, Guo-Qing

    2015-11-01

    Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide-binding elements, and contains three EF-hand motifs (helix-loop-helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double-stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr-h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation. PMID:26280246

  3. Functional redundancy of the kinases MEK1 and MEK2: Rescue of the Mek1 mutant phenotype by Mek2 knock-in reveals a protein threshold effect.

    Science.gov (United States)

    Aoidi, Rifdat; Maltais, Annie; Charron, Jean

    2016-01-26

    The mammalian genome contains two mitogen-activated protein kinase (MAPK) kinase (MEK)-encoding genes, Mek1 and Mek2. MEKs phosphorylate and activate the two extracellular signal-regulated kinase (ERK) isoforms ERK1 and ERK2. Mek1(-/-) embryos die due to placental defects, whereas Mek2(-/-) mice survive with a normal life span and fertility, suggesting that MEK1 has functions not shared by MEK2. However, most Mek1(+/-)Mek2(+/-) embryos also die from placental defects, indicating that both Mek genes contribute to placental development. To assess the functional specificity of the Mek1 and Mek2 genes, we produced a Mek1 knock-in allele in which the Mek2 coding sequences were placed under the control of Mek1 regulatory sequences (Mek1(2) allele). Mek1(2/2) mice were viable with no apparent phenotype, indicating rescue by MEK2 and functional redundancy between the two MEK proteins. However, Mek1(2/-) embryos with Mek2 in only one of the Mek1 alleles and the other Mek1 allele null died from abnormal placenta, suggesting a dosage effect. Analysis of mice from a Mek1 Mek2 allelic series revealed that the occurrence of the placenta phenotype correlated with the amount of MEK protein independently of which MEK isoform was produced. Thus, although MEK1 and MEK2 can substitute for each other, a minimum amount of MEK is critical for placenta development and embryo survival. PMID:26814233

  4. Adhesion and Cohesion

    Directory of Open Access Journals (Sweden)

    J. Anthony von Fraunhofer

    2012-01-01

    Full Text Available The phenomena of adhesion and cohesion are reviewed and discussed with particular reference to dentistry. This review considers the forces involved in cohesion and adhesion together with the mechanisms of adhesion and the underlying molecular processes involved in bonding of dissimilar materials. The forces involved in surface tension, surface wetting, chemical adhesion, dispersive adhesion, diffusive adhesion, and mechanical adhesion are reviewed in detail and examples relevant to adhesive dentistry and bonding are given. Substrate surface chemistry and its influence on adhesion, together with the properties of adhesive materials, are evaluated. The underlying mechanisms involved in adhesion failure are covered. The relevance of the adhesion zone and its importance with regard to adhesive dentistry and bonding to enamel and dentin is discussed.

  5. FRET based quantification and screening technology platform for the interactions of leukocyte function-associated antigen-1 (LFA-1 with intercellular adhesion molecule-1 (ICAM-1.

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    Full Text Available The interaction between leukocyte function-associated antigen-1(LFA-1 and intercellular adhesion molecule-1 (ICAM-1 plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple 'in solution' steady state fluorescence resonance energy transfer (FRET technique to obtain the dissociation constant (Kd of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.

  6. The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

    Directory of Open Access Journals (Sweden)

    Moritz Treeck

    2014-06-01

    Full Text Available Calcium-dependent protein kinases (CDPKs are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC. This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1, as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  7. Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.

    Science.gov (United States)

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2016-05-01

    Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex. PMID:27044741

  8. Syndecans and cell adhesion

    DEFF Research Database (Denmark)

    Couchman, J R; Chen, L; Woods, A

    2001-01-01

    Now that transmembrane signaling through primary cell-matrix receptors, integrins, is being elucidated, attention is turning to how integrin-ligand interactions can be modulated. Syndecans are transmembrane proteoglycans implicated as coreceptors in a variety of physiological processes, including...... cell adhesion, migration, response to growth factors, development, and tumorigenesis. This review will describe this family of proteoglycans in terms of their structures and functions and their signaling in conjunction with integrins, and indicate areas for future research....

  9. A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

    Science.gov (United States)

    Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

    2014-06-15

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

  10. Dancing with the dead: Eph receptors and their kinase-null partners.

    Science.gov (United States)

    Truitt, Luke; Freywald, Andrew

    2011-04-01

    Eph receptor tyrosine kinases and their ligands, ephrins, are membrane proteins coordinating a wide range of biological functions both in developing embryos and in adult multicellular organisms. Numerous studies have implicated Eph receptors in the induction of opposing responses, including cell adhesion or repulsion, support or inhibition of cell proliferation and cell migration, and progression or suppression of multiple malignancies. Similar to other receptor tyrosine kinases, Eph receptors rely on their ability to catalyze tyrosine phosphorylation for signal transduction. Interestingly, however, Eph receptors also actively utilize three kinase-deficient receptor tyrosine kinases, EphB6, EphA10, and Ryk, in their signaling network. The accumulating evidence suggests that the unusual flexibility of the Eph family, allowing it to initiate antagonistic responses, might be partially explained by the influence of the kinase-dead participants and that the exact outcome of an Eph-mediated action is likely to be defined by the balance between the signaling of catalytically potent and catalytically null receptors. We discuss in this minireview the emerging functions of the kinase-dead EphB6, EphA10, and Ryk receptors both in normal biological responses and in malignancy, and analyze currently available information related to the molecular mechanisms of their action in the context of the Eph family. PMID:21455264

  11. Notch-Mediated Cell Adhesion

    OpenAIRE

    Akihiko Murata; Shin-Ichi Hayashi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of...

  12. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

  13. Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis.

    Science.gov (United States)

    Li, Xinjian; Jiang, Yuhui; Meisenhelder, Jill; Yang, Weiwei; Hawke, David H; Zheng, Yanhua; Xia, Yan; Aldape, Kenneth; He, Jie; Hunter, Tony; Wang, Liwei; Lu, Zhimin

    2016-03-01

    It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis.

  14. Structural and Functional Aspects of the Sensor Histidine Kinase PrrB from Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Nowak, E.; Panjikar, S.; Morth, J.P.;

    2006-01-01

    We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both for...

  15. Tailored adhesion behavior of polyelectrolyte thin films deposited on plasma-treated poly(dimethylsiloxane) for functionalized membranes

    Science.gov (United States)

    Bassil, Joelle; Alem, Halima; Henrion, Gérard; Roizard, Denis

    2016-04-01

    Completely homogenous films formed via the layer-by-layer assembly of poly(diallyldimethylammonium chloride) (PDADMAC) and the poly(styrene sulfonate) were successfully obtained on plasma-treated poly(dimethylsiloxane) (PDMS) substrates. To modify the hydrophobicity of the PDMS surface, a cold plasma treatment was previously applied to the membrane, which led to the creation of hydrophilic groups on the surface of the membrane. PDMS wettability and surface morphology were successfully correlated with the plasma parameters. A combination of contact angle measurements, scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis was used to demonstrate that homogeneous and hydrophilic surfaces could be achieved on PDMS cold-plasma-treated membranes. The stability of the assembled PEL layer on the PDMS was evaluated using a combination of pull-off testing and X-ray photoelectron spectroscopy (XPS), which confirmed the relevance of a plasma pre-treatment as the adhesion of the polyelectrolyte multilayers was greatly enhanced when the deposition was completed on an activated PDMS surface at 80 W for 5 min.

  16. In-vivo study of adhesion and bone growth around implanted laser groove/RGD-functionalized Ti-6Al-4V pins in rabbit femurs

    International Nuclear Information System (INIS)

    Titanium surfaces were designed, produced, and evaluated for levels of osseointegration into the femurs of rabbits. A total of 36 Ti-6Al-4V pins (15 mm length, 1.64 mm diameter) were prepared into three experimental groups. These were designed to test the effects of osseointegration on laser grooved, RGD coated, and polished control surfaces, as well as combined effects. Circumferential laser grooves were introduced onto pin surfaces (40 μm spacing) using a UV laser (λ = 355 nm). The tripeptide sequence, Arginine-Glycine-Aspartic acid (RGD), was functionalized onto laser grooved surfaces. Of the prepared samples, surface morphology and chemistry were analyzed using scanning electron microscopy (SEM) and Immunoflourescence (IF) spectroscopy, respectively. The experimental pin surfaces were surgically implanted into rabbit femurs. The samples were then harvested and evaluated histologically. Sections of the sample were preserved in a methylmethacralate mold, sliced via a hard microtome, and polished systematically. In the case of the RGD coated and laser grooved surfaces, histological results showed accelerated bone growth into the implant, pull-out tests were also used to compare the adhesion between bone and the titanium pins with/without laser textures and/or RGD coatings. - Research highlights: → Circumferential laser grooves were introduced onto pin surfaces using a UV laser. → The tripeptide sequence, RGD, was functionalized onto laser grooved surfaces. → The experimental pin surfaces were surgically implanted into rabbit femurs. → RGD coated laser groove surfaces accelerated bone growth into the implant. → RGD coated laser grooved surfaces enhanced the adhesion between the bone and implant.

