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Sample records for adhesion kinase activation

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

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

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    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.

  3. Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion

    Science.gov (United States)

    Ihermann-Hella, Anneliis; Lume, Maria; Miinalainen, Ilkka J.; Pirttiniemi, Anniina; Gui, Yujuan; Peränen, Johan; Charron, Jean; Saarma, Mart; Costantini, Frank; Kuure, Satu

    2014-01-01

    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. PMID:24603431

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    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 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. PMID:26703626

  7. 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.

  8. 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

  9. 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.

  10. Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes

    DEFF Research Database (Denmark)

    Goñi, Guillermina M; Epifano, Carolina; Boskovic, Jasminka

    2014-01-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the p...

  11. Inhibition of tumor necrosis factor alpha-stimulated monocyte adhesion to human aortic endothelial cells by AMP-activated protein kinase.

    Science.gov (United States)

    Ewart, Marie-Ann; Kohlhaas, Christine F; Salt, Ian P

    2008-12-01

    Proatherosclerotic adhesion of leukocytes to the endothelium is attenuated by NO. As AMP-activated protein kinase (AMPK) regulates endothelial NO synthesis, we investigated the modulation of adhesion to cultured human aortic endothelial cells (HAECs) by AMPK. HAECs incubated with the AMPK activator, AICAR, or expressing constitutively active AMPK demonstrated reduced TNFalpha-stimulated adhesion of promonocytic U-937 cells. Rapid inhibition of TNFalpha-stimulated U-937 cell adhesion by AICAR was NO-dependent, associated with unaltered cell surface adhesion molecule expression, and reduced MCP-1 secretion by HAECs. In contrast, inhibition of TNFalpha-stimulated U-937 cell adhesion by prolonged AMPK activation was NO-independent and associated with reduced cell surface adhesion molecule expression. AMPK activation in HAECs inhibits TNFalpha-stimulated leukocyte adhesion by a rapid NO-dependent mechanism associated with reduced MCP-1 secretion and a late NO-independent mechanism whereby adhesion molecule expression, in particular E-selectin, is suppressed.

  12. Indole-3-carbinol inhibits MDA-MB-231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity.

    Science.gov (United States)

    Brew, Christine T; Aronchik, Ida; Kosco, Karena; McCammon, Jasmine; Bjeldanes, Leonard F; Firestone, Gary L

    2009-05-15

    Indole-3-carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent antiproliferative effects in human breast cancer cells and has been shown to decrease metastatic spread of tumors in experimental animals. Using chemotaxis and fluorescent-bead cell motility assays, we demonstrated that I3C significantly decreased the in vitro migration of MDA-MB-231 cells, a highly invasive breast cancer cell line. Immunofluorescence staining of the actin cytoskeleton revealed that concurrent with the loss of cell motility, I3C treatment significantly increased stress fiber formation. Furthermore, I3C induced the localization of the focal adhesion component vinculin and tyrosine-phosphorylated proteins to the cell periphery, which implicates an indole-dependent enhancement of focal adhesions within the outer boundary of the cells. Coimmunoprecipitation analysis of focal adhesion kinase demonstrated that I3C stimulated the dynamic formation of the focal adhesion protein complex without altering the total level of individual focal adhesion proteins. The RhoA-Rho kinase pathway is involved in stress fiber and focal adhesion formation, and I3C treatment stimulated Rho kinase enzymatic activity and cofilin phosphorylation, which is a downstream target of Rho kinase signaling, but did not increase the level of active GTP-bound RhoA. Exposure of MDA-MB-231 cells to the Rho kinase inhibitor Y-27632, or expression of dominant negative RhoA ablated the I3C induced formation of stress fibers and of peripheral focal adhesions. Expression of constitutively active RhoA mimicked the I3C effects on both processes. Taken together, our data demonstrate that I3C induces stress fibers and peripheral focal adhesions in a Rho kinase-dependent manner that leads to an inhibition of motility in human breast cancer cells. (c) 2008 Wiley-Liss, Inc.

  13. Activation of erythropoietin-producing hepatocellular receptor A2 attenuates cell adhesion of human fallopian tube epithelial cells via focal adhesion kinase dephosphorylation.

    Science.gov (United States)

    Yang, Xiao-Yi; Zhu, Wei-Jie; Jiang, Huan

    2012-02-01

    Tyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) and its predominant ligand EphrinA1 have been studied extensively for their roles of mediating cell adhesion in epithelial cells. However, EphA2 signaling in human fallopian tube epithelial cells is poorly understood. In this study, primary cultured fallopian tube epithelial cells were used as a model treated with EphrinA1-Fc or IgG-Fc (control), to explore the role of EphA2 signal and its network involved in the regulation of cell adhesion of tubal epithelia cells. The activation of EphA2 and focal adhesion kinase (FAK) was evaluated by western blotting assay in the cultured fallopian tube epithelia cells, of which the cell adhesion activity was determined by MTT assay. A significantly negative correlation was found between phosphorylated-EphA2 (Pho-EphA2) and phosphorylated-FAK (Pho-FAK) after exposure to EphrinA1-Fc (P = 0.000; r = -0.848). EphrinA1-Fc increased Pho-EphA2 and reduced Pho-FAK in seconds, with the apex level of Pho-EphA2 and the nadir level of Pho-FAK detected at the same time (10 min). Cell adhesion of the cultured cells supplemented with EphrinA1-Fc appeared to be weaker than that of the controls at the later time points of the treatment (from 30 to 120 min) (P < 0.05). Taken together, the EphrinA1 addition directly induces an elevated Pho-EphA2 accompanied by a decreased Pho-FAK in human fallopian tube epithelia cells. Furthermore, activation of EphA2 participates in the regulation of fallopian tube cell adhesion via FAK dephosphorylation.

  14. In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.

    Science.gov (United States)

    Cable, Jennifer; Prutzman, Kirk; Gunawardena, Harsha P; Schaller, Michael D; Chen, Xian; Campbell, Sharon L

    2012-03-20

    Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

  15. RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility

    Czech Academy of Sciences Publication Activity Database

    Vomastek, Tomáš; Iwanicki, M. P.; Schaeffer, J.; J.; Tarcsafalvi, A.; Parsons, J. T.; Weber, M. J.

    2007-01-01

    Roč. 27, č. 23 (2007), s. 8296-8305 ISSN 0270-7306 R&D Projects: GA AV ČR IAA500200716 Institutional research plan: CEZ:AV0Z50200510 Keywords : protein kinase * adhesion * cell Subject RIV: EE - Microbiology, Virology Impact factor: 6.420, year: 2007

  16. 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

    transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor......, 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...

  17. 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)

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-01-01

    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

  18. 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

  19. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling.

    Science.gov (United States)

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2015-01-15

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  20. Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

    Science.gov (United States)

    Zhou, Qingyu; Guo, Xiaofang; Choksi, Riya

    2017-12-01

    Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS- mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS -mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin ( P A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS -mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  1. 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...

  2. 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...

  3. FAK/src-family dependent activation of the Ste20-like kinase SLK is required for microtubule-dependent focal adhesion turnover and cell migration.

    Directory of Open Access Journals (Sweden)

    Simona Wagner

    2008-04-01

    Full Text Available Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.

  4. Titanium dioxide nanoparticles induce human eosinophil adhesion onto endothelial EA.hy926 cells via activation of phosphoinositide 3-kinase/Akt cell signalling pathway.

    Science.gov (United States)

    Murphy-Marion, Maxime; Girard, Denis

    2018-02-01

    The use of nanoparticles (NPs) for developing new therapeutic strategies in a variety of diseases is gaining increasing attention. However, NPs could possess undesired effects, including pro-inflammatory activities. Despite the fact that several studies reported that NPs may induce or exacerbate eosinophilic inflammation in vivo in rodents, the information regarding the direct interaction between NPs and human eosinophils is lacking. In the present study, we test the possibility that NPs could alter the capacity of human eosinophils to adhere onto a cellular substratum. Using a panel of NPs, we found that several were able to increase the adhesion of human eosinophil onto endothelial EA.hy926 cells. Among them, TiO 2 NPs were the most potent and we therefore pursue this study with these NPs. TiO 2 NPs were found to increase the adhesion of eosinophils in a concentration dependent fashion. TiO 2 NPs did not alter the cell surface expression of a panel of cellular adhesion molecules, but CD29. Indeed, a weak to moderate, but significant, decrease of CD29 was observed after 30min but returned to normal levels after 90min. TiO 2 NPs were found to activate Akt, one important target of phosphoinositide 3-kinase (PI3K). However, despite the fact that cells were fully responsive to the cytokine GM-CSF activating both Akt and Erk-1/2, TiO 2 NPs did not activate Erk-1/2. Using a pharmacological approach with the PI3K/Akt inhibitor, wortmannin, the ability of TiO 2 NPs to activate Akt was drastically inhibited and, further, their capacity to increase adhesion of eosinophils was reversed. This study provides insights into the effects of NPs on the biology of human eosinophils indicating that as other agents, NPs, namely TiO 2 NPs, can induce intracellular events associated with a cellular function, adhesion. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    Science.gov (United States)

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

  6. LEFTY2 Controls Migration of Human Endometrial Cancer Cells via Focal Adhesion Kinase Activity (FAK) and miRNA-200a.

    Science.gov (United States)

    Alowayed, Nour; Salker, Madhuri S; Zeng, Ni; Singh, Yogesh; Lang, Florian

    2016-01-01

    LEFTY2, a suppressor of cell proliferation, tumor growth, regulator of stemness and embryonic differentiation, is a negative regulator of cancer cell reprogramming. Malignant transformation may lead to migration requiring loss of adhesion and gain of migratory activity. Signaling involved in the orchestration of migration, proliferation and spreading of cells include focal adhesion kinase (FAK) and adhesion molecule E-cadherin. The present study explored whether LEFTY2 influences the proliferation marker MKi67, FAK activity, E-cadherin abundance and migration of Ishikawa human endometrial carcinoma cells. Moreover, the study explored the involvement of microRNA-200a (miR-200a), which is known to regulate cellular adhesion by targeting E-Cadherin. FAK activity was estimated from FAK phosphorylation quantified by Western blotting, migration utilizing a wound healing assay, miR-200a and MKi67 expression levels utilizing qRT-PCR, cell proliferation and apoptosis using BrdU and Annexin V staining, respectively, and E-Cadherin (E-Cad) abundance, using confocal microscopy. LEFTY2 (25 ng/ml, 48 hours) treatment was followed by decrease of MKi67 expression, FAK activity and migration. LEFTY2 upregulated miRNA-200a and E-Cad protein level in Ishikawa cells. The effect of LEFTY2 on migration was mimicked by FAK inhibitor PF 573228 (50 µM). Addition of LEFTY2 in the presence of PF-573228 did not result in a further significant decline of migration. In conclusion, LEFTY2 down-regulates MKi67 expression and FAK activity, up-regulates miR-200a and E-cadherin, and is thus a powerful negative regulator of endometrial cell proliferation and migration. © 2016 The Author(s) Published by S. Karger AG, Basel.

  7. 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)

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

    2016-01-01

    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...

  8. 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.

  9. Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

    Science.gov (United States)

    Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

    2018-05-02

    Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

  10. Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

    OpenAIRE

    Zhao, Jing; Wei, Jianxin; Bowser, Rachel K; Traister, Russell S; Fan, Ming-Hui; Zhao, Yutong

    2014-01-01

    IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. ST2L, the receptor for IL-33, is expressed on immune effector cells and lung epithelia, and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system, however its upstream internalization has not been studied. Here, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates S...

  11. 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.

  12. Focal adhesion kinase maintains, but not increases the adhesion of dental pulp cells.

    Science.gov (United States)

    Qian, Yuyan; Shao, Meiying; Zou, Wenlin; Wang, Linyan; Cheng, Ran; Hu, Tao

    2017-04-01

    Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.

  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

    The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein...... 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...

  14. 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

    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...... 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...

  15. Copper Deficiency Induced Emphysema Is Associated with Focal Adhesion Kinase Inactivation

    OpenAIRE

    Mizuno, Shiro; Yasuo, Masanori; Bogaard, Harm J.; Kraskauskas, Donatas; Alhussaini, Aysar; Gomez-Arroyo, Jose; Farkas, Daniela; Farkas, Laszlo; Voelkel, Norbert F.

    2012-01-01

    Background Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1?) 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 alveola...

  16. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    International Nuclear Information System (INIS)

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-01-01

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho

  17. Increased Expression of Focal Adhesion Kinase in Thyroid Cancer: Immunohistochemical Study

    OpenAIRE

    Kim, Sang Jin; Park, Jin Woo; Yoon, Ji Sung; Mok, Ji O; Kim, Yeo Joo; Park, Hyeong Kyu; Kim, Chul Hee; Byun, Dong Won; Lee, Yong Jin; Jin, So Young; Suh, Kyo Il; Yoo, Myung Hi

    2004-01-01

    Focal adhesion kinase (FAK) is a tyrosine kinase that is found in cellular structures called focal adhesions. FAK appears to be a key element in signal transduction pathways involved in cell adhesion and locomotion. FAK is overexpressed in various tumors, including tumors derived from regions of the head and neck, colon, breast, prostate, and liver. In this study, we investigated immunohistochemically whether FAK expression was increased in thyroid cancers. FAK staining was not seen in any of...

  18. Integrin-Associated Focal Adhesion Kinase Protects Human Embryonic Stem Cells from Apoptosis, Detachment, and Differentiation

    Directory of Open Access Journals (Sweden)

    Loriana Vitillo

    2016-08-01

    Full Text Available Human embryonic stem cells (hESCs can be maintained in a fully defined niche on extracellular matrix substrates, to which they attach through integrin receptors. However, the underlying integrin signaling mechanisms, and their contribution to hESC behavior, are largely unknown. Here, we show that focal adhesion kinase (FAK transduces integrin activation and supports hESC survival, substrate adhesion, and maintenance of the undifferentiated state. After inhibiting FAK kinase activity we show that hESCs undergo cell detachment-dependent apoptosis or differentiation. We also report deactivation of FAK downstream targets, AKT and MDM2, and upregulation of p53, all key players in hESC regulatory networks. Loss of integrin activity or FAK also induces cell aggregation, revealing a role in the cell-cell interactions of hESCs. This study provides insight into the integrin signaling cascade activated in hESCs and reveals in FAK a key player in the maintenance of hESC survival and undifferentiated state.