  17. In-vivo study of adhesion and bone growth around implanted laser groove/RGD-functionalized Ti-6Al-4V pins in rabbit femurs

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J., E-mail: jianboc@gmail.com [Princeton Institute of Science and Technology of Materials and Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Bly, R.A. [Princeton Institute of Science and Technology of Materials and Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Saad, M.M.; AlKhodary, M.A.; El-Backly, R.M. [Tissue Engineering laboratories, Faculty of Dentistry, Alexandria University, Alexandria (Egypt); Cohen, D.J.; Kattamis, N. [Princeton Institute of Science and Technology of Materials and Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Fatta, M.M.; Moore, W.A. [Tissue Engineering laboratories, Faculty of Dentistry, Alexandria University, Alexandria (Egypt); Arnold, C.B. [Princeton Institute of Science and Technology of Materials and Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States); Marei, M.K. [Tissue Engineering laboratories, Faculty of Dentistry, Alexandria University, Alexandria (Egypt); Soboyejo, W.O. [Princeton Institute of Science and Technology of Materials and Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544 (United States)

    2011-07-20

    Titanium surfaces were designed, produced, and evaluated for levels of osseointegration into the femurs of rabbits. A total of 36 Ti-6Al-4V pins (15 mm length, 1.64 mm diameter) were prepared into three experimental groups. These were designed to test the effects of osseointegration on laser grooved, RGD coated, and polished control surfaces, as well as combined effects. Circumferential laser grooves were introduced onto pin surfaces (40 {mu}m spacing) using a UV laser ({lambda} = 355 nm). The tripeptide sequence, Arginine-Glycine-Aspartic acid (RGD), was functionalized onto laser grooved surfaces. Of the prepared samples, surface morphology and chemistry were analyzed using scanning electron microscopy (SEM) and Immunoflourescence (IF) spectroscopy, respectively. The experimental pin surfaces were surgically implanted into rabbit femurs. The samples were then harvested and evaluated histologically. Sections of the sample were preserved in a methylmethacralate mold, sliced via a hard microtome, and polished systematically. In the case of the RGD coated and laser grooved surfaces, histological results showed accelerated bone growth into the implant, pull-out tests were also used to compare the adhesion between bone and the titanium pins with/without laser textures and/or RGD coatings. - Research highlights: {yields} Circumferential laser grooves were introduced onto pin surfaces using a UV laser. {yields} The tripeptide sequence, RGD, was functionalized onto laser grooved surfaces. {yields} The experimental pin surfaces were surgically implanted into rabbit femurs. {yields} RGD coated laser groove surfaces accelerated bone growth into the implant. {yields} RGD coated laser grooved surfaces enhanced the adhesion between the bone and implant.

  18. Management of adhesive capsulitis

    Directory of Open Access Journals (Sweden)

    Stupay KL

    2015-08-01

    Full Text Available Kristen L Stupay,1 Andrew S Neviaser2 1Tulane University School of Medicine, New Orleans, LA, USA; 2George Washington University Medical Faculty Associates, Washington, DC, USA Abstract: Adhesive capsulitis of the shoulder is a condition of capsular contracture that reduces both active and passive glenohumeral motion. The cause of adhesive capsulitis is not known but it is strongly associated with endocrine abnormalities such as diabetes. Diverse terminology and the absence of definitive criteria for diagnosis make evaluating treatment modalities difficult. Many treatment methods have been reported, most with some success, but few have been proved to alter the natural course of this disease. Most afflicted patients will achieve acceptable shoulder function without surgery. Those who remain debilitated after 8–12 months are reasonable candidates for invasive treatments. Here, the various treatment methods and the data to support their use are reviewed. Keywords: frozen shoulder, stiff shoulder, periarthritis, painful shoulder 

  19. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    Science.gov (United States)

    Lee, Su-Lin; Chou, Chih-Chien; Chuang, Hsiao-Ching; Hsu, En-Chi; Chiu, Po-Chen; Kulp, Samuel K; Byrd, John C; Chen, Ching-Shih

    2013-01-01

    Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA

  20. Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Su-Lin Lee

    Full Text Available Although the rictor-mTOR complex (mTORC2 has been shown to act as phosphoinositide-dependent kinase (PDK2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial

  1. Importance of MAP kinases during protoperithecial morphogenesis in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Alexander Lichius

    Full Text Available In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.

  2. Novel pharmacologic targeting of tight junctions and focal adhesions in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Patrick J Hensley

    Full Text Available Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin, tight junction formation (claudin-11 as well as insulin growth factor binding protein 3 (IGFBP-3 and the angiogenesis modulator thrombospondin 1 (TSP-1. Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer.

  3. Pressure-sensitive adhesives for transdermal drug delivery systems.

    Science.gov (United States)

    Tan; Pfister

    1999-02-01

    Adhesives are a critical component in transdermal drug delivery (TDD) devices. In addition to the usual requirements of functional adhesive properties, adhesives for TDD applications must have good biocompatibility with the skin, chemical compatibility with the drug, various components of the formulation, and provide consistent, effective delivery of the drug. This review discusses the three most commonly used adhesives (polyisobutylenes, polyacrylates and silicones) in TDD devices, and provides an update on recently introduced TDD products and recent developments of new adhesives. PMID:10234208

  4. 蛋白激酶蛋白家族的功能研究进展%Advances in the function of protein kinase D family

    Institute of Scientific and Technical Information of China (English)

    刘思旸; 邹志鹏; 郦俊

    2011-01-01

    蛋白激酶D(protein kinase D,PKD)属丝氨酸/苏氨酸(serine/threonine,Ser/Thr)蛋白质家族,主要包括3个成员:PKD1/PKCμ、PKD2、PKD3/PKCν.其结构相对保守,均具有相似的调节结构域和激酶结构域.近年来的研究表明PKD家族蛋白参与细胞生长、增殖、迁移、分化、凋亡,高尔基体的组织和重建,免疫应答,内分泌调节、增强心肌收缩力等多种生理功能.%Protein kinase D ( PKD) is a kind of Ser/Thr protein kinases including three members : PKD1/PKCμ, PKD2 , PKD3/PKC. All of them have conserved structures and similar regular domains. Recently many investigations have found that PKDs contribute to many cellucar biological functions such as cell survival, migration, differentiation, apoptosis, organization and regeneration of Golgi ' s apparatus, immune responses, regulation of endocrine secretion and myocardial contraccility.

  5. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  6. Modelling the Fracture Behaviour of Adhesively-Bonded Joints as a Function of Test Rate - A Rate Dependent CZM is Required to Predict the Full Range of Behaviour

    OpenAIRE

    Ivankovic, Alojz; Karac, Aleksandar; Blackman, B.R.K.; et al

    2011-01-01

    Adhesive bonding of lightweight, high-performance materials is regarded as a key enabling technology for the development of vehicles with increased crashworthiness, better fuel economy and reduced exhaust emissions. However, as automotive structures can be exposed to impact events during service, it is necessary to gain a sound understanding of the performance of adhesive joints under different rates of loading. Therefore, characterising the behaviour of adhesive joint...

  7. Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

    Directory of Open Access Journals (Sweden)

    Elian Dupré

    2015-03-01

    Full Text Available Two-component systems (TCS represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

  8. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats.

    Science.gov (United States)

    Zhang, Bei; He, Qiang; Li, Ying-Ying; Li, Ce; Bai, Yu-Long; Hu, Yong-Shan; Zhang, Feng

    2015-12-01

    Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT) involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of 'learned non-use' and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model) group, a CIMT + model (CIMT) group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  9. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats

    Directory of Open Access Journals (Sweden)

    Bei Zhang

    2015-01-01

    Full Text Available Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of ′learned non-use′ and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model group, a CIMT + model (CIMT group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  10. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

    OpenAIRE

    Drakou, Christina E.; Malekkou, Anna; Hayes, Joseph M.; Carsten W Lederer; Leonidas, Demetres D.; Oikonomakos, Nikos G.; Lamond, Angus I.; Santama, Niovi; Zographos, Spyros E.

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. D...

  11. Structural and Functional Analysis of G Protein–Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

    OpenAIRE

    Homan, Kristoff T.; Wu, Emily; Wilson, Michael W.; Singh, Puja; Larsen, Scott D.; Tesmer, John J.G.

    2014-01-01

    Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein–coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanism...

  12. Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases.

    Science.gov (United States)

    Floss, Doreen M; Klöcker, Tobias; Schröder, Jutta; Lamertz, Larissa; Mrotzek, Simone; Strobl, Birgit; Hermanns, Heike; Scheller, Jürgen

    2016-07-15

    The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable.

  13. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    Energy Technology Data Exchange (ETDEWEB)

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  14. Functional and spatial analysis of C. elegans SYG-1 and SYG-2, orthologs of the Neph/nephrin cell adhesion module directing selective synaptogenesis.