  19. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis; Goldmann, Wolfgang H.

    2009-01-01

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  20. Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration

    International Nuclear Information System (INIS)

    Wu, C.-C.; Su, H.-W.; Lee, C.-C.; Tang, M.-J.; Su, F.-C.

    2005-01-01

    Focal adhesion kinase (FAK) is a critical protein for the regulation of integrin-mediated cellular functions and it can enhance cell motility in Madin-Darby canine kidney (MDCK) cells by hepatocyte growth factor (HGF) induction. We utilized optical trapping and cytodetachment techniques to measure the adhesion force between pico-Newton and nano-Newton (nN) for quantitatively investigating the effects of FAK on adhesion force during initial binding (5 s), beginning of spreading (30 min), spreadout (12 h), and migration (induced by HGF) in MDCK cells with overexpressed FAK (FAK-WT), FAK-related non-kinase (FRNK), as well as normal control cells. Optical tweezers was used to measure the initial binding force between a trapped cell and glass coverslide or between a trapped bead and a seeded cell. In cytodetachment, the commercial atomic force microscope probe with an appropriate spring constant was used as a cyto-detacher to evaluate the change of adhesion force between different FAK expression levels of cells in spreading, spreadout, and migrating status. The results demonstrated that FAK-WT significantly increased the adhesion forces as compared to FRNK cells throughout all the different stages of cell adhesion. For cells in HGF-induced migration, the adhesion force decreased to almost the same level (∼600 nN) regardless of FAK levels indicating that FAK facilitates cells to undergo migration by reducing the adhesion force. Our results suggest FAK plays a role of enhancing cell adhesive ability in the binding and spreading, but an appropriate level of adhesion force is required for HGF-induced cell migration

  1. 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.

  2. Cell adhesion and EGFR activation regulate EphA2 expression in cancer

    DEFF Research Database (Denmark)

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-01-01

    largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src...

  3. Focal adhesion kinase, a downstream mediator of Raf-1 signaling, suppresses cellular adhesion, migration, and neuroendocrine markers in BON carcinoid cells.

    Science.gov (United States)

    Ning, Li; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-05-01

    We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells. (c)2010 AACR.

  4. Chitin and stress induced protein kinase activation

    DEFF Research Database (Denmark)

    Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

    2017-01-01

    The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

  5. Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

    Science.gov (United States)

    Wu, Jui-Chung; Chen, Yu-Chen; Kuo, Chih-Ting; Wenshin Yu, Helen; Chen, Yin-Quan; Chiou, Arthur; Kuo, Jean-Cheng

    2015-01-01

    Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration. PMID:26681405

  6. Cell adhesion and EGFR activation regulate EphA2 expression in cancer

    DEFF Research Database (Denmark)

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-01-01

    largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src...... family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability...

  7. Cell adhesion and EGFR activation regulate EphA2 expression in cancer

    DEFF Research Database (Denmark)

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-01-01

    largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src...... family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability....... These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated...

  8. Focal Adhesion Kinase: Insight into Molecular Roles and Functions in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Nadia Panera

    2017-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of cancer-related death worldwide. Due to the high incidence of post-operative recurrence after current treatments, the identification of new and more effective drugs is required. In previous years, new targetable genes/pathways involved in HCC pathogenesis have been discovered through the help of high-throughput sequencing technologies. Mutations in TP53 and β-catenin genes are the most frequent aberrations in HCC. However, approaches able to reverse the effect of these mutations might be unpredictable. In fact, if the reactivation of proteins, such as p53 in tumours, holds great promise as anticancer therapy, there are studies arguing that chronic activation of these types of molecules may be deleterious. Thus, recently the efforts on potential targets have focused on actionable mutations, such as those occurring in the gene encoding for focal adhesion kinase (FAK. This tyrosine kinase, localized to cellular focal contacts, is over-expressed in a variety of human tumours, including HCC. Moreover, several lines of evidence demonstrated that FAK depletion or inhibition impair in vitro and in vivo HCC growth and metastasis. Here, we provide an overview of FAK expression and activity in the context of tumour biology, discussing the current evidence of its connection with HCC development and progression.

  9. 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...

  10. Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling.

    Science.gov (United States)

    Kim, Yong-Chul; Gonzalez-Nieves, Reyda; Cutler, Mary L

    2015-01-01

    The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α-parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.

  11. Activation of the canonical Wnt/β-catenin pathway enhances monocyte adhesion to endothelial cells

    International Nuclear Information System (INIS)

    Lee, Dong Kun; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-01-01

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/β-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3β or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/β-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/β-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules

  12. Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma.

    Science.gov (United States)

    Kanteti, Rajani; Mirzapoiazova, Tamara; Riehm, Jacob J; Dhanasingh, Immanuel; Mambetsariev, Bolot; Wang, Jiale; Kulkarni, Prakash; Kaushik, Garima; Seshacharyulu, Parthasarathy; Ponnusamy, Moorthy P; Kindler, Hedy L; Nasser, Mohd W; Batra, Surinder K; Salgia, Ravi

    2018-04-03

    The non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

  13. Focal adhesion kinase and paxillin promote migration and adhesion to fibronectin by swine skeletal muscle satellite cells

    Science.gov (United States)

    Wang, Dan; Gao, Chun-qi; Chen, Rong-qiang; Jin, Cheng-long; Li, Hai-chang; Yan, Hui-chao; Wang, Xiu-qi

    2016-01-01

    The focal adhesion kinase (FAK) signaling pathway contributes to the cell migration and adhesion that is critical for wound healing and regeneration of damaged muscle, but its function in skeletal muscle satellite cells (SCs) is less clear. We compared the migration and adhesion of SCs derived from two species of pig (Lantang and Landrace) in vitro, and explored how FAK signaling modulates the two processes. The results showed that Lantang SCs had greater ability to migrate and adhere to fibronection (P < 0.05) than Landrace SCs. Compared to Landrace SCs, Lantang SCs expressed many more focal adhesion (FA) sites, which were indicated by the presence of p-paxillin (Tyr118), and exhibited less F-actin reorganization 24 h after seeding onto fibronectin. Levels of p-FAK (Tyr397) and p-paxillin (Tyr118) were greater (P < 0.05) in Lantang SCs than Landrace SCs after migration for 24 h. Similarly, Lantang SCs showed much higher levels of p-FAK (Tyr397), p-paxillin (Tyr118) and p-Akt (Ser473) than Landrace SCs 2 h after adhesion. Treatment with the FAK inhibitor PF-573228 (5 or 10 μmol/L) inhibited Lantang SC migration and adhesion to fibronectin (P < 0.05), decreased levels of p-paxillin (Tyr118) and p-Akt (Ser473) (P < 0.05), and suppressed the formation of FA sites on migrating SCs. Thus FAK appears to play a key role in the regulation of SC migration and adhesion necessary for muscle regeneration. PMID:27127174

  14. 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.

  15. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Shiro Mizuno

    Full Text Available 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.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.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.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.

  18. Measuring Kinase Activity-A Global Challenge.

    Science.gov (United States)

    Cann, Marissa L; McDonald, Ian M; East, Michael P; Johnson, Gary L; Graves, Lee M

    2017-11-01

    The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. HGF induces FAK activation and integrin-mediated adhesion in MTLn3 breast carcinoma cells.

    Science.gov (United States)

    Beviglia, L; Kramer, R H

    1999-11-26

    Expression of hepatocyte growth factor (HGF) and its tyrosine kinase receptor, c-Met, is positively correlated with breast carcinoma progression. We found that in invasive and metastatic MTLn3 breast carcinoma cells, HGF stimulated both initial adhesion to and motility on the extracellular matrix (ECM) ligands laminin 1, type I collagen, and fibronectin. Next, analysis with function-perturbing antibodies showed that adhesion to the different ECM proteins was mediated through specific beta1 integrins. In MTLn3 cells, HGF induced rapid tyrosine phosphorylation and activation of both c-Met and focal adhesion kinase (FAK). Cell anchorage and adhesion to the ECM substrates was required for HGF-induced FAK activation, since HGF failed to trigger tyrosine phosphorylation of FAK in suspended cells. Our results provide evidence that the 2 signaling pathways, integrin/ECM and c-Met/HGF, cooperate synergistically to induce FAK activation in an adhesion-dependent manner, leading to enhanced cell adhesion and motility. Moreover, we found that a FRNK (the FAK-related non-kinase)-like molecule is expressed in MTLn3 cells. Since FRNK acts as a competitive inhibitor of FAK function, our results suggest that a FRNK-like protein could facilitate disassembly of focal adhesions and likely be responsible for the HGF-induced scattering and motility of MTLn3 cells. Copyright 1999 Wiley-Liss, Inc.

  20. Focal Adhesion Kinase as a Potential Target in AML and MDS.

    Science.gov (United States)

    Carter, Bing Z; Mak, Po Yee; Wang, Xiangmeng; Yang, Hui; Garcia-Manero, Guillermo; Mak, Duncan H; Mu, Hong; Ruvolo, Vivian R; Qiu, Yihua; Coombes, Kevin; Zhang, Nianxiang; Ragon, Brittany; Weaver, David T; Pachter, Jonathan A; Kornblau, Steven; Andreeff, Michael

    2017-06-01

    Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics ( P = 2 × 10 -4 ) and relapse ( P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3 -ITD ( P = 0.0024) or RAS ( P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34 + ( P = 5.42 × 10 -20 ) and CD34 + CD38 - MDS ( P = 7.62 × 10 -9 ) cells compared with normal CD34 + cells. MDS patients with higher FAK in CD34 + cells tended to have better overall survival ( P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR . ©2017 American Association for Cancer Research.

  1. Expression of MLN64 influences cellular matrix adhesion of breast cancer cells, the role for focal adhesion kinase.

    Science.gov (United States)

    Cai, Wei; Ye, Lin; Sun, Jiabang; Mansel, Robert E; Jiang, Wen G

    2010-04-01

    The metastatic lymph node 64 (MLN64) gene was initially identified as highly expressed in the metastatic lymph node from breast cancer. It is localized in q12-q21 of the human chromosome 17 and is often co-amplified with erbB-2. However, the role played by MLN64 in breast cancer remains unclear. In the present study, the expression of MLN64 was examined in a breast cancer cohort using quantitative real-time PCR and immunohistochemical staining. It demonstrated that MLN64 was highly expressed in breast tumours compared to corresponding background tissues at both transcript level and protein level. The elevated level of MLN64 transcripts was correlated with the poor prognosis and overall survival of the patients. A panel of breast cancer cell sublines was subsequently developed by knockdown of MLN64 expression. Loss of MLN64 expression in MCF-7 cells resulted in a significant reduction of cell growth (absorbance for MCF-7DeltaMLN64 being 0.87+/-0.07, Padhesion assay, MDA-MB-231DeltaMLN64 cells showed a significant increase in adhesion (86+/-14), padhesion kinase (FAK) in MDA-MB-231DeltaMLN64 cells using Western blot analysis and immunofluorescent staining of FAK. Moreover, addition of FAK inhibitor to these cells diminished the effect of MLN64 on cell-matrix adhesion, suggesting that FAK contributed to the increased adhesion in MDA-MB-231DeltaMLN64 cells. In conclusion, MLN64 is overexpressed in breast cancer, and its level correlates with poor prognosis and patient survival. MLN64 contributes to the development and progression of breast cancer through the regulation of cell proliferation and adhesive capacity.

  2. The new InsP3Kinase inhibitor BIP-4 is competitive to InsP3 and blocks proliferation and adhesion of lung cancer cells.

    Science.gov (United States)

    Schröder, Dominik; Tödter, Klaus; Gonzalez, Beatriz; Franco-Echevarría, Elsa; Rohaly, Gabor; Blecher, Christine; Lin, Hong-Ying; Mayr, Georg W; Windhorst, Sabine

    2015-07-15

    As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 μM or 2 μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Syndecans: synergistic activators of cell adhesion

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1998-01-01

    Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual...... proteoglycan species in cellular signalling pathways are being determined. This review describes some of the recent advances in our understanding of the major transmembrane group of heparan sulfate proteoglycans, the syndecans, including evidence that they play an important role as accessory signalling...... molecules modulating integrin-based adhesion....

  4. Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Yuenv Wu

    2017-08-01

    Full Text Available The pathogenic role of mesenchymal stromal cells (MSCs in myelodysplastic syndromes (MDS development and progression has been investigated by numerous studies, yet, it remains controversial in some aspects (1, 2. In the present study, we found distinct features of MSCs from low-risk (LR-MDS stromal microenvironment as compared to those from healthy subjects. At the molecular level, focal adhesion kinase, a key tyrosine kinase in control of cell proliferation, survival, and adhesion process, was found profoundly suppressed in expression and activation in LR-MDS MSC. At a functional level, LR-MDS MSCs showed impaired growth and clonogenic capacity, which were independent of cellular senescence and apoptosis. The pro-adipogenic differentiation and attenuated osteogenic capacity along with reduced SDF-1 expression could be involved in creating an unfavorable microenvironment for hematopoiesis. In conclusion, our experiments support the theory that the stromal microenvironment is fundamentally altered in LR-MDS, and these preliminary data offer a new perspective on LR-MDS pathophysiology.

  5. The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Asma Begum

    Full Text Available Cancer stem cells (CSCs play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC. A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM proteins on PDAC CSCs. We quantified the effect of ECM proteins, β1-integrin, and focal adhesion kinase (FAK on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

  6. New Treatment Opportunities in Phosphatase and Tensin Homolog (PTEN-Deficient Tumors: Focus on PTEN/Focal Adhesion Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Roberta Alfieri

    2017-08-01

    Full Text Available Deep genetic studies revealed that phosphatase and tensin homolog (PTEN mutations or loss of expression are not early events in cancer development but characterize tumor progression and invasion. Loss of PTEN function causes a full activation of the prosurvival phosphoinositide 3-kinase (PI3K/AKT/mTOR pathway, but the treatment with specific inhibitors of PI3K/AKT/mTOR did not produce the expected results. One of the alternative targets of PTEN is the focal adhesion kinase (FAK kinase, mainly involved in the control of cancer cell spread. The connection between PTEN and FAK has been demonstrated in different tumor types, with reduced PTEN activity often correlated with increased expression and phosphorylation of FAK. FAK inhibition may thus represent a promising strategy, and some clinical trials are testing FAK inhibitors alone or combined with other agents in a number of solid tumors. However, only few preclinical and clinical data described the effects of the combination of PI3K/AKT/mTOR and FAK inhibitors. Increasing knowledge on the PTEN/FAK connection could confirm PTEN as a good prognostic marker for a combination strategy based on concomitant inhibition of PI3K/AKT and FAK signaling, in advanced metastatic malignancies with altered or reduced PTEN expression.

  7. Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion.

    Science.gov (United States)

    Bharadwaj, Shantaram; Thanawala, Ruchi; Bon, Giulia; Falcioni, Rita; Prasad, G L

    2005-12-15

    Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis. Oncogene (2005) 24, 8291-8303. doi:10.1038/sj.onc.1208993; published online 19 September 2005.

  8. 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.

  9. Kinase activity and specificity assay using synthetic peptides.

    Science.gov (United States)

    Wu, Xu Na; Schulze, Waltraud X

    2015-01-01

    Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.

  10. c-Src, Insulin-Like Growth Factor I Receptor, G-Protein-Coupled Receptor Kinases and Focal Adhesion Kinase are Enriched Into Prostate Cancer Cell Exosomes.

    Science.gov (United States)

    DeRita, Rachel M; Zerlanko, Brad; Singh, Amrita; Lu, Huimin; Iozzo, Renato V; Benovic, Jeffrey L; Languino, Lucia R

    2017-01-01

    It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (Src pY416 ) is co-expressed in Exo with phosphorylated FAK (FAK pY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, Src pY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles.

    Science.gov (United States)

    Xie, Yeming; Han, Koon Hee; Grainger, Nathan; Li, Wen; Corrigan, Robert D; Perrino, Brian A

    2018-03-12

    Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. CPI-17 and MYPT1 are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including FAK, to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of L-NAME and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the EFS-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca 2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. PDBu or calyculin A contracted gastric fundus smooth muscle strips Ca 2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction, and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

    OpenAIRE

    Gardner, A M; Vaillancourt, R R; Lange-Carter, C A; Johnson, G L

    1994-01-01

    MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphory...

  13. Activation and extinction models for platelet adhesion.

    Science.gov (United States)

    David, T; Walker, P G

    2002-01-01

    Adherent platelets are an important part of both thrombus formation and in certain stages of atherogenesis. Platelets can be activated by potent chemicals released from adherent platelets and adhere far more readily than unactivated ones. An analytical and numerical model is presented utilising high Peclet number for the activation and adhesion of platelets in shear flows. The model uses a similarity transformation, which characterises the relationship between convective, diffusive transport and the bulk platelet activating reaction mechanism. A first order surface reaction mechanism is used to model platelet adhesion at the wall (cell) surface. The reduced Damköhler number, M, characterises the importance of the bulk reaction and includes both convective and diffusive terms. For a high rate of blood flow (M-->0) the activation of platelets can effectively be terminated. In contrast, for (M-->infinity) an inner layer of activated platelets exists with an infinitesimally thin reaction sheet separating activated and non-activated platelets. This characterisation by the Damköhler number highlights results found clinically, in that thrombus forms in areas of low shear (high M) and in some cases an increased blood flow (low M) can inhibit the activation of platelets completely. The model shows the critical balance that exists between convection, diffusion and reaction.

  14. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  15. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  16. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

    Science.gov (United States)

    Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

    2016-04-14

    Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

  17. Role of Adhesion Molecules in Eosinophil Activation: A Comparative Study on the Effect of Adhesion Molecules on Eosinophil Survival

    Directory of Open Access Journals (Sweden)

    Kazutoshi Yamaguchi

    2004-01-01

    Conclusions: The regulation of adhesion molecules, by not only preventing eosinophil adhesion but also eosinophil activation, may be a potential target in the treatment of allergic inflammatory disorders.

  18. Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling

    NARCIS (Netherlands)

    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; Sears, Rosalie C.; Clarke, Alan R.; Frame, Margaret C.; Sansom, Owen J.

    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

  19. 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

  20. The role of focal adhesion kinase in the regulation of cellular mechanical properties

    International Nuclear Information System (INIS)

    Mierke, Claudia Tanja

    2013-01-01

    The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation. (paper)

  1. p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

    DEFF Research Database (Denmark)

    Wang, Zhipeng; Fu, Meng; Wang, Lifeng

    2013-01-01

    204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...

  2. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

    Brewster, Lizzy M.; Mairuhu, Gideon; Bindraban, Navin R.; Koopmans, Richard P.; Clark, Joseph F.; van Montfrans, Gert A.

    2006-01-01

    BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular

  3. 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

    beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...... 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...... 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 fak...

  4. The PI3-kinase delta inhibitor idelalisib (GS-1101 targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL cell to endothelial and marrow stromal cells.

    Directory of Open Access Journals (Sweden)

    Stefania Fiorcari

    Full Text Available CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC. We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106 expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

  5. 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.

  6. Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

    Energy Technology Data Exchange (ETDEWEB)

    Schneck, Jessica L.; Briand, Jacques; Chen, Stephanie; Lehr, Ruth; McDevitt, Patrick; Zhao, Baoguang; Smallwood, Angela; Concha, Nestor; Oza, Khyati; Kirkpatrick, Robert; Yan, Kang; Villa, James P.; Meek, Thomas D.; Thrall, Sara H. (Chemizon); (GSKPA)

    2010-12-07

    Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH{sub 2}, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k{sub cat} (0.052 {+-} 0.001 s{sup -1}), K{sub MgATP} (1.2 {+-} 0.1 {micro}M), K{sub iMgATP} (1.3 {+-} 0.2 {micro}M), K{sub FAK-tide} (5.6 {+-} 0.4 {micro}M), and K{sub iFAK-tide} (6.1 {+-} 1.1 {micro}M) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the {beta}{gamma}-bridge:{beta}-nonbridge positional oxygen exchange of [{gamma}-{sup 18}O{sub 4}]ATP in the presence of 1 mM [{gamma}-{sup 18}O{sub 4}]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k{sub x}/k{sub cat} = 0.14 {+-} 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k{sub cat}/K{sub FAK-tide}, while k{sub cat} and k{sub cat}/K{sub MgATP} were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k{sub cat}/K{sub m} for MgATP but not on k{sub cat}/K{sub m} for FAK-tide. From the positional isotope exchange, viscosity, and

  7. Nitric oxide-induced activation of the AMP-activated protein kinase α2 subunit attenuates IκB kinase activity and inflammatory responses in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Elke Bess

    Full Text Available BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO. METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2(-/- mice the interleukin (IL-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2(-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK. Indeed, IKK (more specifically residues Ser177 and Ser181 was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2(+/+ versus AMPKα2(-/- mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.

  8. Disruption of laminin-integrin-CD151-focal adhesion kinase axis sensitizes breast cancer cells to ErbB2 antagonists.

    Science.gov (United States)

    Yang, Xiuwei H; Flores, Ludmila M; Li, Qinglin; Zhou, Pengcheng; Xu, Fenghui; Krop, Ian E; Hemler, Martin E

    2010-03-15

    Resistance to anti-ErbB2 agents is a significant problem in the treatment of human ErbB2+ breast cancers. We show here that adhesion of human ErbB2+ breast cancer cells to basement membrane laminin-5 provides substantial resistance to trastuzumab and lapatinib, agents that respectively target the extracellular and kinase domains of ErbB2. Knockdown of laminin-binding integrins (alpha6beta4, alpha3beta1) or associated tetraspanin protein CD151 reversed laminin-5 resistance and sensitized ErbB2+ cells to trastuzumab and lapatinib. CD151 knockdown, together with trastuzumab treatment, inhibited ErbB2 activation and downstream signaling through Akt, Erk1/2, and focal adhesion kinase (FAK). Hence, ErbB2 function in mammary tumor cells is promoted by integrin-mediated adhesion to laminin-5, with strong support by CD151, leading to signaling through FAK. Consequently, removal or inhibition of any of these components (laminin-5, integrin, CD151, FAK) markedly sensitizes cells to anti-ErbB2 agents. These new insights should be useful when devising strategies for overcoming drug resistance in ErbB2+ cancers.

  9. Multi-targeted molecular therapeutic approach in aggressive neuroblastoma: the effect of Focal Adhesion Kinase-Src-Paxillin system.

    Science.gov (United States)

    Kratimenos, Panagiotis; Koutroulis, Ioannis; Marconi, Dante; Syriopoulou, Vasiliki; Delivoria-Papadopoulos, Maria; Chrousos, George P; Theocharis, Stamatios

    2014-12-01

    Nonreceptor tyrosine kinases play key roles in the integrin system. Located at the focal adhesions, they consist of large protein complexes through which the cytoskeleton connects to the extracellular matrix. The focal adhesion kinase (FAK)-Src-paxillin complex, a major mediator of the integrin pathway, contributes to cell migration and motility. Its overexpression is increased in children with advanced neuroblastoma (NB), one of the most common malignancies of childhood, with poor survival. We review the most recent data on FAK-Src-paxillin and their implications in NB, the molecular structure and the regulatory mechanisms of each molecule and their interactions and up-to-date information on their use as the newest biomarkers and their potential use as therapeutic targets in NB. Based on the current literature, we hypothesize that combined and concurrent inhibition of the FAK-Src-Paxillin system may result in significant tumor suppression and prevention or delay of metastasis.

  10. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  11. Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

    Science.gov (United States)

    Wong, Victor W; Garg, Ravi K; Sorkin, Michael; Rustad, Kristine C; Akaishi, Satoshi; Levi, Kemal; Nelson, Emily R; Tran, Misha; Rennert, Robert; Liu, Wei; Longaker, Michael T; Dauskardt, Reinhold H; Gurtner, Geoffrey C

    2014-12-01

    To investigate how epithelial mechanotransduction pathways impact wound repair. Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  12. Activation of oocyte phosphatidylinositol kinase by polyamines

    International Nuclear Information System (INIS)

    Allende, J.E.; Carrasco, D.; Allende, C.C.

    1987-01-01

    Membrane bound phosphatidylinositol is phosphorylated by a specific membrane enzyme to form phosphatidylinositol 4 phosphate (PIP) which in turn is again phosphorylated to generate phosphatidylinositol 4,5 biphosphate (PIPP). The regulation of phosphatidylinositol phosphorylation and hydrolysis is relevant to the possible role of inositol phosphates as second messengers of hormone action. The membranes of Xenopus laevis oocytes contain a phosphatidylinositol kinase that can generate radioactive PIP after incubation with [ 32 ATP]. The radioactive product is extracted with methanol-chloroform and isolated by thin layer chromatography. The oocyte enzyme has an app Km for ATP of 80 μM and cannot use GTP as a phosphate donor. The formation of PIP is greatly stimulated by the addition of synthetic peptides containing clusters of polylysine at concentrations 0.5 mM. A similar effect is observed with a lysine rich peptide that corresponds to the 14 amino acids of the carboxyl terminus of the Kirstein ras 2 protein and also by polyornithine. Polyarginine and histone H 1 have much lower effects. Peptides containing polylysine clusters have also been found to affect the activity of other key membrane enzymes such as protein kinases and adenylate cyclase

  13. Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Shimotohno, A.; Ohno, R.; Bišová, Kateřina; Sakaguchi, N.; Huang, J.; Koncz, C.; Uchimiya, H.; Umeda, M.

    2006-01-01

    Roč. 47, - (2006), s. 701-710 ISSN 0960-7412 Institutional research plan: CEZ:AV0Z50200510 Keywords : cyclin -dependent kinase * cdk-activating kinase * cyclin Subject RIV: EE - Microbiology, Virology Impact factor: 6.565, year: 2006

  14. 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.

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

    International Nuclear Information System (INIS)

    Xiong, Xiangyang; Wang, Yao; Liu, Chengmei; Lu, Quqin; Liu, Tao; Chen, Guoan; Rao, Hai; Luo, Shiwen

    2014-01-01

    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

  16. Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain.

    Science.gov (United States)

    Siljander, P; Farndale, R W; Feijge, M A; Comfurius, P; Kos, S; Bevers, E M; Heemskerk, J W

    2001-04-01

    In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

  17. 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

  18. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  19. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    Directory of Open Access Journals (Sweden)

    Wymke Ockenga

    2014-11-01

    Full Text Available Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR, which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.

  20. Quantal concept of T-cell activation: adhesion domains as immunological synapses

    International Nuclear Information System (INIS)

    Sackmann, Erich

    2011-01-01

    Adhesion micro-domains (ADs) formed during encounters of lymphocytes with antigen-presenting cells (APC) mediate the genetic expression of quanta of cytokines interleukin-2 (IL-2). The IL-2-induced activation of IL-2 receptors promotes the stepwise progression of the T-cells through the cell cycle, hence their name, immunological synapses. The ADs form short-lived reaction centres controlling the recruitment of activators of the biochemical pathway (the kinases Lck and ZAP) while preventing the access of inhibitors (phosphatase CD45) through steric repulsion forces. CD45 acts as the generator of adhesion domains and, through its role as a spacer protein, also as the promoter of the reaction. In a second phase of T-cell-APC encounters, long-lived global reaction spaces (called supramolecular activation complexes (SMAC)) form by talin-mediated binding of the T-cell integrin (LFA-1) to the counter-receptor ICAM-1, resulting in the formation of ring-like tight adhesion zones (peripheral SMAC). The ADs move to the centre of the intercellular adhesion zone forming the central SMAC, which serve in the recycling of the AD. We propose that cell stimulation is triggered by integrating the effect evoked by the short-lived adhesion domains. Similar global reaction platforms are formed by killer cells to destruct APC. We present a testable mechanical model showing that global reaction spaces (SMAC or dome-like contacts between cytotoxic cells and APC) form by self-organization through delayed activation of the integrin-binding affinity and stabilization of the adhesion zones by F-actin recruitment. The mechanical stability and the polarization of the adhering T-cells are mediated by microtubule-actin cross-talk.