    Directory of Open Access Journals (Sweden)

    Nicola Wanner

    Full Text Available The assembly of specific synaptic connections represents a prime example of cellular recognition. Members of the Ig superfamily are among the most ancient proteins represented in the genomes of both mammalian and invertebrate organisms, where they constitute a trans-synaptic adhesion system. The correct connectivity patterns of the highly conserved immunoglobulin superfamily proteins nephrin and Neph1 are crucial for the assembly of functional neuronal circuits and the formation of the kidney slit diaphragm, a synapse-like structure forming the filtration barrier. Here, we utilize the nematode C. elegans model for studying the requirements of synaptic specificity mediated by nephrin-Neph proteins. In C. elegans, the nephrin/Neph1 orthologs SYG-2 and SYG-1 form intercellular contacts strictly in trans between epithelial guidepost cells and neurons specifying the localization of synapses. We demonstrate a functional conservation between mammalian nephrin and SYG-2. Expression of nephrin effectively compensated loss of syg-2 function in C. elegans and restored defective synaptic connectivity further establishing the C. elegans system as a valuable model for slit diaphragm proteins. Next, we investigated the effect of SYG-1 and SYG-2 trans homodimerization respectively. Strikingly, synapse assembly could be induced by homophilic SYG-1 but not SYG-2 binding indicating a critical role of SYG-1 intracellular signalling for morphogenetic events and pointing toward the dynamic and stochastic nature of extra- and intracellular nephrin-Neph interactions to generate reproducible patterns of synaptic connectivity.

  15. Pentosan polysulfate treatment ameliorates motor function with increased serum soluble vascular cell adhesion molecule-1 in HTLV-1-associated neurologic disease.

    Science.gov (United States)

    Nakamura, Tatsufumi; Satoh, Katsuya; Fukuda, Taku; Kinoshita, Ikuo; Nishiura, Yoshihiro; Nagasato, Kunihiko; Yamauchi, Atsushi; Kataoka, Yasufumi; Nakamura, Tadahiro; Sasaki, Hitoshi; Kumagai, Kenji; Niwa, Masami; Noguchi, Mitsuru; Nakamura, Hideki; Nishida, Noriyuki; Kawakami, Atsushi

    2014-06-01

    The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-α, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP.

  16. Phylogeny of plant calcium and calmodulin-dependent protein kinases (CCaMKs and functional analyses of tomato CCaMK in disease resistance

    Directory of Open Access Journals (Sweden)

    Ji-Peng eWang

    2015-12-01

    Full Text Available Calcium and calmodulin-dependent protein kinase (CCaMK is a member of calcium/calmodulin-dependent protein kinase superfamily and is essential to microbe- plant symbiosis. To date, the distribution of CCaMK gene in plants has not yet been completely understood, and its function in plant disease resistance remains unclear. In this study, we systemically identified the CCaMK genes in genomes of 44 plant species in Phytozome and analyzed the function of tomato CCaMK (SlCCaMK in resistance to various pathogens. CCaMKs in 18 additional plant species were identified, yet the absence of CCaMK gene in green algae and cruciferous species was confirmed. Sequence analysis of full-length CCaMK proteins from 44 plant species demonstrated that plant CCaMKs are highly conserved across all domains. Most of the important regulatory amino acids are conserved throughout all sequences, with the only notable exception being observed in N-terminal autophosphorylation site corresponding to Ser 9 in the Medicago truncatula CCaMK. CCaMK gene structures are similar, mostly containing six introns with a phase profile of 200200 and the exception was only noticed at the first exons. Phylogenetic analysis demonstrated that CCaMK lineage is likely to have diverged early from a calcium-dependent protein kinase (CDPK gene in the ancestor of all nonvascular plant species. The SlCCaMK gene was widely and differently responsive to diverse pathogenic stimuli. Furthermore, knock-down of SlCCaMK reduced tomato resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. tomato (Pst DC3000 and decreased H2O2 accumulation in response to Pst DC3000 inoculation. Our results reveal that SlCCaMK positively regulates disease resistance in tomato via promoting H2O2 accumulation. SlCCaMK is the first CCaMK gene proved to function in plant disease resistance.

  17. Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1.

    Science.gov (United States)

    Jamros, Michael A; Aubol, Brandon E; Keshwani, Malik M; Zhang, Zhaiyi; Stamm, Stefan; Adams, Joseph A

    2015-07-10

    Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis. PMID:26013829

  18. Chk2蛋白激酶生物学功能研究进展%Research Progression of Biological Function of Chk2 Protein Kinase

    Institute of Scientific and Technical Information of China (English)

    李傲航

    2014-01-01

    Checkpoint kinase 2(Chk2)是一种多功能蛋白激酶,不仅在DNA损伤诱导的细胞周期阻滞、DNA修复和细胞凋亡过程中扮演着重要角色,而且最新研究发现,其在有丝分裂过程中对纺锤体的组装和染色体稳定性的维持也是必须的。越来越多的证据显示,Chk2和人类肿瘤存在着密切关系,其被视为肿瘤治疗新的分子靶标,在抑制人类肿瘤发生发展中起着关键作用。本文就Chk2蛋白激酶的主要生物学功能作一综述。%Checkpoint kinase 2 (Chk2) isa multifunctional enzyme whose functions are central to the induction of cel cycle arrest,DNA repair and apoptosis by DNA damage.In addition,most recent work has revealed another function of Chk2,independent of DNA damage,that is required for the proper mitotic spindle assembly and for the maintenance of chromosomal stability.The relationship of Chk2 to human cancer studies are developing rapidlywith increasing evidence that Chk2 plays a vital role in tumor suppression and wil be viewed as a new anti-cancer therapy molecular target.The research progression of biological function of Chk2 protein kinase is reviewed in this article.

  19. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  20. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    During cell-matrix adhesion, both tyrosine and serine/threonine kinases are activated. Integrin ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface...... adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans....

  1. Adhesion in microelectronics

    CERN Document Server

    Mittal, K L

    2014-01-01

    This comprehensive book will provide both fundamental and applied aspects of adhesion pertaining to microelectronics in a single and easily accessible source. Among the topics to be covered include; Various theories or mechanisms of adhesionSurface (physical or chemical) characterization of materials as it pertains to adhesionSurface cleaning as it pertains to adhesionWays to improve adhesionUnraveling of interfacial interactions using an array of pertinent techniquesCharacterization of interfaces / interphasesPolymer-polymer adhesionMetal-polymer adhesion  (metallized polymers)Polymer adhesi

  2. Application of the Blister Test in Study of Epoxy Adhesive

    Institute of Scientific and Technical Information of China (English)

    Fei Xiong; Ingegerd Annergren

    2000-01-01

    Shaft-loaded blister test technique is used as an effective quantitative tool to measure adhesion strength. Investigation on conductive adhesive was done by modified blister test. It is found that shaftloaded blister test can be a good solution for the debonding of thin film adhesion. The intrinsic stable interface debonding process has been proved an attractive alternative to the conventional adhesion measurement techniques. In our study, epoxy matrix adhesive was studied using blister test technique in comparison with the traditional test-lap shear test. Adhesion strength was studied as a function of surface treatment and the metallization of substrate. It was found that surface conditions of substrate have significant impact on adhesion behaviour. The oxidation of surface is responsible for the poor adhesion. Activating chemical treatment and Plasma cleaning on substrate surface has been found to be a way of dreamatically improving adhesion strength of electronic conductive adhesive.

  3. Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases.

    Science.gov (United States)

    Floss, Doreen M; Klöcker, Tobias; Schröder, Jutta; Lamertz, Larissa; Mrotzek, Simone; Strobl, Birgit; Hermanns, Heike; Scheller, Jürgen

    2016-07-15

    The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable. PMID:27193299

  4. Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

    Directory of Open Access Journals (Sweden)

    Antonia Terriza

    2014-03-01

    Full Text Available New biomaterials for Guided Bone Regeneration (GBR, both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB® HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide (PLGA membrane foil functionalized by a very thin film (around 15 nm of TiO2 (i.e., TiO2/PLGA membranes, designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors.

  5. IL-2 inducible T cell kinase (ITK) tunes T regulatory cell development and is required for suppressive function1

    OpenAIRE

    Huang, Weishan; Jeong, Ah-Reum; Kannan, Arun K.; Huang, Lu; August, Avery

    2014-01-01

    ITK is a key signaling mediator downstream of TcR, mediating T cell positive selection, innate T cell and CD4+ Th2/Th17 differentiation. Here we show that ITK also negatively tunes IL-2-induced expansion of Foxp3+ regulatory T cells (Treg). In vivo, Treg abundance is inversely correlated with ITK expression, and iTreg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of I...

  6. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    , no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-specific expression of CK-2 at the mRNA and at the protein level has also been given attention. The fact that the enzyme activity is surprisingly high in brain and low in heart and lung may be indicative of involvement of CK-2 in processes other than proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)...