  1. Quantal concept of T-cell activation: adhesion domains as immunological synapses

    Energy Technology Data Exchange (ETDEWEB)

    Sackmann, Erich, E-mail: sackmann@ph.tum.de [Physics Department E22, Technical University Munich, D-85748 Garching (Germany)

    2011-06-15

    Adhesion micro-domains (ADs) formed during encounters of lymphocytes with antigen-presenting cells (APC) mediate the genetic expression of quanta of cytokines interleukin-2 (IL-2). The IL-2-induced activation of IL-2 receptors promotes the stepwise progression of the T-cells through the cell cycle, hence their name, immunological synapses. The ADs form short-lived reaction centres controlling the recruitment of activators of the biochemical pathway (the kinases Lck and ZAP) while preventing the access of inhibitors (phosphatase CD45) through steric repulsion forces. CD45 acts as the generator of adhesion domains and, through its role as a spacer protein, also as the promoter of the reaction. In a second phase of T-cell-APC encounters, long-lived global reaction spaces (called supramolecular activation complexes (SMAC)) form by talin-mediated binding of the T-cell integrin (LFA-1) to the counter-receptor ICAM-1, resulting in the formation of ring-like tight adhesion zones (peripheral SMAC). The ADs move to the centre of the intercellular adhesion zone forming the central SMAC, which serve in the recycling of the AD. We propose that cell stimulation is triggered by integrating the effect evoked by the short-lived adhesion domains. Similar global reaction platforms are formed by killer cells to destruct APC. We present a testable mechanical model showing that global reaction spaces (SMAC or dome-like contacts between cytotoxic cells and APC) form by self-organization through delayed activation of the integrin-binding affinity and stabilization of the adhesion zones by F-actin recruitment. The mechanical stability and the polarization of the adhering T-cells are mediated by microtubule-actin cross-talk.

  2. Quantal concept of T-cell activation: adhesion domains as immunological synapses

    Science.gov (United States)

    Sackmann, Erich

    2011-06-01

    Adhesion micro-domains (ADs) formed during encounters of lymphocytes with antigen-presenting cells (APC) mediate the genetic expression of quanta of cytokines interleukin-2 (IL-2). The IL-2-induced activation of IL-2 receptors promotes the stepwise progression of the T-cells through the cell cycle, hence their name, immunological synapses. The ADs form short-lived reaction centres controlling the recruitment of activators of the biochemical pathway (the kinases Lck and ZAP) while preventing the access of inhibitors (phosphatase CD45) through steric repulsion forces. CD45 acts as the generator of adhesion domains and, through its role as a spacer protein, also as the promoter of the reaction. In a second phase of T-cell-APC encounters, long-lived global reaction spaces (called supramolecular activation complexes (SMAC)) form by talin-mediated binding of the T-cell integrin (LFA-1) to the counter-receptor ICAM-1, resulting in the formation of ring-like tight adhesion zones (peripheral SMAC). The ADs move to the centre of the intercellular adhesion zone forming the central SMAC, which serve in the recycling of the AD. We propose that cell stimulation is triggered by integrating the effect evoked by the short-lived adhesion domains. Similar global reaction platforms are formed by killer cells to destruct APC. We present a testable mechanical model showing that global reaction spaces (SMAC or dome-like contacts between cytotoxic cells and APC) form by self-organization through delayed activation of the integrin-binding affinity and stabilization of the adhesion zones by F-actin recruitment. The mechanical stability and the polarization of the adhering T-cells are mediated by microtubule-actin cross-talk.

  3. Mitogen-activated protein kinase signaling in plants

    DEFF Research Database (Denmark)

    Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

    2010-01-01

    Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation of subs...... the Arabidopsis thaliana MAPKs MPK3, 4, and 6 and MAP2Ks MKK1, 2, 4, and 5. Future work needs to focus on identifying substrates of MAPKs, and on understanding how specificity is achieved among MAPK signaling pathways.......Eukaryotic mitogen-activated protein kinase (MAPK) cascades have evolved to transduce environmental and developmental signals into adaptive and programmed responses. MAPK cascades relay and amplify signals via three types of reversibly phosphorylated kinases leading to the phosphorylation...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

  4. A Molecular Mechanism for the Requirement of PAT-4 (Integrin-linked Kinase (ILK)) for the Localization of UNC-112 (Kindlin) to Integrin Adhesion Sites*

    Science.gov (United States)

    Qadota, Hiroshi; Moerman, Donald G.; Benian, Guy M.

    2012-01-01

    Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (β-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3. PMID:22761445

  5. A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (Kindlin) to integrin adhesion sites.

    Science.gov (United States)

    Qadota, Hiroshi; Moerman, Donald G; Benian, Guy M

    2012-08-17

    Caenorhabditis elegans muscle cells attach to basement membrane through adhesion plaques. PAT-3 (β-integrin), UNC-112 (kindlin), and PAT-4 (integrin-linked kinase) are associated with these structures. Genetic analysis indicated that PAT-4 is required for UNC-112 to be properly localized. We investigated the molecular basis of this requirement. We show that the cytoplasmic tail of PAT-3 binds to full-length UNC-112 and that the N- and C-terminal halves of UNC-112 bind to each other. We demonstrate competition between the UNC-112 C-terminal half and PAT-4 for binding to the UNC-112 N-terminal half. The D382V mutation results in lack of binding to PAT-4 and lack of localization to adhesion structures. T346A or E349K mutations, which abolish interaction of the N- and C-terminal halves, permit D382V UNC-112 to localize to adhesion structures. The following model is proposed. UNC-112 exists in closed inactive and open active conformations, and upon binding of PAT-4 to the UNC-112 N-terminal half, UNC-112 is converted into the open state, able to bind to PAT-3.

  6. 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.

  7. Shoulder activity level in patients with idiopathic adhesive capsulitis.

    Science.gov (United States)

    Lamplot, Joseph D; Lillegraven, Olivia; Brophy, Robert H

    2017-09-01

    Idiopathic adhesive capsulitis is a common condition resulting in painful multidirectional restriction of motion. Adhesive capsulitis may inhibit shoulder activity level, but this relationship has not been previously studied. This study tested the hypothesis that patients with idiopathic adhesive capsulitis have lower shoulder activity than sex- and age-matched controls. Seventy-two eligible patients (37 men and 35 women) with idiopathic adhesive capsulitis completed a validated shoulder activity scale that was compared with sex- and age-matched norms from a healthy population with no history of shoulder disorders. The association of shoulder activity level with patient age, sex, and American Shoulder and Elbow Surgeons and Simple Shoulder Test (SST) scores was evaluated. Overall, 58% of patients actually had higher shoulder activity scores than sex- and age-matched controls. Among patients aged 51 to 70 years, 68% of patients (73% of men and 63% of women) demonstrated higher Shoulder Activity Scale scores compared with controls. The activity level was higher among all patients aged 51 to 70 years compared with controls (10.3 ± 1.48 vs. 8 ± 0.52, P = .0067). The difference was significant for men in this age group (12.2 ± 1.7 vs. 9 ± 0.75, P = .0042). There was a statistically significant positive correlation of the Shoulder Activity Scale score with the SST score (r = 0.31, P = .009). Patients with idiopathic adhesive capsulitis do not have a lower shoulder activity level than sex- and age-matched controls, and older men may actually have a higher level of shoulder activity than controls. Shoulder activity level is correlated with the SST score in patients with idiopathic adhesive capsulitis. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  8. 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.

  9. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are ...

  10. Mitogen-activated protein kinases mediate Mycobacterium

    Indian Academy of Sciences (India)

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are ...

  11. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Science.gov (United States)

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  12. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Directory of Open Access Journals (Sweden)

    Westa Domanova

    Full Text Available In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds, making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same

  13. Dentine bond strength and antimicrobial activity evaluation of adhesive systems.

    Science.gov (United States)

    André, Carolina Bosso; Gomes, Brenda Paula Figueiredo Almeida; Duque, Thais Mageste; Stipp, Rafael Nobrega; Chan, Daniel Chi Ngai; Ambrosano, Glaucia Maria Bovi; Giannini, Marcelo

    2015-04-01

    This study evaluated the dentine bond strength (BS) and the antibacterial activity (AA) of six adhesives against strict anaerobic and facultative bacteria. Three adhesives containing antibacterial components (Gluma 2Bond (glutaraldehyde)/G2B, Clearfil SE Protect (MDPB)/CSP and Peak Universal Bond (PUB)/chlorhexidine) and the same adhesive versions without antibacterial agents (Gluma Comfort Bond/GCB, Clearfil SE Bond/CSB and Peak LC Bond/PLB) were tested. The AA of adhesives and control groups was evaluated by direct contact method against four strict anaerobic and four facultative bacteria. After incubation, according to the appropriate periods of time for each microorganism, the time to kill microorganisms was measured. For BS, the adhesives were applied according to manufacturers' recommendations and teeth restored with composite. Teeth (n=10) were sectioned to obtain bonded beams specimens, which were tested after artificial saliva storage for one week and one year. BS data were analyzed using two-way ANOVA and Tukey test. Saliva storage for one year reduces the BS only for GCB. In general G2B and GCB required at least 24h for killing microorganisms. PUB and PLB killed only strict anaerobic microorganisms after 24h. For CSP the average time to eliminate the Streptococcus mutans and strict anaerobic oral pathogens was 30 min. CSB showed no AA against facultative bacteria, but had AA against some strict anaerobic microorganisms. Storage time had no effect on the BS for most of the adhesives. The time required to kill bacteria depended on the type of adhesive and never was less than 10 min. Most of the adhesives showed stable bond strength after one year and the Clearfil SE Protect may be a good alternative in restorative procedures performed on dentine, considering its adequate bond strength and better antibacterial activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases.

    Science.gov (United States)

    Marcotte, Douglas J; Liu, Yu-Ting; Arduini, Robert M; Hession, Catherine A; Miatkowski, Konrad; Wildes, Craig P; Cullen, Patrick F; Hong, Victor; Hopkins, Brian T; Mertsching, Elisabeth; Jenkins, Tracy J; Romanowski, Michael J; Baker, Darren P; Silvian, Laura F

    2010-03-01

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 A resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 A resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  15. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    Science.gov (United States)

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Focal Adhesion Kinase (FAK) Binds RET Kinase via Its FERM Domain, Priming a Direct and Reciprocal RET-FAK Transactivation Mechanism

    NARCIS (Netherlands)

    Plaza-Menacho, Ivan; Morandi, Andrea; Mologni, Luca; Boender, Piet; Gambacorti-Passerini, Carlo; Magee, Anthony I.; Hofstra, Robert M. W.; Knowles, Phillip; McDonald, Neil Q.; Isacke, Clare M.

    2011-01-01

    Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely

  17. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Mikkelsen, N.E.; Clausen, A.R.

    2009-01-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 angstrom resolution...

  18. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Hochachka and Somero 2002). Therefore, some animals have to initiate anaerobic metabolism to meet part of energy needs (Costanzo et al. 2004; Colson-Proch et al. 2009). Adenosine monophosphate-activated protein kinase.

  19. Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

    DEFF Research Database (Denmark)

    Peraldi, P; Frödin, M; Barnier, J V

    1995-01-01

    In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the r...

  20. Auto-thiophosphorylation activity of Src tyrosine kinase.

    Science.gov (United States)

    Cabail, M Zulema; Chen, Emily I; Koller, Antonius; Miller, W Todd

    2016-07-07

    Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low. Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni(2+), resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni(2+). Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase. Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni(2+). Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.

  1. Rapamycin Induces Mitogen-activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Expression through Activation of Protein Kinase B and Mitogen-activated Protein Kinase Kinase Pathways*

    Science.gov (United States)

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J.; Samavati, Lobelia

    2013-01-01

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition. PMID:24126911

  2. Profiling bacterial kinase activity using a genetic circuit

    DEFF Research Database (Denmark)

    van der Helm, Eric; Bech, Rasmus; Lehning, Christina Eva

    subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously...... reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool....

  3. Expression, purification and kinase activity analysis of maize ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... Kinase activity is essential for a protein kinase to perform its biological function. In previous study we have cloned a novel plant SnRK2 subfamily gene from maize and named it as ZmSPK1. In this study the. cDNA of ZmSPK1 with dHA-His6 tag was amplified by PCR and was subcloned into the yeast.

  4. Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

    DEFF Research Database (Denmark)

    Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

    2012-01-01

    , but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα...... DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization....

  5. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  6. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    Energy Technology Data Exchange (ETDEWEB)

    Knecht, Wolfgang [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Mikkelsen, Nils Egil [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Clausen, Anders Ranegaard [Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden); Willer, Mette [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Centre, SE-751 24 Uppsala (Sweden); Gojkovic, Zoran [ZGene A/S, Agern Alle 7, DK-2970 Horsholm (Denmark); Piskur, Jure, E-mail: Jure.Piskur@cob.lu.se [BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby (Denmark); Cell and Organism Biology, Lund University, Soelvegatan 35, SE-22362 Lund (Sweden)

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  7. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... upstream activator of ERK1 in the MAP kinase cascade. Supporting this view, forskolin and a cAMP analogue were found to increase the activity of MAP kinase kinase in PC12 cells, alone as well as in combination with phorbol ester. PACAP38 also stimulated in vivo 32P-labeling of ERK1 and MAP kinase kinase...... activity. Finally, cAMP or PACAP38 increased by 3-fold nerve growth factor-stimulated neurite formation in PC12 cells, which may be correlated with the potentiating effect of these agents on nerve growth factor-stimulated ERK1 activity....

  8. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    International Nuclear Information System (INIS)

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-01-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification

  9. 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.

  10. Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

    KAUST Repository

    Kwezi, Lusisizwe

    2018-01-22

    Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

  11. The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

    Science.gov (United States)

    Liu, Chanjuan; Li, Yinan; Xing, Yang; Cao, Benjin; Yang, Fan; Yang, Tianxiao; Ai, Zhilong; Wei, Yuanyan; Jiang, Jianhai

    2016-07-22

    CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. An active form of calcium and calmodulin dependant protein kinase ...

    African Journals Online (AJOL)

    The removal of the auto-inhibitory domain that negatively regulates the kinase activity in M. truncatula results in a constitutively-active form, inducing symbiotic responses in the absence of bacterial signals. In this study, we verified the functionality of a DMI3 variant and its ability to induce spontaneous nodules in M.

  13. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  14. Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Sayed, M; Kim, S O; Salh, B S

    2000-01-01

    Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities....

  15. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3......Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray......, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth...

  16. Phosphatidylinositol 4-phosphate 5-kinases in the regulation of T cell activation

    Directory of Open Access Journals (Sweden)

    Loretta eTuosto

    2016-05-01

    Full Text Available Phosphatidylinositol 4,5-biphosphate kinases (PIP5K are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2. PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen presenting cells (APC, spatial organization of the immunological synapse (IS, and costimulation. Moreover, PIP2 serves also as a precursor for the second messengers inositol triphosphate (IP3, diacylglycerol (DAG and phosphatidylinositol 3,4,5-triphosphate (PIP3, which are essential for the activation of signalling pathways regulating cytokine production, cell cycle progression, survival, metabolism and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation.

  17. p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I.

    Science.gov (United States)

    Brzeska, H; Knaus, U G; Wang, Z Y; Bokoch, G M; Korn, E D

    1997-02-18

    Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

  18. Evaluation of Creatine Kinase Activity and Inorganic Phosphate ...

    African Journals Online (AJOL)

    subjects presenting with major VOC. Keywords: Serum creatine kinase activity, Serum inorganic phosphate concentration, Sickle cell disease,. Steady state, Vaso‑occlusive crisis. Original Article. Address for correspondence: Dr. John C Aneke,. Department of Hematology,. Nnamdi Azikiwe University Teaching. Hospital ...

  19. Differences in muscle pain and plasma creatine kinase activity after ...

    African Journals Online (AJOL)

    Objective. The aim of this study was to compare the acute changes in muscle pain and plasma creatine kinase (CK) activity following the 'up' and 'down' Comrades marathon. Design. This was a quasi-experimental design. Eleven male runners (39.7±9.3 years) completed the 'up' Comrades marathon, and 11 male runners ...

  20. VHH Activators and Inhibitors for Protein Kinase C Epsilon

    NARCIS (Netherlands)

    Summanen, M.M.I.

    2012-01-01

    Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are

  1. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... ... Journal of Genetics; Volume 95; Issue 4. Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG KUN YU HUIYANG HUANG HAIHUI YE. RESEARCH ARTICLE Volume 95 Issue 4 December 2016 pp ...

  2. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    Background and Objectives: There is the recognition of a pattern of elevations of serum enzymes in hyperthyroid and hypothyroid patients. The aims of this study were to determine the activities of serum creatine kinase (CK) and lactate deydrogenase (LDH) in thyroid disorders, and to evaluate the relationship between CK, ...

  3. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    P = 0.002). Conclusion: The significant elevation in serum CK and LDH activities indicates that these can be used as parameters for screening hypothyroid patients but not hyperthyroid patients. Key words: Hyperthyroidism, hypothyroidism, lactate dehydrogenase, serum creatine kinase. Date of Acceptance: 28-Aug-2011.

  4. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    CHENCUI HUANG

    Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. CHENCUI HUANG1, KUN YU1, HUIYANG HUANG1,2 and HAIHUI YE1,2∗. 1College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, People's Republic of China.

  5. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain. [Huang C., Yu K., Huang H. and Ye H. 2016 Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress. J. Genet.

  6. Evaluation of Creatine Kinase Activity and Inorganic Phosphate ...

    African Journals Online (AJOL)

    Background: Biochemical parameters vary in subjects with different hemoglobin phenotypes, compared with normal controls. Aim: The aim was to evaluate serum creatine kinase (CK) activity and inorganic phosphate concentrations in Nigerian adults with homozygous and heterozygous hemoglobin phenotypes. Subjects ...

  7. Mitogen-activated protein kinases mediate Mycobacterium ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... heat shock, UV irradiation and also to inflammatory cytokines. ERK is mainly activated by growth factors and phorbol esters. (Lewis et al. 1998; Cowan and Storey 2003). The activation of some MAPK family members by. M. tuberculosis H37Rv in human monocytes has already been reported. Song et al.

  8. MAP kinase activity increases during mitosis in early sea urchin embryos.

    Science.gov (United States)

    Philipova, R; Whitaker, M

    1998-09-01

    A MBP kinase activity increases at mitosis during the first two embryonic cell cycles of the sea urchin embryo. The activity profile of the MBP kinase is the same both in whole cell extracts and after immunoprecipitation with an anti-MAP kinase antibody (2199). An in-gel assay of MBP activity also shows the same activity profile. The activity is associated with the 44 kDa protein that cross-reacts with anti-MAP kinase antibodies. The 44 kDa protein shows cross-reactivity to anti-phosphotyrosine and MAP kinase-directed anti-phosphotyrosine/phosphothreonine antibodies at the times that MBP kinase activity is high. The 2199 antibody co-precipitates some histone H1 kinase activity, but the MBP kinase activity cannot be accounted for by histone H1 kinase-dependent phosphorylation of MBP. The MAP kinase 2199 antibody was used to purify the MBP kinase activity. Peptide sequencing after partial digestion shows the protein to be homologous to MAP kinases from other species. These data demonstrate that MAP kinase activation during nuclear division is not confined to meiosis, but also occurs during mitotic cell cycles. MAP kinase activity in immunoprecipitates also increases immediately after fertilization, which in the sea urchin egg occurs at interphase of the cell cycle. Treating unfertilized eggs with the calcium ionophore A23187 stimulates the increase in MAP kinase activity, demonstrating that a calcium signal can activate MAP kinase and suggesting that the activation of MAP kinase at fertilization is due to the fertilization-induced increase in cytoplasmic free calcium concentration. This signalling pathway must differ from the pathway responsible for calcium-induced inactivation of MAP kinase activity that is found in eggs that are fertilized in meiotic metaphase.

  9. Inhibiting MAP kinase activity prevents calcium transients and mitosis entry in early sea urchin embryos.

    Science.gov (United States)

    Philipova, Rada; Larman, Mark G; Leckie, Calum P; Harrison, Patrick K; Groigno, Laurence; Whitaker, Michael

    2005-07-01

    A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently, MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos, both kinases show a similar activation profile, peaking at the time of mitosis entry. We tested whether the activity of both kinases is required for mitosis entry and whether either kinase controls mitotic calcium signals. We found that reducing the activity of either mitotic kinase prevents nuclear envelope breakdown, despite the presence of a calcium transient, when cdk1/cyclin B kinase activity is alone inhibited. When MAP kinase activity alone was inhibited, the calcium signal was absent, suggesting that MAP kinase activity is required to generate the calcium transient that triggers nuclear envelope breakdown. However, increasing intracellular free calcium by microinjection of calcium buffers or InsP(3) while MAP kinase was inhibited did not itself induce nuclear envelope breakdown, indicating that additional MAP kinase-regulated events are necessary. After MAP kinase inhibition early in the cell cycle, the early events of the cell cycle (pronuclear migration/fusion and DNA synthesis) were unaffected, but chromosome condensation and spindle assembly are prevented. These data indicate that in sea urchin embryos, MAP kinase activity is part of a signaling complex alongside two components previously shown to be essential for entry into mitosis: the calcium transient and the increase in cdk1/cyclinB kinase activity.

  10. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, Cicerone; te Riet, J.; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen Wenger, J.; Ameloot, M.; Holt, M.; Kanger, Johannes S.; Figdor, Carl; Cambi, A.; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  11. [Fibrinolytic activity and adhesive syndromes of the lumbar vertebral canal].

    Science.gov (United States)

    Greco, F; De Palma, L; Santucci, A; Marra, R; Pagano, L; Storti, S

    1989-01-01

    The recent studies, related in medical literature, indicate a reduced activity of fibrinolysis in patients with low back-pain caused by multiple factors (lumbar disc disease, post-laminectomy, post myelography) and suggest a new pathogenetic hypothesis of this disease and new therapeutic implications. The authors conducted a research based on the study of fibrinolysis in patients affected by post-surgical lumbar adhesive syndrome, with the aim of finding, eventually, a relationship between reduced fibrinolytic activity and clinical symptoms. The results are referred and discussed.

  12. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu

    2011-05-01

    Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  13. Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

    Science.gov (United States)

    Zheng, Xu; Bao, Wenlei; Yang, Jiaofu; Zhang, Tao; Sun, Dongsheng; Liang, Yan; Li, Shuyu; Wang, Yanfeng; Feng, Xue; Hao, Huifang; Wang, Zhigang

    2016-09-01

    Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs.

  14. Activation of ZmMKK10, a maize mitogen-activated protein kinase kinase, induces ethylene-dependent cell death.

    Science.gov (United States)

    Chang, Ying; Yang, Hailian; Ren, Dongtao; Li, Yuan

    2017-11-01

    Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant growth, development and stress responses. Here, we report that ZmMKK10, a maize MAP kinase kinase, positively regulates cell death. Sequence comparison to Arabidopsis MKKs has led to ZmMKK10 being classified as a group D MKK. Kinase activity analysis of recombinant ZmMKK10 showed that the Mg 2+ ion was required for its kinase activity. Transient expression of ZmMKK10 WT or ZmMKK10 DD (the active form of ZmMKK10) in maize mesophyll protoplast significantly increased the cell death rate. Inducible expression of ZmMKK10 WT or ZmMKK10 DD in Arabidopsis transgenic plants caused rapid HR-like cell death, whereas induction of ZmMKK10 KR (the inactive form of ZmMKK10) expression in transgenic plants did not yield the same phenotype. Genetic and pharmacological analysis revealed that ZmMKK10-induced cell death in transgenic plants requires the activation of Arabidopsis MPK3 and MPK6 and that it partially depended on ethylene biosynthesis. ZmMPK3 and ZmMPK7, the orthologues of Arabidopsis MPK3 and MPK6, interacted with ZmMKK10 in yeast and ZmMKK10 phosphorylated them both in vitro. Our results demonstrate that ZmMKK10 induces cell death in an ethylene-dependent manner. Furthermore, ZmMPK3 and ZmMPK7 may be the downstream MAPKs in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. 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.

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

    International Nuclear Information System (INIS)

    Leich, E; Weißbach, S; Klein, H-U; Grieb, T; Pischimarov, J; Stühmer, T; Chatterjee, M; Steinbrunn, T; Langer, C; Eilers, M; Knop, S; Einsele, H; Bargou, R; Rosenwald, A

    2013-01-01

    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

  17. Adhesive disbond detection using piezoelectric wafer active sensors

    Science.gov (United States)

    Roth, William; Giurgiutiu, Victor

    2015-04-01

    The aerospace industry continues to increase the use of adhesives for structural bonding due to the increased joint efficiency (reduced weight), even distribution of the load path and decreases in stress concentrations. However, the limited techniques for verifying the strength of adhesive bonds has reduced its use on primary structures and requires an intensive inspection schedule. This paper discusses a potential structural health monitoring (SHM) technique for the detection of disbonds through the in situ inspection of adhesive joints. This is achieved through the use of piezoelectric wafer active sensors (PWAS), thin unobtrusive sensors which are permanently bonded to the aircraft structure. The detection method discussed in this study is electromechanical impedance spectroscopy (EMIS), a local vibration method. This method detects disbonds from the change in the mechanical impedance of the structure surrounding the disbond. This paper will discuss how predictive modeling can provide valuable insight into the inspection method, and provide better results than empirical methods alone. The inspection scheme was evaluated using the finite element method, and the results were verified experimentally using a large aluminum test article, and included both pristine and disbond coupons.

  18. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    Directory of Open Access Journals (Sweden)

    Koul Sweaty

    2004-06-01

    Full Text Available Abstract The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.

  19. Emerging Roles of AMP-Activated Protein Kinase

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel

    The cellular energy sensor AMP-activated protein kinase (AMPK) is activated, when the energy balance of the cell decreases. AMPK has been proposed to regulate multiple metabolic processes. However, much of the evidence for these general effects of AMPK relies on investigations in cell systems...... exercise appears to inhibit pyruvate dehydrogenase (PDH) activity by an immediate up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) protein content. Consequently, this may inhibit glucose oxidation and thereby generate conditions for increased FA oxidation and glycogen resynthesis in skeletal muscle...... be of importance for prioritising energy dissipation, inhibition of lipid storage pathways and regulation of mitochondrial and metabolic proteins, but this needs further investigations. In addition, we provide evidence that AMPK is regulating autophagic signalling in skeletal muscle. Thus, in skeletal muscle AMPK...

  20. ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead.

    Science.gov (United States)

    Huang, Tsai-Lien; Huang, Hao-Jen

    2008-04-01

    Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

  1. The catalytic domain of acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK).

    Science.gov (United States)

    Brzeska, H; Szczepanowska, J; Hoey, J; Korn, E D

    1996-10-25

    Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca2+-calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C- and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P+1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.

  2. Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

    Directory of Open Access Journals (Sweden)

    K. Pollock

    1993-01-01

    Full Text Available The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK and protein kinase C (PKC, respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP induced generation of superoxide anion and thromboxane B2 (TXB2 in guinea-pig alveolar macrophages (AM. Genistein (3–100 μM dose dependently inhibited FMLP (3 nM induced superoxide generation in non-primed AM and TXB2 release in non-primed or in lipopolysaccharide (LPS (10 ng/ml primed AM to a level > 80% but had litle effect up to 100 μM on phorbol myristate acetate (PMA (10 nM induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC50 0.21 ± 0.10 μM but had no effect on or potentiated (at 3 and 10 μM FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 μM inhibited primed TXB2 release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.

  3. Heart 6-phosphofructo-2-kinase activation by insulin requires PKB (protein kinase B), but not SGK3 (serum- and glucocorticoid-induced protein kinase 3).

    OpenAIRE

    Mouton, Veronique; Toussaint, Louise; Vertommen, Didier; Gueuning, Marie-Agnes; Maisin, Liliane; Havaux, Xavier; Sanchez-Canedo, Cossette; Bertrand, Luc; Dequiedt, Franck; Hemmings, Brian A; Hue, Louis; Rider, Mark H

    2010-01-01

    On the basis of transfection experiments using a dominant-negative approach, our previous studies suggested that PKB (protein kinase B) was not involved in heart PFK-2 (6-phosphofructo2-kinase) activation by insulin. Therefore we first tested whether SGK3 (serum- and glucocorticoid-induced protein kinase 3) might be involved in this effect. Treatment of recombinant heart PFK-2 with [gamma-32P]ATP and SGK3 in vitro led to PFK-2 activation and phosphorylation at Ser466 and Ser483. However, in H...

  4. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    Science.gov (United States)

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. K63-Linked Ubiquitination in Kinase Activation and Cancer

    International Nuclear Information System (INIS)

    Wang, Guocan; Gao, Yuan; Li, Liren; Jin, Guoxiang; Cai, Zhen; Chao, Jui-I; Lin, Hui-Kuan

    2012-01-01

    Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.