  7. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Institute of Scientific and Technical Information of China (English)

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng

    2009-01-01

    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  8. Signaling transduction pathways involved in basophil adhesion and histamine release

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Little is known about basophil with respect to the different signaling transduction pathways involved in spontaneous, cytokine or anti-IgE induced adhesion and how this compares to IgE-dependent and IgE-independent mediator secretion. The purpose of the present study was to investigate the roles of β1 andβ2 integrins in basophil adhesion as well as hosphatidylinositol 3-kinase (PI3K), src-kinases and extracellular signal regulated kinase (ERK)1/2 in basophil adhesion and histamine release (HR). Methods Basophils (purity of 10%-50%) were preincubated with anti-CD29 or anti-CD18 blocking antibodies before used for adhesion study. Basophils were preincubated with the pharmacological inhibitors wortmannin, PP1, PD98059 before used for adhesion and HR study. Cell adherence to bovine serum albumin (BSA) or fibronectin (Fn) was monitored using cell associated histamine as a basophil marker and the histamine was measured by the glass fiber assay.Results Basophil spontaneous adhesion to Fn was inhibited by anti-CD29. Interleukin (IL)-3, granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA was inhibited by anti-CD18. Wortmannin at 1 μmol/L and PP1 at 20 μmol/L strongly interfered with, whereas PD98059 at 50 μmol/L weakly inhibited basophil spontaneous adhesion to Fn. One μmol/L wortmannin strongly inhibited IL-3, IL-5, GM-CSF and anti-IgE induced adhesion to BSA. PP1 at 20 μmol/L partly inhibited anti-IgE induced adhesion. Fifty μmol/L PD98059 marginally inhibited IL-5, weakly inhibited anti-IgE, partly inhibited GM-CSF induced adhesion. Wortmannin, PP1 and PD98059 inhibited anti-IgE (1:100 or 1:1000) induced basophil HR in a dose dependent manner. They inhibited calcium ionophore A23187 (10 μmol/L, 5 μmol/L) induced basophil HR in a dose dependent manner, but to different extend with PP1 being the most efficient.Conclusions Basophil spontaneous adhesion to Fn is mediated by β1-integrins whereas cytokine induced adhesion

  9. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  10. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell

    KAUST Repository

    Xue, Shaowu

    2011-03-18

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

  11. Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system.

    Science.gov (United States)

    Wang, Jin-Lei; Huang, Si-Yang; Li, Ting-Ting; Chen, Kai; Ning, Hong-Rui; Zhu, Xing-Quan

    2016-02-01

    Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii. PMID:26499803

  12. Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

    Science.gov (United States)

    Svensson, Mattias N D; Andersson, Karin M E; Wasén, Caroline; Erlandsson, Malin C; Nurkkala-Karlsson, Merja; Jonsson, Ing-Marie; Brisslert, Mikael; Bemark, Mats; Bokarewa, Maria I

    2015-12-01

    Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1. PMID:26627255

  13. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  14. Determinants of homodimerization specificity in histidine kinases

    OpenAIRE

    Ashenberg, Orr; Rozen-Gagnon, Kathryn; Laub, Michael T; Keating, Amy E.

    2011-01-01

    Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and...

  15. Adhesion- and Degranulation-Promoting Adapter Protein Promotes CD8 T Cell Differentiation and Resident Memory Formation and Function during an Acute Infection.

    Science.gov (United States)

    Fiege, Jessica K; Beura, Lalit K; Burbach, Brandon J; Shimizu, Yoji

    2016-09-15

    During acute infections, naive Ag-specific CD8 T cells are activated and differentiate into effector T cells, most of which undergo contraction after pathogen clearance. A small population of CD8 T cells persists as memory to protect against future infections. We investigated the role of adhesion- and degranulation-promoting adapter protein (ADAP) in promoting CD8 T cell responses to a systemic infection. Naive Ag-specific CD8 T cells lacking ADAP exhibited a modest expansion defect early after Listeria monocytogenes or vesicular stomatitis virus infection but comparable cytolytic function at the peak of response. However, reduced numbers of ADAP-deficient CD8 T cells were present in the spleen after the peak of the response. ADAP deficiency resulted in a greater frequency of CD127(+) CD8 memory precursors in secondary lymphoid organs during the contraction phase. Reduced numbers of ADAP-deficient killer cell lectin-like receptor G1(-) CD8 resident memory T (TRM) cell precursors were present in a variety of nonlymphoid tissues at the peak of the immune response, and consequently the total numbers of ADAP-deficient TRM cells were reduced at memory time points. TRM cells that did form in the absence of ADAP were defective in effector molecule expression. ADAP-deficient TRM cells exhibited impaired effector function after Ag rechallenge, correlating with defects in their ability to form T cell-APC conjugates. However, ADAP-deficient TRM cells responded to TGF-β signals and recruited circulating memory CD8 T cells. Thus, ADAP regulates CD8 T cell differentiation events following acute pathogen challenge that are critical for the formation and selected functions of TRM cells in nonlymphoid tissues. PMID:27521337

  16. Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs, named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig domains 1-4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn domains 1-3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H(2O(2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1-4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1-3 trigger L1 functions of cultured neuroblastoma cells.

  17. Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans.

    Science.gov (United States)

    Saputo, Sarah; Norman, Kaitlyn L; Murante, Thomas; Horton, Brooke N; Diaz, Jacinto De La Cruz; DiDone, Louis; Colquhoun, Jennifer; Schroeder, Jeremy W; Simmons, Lyle A; Kumar, Anuj; Krysan, Damian J

    2016-07-01

    Although the analysis of genetic interactions and networks is a powerful approach to understanding biology, it has not been applied widely to the pathogenic yeast Candida albicans Here, we describe the use of both screening and directed genetic interaction studies based on complex haploinsufficiency to probe the function of the R: egulation of A: ce2 and M: orphogenesis (RAM) pathway in C. albicans A library of 5200 Tn7-mutagenized derivatives of a parental strain heterozygous at CBK1, the key kinase in the RAM pathway, was screened for alterations in serum-induced filamentation. Following confirmation of phenotypes and identification of insertion sites by sequencing, a set of 36 unique double heterozygous strains showing complex haploinsufficiency was obtained. In addition to a large set of genes regulated by the RAM transcription factor Ace2, genes related to cell wall biosynthesis, cell cycle, polarity, oxidative stress, and nitrogen utilization were identified. Follow-up analysis led to the first demonstration that the RAM pathway is required for oxidative stress tolerance in a manner related to the two-component-regulated kinase Chk1 and revealed a potential direct connection between the RAM pathway and the essential Mps1 spindle pole-related kinase. In addition, genetic interactions with CDC42-related genes MSB1, a putative scaffold protein, and RGD3, a putative Rho GTPase-activating protein (GAP) were identified. We also provide evidence that Rgd3 is a GAP for Cdc42 and show that its localization and phosphorylation are dependent on Cbk1. PMID:27206715

  18. Intercellular adhesion molecule-1 clusters during osteoclastogenesis

    NARCIS (Netherlands)

    V. Bloemen; T.J. de Vries; T. Schoenmaker; V. Everts

    2009-01-01

    Adhesion between osteoblasts and osteoclast precursors is established via an interaction involving intercellular adhesion molecule-1 (ICAM-1) on osteoblasts and leukocyte function-associated antigen-1 (LFA-1) on osteoclast precursors. The latter cells also express ICAM-1, but little is known about t

  19. Mechanisms of temporary adhesion in benthic animals

    NARCIS (Netherlands)

    Dodou, D.; Breedveld, P.; Winter, J.C.F.; Dankelman, J.; Leeuwen, van J.L.

    2011-01-01

    Adhesive systems are ubiquitous in benthic animals and play a key role in diverse functions such as locomotion, food capture, mating, burrow building, and defence. For benthic animals that release adhesives, surface and material properties and external morphology have received little attention compa

  20. Design and fabrication of polymer based dry adhesives inspired by the gecko adhesive system

    Science.gov (United States)

    Jin, Kejia

    There has been significant interest in developing dry adhesives mimicking the gecko adhesive system, which offers several advantages compared to conventional pressure sensitive adhesives. Specifically, gecko adhesive pads have anisotropic adhesion properties: the adhesive pads (spatulae) stick strongly when sheared in one direction but are non-adherent when sheared in the opposite direction. This anisotropy property is attributed to the complex topography of the array of fine tilted and curved columnar structures (setae) that bear the spatulae. In this thesis, easy, scalable methods, relying on conventional and unconventional techniques are presented to incorporate tilt in the fabrication of synthetic polymer-based dry adhesives mimicking the gecko adhesive system, which provide anisotropic adhesion properties. In the first part of the study, the anisotropic adhesion and friction properties of samples with various tilt angles to test the validity of a nanoscale tape-peeling model of spatular function are measured. Consistent with the Peel Zone model, samples with lower tilt angles yielded larger adhesion forces. Contact mechanics of the synthetic array were highly anisotropic, consistent with the frictional adhesion model and gecko-like. Based on the original design, a new design of gecko-like dry adhesives was developed which showed superior tribological properties and furthermore showed anisotropic adhesive properties without the need for tilt in the structures. These adhesives can be used to reversibly suspend weights from vertical surfaces (e.g., walls) and, for the first time to our knowledge, horizontal surfaces (e.g., ceilings) by simultaneously and judiciously activating anisotropic friction and adhesion forces. Furthermore, adhesion properties between artificial gecko-inspired dry adhesives and rough substrates with varying roughness are studied. The results suggest that both adhesion and friction forces on a rough substrate depends significantly on the

  1. Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

    LENUS (Irish Health Repository)

    Olivieri, Aldo

    2010-04-01

    The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO\\/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.

  2. Functional assignment by Chimera construction of the domain affecting heterotropic activation of deoxyadenosine kinase from Lactobacillus acidophilus R-26.

    Science.gov (United States)

    Guo, S; Ives, D H

    1998-10-01

    The heterodimeric subunits of deoxyadenosine kinase (dAK)-deoxyguanosine kinase (dGK) from Lactobacillus acidophilus R-26 exhibit contrasting conformations manifested in the nearly unidirectional heterotropic activation of dAK when dGK binds deoxyguanosine. This is mediated, in part, by the conserved Ras switch I-like sequence (residues 153-161) [Guo et al. (1997) J. Biol. Chem. 272, 6890-6897]. In an attempt to identify domains differentiating the specificities of dAK and dGK, we constructed several chimeras splicing heterodimeric dAK within this region. In Chimera-III, dAK residues 120-170 were replaced by the homologous section of dGK. dAK activity was elevated 40%, but although it retained its original specificity and Km values, it could no longer be activated by deoxyguanosine. Moreover, both the activated dAK and the "dAK" of Chimera-III exhibited (i) an increased Ks for the leading substrate ATP-Mg2+, suggesting the formation of intermediate enzyme species along their respective kinetic pathways, and (ii) broadened and lower pH optima for the dAK activities. These observations further indicate the importance of dAK residues 120-170, including the Ras-like segment, in catalysis and heterotropic activation. The other conformational properties of dAK (e.g. self-inactivity and MgATP being the leading substrate) were unaltered by this substitution, thus localizing the responsible domains even further upstream.

  3. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth.

    Science.gov (United States)

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S; Grossniklaus, Ueli

    2015-09-29

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRI(R240C) mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  4. Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion.

    Science.gov (United States)

    Cheung, Samuel M S; Ostergaard, Hanne L

    2016-09-01

    Protein tyrosine kinase 2 (Pyk2) is required for T cell adhesion to ICAM-1; however, the mechanism by which it regulates adhesion remains unexplored. Pyk2 function in murine CTL clones and activated ex vivo CD8(+) T cells was disrupted by pharmacological inhibition, knockdown of expression with small interfering RNA, or expression of the dominant-negative C-terminal domain. We found that Pyk2 is not absolutely required for adhesion of CTL to ICAM-1, but rather delays the initial adhesion. Disruption of Pyk2 function caused cells to display an unusual elongated appearance after 1 h on ICAM-1, consistent with abnormally strong adhesion. Furthermore, the random mobility of CTL on ICAM-1 was severely compromised using all three methods of disrupting Pyk2 function. Live-cell imaging studies revealed that the decreased migration is the result of a defect in the detachment from ICAM-1 at the trailing edge when Pyk2 function is inhibited. Examination of Pyk2 tyrosine phosphorylation in normal polarized cells demonstrated that Pyk2 phosphorylated at Y579 and Y580 preferentially localizes to the leading edge, whereas Y881-phosphorylated Pyk2 is enriched at the trailing edge, suggesting that the tyrosine phosphorylation of Pyk2 is spatially regulated in migrating CTL. Additionally, inhibition of Pyk2 caused cells to form multiple LFA-1-rich tails at the trailing edge, most likely resulting from a defect in LFA-1 release required for forward movement. Our results show that Pyk2 contributes to CTL migration by regulating detachment of CTL at the trailing edge, which could explain why Pyk2 is important for chemotactic and migratory responses. PMID:27456486

  5. T Cell-Mediated Modulation of Mast Cell Function: Heterotypic Adhesion-Induced Stimulatory or Inhibitory Effects

    Directory of Open Access Journals (Sweden)

    Yoseph A. Mekori

    2012-01-01

    Full Text Available Close physical proximity between mast cells and T cells has been demonstrated in several T cell mediated inflammatory processes such as rheumatoid arthritis and sarcoidosis. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We have identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells, and showed that this pathway is associated with degranulation and cytokine release. The signaling events associated with this pathway of mast cell activation have also been elucidated confirming the activation of the Ras MAPK systems. More recently, we hypothesized and demonstrated that mast cells may also be activated by microparticles released from activated T cells that are considered as miniature version of a cell. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site. Recent works have also focused on the effects of regulatory T cells on mast cells. These reports highlighted the importance of the cytokines IL-2 and IL-9, produced by mast cells and T cells, respectively, in obtaining optimal immune suppression. Finally, physical contact, associated by OX40-OX40L engagement has been found to underlie the down-regulatory effects exerted by regulatory T cells on mast cell function.

  6. Cytokine-induced loss of glucocorticoid function: effect of kinase inhibitors, long-acting β(2-adrenoceptor [corrected] agonist and glucocorticoid receptor ligands.

    Directory of Open Access Journals (Sweden)

    Christopher F Rider

    Full Text Available Acting on the glucocorticoid receptor (NR3C1, glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2 × glucocorticoid response element (GRE reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3

  7. In-vitro rescue and recovery studies of human melanoma (BLM) cell growth, adhesion and migration functions after treatment with progesterone

    OpenAIRE

    Leder, Douglas C; Brown, Jason R; Ramaraj, Pandurangan

    2015-01-01

    Treatment of human melanoma (BLM) cells for 48 hrs with progesterone resulted in a significant inhibition of cell growth. The mechanism of growth inhibition was due to autophagy and this action of progesterone was not mediated through progesterone receptor. As cells were floating during treatment, adhesion assay was performed, which showed complete loss of adhesion. When cells were allowed to recover after treatment by culturing in growth medium without progesterone, there was recovery in cel...

  8. Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Berezin, Vladimir; Bock, Elisabeth

    2008-01-01

    The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on...... this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does...

  9. Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering.

    Science.gov (United States)

    Vitte, Joana; Benoliel, Anne-Marie; Eymeric, Philippe; Bongrand, Pierre; Pierres, Anne

    2004-06-01

    The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor association. This interpretation accounts for the functional importance of integrin clustering.

  10. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1994-01-01

    Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs...

  11. Loss of MLCK leads to disruption of cell-cell adhesion and invasive behavior of breast epithelial cells via increased expression of EGFR and ERK/JNK signaling.

    Science.gov (United States)

    Kim, D Y; Helfman, D M

    2016-08-25

    Myosin light chain kinase (MLCK) expression is downregulated in breast cancer, including invasive ductal carcinoma compared with ductal breast carcinoma in situ and metastatic breast tumors. However, little is known about how loss of MLCK expression contributes to tumor progression. MLCK is a component of the actin cytoskeleton and its known role is the phosphorylation of the regulatory light chain of myosin II. To gain insights into the role of MLCK in breast cancer, we perturbed its function using small interfering RNA (siRNA) or pharmacological inhibition in untransformed breast epithelial cells (MCF10A). Loss of MLCK by siRNAs led to increased cell migration and invasion, disruption of cell-cell adhesions and enhanced formation of focal adhesions at the leading edge of migratory cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and low levels of MLCK is associated with a poor prognosis in HER2-positive breast cancer patients. Associated with these altered migratory behaviors were increased expression of epidermal growth factor receptor and activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways in MLCK downregulated MCF10A cells. By contrast, inhibition of the kinase function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affect cellular adhesions. Our results show that loss of MLCK contributes to the migratory properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased epidermal growth factor receptor signaling. These findings suggest that decreased expression of MLCK may have a critical role during tumor progression by facilitating the metastatic potential of tumor cells. PMID:26876209

  12. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Robert S., E-mail: Robert.Krauss@mssm.edu [Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029 (United States)

    2010-11-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre and Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed.

  13. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  14. Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

    Science.gov (United States)

    Miron, S; Kachalsky, S G; Hershkoviz, R; Lider, O

    1997-09-01

    The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins. PMID:9307082

  15. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  16. Functionally Different Pads on the Same Foot Allow Control of Attachment: Stick Insects Have Load-Sensitive “Heel” Pads for Friction and Shear-Sensitive “Toe” Pads for Adhesion

    Science.gov (United States)

    Labonte, David; Federle, Walter

    2013-01-01

    Stick insects (Carausius morosus) have two distinct types of attachment pad per leg, tarsal “heel” pads (euplantulae) and a pre-tarsal “toe” pad (arolium). Here we show that these two pad types are specialised for fundamentally different functions. When standing upright, stick insects rested on their proximal euplantulae, while arolia were the only pads in surface contact when hanging upside down. Single-pad force measurements showed that the adhesion of euplantulae was extremely small, but friction forces strongly increased with normal load and coefficients of friction were 1. The pre-tarsal arolium, in contrast, generated adhesion that strongly increased with pulling forces, allowing adhesion to be activated and deactivated by shear forces, which can be produced actively, or passively as a result of the insects' sprawled posture. The shear-sensitivity of the arolium was present even when corrected for contact area, and was independent of normal preloads covering nearly an order of magnitude. Attachment of both heel and toe pads is thus activated partly by the forces that arise passively in the situations in which they are used by the insects, ensuring safe attachment. Our results suggest that stick insect euplantulae are specialised “friction pads” that produce traction when pressed against the substrate, while arolia are “true” adhesive pads that stick to the substrate when activated by pulling forces. PMID:24349156

  17. Functionally different pads on the same foot allow control of attachment: stick insects have load-sensitive "heel" pads for friction and shear-sensitive "toe" pads for adhesion.