  6. INHIBITING MAP KINASE ACTIVITY PREVENTS CALCIUM TRANSIENTS AND MITOSIS ENTRY IN EARLY SEA URCHIN EMBRYOS

    OpenAIRE

    Philipova, Rada; Larman, Mark G.; Leckie, Calum P.; Harrison, Patrick K.; Groigno, Laurence; Whitaker, Michael

    2005-01-01

    A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos both kinases show a similar activation profile, peaking at the time of mitosis entry.

  7. Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

    Science.gov (United States)

    Sun, Yun; Wang, Chen; Yang, Bo; Wu, Feifei; Hao, Xueyu; Liang, Wanwan; Niu, Fangfang; Yan, Jingli; Zhang, Hanfeng; Wang, Boya; Deyholos, Michael K; Jiang, Yuan-Qing

    2014-05-01

    Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.

  8. Mitogen-activated protein kinases in the acute diabetic myocardium

    Czech Academy of Sciences Publication Activity Database

    Strnisková, M.; Barančík, M.; Neckář, Jan; Ravingerová, T.

    2003-01-01

    Roč. 249, 1-2 (2003), s. 59-65 ISSN 0300-8177 R&D Projects: GA MŠk LN00A069 Grant - others:VEGA(SK) 2/2063/22 Institutional research plan: CEZ:AV0Z5011922 Keywords : experimental diabetes * ischemia * mitogen-activated protein kinases (MAPK) Subject RIV: ED - Physiology Impact factor: 1.763, year: 2003

  9. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...... reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12...

  10. Expression of focal adhesion kinase in uveal melanoma and the effects of Hsp90 inhibition by 17-AAG.

    Science.gov (United States)

    Faingold, Dana; Filho, Vasco Bravo; Fernandes, Bruno; Jagan, Lisa; de Barros, Alexandre M; Orellana, Maria Eugenia; Antecka, Emilia; Burnier, Miguel N

    2014-11-01

    Focal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells. FAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10 μmol/L of 17-AAG. FAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24 h of exposure. FAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. A BCR-ABL Kinase Activity-Independent Signaling Pathway in Chronic Myelogenous Leukemia

    National Research Council Canada - National Science Library

    Li, Shaoguang

    2008-01-01

    .... We identified Src kinases as key molecules in this BCR- ABL kinase activity-independent pathway and they are essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis...

  12. Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

    DEFF Research Database (Denmark)

    Hauge, Camilla; Antal, Torben L; Hirschberg, Daniel

    2007-01-01

    The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We...... investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within...... the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S...

  13. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

    Directory of Open Access Journals (Sweden)

    Silviya R Stateva

    Full Text Available Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7 inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR, in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

  14. Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Puja; Wang, Benlian; Maeda, Tadao; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2008-10-08

    G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1{center_dot}(Mg{sup 2+}){sub 2} {center_dot}ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.

  15. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    KAUST Repository

    Zourelidou, Melina

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  16. Activation of the Antiviral Kinase PKR and Viral Countermeasures

    Directory of Open Access Journals (Sweden)

    Bianca Dauber

    2009-10-01

    Full Text Available The interferon-induced double-stranded (dsRNA-dependent protein kinase (PKR limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5’-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR.

  17. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline...... and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction...... of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant...

  18. Overexpression of Populus trichocarpa Mitogen-Activated Protein Kinase Kinase4 Enhances Salt Tolerance in Tobacco

    Directory of Open Access Journals (Sweden)

    Chengjun Yang

    2017-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK is one of the factors of cascade reactions affecting responses to signal pathway of environmental stimuli. Throughout the life of plants, MAPK family members participate in signal transduction pathways and regulate various intracellular physiological and metabolic reactions. To gain insights into regulatory function of MAPK kinase (MAPKK in Populus trichocarpa under salt stress, we obtained full-length cDNA of PtMAPKK4 and analyzed different expression levels of PtMAPKK4 gene in leaves, stems, and root organs. The relationship between PtMAPKK4 and salt stress was studied by detecting expression characteristics of mRNA under 150 mM NaCl stress using real-time quantitative polymerase chain reaction. The results showed that expression of PtMAPKK4 increased under salt (NaCl stress in leaves but initially reduced and then increased in roots. Thus, salt stress failed to induce PtMAPKK4 expression in stems. PtMAPKK4 possibly participates in regulation of plant growth and metabolism, thereby improving its salt tolerance. We used Saccharomyces cerevisiae strain INVScI to verify subcellular localization of PtMAPKK4 kinase. The yeast strains containing pYES2-PtMAPKK4-GFP plasmid expressed GFP fusion proteins under the induction of d-galactose, and the products were located in nucleus. These results were consistent with network prediction and confirmed location of PtMAPKK4 enzyme in the nucleus. We tested NaCl tolerance in transgenic tobacco lines overexpressing PtMAPKK4 under the control of 35S promoter at germination stage to detect salt tolerance function of PtMAPKK4. Compared withK326 (a wild-type tobacco, lines overexpressing PtMAPKK4 showed a certain degree of improvement in tolerance, germination, and growth. NaCl inhibited growth of overexpressed line and K326 at the seedling stage. However, statistical analysis showed longer root length, higher fresh weight, and lower MDA content in transgenic lines in

  19. Fibroblast adhesion and activation onto micro-machined titanium surfaces.

    Science.gov (United States)

    Guillem-Marti, J; Delgado, L; Godoy-Gallardo, M; Pegueroles, M; Herrero, M; Gil, F J

    2013-07-01

    Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 μm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 μm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (micro-grooved surfaces could improve implant integration at the gingival site with respect to polished surfaces. Micro-grooved surfaces enhance early fibroblast adhesion and activation, which could be critical for the formation of a biological seal and finally promote tissue integration. Surfaces with wider grooves (≥50 μm) seem to be more

  20. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    Science.gov (United States)

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  1. Activation of c-Src and Fyn kinases by protein tyrosine phosphatase RPTPalpha is substrate-specific and compatible with lipid raft localization

    DEFF Research Database (Denmark)

    Vacaresse, Nathalie; Møller, Bente; Danielsen, Erik Michael

    2008-01-01

    Tyrosine kinases of the Src family (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein tyrosine phosphatases (PTPs), among which RPTPa and SHP2 are the best established. We...... studied how RPTPa affects substrate specificity and regulation of c-Src and Fyn in response to EGF and PDGF. We find that RPTPa, in a growth factor-specific manner, directs the specificity of these kinases towards a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin...

  2. Gene therapy targeting hepatocellular carcinoma by a dual-regulated oncolytic adenovirus harboring the focal adhesion kinase shRNA.

    Science.gov (United States)

    Gao, Yang; Zhu, Yayun; Huang, Xinyu; Ai, Kaixing; Zheng, Qi; Yuan, Zhou

    2015-08-01

    Cancer targeting gene-viro-therapy (CTGVT) approach has become a hotspot and a trend in the field of cancer biotherapy and oncolytic adenovirus is an ideal vector to carry the targeting genes. In this study, we used human telomerase reverse transcriptase (hTERT) promoter to control the adenovirus early region 1a (E1A) and the human α-fetoprotein (AFP) promoter integrated with hypoxia response element (HRE) to control the adenovirus early region 1b (E1B). Then the novel double-regulated adenovirus Ad-hTERT-HREAF (named SG505) was engineered. The short-hairpin RNA against focal adhesion kinase (FAK) was inserted into SG505 and thus forming Ad-hTERT-HREAF-shRNA (called SG505‑siFAK). Then various oncolytic adenoviruses were examined to verify whether they could suppress liver cancer cells selectively and efficiently both in vitro and in vivo. Both replicative and replication-defective adenoviruses carrying FAK-shRNA significantly inhibited the expression of FAK in Hep3B and SMMC-7721 cell lines and efficiently suppressed the growth of liver cancer cell lines with minor effect to normal cells. Furthermore, the recombined oncolytic adenoviruses, SG505-siFAK, SG505-EGFP and SG505 were able to selectively propagate in AFP-positive liver cancer cells in vitro and the SG505-siFAK efficiently suppressed the expression of FAK. SG505-siFAK showed the most potent tumor inhibition capability among the three recombined adenovirus with IC50 levels of 0.092±0.009 and 0.424±0.414 pfu/cell in the Hep3B and HepG2 cell line, respectively. Animal experiment further confirmed that SG505-siFAK achieved the most significant tumor inhibition of Hep3B liver cancer xenografted growth by intratumoral injection comparing to the intravenous injection among the three recombined viruses. Immunohistochemical results indicated that FAK expression was downregulated significantly in the tumors treated with SG505-siFAK. The dual-regulated oncolytic adenovirus SG505-siFAK was proven to inhibit the

  3. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    Science.gov (United States)

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  4. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...... remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim...... was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease...

  5. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Directory of Open Access Journals (Sweden)

    Belén Mezquita

    2014-02-01

    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  6. v-Src SH3-enhanced Interaction with Focal Adhesion Kinase at Beta1 Integrin-containing Invadopodia Promotes Cell Invasion

    OpenAIRE

    Hauck, Christof R.; Hsia, Datsun A.; Ilić, Du ko; Schlaepfer, David D.

    2002-01-01

    In viral Src (v-Src) transformed cells, focal adhesion kinase (FAK) associates in a stable signaling complex with v-Src that is mediated by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here, we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point-mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited...

  7. Activation of mitogen-activated protein kinase by heat shock treatment in Drosophila.

    OpenAIRE

    Chen, F; Torres, M; Duncan, R F

    1995-01-01

    Heat shock treatment of Drosophila melanogaster tissue culture cells causes increased tyrosine phosphorylation of several 44 kDa proteins, which are identified as Drosophila mitogen-activated protein (MAP) kinases. Tyrosine phosphorylation occurs within 5 min, and is maintained at high levels during heat shock. It decreases to basal levels during recovery, concurrent with the repression of heat shock transcription and heat-shock-protein synthesis. The increased MAP kinase tyrosine phosphoryla...

  8. Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

    Science.gov (United States)

    Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

    2018-02-11

    Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2017. Published by Elsevier Inc.

  9. Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens

    OpenAIRE

    Singh, Tejinder P.; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K.

    2017-01-01

    Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were ...

  10. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

    2013-01-01

    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....

  11. TPX2 Protein of Arabidopsis Activates Aurora Kinase 1, But Not Aurora Kinase 3 In Vitro

    Czech Academy of Sciences Publication Activity Database

    Tomaštíková, Eva; Demidov, D.; Jeřábková, Hana; Binarová, Pavla; Houben, A.; Doležel, Jaroslav; Petrovská, Beáta

    2015-01-01

    Roč. 33, č. 6 (2015), s. 1988-1995 ISSN 0735-9640 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204; GA ČR GAP501/12/2333 Institutional support: RVO:61389030 ; RVO:61388971 Keywords : Aurora kinase * Targeting protein for Xklp2 * In vitro kinase assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.304, year: 2015

  12. High quality, small molecule-activity datasets for kinase research [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rajan Sharma

    2016-10-01

    Full Text Available Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR data. Bioactivity databases such as the Kinase Knowledgebase (KKB, WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note.

  13. High quality, small molecule-activity datasets for kinase research [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rajan Sharma

    2016-06-01

    Full Text Available Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR data. Bioactivity databases such as the Kinase Knowledgebase (KKB, WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note.

  14. High quality, small molecule-activity datasets for kinase research [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rajan Sharma

    2016-07-01

    Full Text Available Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR data. Bioactivity databases such as the Kinase Knowledgebase (KKB, WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note.

  15. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  16. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that ...

  17. Polo-Like Kinase-1 Controls Aurora A Destruction by Activating APC/C-Cdh1

    NARCIS (Netherlands)

    van Leuken, Renske; Clijsters, Linda; van Zon, Wouter; Lim, Dan; Yao, XueBiao; Wolthuis, Rob M. F.; Yaffe, Michael B.; Medema, Rene H.; van Vugt, Marcel A. T. M.

    2009-01-01

    Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-beta TrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A

  18. Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Jarrett T.; Wang, Lei; Chen, Jianming; Metts, Meagan E.; Nasser, Taj A.; McGoldrick, Liam J. [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); Bridges, Lance C., E-mail: bridgesl@ecu.edu [Department of Biochemistry and Molecular Biology, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States); East Carolina Diabetes and Obesity Institute, The Brody School of Medicine at East Carolina University, Greenville, NC 27834 (United States)

    2014-11-28

    Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.

  19. 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

  20. Examining Potential Active Tempering of Adhesive Curing by Marine Mussels

    Directory of Open Access Journals (Sweden)

    Natalie A. Hamada

    2017-08-01

    Full Text Available Mussels generate adhesives for staying in place when faced with waves and turbulence of the intertidal zone. Their byssal attachment assembly consists of adhesive plaques connected to the animal by threads. We have noticed that, every now and then, the animals tug on their plaque and threads. This observation had us wondering if the mussels temper or otherwise control catechol chemistry within the byssus in order to manage mechanical properties of the materials. Here, we carried out a study in which the adhesion properties of mussel plaques were compared when left attached to the animals versus detached and exposed only to an aquarium environment. For the most part, detachment from the animal had almost no influence on the mechanical properties on low-energy surfaces. There was a slight, yet significant difference observed with attached versus detached adhesive properties on high energy surfaces. There were significant differences in the area of adhesive deposited by the mussels on a low- versus a high-energy surface. Mussel adhesive plaques appear to be unlike, for example, spider silk, for which pulling on the material is needed for assembly of proteinaceous fibers to manage properties.

  1. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

    Science.gov (United States)

    Sroka, Robert; Van Lint, Johan; Katz, Sarah-Fee; Schneider, Marlon R; Kleger, Alexander; Paschke, Stephan; Seufferlein, Thomas; Eiseler, Tim

    2016-06-15

    Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. © 2016. Published by The Company of Biologists Ltd.

  2. Skeletal muscle mitogen-activated protein kinases and ribosomal S6 kinases. Suppression in chronic diabetic rats and reversal by vanadium.