    Directory of Open Access Journals (Sweden)

    David Labonte

    Full Text Available Stick insects (Carausius morosus have two distinct types of attachment pad per leg, tarsal "heel" pads (euplantulae and a pre-tarsal "toe" pad (arolium. Here we show that these two pad types are specialised for fundamentally different functions. When standing upright, stick insects rested on their proximal euplantulae, while arolia were the only pads in surface contact when hanging upside down. Single-pad force measurements showed that the adhesion of euplantulae was extremely small, but friction forces strongly increased with normal load and coefficients of friction were [Formula: see text] 1. The pre-tarsal arolium, in contrast, generated adhesion that strongly increased with pulling forces, allowing adhesion to be activated and deactivated by shear forces, which can be produced actively, or passively as a result of the insects' sprawled posture. The shear-sensitivity of the arolium was present even when corrected for contact area, and was independent of normal preloads covering nearly an order of magnitude. Attachment of both heel and toe pads is thus activated partly by the forces that arise passively in the situations in which they are used by the insects, ensuring safe attachment. Our results suggest that stick insect euplantulae are specialised "friction pads" that produce traction when pressed against the substrate, while arolia are "true" adhesive pads that stick to the substrate when activated by pulling forces.

  18. Mouse Genetic Models Reveal Surprising Functions of IκB Kinase Alpha in Skin Development and Skin Carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Xiaojun [The Methodist Hospital Research Institute, Houston, TX 77030 (United States); Park, Eunmi [Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 (United States); Fischer, Susan M. [Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78967 (United States); Hu, Yinling, E-mail: huy2@mail.nih.gov [Cancer and Inflammation Program, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21701 (United States)

    2013-02-15

    Gene knockout studies unexpectedly reveal a pivotal role for IκB kinase alpha (IKKα) in mouse embryonic skin development. Skin carcinogenesis experiments show that Ikkα heterozygous mice are highly susceptible to chemical carcinogen or ultraviolet B light (UVB) induced benign and malignant skin tumors in comparison to wild-type mice. IKKα deletion mediated by keratin 5 (K5).Cre or K15.Cre in keratinocytes induces epidermal hyperplasia and spontaneous skin squamous cell carcinomas (SCCs) in Ikkα floxed mice. On the other hand, transgenic mice overexpressing IKKα in the epidermis, under the control of a truncated loricrin promoter or K5 promoter, develop normal skin and show no defects in the formation of the epidermis and other epithelial organs, and the transgenic IKKα represses chemical carcinogen or UVB induced skin carcinogenesis. Moreover, IKKα deletion mediated by a mutation, which generates a stop codon in the Ikkα gene, has been reported in a human autosomal recessive lethal syndrome. Downregulated IKKα and Ikkα mutations and deletions are found in human skin SCCs. The collective evidence not only highlights the importance of IKKα in skin development, maintaining skin homeostasis, and preventing skin carcinogenesis, but also demonstrates that mouse models are extremely valuable tools for revealing the mechanisms underlying these biological events, leading our studies from bench side to bedside.

  19. A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

    Science.gov (United States)

    Casey, Nicholas Paul; Fujiwara, Hiroshi; Tanimoto, Kazushi; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Yasukawa, Masaki

    2016-01-01

    Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene

  20. A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

    Directory of Open Access Journals (Sweden)

    Nicholas Paul Casey

    Full Text Available Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell

  1. Particle adhesion and removal

    CERN Document Server

    Mittal, K L

    2015-01-01

    The book provides a comprehensive and easily accessible reference source covering all important aspects of particle adhesion and removal.  The core objective is to cover both fundamental and applied aspects of particle adhesion and removal with emphasis on recent developments.  Among the topics to be covered include: 1. Fundamentals of surface forces in particle adhesion and removal.2. Mechanisms of particle adhesion and removal.3. Experimental methods (e.g. AFM, SFA,SFM,IFM, etc.) to understand  particle-particle and particle-substrate interactions.4. Mechanics of adhesion of micro- and  n

  2. Protein Kinase D family kinases

    OpenAIRE

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lo...

  3. Activation of AKT negatively regulates the pro-apoptotic function of death-associated protein kinase 3 (DAPK3) in prostate cancer.

    Science.gov (United States)

    Das, Trinath P; Suman, Suman; Papu John, A M Sashi; Pal, Deeksha; Edwards, Angelena; Alatassi, Houda; Ankem, Murali K; Damodaran, Chendil

    2016-07-28

    The activation of AKT governs many signaling pathways and promotes cell growth and inhibits apoptosis in human malignancies including prostate cancer (CaP). Here, we investigated the molecular association between AKT activation and the function of death-associated protein kinase 3 (DAPK3) in CaP. An inverse correlation of pAKT and DAPK3 expression was seen in a panel of CaP cell lines. Inhibition of AKT by wortmannin/LY294002 or overexpression of DAPK3 reverts the proliferative function of AKT in CaP cells. On the other hand, ectopic expression of AKT inhibited DAPK3 function and induced proliferation of CaP cells. In addition, AKT over-expressed tumors exhibit aggressive growth when compared to control vector in xenograft models. The immunohistochemistry results revealed a down-regulation of DAPK3 expression in AKT over-expressed tumors as compared to control tumors. Finally, we examined the expression pattern of AKT and DAPK3 in human CaP specimens - the expected gradual increase and nuclear localization of pAKT was seen in higher Gleason score samples versus benign hyperplasia (BPH). On the contrary, reduced expression of DAPK3 was seen in higher Gleason stages versus BPH. This suggests that inhibition of DAPK3 may be a contributing factor to the carcinogenesis of the prostate. Understanding the mechanism by which AKT negatively regulates DAPK3 function may suggest whether DAPK3 can be a therapeutic target for CaP. PMID:27126362

  4. Epithelial cell adhesion molecule aptamer functionalized PLGA-lecithin-curcumin-PEG nanoparticles for targeted drug delivery to human colorectal adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Li L

    2014-02-01

    Full Text Available Lei Li,1,* Dongxi Xiang,2,* Sarah Shigdar,2 Wenrong Yang,3 Qiong Li,2 Jia Lin,4 Kexin Liu,1 Wei Duan2 1College of Pharmacy, Dalian Medical University, Dalian, People's Republic of China; 2School of Medicine, Faculty of Health, Deakin University, Waurn Ponds, VIC, Australia; 3School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, VIC, Australia; 4Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, People's Republic of China *These authors contributed equally to this work Abstract: To improve the efficacy of drug delivery, active targeted nanotechnology-based drug delivery systems are gaining considerable attention as they have the potential to reduce side effects, minimize toxicity, and improve efficacy of anticancer treatment. In this work CUR-NPs (curcumin-loaded lipid-polymer-lecithin hybrid nanoparticles were synthesized and functionalized with ribonucleic acid (RNA Aptamers (Apts against epithelial cell adhesion molecule (EpCAM for targeted delivery to colorectal adenocarcinoma cells. These CUR-encapsulated bioconjugates (Apt-CUR-NPs were characterized for particle size, zeta potential, drug encapsulation, stability, and release. The in vitro specific cell binding, cellular uptake, and cytotoxicity of Apt-CUR-NPs were also studied. The Apt-CUR-NP bioconjugates exhibited increased binding to HT29 colon cancer cells and enhancement in cellular uptake when compared to CUR-NPs functionalized with a control Apt (P<0.01. Furthermore, a substantial improvement in cytotoxicity was achieved toward HT29 cells with Apt-CUR-NP bioconjugates. The encapsulation of CUR in Apt-CUR-NPs resulted in the increased bioavailability of delivered CUR over a period of 24 hours compared to that of free CUR in vivo. These results show that the EpCAM Apt-functionalized CUR-NPs enhance the targeting and drug

  5. The effect of midazolam on neutrophil mitogen-activated protein kinase.

    LENUS (Irish Health Repository)

    Ghori, Kamran

    2010-06-01

    Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b\\/CD18.

  6. Structural Studies on the Extracellular Domain of Sensor Histidine Kinase YycG from Staphylococcus aureus and Its Functional Implications.

    Science.gov (United States)

    Kim, Truc; Choi, Jongkeun; Lee, Sangho; Yeo, Kwon Joo; Cheong, Hae-Kap; Kim, Kyeong Kyu

    2016-07-31

    Bacterial two-component signal transduction systems are used to adapt to fluctuations in the environment. YycG, a key two-component histidine kinase in Staphylococcus aureus, plays an essential role in cell viability and regulates cell wall metabolism, biofilm formation, virulence, and antibiotic resistance. For these reasons, YycG is considered a compelling target for the development of novel antibiotics. However, to date, the signaling mechanism of YycG and its stimulus are poorly understood mainly because of a lack of structural information on YycG. To address this deficiency, we determined the crystal structure of the extracellular domain of S. aureus YycG (YycGex) at 2.0-Å resolution. The crystal structure indicated two subunits with an extracellular Per-Arnt-Sim (PAS) topology packed into a dimer with interloop interactions. Disulfide scanning using cysteine-substituted mutants revealed that YycGex possessed dimeric interfaces not only in the loop but also in the helix α1. Cross-linking studies using intact YycG demonstrated that it was capable of forming high molecular weight oligomers on the cell membrane. Furthermore, we also observed that two auxiliary proteins of YycG, YycH and YycI, cooperatively interfered with the multimerization of YycG. From these results, we propose that signaling through YycG is regulated by multimerization and binding of YycH and YycI. These structural studies, combined with biochemical analyses, provide a better understanding of the signaling mechanism of YycG, which is necessary for developing novel antibacterial drugs targeting S. aureus. PMID:27389096

  7. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    Science.gov (United States)

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  8. Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

    DEFF Research Database (Denmark)

    Whiteford, James; Behrends, Volker; Kirby, Hishani;

    2007-01-01

    to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence...

  9. Cloning of a conserved receptor-like protein kinase gene and its use as a functional marker for homoeologous group-2 chromosomes of the triticeae species.

    Directory of Open Access Journals (Sweden)

    Bi Qin

    Full Text Available Receptor-like kinases (RLKs play broad biological roles in plants. We report on a conserved receptor-like protein kinase (RPK gene from wheat and other Triticeae species. The TaRPK1 was isolated from the Triticum aestivum cv. Prins - Triticum timopheevii introgression line IGVI-465 carrying the powdery mildew resistance gene Pm6. The TaRPK1 was mapped to homoeologous chromosomes 2A (TaRPK1-2A, 2D (TaRPK1-2D and the Pm6-carrier chromosome 2G (TaRPK1-2G of IGVI-465. Under the tested conditions, only the TaRPK1-2G allele was actively transcribed, producing two distinct transcripts via alternative splicing. The predicted 424-amino acid protein of TaRPK1-2G contained a signal peptide, a transmembrane domain and an intracellular serine/threonine kinase domain, but lacked a typical extracellular domain. The expression of TaRPK1-2G gene was up-regulated upon the infection by Blumeria graminis f.sp. tritici (Bgt and treatment with methyl jasmonate (MeJA, but down-regulated in response to treatments of SA and ABA. Over-expression of TaRPK1-2G in the powdery mildew susceptible wheat variety Prins by a transient expression assay showed that it slightly reduced the haustorium index of the infected Bgt. These data indicated that TaRPK1-2G participated in the defense response to Bgt infection and in the JA signaling pathway. Phylogenetic analysis indicated that TaRPK1-2G was highly conserved among plant species, and the amino acid sequence similarity of TaRPK1-2G among grass species was more than 86%. Based on its conservation, the RPK gene-based STS primers were designed, and used to amplify the RPK orthologs from the homoeologous group-2 chromosomes of all the tested Triticeae species, such as chromosome 2G of T. timopheevii, 2R of Secale cereale, 2H of Hordeum vulgare, 2S of Aegilops speltoides, 2S(l of Ae. longissima, 2M(g of Ae. geniculata, 2S(p and 2U(p of Ae. peregrina. The developed STS markers serve as conserved functional markers for the

  10. Couplings between hierarchical conformational dynamics from multi-time correlation functions and two-dimensional lifetime spectra: Application to adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Junichi [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); Takada, Shoji [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan); Saito, Shinji, E-mail: shinji@ims.ac.jp [Department of Theoretical and Computational Molecular Science, Institute for Molecular Science, Okazaki 444-8585 (Japan); The Graduate University for Advanced Studies, Okazaki 444-8585 (Japan)

    2015-06-07

    An analytical method based on a three-time correlation function and the corresponding two-dimensional (2D) lifetime spectrum is developed to elucidate the time-dependent couplings between the multi-timescale (i.e., hierarchical) conformational dynamics in heterogeneous systems such as proteins. In analogy with 2D NMR, IR, electronic, and fluorescence spectroscopies, the waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra can provide a quantitative description of the dynamical correlations between the conformational motions with different lifetimes. The present method is applied to intrinsic conformational changes of substrate-free adenylate kinase (AKE) using long-time coarse-grained molecular dynamics simulations. It is found that the hierarchical conformational dynamics arise from the intra-domain structural transitions among conformational substates of AKE by analyzing the one-time correlation functions and one-dimensional lifetime spectra for the donor-acceptor distances corresponding to single-molecule Förster resonance energy transfer experiments with the use of the principal component analysis. In addition, the complicated waiting-time dependence of the off-diagonal peaks in the 2D lifetime spectra for the donor-acceptor distances is attributed to the fact that the time evolution of the couplings between the conformational dynamics depends upon both the spatial and temporal characters of the system. The present method is expected to shed light on the biological relationship among the structure, dynamics, and function.

  11. The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sergi Portolés

    2010-11-01

    Full Text Available Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1. CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2 are essential for clock temperature compensation in Arabidopsis.

  12. Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells

    Directory of Open Access Journals (Sweden)

    Schmidt Stefan

    2010-11-01

    Full Text Available Abstract Background Glucocorticoids (GCs cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL. PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2, a kinase controlling glucose metabolism, was identified by us previously as a GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives, PFKFB1, 3, and 4, were further analyzed. Methods Gene expression analyses of isolated lymphoblasts were performed on Affymetrix HGU133 Plus 2.0 microarrays. GCRMA normalized microarray data were analyzed using R-Bioconductor packages version 2.5. Functional gene analyses of PFKFB2-15A and -15B isoforms were performed by conditional gene over-expression experiments in the GC-sensitive T-ALL model CCRF-CEM. Results Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly T-ALL cells. The 3 other family members, in contrast, were either absent or only weakly expressed (PFKFB1 and 4 or not induced by GC (PFKFB3. Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (PFKFB2-15A and PFKFB2-15B had no detectable effect on cell survival. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Conclusions Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC.

  13. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    Science.gov (United States)

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  14. Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells

    International Nuclear Information System (INIS)

    Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as a GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives, PFKFB1, 3, and 4, were further analyzed. Gene expression analyses of isolated lymphoblasts were performed on Affymetrix HGU133 Plus 2.0 microarrays. GCRMA normalized microarray data were analyzed using R-Bioconductor packages version 2.5. Functional gene analyses of PFKFB2-15A and -15B isoforms were performed by conditional gene over-expression experiments in the GC-sensitive T-ALL model CCRF-CEM. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly T-ALL cells. The 3 other family members, in contrast, were either absent or only weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (PFKFB2-15A and PFKFB2-15B) had no detectable effect on cell survival. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC

  15. Functional and structural comparison of pyrrolnitrin- and iprodione-induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea.

    Directory of Open Access Journals (Sweden)

    Sabine Fillinger

    Full Text Available Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade.In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure

  16. The evolution of adhesiveness as a social adaptation.

    Science.gov (United States)

    Garcia, Thomas; Doulcier, Guilhem; De Monte, Silvia

    2015-11-27

    Cellular adhesion is a key ingredient to sustain collective functions of microbial aggregates. Here, we investigate the evolutionary origins of adhesion and the emergence of groups of genealogically unrelated cells with a game-theoretical model. The considered adhesiveness trait is costly, continuous and affects both group formation and group-derived benefits. The formalism of adaptive dynamics reveals two evolutionary stable strategies, at each extreme on the axis of adhesiveness. We show that cohesive groups can evolve by small mutational steps, provided the population is already endowed with a minimum adhesiveness level. Assortment between more adhesive types, and in particular differential propensities to leave a fraction of individuals ungrouped at the end of the aggregation process, can compensate for the cost of increased adhesiveness. We also discuss the change in the social nature of more adhesive mutations along evolutionary trajectories, and find that altruism arises before directly beneficial behavior, despite being the most challenging form of cooperation.

  17. Strong, reversible underwater adhesion via gecko-inspired hydrophobic fibers.

    Science.gov (United States)

    Soltannia, Babak; Sameoto, Dan

    2014-12-24

    Strong, reversible underwater adhesion using gecko-inspired surfaces is achievable through the use of a hydrophobic structural material and does not require surface modification or suction cup effects for this adhesion to be effective. Increased surface energy can aid in dry adhesion in an air environment but strongly degrades wet adhesion via reduction of interfacial energy underwater. A direct comparison of structurally identical but chemically different mushroom shaped fibers shows that strong, reversible adhesion, even in a fully wetted, stable state, is feasible underwater if the structural material of the fibers is hydrophobic and the mating surface is not strongly hydrophilic. The exact adhesion strength will be a function of the underwater interfacial energy between surfaces and the specific failure modes of individual fibers. This underwater adhesion has been calculated to be potentially greater than the dry adhesion for specific combinations of hydrophobic surfaces.