    Science.gov (United States)

    Hei, Y J; Chen, X; Pelech, S L; Diamond, J; McNeill, J H

    1995-10-01

    The mitogen-activated protein (MAP) kinases and ribosomal S6 protein kinases in the skeletal muscle of insulin-resistant long-term (2 and 6 months' duration) diabetic rats were investigated to understand further the changes in insulin intracellular signaling pathways that accompany diabetes. The effects of insulin-mimetic vanadium compounds on the activity of these kinases were also examined. In the insulin-resistant 2-month diabetic rats, the basal activities of MAP kinases were relatively unchanged, while the basal activities of S6 kinases were significantly increased. Intravenous injection of insulin moderately activated both the 42-kDa MAP kinase (p42mapk) and a 44-kDa MAP kinase (p44erk1) in the 2-month control rats but not in the 2-month diabetic rats. Insulin treatment markedly stimulated the activity of a novel 31-kDa S6 kinase and the previously described 90-kDa ribosomal S6 kinase encoded by one of the rsk genes (p90rsk) in the 2-month control rats, while the effect was substantially reduced in the diabetic rats. In the 6-month diabetic rats, the basal phosphotransferase activities of both MAP kinases were depressed threefold or greater. This correlated with reductions in the amount of immunoreactive p42mapk and p44erk1 proteins in extracts from the diabetic rats. The basal activity of the 31-kDa S6 kinase activity was also reduced fourfold in the 6-month diabetic rats. Treatment of the 2-month diabetic rats with vanadyl sulfate resulted in euglycemia, prevented the increase in the basal activity of S6 kinase, and improved the activation of S6 kinase by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Anomalous constitutive Src kinase activity promotes B lymphoma survival and growth

    OpenAIRE

    Ke, Jiyuan; Chelvarajan, R Lakshman; Sindhava, Vishal; Robertson, Darrell A; Lekakis, Lazaros; Jennings, C Darrell; Bondada, Subbarao

    2009-01-01

    Abstract Background Previously we have shown that B cell receptor (BCR) expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK), ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs) like Lyn are known to be...

  4. Subtype activation and interaction of protein kinase C and mitogen-activated protein kinase controlling receptor expression in cerebral arteries and microvessels after subarachnoid hemorrhage

    DEFF Research Database (Denmark)

    Ansar, S.; Edvinsson, L.

    2008-01-01

    BACKGROUND AND PURPOSE: The pathogenesis of cerebral ischemia associated with subarachnoid hemorrhage (SAH) still remains elusive. The aim of this study was to examine the involvement of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) subtypes in the pathophysiology of cerebral...... ischemia after SAH in cerebral arteries and microvessels and to examine temporal activation of the kinases. We hypothesize that treatment with a MAPK or PKC inhibitor will prevent the SAH-induced kinase activation in brain vessels. METHODS: SAH was induced by injecting 250 microL blood...... into the prechiasmatic cistern in the rat. The activation of different MAPK and PKC isotypes in large circle of Willis cerebral arteries and intracerebral microvessels was examined at 0, 1, 3, 6, 12, 24, and 48 hours after SAH and after intrathecal treatment with PKC or MAPK inhibitor by use of Western blot. RESULTS...

  5. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  6. A switch in nucleotide affinity governs activation of the Src and Tec family kinases.

    Science.gov (United States)

    von Raußendorf, Freia; de Ruiter, Anita; Leonard, Thomas A

    2017-12-12

    The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases.

  7. The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

    Directory of Open Access Journals (Sweden)

    Doron Teper

    2018-01-01

    Full Text Available The Gram-negative bacterium Xanthomonas euvesicatoria (Xe is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

  8. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    Science.gov (United States)

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  9. Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF).

    Science.gov (United States)

    Dutra, Patricia M L; Vieira, Danielle P; Meyer-Fernandes, Jose R; Silva-Neto, Mario A C; Lopes, Angela H

    2009-09-01

    Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

  10. Does active application of universal adhesives to enamel in self-etch mode improve their performance?

    Science.gov (United States)

    Loguercio, Alessandro D; Muñoz, Miguel Angel; Luque-Martinez, Issis; Hass, Viviane; Reis, Alessandra; Perdigão, Jorge

    2015-09-01

    To evaluate the effect of adhesion strategy on the enamel microshear bond strengths (μSBS), etching pattern, and in situ degree of conversion (DC) of seven universal adhesives. 84 extracted third molars were sectioned in four parts (buccal, lingual, proximal) and divided into 21 groups, according to the combination of the main factors adhesive (AdheSE Universal [ADU], All-Bond Universal [ABU], Clearfil Universal [CFU], Futurabond U [FBU], G-Bond Plus [GBP], Prime&Bond Elect (PBE), and Scotchbond Universal Adhesive [SBU]), and adhesion strategy (etch-and-rinse, active self-etch, and passive self-etch). Specimens were stored in water (37°C/24h) and tested at 1.0mm/min (μSBS). Enamel-resin interfaces were evaluated for DC using micro-Raman spectroscopy. The enamel-etching pattern was evaluated under a field-emission scanning electron microscope (direct and replica techniques). Data were analyzed with two-way ANOVA and Tukey's test (α=0.05). Active self-etch application increased μSBS and DC for five out of the seven universal adhesives when compared to passive application (pself-etch application compared to that of passive self-etch application. Replicas of GBP and PBE applied in active self-etch mode displayed morphological features compatible with water droplets. The DC of GBP and PBE were not affected by the application/strategy mode. In light of the improved performance of universal adhesives when applied actively in SE mode, selective enamel etching with phosphoric acid may not be crucial for their adhesion to enamel. The active application of universal adhesives in self-etch mode may be a practical alternative to enamel etching in specific clinical situations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition

    DEFF Research Database (Denmark)

    Soundararajan, M.; Roos, A.K.; Savitsky, P.

    2013-01-01

    Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinases (DYRKs) play key roles in brain development, regulation of splicing, and apoptosis, and are potential drug targets for neurodegenerative diseases and cancer. We present crystal structures of one representative member of each DYRK sub...

  12. Roles of Mitogen-Activating Protein Kinase Kinase Kinase Kinase-3 (MAP4K3) in Preterm Skeletal Muscle Satellite Cell Myogenesis and Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Regulation.

    Science.gov (United States)

    Guo, Chu-Yi; Yu, Mu-Xue; Dai, Jie-Min; Pan, Si-Nian; Lu, Zhen-Tong; Qiu, Xiao-Shan; Zhuang, Si-Qi

    2017-07-21

    BACKGROUND Preterm skeletal muscle genesis is a paradigm for myogenesis. The role of mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) in preterm skeletal muscle satellite cells myogenesis or its relationship to mammalian target of rapamycin complex 1 (mTORC1) activity have not been previously elaborated. MATERIAL AND METHODS Small interfering RNA (siRNA) interference technology was used to inhibit MAP4K3 expression. Leucine stimulation experiments were performed following MAP4K3-siRNA interference. The differentiation of primary preterm skeletal muscle satellite cells was observed after siRNA-MAP4K3 interference. Western blot analysis was used to determine the expression of MAP4K3, MyHC, MyoD, myogenin, p-mTOR, and p-S6K1. The immunofluorescence fusion index of MyHC and myogenin were detected. MAP4K3 effects on preterm rat satellite cells differentiation and its relationship to mTORC1 activity are reported. RESULTS MAP4K3 siRNA knockdown inhibited myotube formation and both MyoD and myogenin expression in primary preterm rat skeletal muscle satellite cells, but MAP4K3 siRNA had no effect on the activity of mTORC1. In primary preterm rat skeletal muscle satellite cells, MAP4K3 knockdown resulted in significantly weaker, but not entirely blunted, leucine-induced mTORC1 signaling. CONCLUSIONS MAP4K3 positively regulates preterm skeletal muscle satellite cell myogenesis, but may not regulate mTORC1 activity. MAP4K3 may play a role in mTORC1 full activation in response to leucine.

  13. Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of β1 integrin-null cells

    International Nuclear Information System (INIS)

    Kikkawa, Yamato; Yu, Hao; Genersch, Elke; Sanzen, Noriko; Sekiguchi, Kiyotoshi; Faessler, Reinhard; Campbell, Kevin P.; Talts, Jan F.; Ekblom, Peter

    2004-01-01

    The presence of many laminin receptors of the β1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin α6β4 and dystroglycan. We therefore tested the binding of a β1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin α6Aβ4A variant. GD25 β1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin α6 antibody, but not by a dystroglycan antibody. Hence, integrin α6Aβ4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin α6Aβ4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin α6Aβ4A

  14. Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

    International Nuclear Information System (INIS)

    Jeffrey, W.H.; Paul, J.H.

    1990-01-01

    One assumption made in bacterial production estimates from [ 3 H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported the thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibro sp. strain DI9, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems

  15. Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3.

    Science.gov (United States)

    Bassi, Rekha; Burgoyne, Joseph R; DeNicola, Gian F; Rudyk, Olena; DeSantis, Vittorio; Charles, Rebecca L; Eaton, Philip; Marber, Michael S

    2017-09-29

    The kinase p38α MAPK (p38α) plays a pivotal role in many biological processes. p38α is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38α. p38α was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H 2 O 2 This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38α, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H 2 O 2 Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38α C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38α C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO 2 -OA), a component of the Mediterranean diet, reduced p38α activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO 2 -OA reduced the dephosphorylation of p38α by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO 2 -OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H 2 O 2 -induced contractile dysfunction. Our findings suggest that cysteine residues within p38α act as redox sensors that can dynamically regulate the association between p38 and MKK3. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  17. The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

    Directory of Open Access Journals (Sweden)

    Galo L. Mejia

    2016-07-01

    Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

  18. Effects of FGFR2 kinase activation loop dynamics on catalytic activity.

    Directory of Open Access Journals (Sweden)

    Jerome M Karp

    2017-02-01

    Full Text Available The structural mechanisms by which receptor tyrosine kinases (RTKs regulate catalytic activity are diverse and often based on subtle changes in conformational dynamics. The regulatory mechanism of one such RTK, fibroblast growth factor receptor 2 (FGFR2 kinase, is still unknown, as the numerous crystal structures of the unphosphorylated and phosphorylated forms of the kinase domains show no apparent structural change that could explain how phosphorylation could enable catalytic activity. In this study, we use several enhanced sampling molecular dynamics (MD methods to elucidate the structural changes to the kinase's activation loop that occur upon phosphorylation. We show that phosphorylation favors inward motion of Arg664, while simultaneously favoring outward motion of Leu665 and Pro666. The latter structural change enables the substrate to bind leading to its resultant phosphorylation. Inward motion of Arg664 allows it to interact with the γ-phosphate of ATP as well as the substrate tyrosine. We show that this stabilizes the tyrosine and primes it for the catalytic phosphotransfer, and it may lower the activation barrier of the phosphotransfer reaction. Our work demonstrates the value of including dynamic information gleaned from computer simulation in deciphering RTK regulatory function.

  19. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Blaha, Milan

    2015-01-01

    Roč. 61, č. 6 (2015), s. 495-502 ISSN 0916-8818 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : cumulus oocyte complexes * meiosis resumption * mitogen-activated protein kinase 3/1 (MAPK3/1) Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.453, year: 2015

  20. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    DEFF Research Database (Denmark)

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested o...

  1. Stromal serine protein kinase activity in spinach chloroplasts

    International Nuclear Information System (INIS)

    Cortez, N.; Lucero, H.A.; Vallejos, R.H.

    1987-01-01

    At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

  2. 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

    approach revealed regulated phosphorylated proteins in SCCs, which were derived from DFCs after 24 and 48 h in SFM. These regulated proteins were categorized using the Kyoto encyclopedia of genes and genomes program. Here, cellular processes and signaling pathway were identified such as the focal adhesion...

  3. Function of adhesion molecules lymphocyte function-associated antigen-3 and intercellular adhesion molecule-1 on human epidermal Langerhans cells in antigen-specific T cell activation

    NARCIS (Netherlands)

    Teunissen, M. B.; Rongen, H. A.; Bos, J. D.

    1994-01-01

    In addition to the interaction between the TCR and the MHC/Ag complex on the APC, optimal T cell activation also requires interaction between adhesion molecules on the APC and their ligands on T cells. We determined the presence of adhesion molecules on human epidermal Langerhans cells (LC) and

  4. Rho-kinase inhibition ameliorates metabolic disorders through activation of AMPK pathway in mice.

    Directory of Open Access Journals (Sweden)

    Kazuki Noda

    Full Text Available BACKGROUND: Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK, a key molecule of metabolic conditions. METHODS AND RESULTS: Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1, with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C. CONCLUSIONS: These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that

  5. Adhesive ability and biofilm metabolic activity of Listeria ...

    African Journals Online (AJOL)

    Listeria monocytogenes is an important pathogen responsible for major outbreaks associated with food products. Adhesion to surfaces leads to significant modifications in cell physiology. In this work, the ability of L. monocytogenes to produce biofilm and its ability to adhere to abiotic surfaces under cold stress were ...

  6. OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc | Office of Cancer Genomics

    Science.gov (United States)

    Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network.

  7. S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; Liu, Zhen; Qiu, Wen-Li; Whitham, Steven A.; Qian, Wei-Jun

    2017-09-29

    It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse

  8. Diminished Antimicrobial Peptide and Antifungal Antibiotic Activities against Candida albicans in Denture Adhesive

    Directory of Open Access Journals (Sweden)

    Amber M. Bates

    2017-02-01

    Full Text Available The underlying causes of denture stomatitis may be related to the long-term use of adhesives, which may predispose individuals to oral candidiasis. In this study, we hypothesize that antimicrobial peptides and antifungal antibiotics have diminished anti-Candida activities in denture adhesive. To show this, nine antimicrobial peptides and five antifungal antibiotics with and without 1.0% denture adhesive were incubated with Candida albicans strains ATCC 64124 and HMV4C in radial diffusion assays. In gels with 1.0% adhesive, HNP-1, HBD2, HBD3, IP-10, LL37 (only one strain, histatin 5 (only one strain, lactoferricin B, and SMAP28 showed diminished activity against C. albicans. In gels with 1.0% adhesive, amphotericin B and chlorhexidine dihydrochloride were active against both strains of C. albicans. These results suggest that denture adhesive may inactivate innate immune mediators in the oral cavity increasing the risk of C. albicans infections, but inclusion of antifungal antibiotics to denture adhesive may aid in prevention or treatment of Candida infections and denture stomatitis.

  9. Controlling the activity of the Tec kinase Itk by mutation of the phenylalanine gatekeeper residue.

    Science.gov (United States)

    Joseph, Raji E; Andreotti, Amy H

    2011-01-18

    The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

  10. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  11. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  12. The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells.