  18. The evolution of adhesiveness as a social adaptation.

    Science.gov (United States)

    Garcia, Thomas; Doulcier, Guilhem; De Monte, Silvia

    2015-01-01

    Cellular adhesion is a key ingredient to sustain collective functions of microbial aggregates. Here, we investigate the evolutionary origins of adhesion and the emergence of groups of genealogically unrelated cells with a game-theoretical model. The considered adhesiveness trait is costly, continuous and affects both group formation and group-derived benefits. The formalism of adaptive dynamics reveals two evolutionary stable strategies, at each extreme on the axis of adhesiveness. We show that cohesive groups can evolve by small mutational steps, provided the population is already endowed with a minimum adhesiveness level. Assortment between more adhesive types, and in particular differential propensities to leave a fraction of individuals ungrouped at the end of the aggregation process, can compensate for the cost of increased adhesiveness. We also discuss the change in the social nature of more adhesive mutations along evolutionary trajectories, and find that altruism arises before directly beneficial behavior, despite being the most challenging form of cooperation. PMID:26613415

  19. Adhesive loose packings of small dry particles

    Science.gov (United States)

    Liu, Wenwei; Li, Shuiqing; Baule, Adrian; Makse, Hernán A.

    We explore adhesive loose packings of dry small spherical particles of micrometer size using 3D discrete-element simulations with adhesive contact mechanics. A dimensionless adhesion parameter ($Ad$) successfully combines the effects of particle velocities, sizes and the work of adhesion, identifying a universal regime of adhesive packings for $Ad>1$. The structural properties of the packings in this regime are well described by an ensemble approach based on a coarse-grained volume function that includes correlations between bulk and contact spheres. Our theoretical and numerical results predict: (i) An equation of state for adhesive loose packings that appears as a continuation from the frictionless random close packing (RCP) point in the jamming phase diagram; (ii) The existence of a maximal loose packing point at the coordination number $Z=2$ and packing fraction $\\phi=1/2^{3}$. Our results highlight that adhesion leads to a universal packing regime at packing fractions much smaller than the random loose packing, which can be described within a statistical mechanical framework. We present a general phase diagram of jammed matter comprising frictionless, frictional, adhesive as well as non-spherical particles, providing a classification of packings in terms of their continuation from the spherical frictionless RCP.

  20. Different pH-dependencies of the two synaptic adhesion molecules N-cadherin and cadherin-11 and the possible functional implication for long-term potentiation

    DEFF Research Database (Denmark)

    Baumgartner, Werner; Osmanagic, Armin; Gebhard, Marita;

    2013-01-01

    Ca(2+) -dependent adhesion molecules, cadherins, localised at synaptic sites are critically involved in long-term potentiation (LTP). N-cadherin is thought to promote LTP whereas cadherin-11 seems to counteract LTP. Since high synaptic activity is accompanied by local transient changes of the p...

  1. Evolutionary Reconstruction and Population Genetics Analysis of Aurora Kinases

    OpenAIRE

    Balu Kamaraj; Ambuj Kumar; Rituraj Purohit

    2013-01-01

    BACKGROUND: Aurora kinases belong to the highly conserved kinase family and play a vital role in cell cycle regulation. The structure and function of these kinases are inter-related and sometimes they also act as substitutes in case of knockdown of other aurora kinases. METHOD: In this work we carried out the evolutionary reconstruction and population genetic studies of aurora kinase proteins. Substitution saturation test, CAI (Codon adaptation index), gene expression and RSCU (Relative synon...

  2. Reinforcement of epithelial cell adhesion to basement membrane by a bacterial pathogen as a new infectious stratagem.

    Science.gov (United States)

    Kim, Minsoo; Ogawa, Michinaga; Mimuro, Hitomi; Sasakawa, Chihiro

    2010-01-01

    The intestinal epithelium undergoes a rapid turnover in addition to rapid exfoliation in response to bacterial infection, thus acting as an intrinsic defense against microbial intruders. It has long been questioned how mucosal pathogens can circumvent the intestinal defense systems. Our recent discovery of a bacterial ploy used by Shigella provided us with fresh insight. Shigella delivers OspE via the type III secretion system during multiplication within epithelial cells. This effector protein reinforces epithelial adherence to the basement membrane by interacting with integrin-linked kinase (ILK), a unique intracellular Ser/Thr kinase that links the cell-adhesion receptors, integrin, and growth factors to the actin cytoskeleton. The interaction between OspE and ILK increased formation of focal adhesions (FAs) and surface levels of b1-integrin, while suppressing phosphorylation of FAK and paxillin, thus suppressing rapid turnover of FAs, reducing cell motility and promoting cell adhesion to extracellular matrix. The impact of this OspE-ILK interplay was demonstrated both in vitro and in vivo by infecting polarized epithelial cell monolayers and guinea pig colons with Shigella possessing or lacking the ospE gene. The findings thus establish a new class of virulence-associated factors, and provide new insight into the functioning of the intestinal barrier and bacterial strategies for circumventing it. PMID:21178415

  3. Adverse effects of AMP-activated protein kinase alpha2-subunit deletion and high-fat diet on heart function and ischemic tolerance in aged female mice.

    Science.gov (United States)

    Slámová, K; Papoušek, F; Janovská, P; Kopecký, J; Kolář, F

    2016-03-14

    AMP-activated protein kinase (AMPK) plays a role in metabolic regulation under stress conditions, and inadequate AMPK signaling may be also involved in aging process. The aim was to find out whether AMPK alpha2-subunit deletion affects heart function and ischemic tolerance of adult and aged mice. AMPK alpha2(-/-) (KO) and wild type (WT) female mice were compared at the age of 6 and 18 months. KO mice exhibited subtle myocardial AMPK alpha2-subunit protein level, but no difference in AMPK alpha1-subunit was detected between the strains. Both alpha1- and alpha2-subunits of AMPK and their phosphorylation decreased with advanced age. Left ventricular fractional shortening was lower in KO than in WT mice of both age groups and this difference was maintained after high-fat feeding. Infarct size induced by global ischemia/reperfusion of isolated hearts was similar in both strains at 6 months of age. Aged WT but not KO mice exhibited improved ischemic tolerance compared with the younger group. High-fat feeding for 6 months during aging abolished the infarct size-reduction in WT without affecting KO animals; nevertheless, the extent of injury remained larger in KO mice. The results demonstrate that adverse effects of AMPK alpha2-subunit deletion and high-fat feeding on heart function and myocardial ischemic tolerance in aged female mice are not additive. PMID:26596312

  4. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    Science.gov (United States)

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  5. Fusion tyrosine kinase NPM-ALK Deregulates MSH2 and suppresses DNA mismatch repair function novel insights into a potent oncoprotein.

    Science.gov (United States)

    Young, Leah C; Bone, Kathleen M; Wang, Peng; Wu, Fang; Adam, Benjamin A; Hegazy, Samar; Gelebart, Pascal; Holovati, Jelena; Li, Liang; Andrew, Susan E; Lai, Raymond

    2011-07-01

    The fusion tyrosine kinase NPM-ALK is central to the pathogenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL). We recently identified that MSH2, a key DNA mismatch repair (MMR) protein integral to the suppression of tumorigenesis, is an NPM-ALK-interacting protein. In this study, we found in vitro evidence that enforced expression of NPM-ALK in HEK293 cells suppressed MMR function. Correlating with these findings, six of nine ALK(+)ALCL tumors displayed evidence of microsatellite instability, as opposed to none of the eight normal DNA control samples (P = 0.007, Student's t-test). Using co-immunoprecipitation, we found that increasing levels of NPM-ALK expression in HEK293 cells resulted in decreased levels of MSH6 bound to MSH2, whereas MSH2·NPM-ALK binding was increased. The NPM-ALK·MSH2 interaction was dependent on the activation/autophosphorylation of NPM-ALK, and the Y191 residue of NPM-ALK was a crucial site for this interaction and NPM-ALK-mediated MMR suppression. MSH2 was found to be tyrosine phosphorylated in the presence of NPM-ALK. Finally, NPM-ALK impeded the expected DNA damage-induced translocation of MSH2 out of the cytoplasm. To conclude, our data support a model in which the suppression of MMR by NPM-ALK is attributed to its ability to interfere with normal MSH2 biochemistry and function.

  6. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    Science.gov (United States)

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  7. Reversible Thermoset Adhesives

    Science.gov (United States)

    Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

    2016-01-01

    Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

  8. Electro-dry-adhesion.

    Science.gov (United States)

    Krahn, Jeffrey; Menon, Carlo

    2012-03-27

    This work presents novel conductive bioinspired dry adhesives with mushroom caps that enable the use of a synergistic combination of electrostatic and van der Waals forces (electro-dry-adhesion). An increase in shear adhesion bond strength of up to 2046% on a wide range of materials is measured when a maximum electrical field of 36.4 V μm(-1) is applied. A suction effect, due to the shape of the dry adhesive fibers, on overall adhesion was not noted for electro-dry-adhesives when testing was performed at both atmospheric and reduced pressure. Utilization of electrostatics to apply a preloading force to dry adhesive fiber arrays allows increased adhesion even after electrostatic force generation has been halted by ensuring the close contact necessary for van der Waals forces to be effective. A comparison is made between self-preloading of the electro-dry-adhesives and the direct application of a normal preloading pressure resulting in nearly the same shear bond strength with an applied voltage of 3.33 kV on the same sample.

  9. High-performance mussel-inspired adhesives of reduced complexity

    Science.gov (United States)

    Ahn, B. Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R.;