    Science.gov (United States)

    Semini, Geo; Hildmann, Annette; Reissig, Hans-Ulrich; Reutter, Werner; Danker, Kerstin

    2011-04-15

    New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell-matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway.

    Science.gov (United States)

    Cheng, Lihong; Ye, Ying; Xiang, Lan; Osada, Hiroyuki; Qi, Jianhua

    2017-01-15

    Neurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood-brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases. The present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance. Extract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments. The ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal

  14. Tyrosine kinase Btk regulates E-selectin–mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) γ2 and PI3Kγ pathways

    Science.gov (United States)

    Mueller, Helena; Stadtmann, Anika; Van Aken, Hugo; Hirsch, Emilio; Wang, Demin; Ley, Klaus

    2010-01-01

    Selectins mediate leukocyte rolling, trigger β2-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)–dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) γ2– and phosphoinositide 3-kinase (PI3K) γ–dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP3), and inducing E-selectin–mediated slow rolling. Inhibition of this signal-transduction pathway diminished Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Gαi-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk−/− and Plcg2−/− mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement. PMID:20167705

  15. HCLK2 is required for activity of the DNA damage response kinase ATR

    DEFF Research Database (Denmark)

    Rendtlew Danielsen, Jannie M; Larsen, Dorthe Helena; Schou, Kenneth Bødtker

    2008-01-01

    of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its...... in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation....

  16. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  17. Micro–adhesion rings surrounding TCR microclusters are essential for T cell activation

    Science.gov (United States)

    Sakuma, Machie; Yokosuka, Tadashi

    2016-01-01

    The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro–adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro–adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro–adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro–adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals. PMID:27354546

  18. Skeletal muscle Ca(2+)-independent kinase activity increases during either hypertrophy or running

    Science.gov (United States)

    Fluck, M.; Waxham, M. N.; Hamilton, M. T.; Booth, F. W.

    2000-01-01

    Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

  19. TrkB kinase activity maintains synaptic function and structural integrity at adult neuromuscular junctions

    Science.gov (United States)

    Stowe, Jessica M.; Sieck, Dylan C.; Ermilov, Leonid G.; Greising, Sarah M.; Zhang, Chao; Shokat, Kevan M.; Sieck, Gary C.

    2014-01-01

    Activation of the tropomyosin-related kinase receptor B (TrkB) by brain-derived neurotrophic factor acutely regulates synaptic transmission at adult neuromuscular junctions (NMJs). The role of TrkB kinase activity in the maintenance of NMJ function and structure at diaphragm muscle NMJs was explored using a chemical-genetic approach that permits reversible inactivation of TrkB kinase activity in TrkBF616A mice by 1NMPP1. Inhibiting TrkB kinase activity for 7 days resulted in significant, yet reversible, impairments in neuromuscular transmission at diaphragm NMJs. Neuromuscular transmission failure following 2 min of repetitive phrenic nerve stimulation increased from 42% in control to 59% in 1NMPP1-treated TrkBF616A mice (P = 0.010). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment improved neuromuscular transmission (P = 0.006). Electrophysiological measurements at individual diaphragm NMJs documented lack of differences in quantal content in control and 1NMPP1-treated mice (P = 0.845). Morphological changes at diaphragm NMJs were modest following inhibition and recovery of TrkB kinase activity. Three-dimensional reconstructions of diaphragm NMJs revealed no differences in volume at motor end plates (labeled by α-bungarotoxin; P = 0.982) or presynaptic terminals (labeled by synaptophysin; P = 0.515). Inhibition of TrkB kinase activity by 1NMPP1 resulted in more compact NMJs, with increased apposition of presynaptic terminals and motor end plates (P = 0.017) and reduced fragmentation of motor end plates (P = 0.005). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment resulted in postsynaptic remodeling likely reflecting increased gutter depth (P = 0.007), without significant presynaptic changes. These results support an essential role for TrkB kinase activity in maintaining synaptic function and structural integrity at NMJs in the adult mouse diaphragm muscle. PMID:25170066

  20. Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

    Science.gov (United States)

    Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

    2011-02-25

    FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

  1. Shedding of APP limits its synaptogenic activity and cell adhesion properties

    Science.gov (United States)

    Stahl, Ronny; Schilling, Sandra; Soba, Peter; Rupp, Carsten; Hartmann, Tobias; Wagner, Katja; Merdes, Gunter; Eggert, Simone; Kins, Stefan

    2014-01-01

    The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules. PMID:25520622

  2. Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

    Energy Technology Data Exchange (ETDEWEB)

    Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn; Easterly,Evangeline; Barcellos-Hoff, Mary Helen; Yingling, Jonathan M.; Zent, Roy; Arteaga, Carlos L.

    2005-01-02

    The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3. Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed in

  3. 2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors.

    Science.gov (United States)

    Nkizinkiko, Yves; Desantis, Jenny; Koivunen, Jarkko; Haikarainen, Teemu; Murthy, Sudarshan; Sancineto, Luca; Massari, Serena; Ianni, Federica; Obaji, Ezeogo; Loza, Maria I; Pihlajaniemi, Taina; Brea, Jose; Tabarrini, Oriana; Lehtiö, Lari

    2018-01-26

    Tankyrases (TNKSs) are enzymes specialized in catalyzing poly-ADP-ribosylation of target proteins. Several studies have validated TNKSs as anti-cancer drug targets due to their regulatory role in Wnt/β-catenin pathway. Recently a lot of effort has been put into developing more potent and selective TNKS inhibitors and optimizing them towards anti-cancer agents. We noticed that some 2-phenylquinazolinones (2-PQs) reported as CDK9 inhibitors were similar to previously published TNKS inhibitors. In this study, we profiled this series of 2-PQs against TNKS and selected kinases that are involved in the Wnt/β-catenin pathway. We found that they were much more potent TNKS inhibitors than they were CDK9/kinase inhibitors. We evaluated the compound selectivity to tankyrases over the ARTD enzyme family and solved co-crystal structures of the compounds with TNKS2. Comparative structure-based studies of the catalytic domain of TNKS2 with selected CDK9 inhibitors and docking studies of the inhibitors with two kinases (CDK9 and Akt) revealed important structural features, which could explain the selectivity of the compounds towards either tankyrases or kinases. We also discovered a compound, which was able to inhibit tankyrases, CDK9 and Akt kinases with equal µM potency.

  4. Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

    Science.gov (United States)

    2010-01-01

    mediated activation ( Abo et al., 1998; Cau et al., 2001; Lee et al., 2002), the basal kinase activity of the group II Paks was not stimulated by Cdc42...Received: November 15, 2007 Revised: February 25, 2008 Accepted: March 3, 2008 Published: April 18, 2008 REFERENCES Abo , A., Qu, J., Cammarano, M.S...et al. Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat Neurosci 2006;9(2):234-42. 53. Reutershan J

  5. Effect of cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle through Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2.

    Science.gov (United States)

    Wu, Tao; Chen, Lin; Wei, Tangqiang; Wang, Yan; Xu, Feng; Wang, Kunjie

    2012-09-01

    To examine the role of Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 in the cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle cells. Human bladder smooth muscle cells were exposed to cyclic hydrodynamic pressures in vitro with defined parameters (static, 100 cmH(2) O, 200 cmH(2) O and 300 cmH(2) O pressure) for 24 h. The proliferation of cells was assessed by flow cytometry. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 messenger ribonucleic acid, and protein expression was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the Rac1 was determined with real-time polymerase chain reaction and Western blot technique with small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766). The proliferation of human bladder smooth muscle cells was increased. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 were activated by 200 and 300 cmH(2) O cyclic hydrodynamic pressure compared with static and 100 cmH(2) O pressure. The "knockdown" of activation of Rac1 using target small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766) decreased proliferation of human bladder smooth muscle cells, and downregulated mitogen-activated protein kinase kinase 1/2, extracellular regulated protein kinases 1/2. The Rac1 pathway is activated in mechanotransduction and regulation of human bladder smooth muscle cell proliferation in response to cyclic hydrodynamic pressure. © 2012 The Japanese Urological Association.

  6. Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

    Science.gov (United States)

    Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

    2018-03-15

    Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. IKAP: A heuristic framework for inference of kinase activities from Phosphoproteomics data.

    Science.gov (United States)

    Mischnik, Marcel; Sacco, Francesca; Cox, Jürgen; Schneider, Hans-Christoph; Schäfer, Matthias; Hendlich, Manfred; Crowther, Daniel; Mann, Matthias; Klabunde, Thomas

    2016-02-01

    Phosphoproteomics measurements are widely applied in cellular biology to detect changes in signalling dynamics. However, due to the inherent complexity of phosphorylation patterns and the lack of knowledge on how phosphorylations are related to functions, it is often not possible to directly deduce protein activities from those measurements. Here, we present a heuristic machine learning algorithm that infers the activities of kinases from Phosphoproteomics data using kinase-target information from the PhosphoSitePlus database. By comparing the estimated kinase activity profiles to the measured phosphosite profiles, it is furthermore possible to derive the kinases that are most likely to phosphorylate the respective phosphosite. We apply our approach to published datasets of the human cell cycle generated from HeLaS3 cells, and insulin signalling dynamics in mouse hepatocytes. In the first case, we estimate the activities of 118 at six cell cycle stages and derive 94 new kinase-phosphosite links that can be validated through either database or motif information. In the second case, the activities of 143 kinases at eight time points are estimated and 49 new kinase-target links are derived. The algorithm is implemented in Matlab and be downloaded from github. It makes use of the Optimization and Statistics toolboxes. https://github.com/marcel-mischnik/IKAP.git. marcel.mischnik@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Troglitazone and Δ2Troglitazone Enhance Adiponectin Expression in Monocytes/Macrophages through the AMP-Activated Protein Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Jaw-Shiun Tsai

    2014-01-01

    Full Text Available Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for inflammation and cardiovascular disorders. Here, we tested the effect of troglitazone (TG and its newly synthesized derivative, 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy-benzylidene]-2,4-thiazolidinedione (Δ2troglitazone, (Δ2TG, on the adiponectin expression in monocytes/macrophages and the relative mechanisms. The expression of adiponectin was located in macrophages of atherosclerotic lesions from patients and cholesterol-fed rabbits. TG and Δ2TG enhanced adiponectin mRNA and protein expression in THP-1 cells by quantitative real-time PCR, Western blot, and immunocytochemistry. TG induced adiponectin mRNA expression through a PPARγ-dependent pathway whereas Δ2TG enhanced adiponectin mRNA expression through a PPARγ-independent pathway in THP-1 cells. Both TG and Δ2TG enhanced adiponectin mRNA expression through AMP-activated protein kinase (AMPK activation. TG and Δ2TG decreased the adhesion of THP-1 cells to TNF-α-treated HUVECs and the inhibitory effect was abolished by specific antiadiponectin antibodies. TG- and Δ2TG-induced suppression on monocyte adhesion were inhibited by a selective AMPK inhibitor compound C. Our data suggest that the inhibitory effect of TG and Δ2TG on monocyte adhesion might be at least in part through de novo adiponectin expression and activation of an AMPK-dependent pathway, which might play an important role in anti-inflammation and antiatherosclerosis.

  9. Mechanical Activation of Wood for Adhesive-free board Production

    Science.gov (United States)

    Ermolin, V. N.; Bayandin, M. A.; Kazitsin, S. N.

    2016-11-01

    This paper proposes to use hydrodynamic treatment of wood for the manufacture of wood-based panels from sawdust without using adhesive materials. It was found that such a treatment of wood particles (sawdust, dust, wood powder) allows producing panels with high physical-mechanical properties and water resistance. It is proved that the hydrodynamic treatment allows providing maximum energy of autoadhesion interaction in the moulding material due to increase of specific surface with small changes of geometric size of particles in comparison with mechanical methods of milling.

  10. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

    Science.gov (United States)

    Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

    2017-03-01

    Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

  11. CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

    International Nuclear Information System (INIS)

    Dey, Nandini; Howell, Brian W.; De, Pradip K.; Durden, Donald L.

    2005-01-01

    Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation

  12. Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes

    Directory of Open Access Journals (Sweden)

    Ndifon Nsah

    2010-05-01

    Full Text Available Abstract Background MAP (mitogen-activated protein kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known. Results We investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species. Conclusions We present three alternative interpretations to explain our findings. (1 MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2 Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short intervall although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3 The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is

  13. Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

    Science.gov (United States)

    Silliman, Christopher C; Kelher, Marguerite R; Khan, Samina Y; West, F Bernadette; McLaughlin, Nathan J D; Elzi, David J; England, Kelly; Bjornsen, Jason; Kuldanek, Susan A; Banerjee, Anirban

    2017-11-01

    Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%] FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. The supernatants [10%-40%] FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%] FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of β-arrestin-1 with BLT2, protein kinase C (PKC)β I , and PKCδ and served as the first event in a two-event rodent model of ALI. Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients. © 2017 AABB.

  14. Assessment of creatine kinase and lactate dehydrogenase activities ...

    African Journals Online (AJOL)

    Ina bid to investigate the influence of menopausal on coronary heart disease, plasma creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes were analysed on a prospective cohort of 100 women attending Irrua Specialist Teaching Hospital (ISTH), Irrua, Edo state-Nigeria. They were divided into two groups; ...

  15. Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, Anders; On, Stephen L. W.

    2007-01-01

    bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens...

  16. Aspirin Augments IgE-Mediated Histamine Release from Human Peripheral Basophils via Syk Kinase Activation

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2013-01-01

    Conclusions: Aspirin enhanced histamine release from basophils via increased Syk kinase activation, and that the augmentation of histamine release by NSAIDs or FAs may be one possible cause of worsening symptoms in patients with chronic urticaria and FDEIA.

  17. 2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

    Science.gov (United States)

    Dow, Robert L; Ammirati, Mark; Bagley, Scott W; Bhattacharya, Samit K; Buckbinder, Leonard; Cortes, Christian; El-Kattan, Ayman F; Ford, Kristen; Freeman, Gary B; Guimarães, Cristiano R W; Liu, Shenping; Niosi, Mark; Skoura, Athanasia; Tess, David

    2018-04-12

    Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

  18. Discovery of a Stress-Activated Protein Kinase Inhibitor for Lymphatic Filariasis.

    Science.gov (United States)

    Tummalapalli, Sreedhar R; Bhat, Rohit; Chojnowski, Agnieszka; Prorok, Monika; Kreiss, Tamara; Goldberg, Ronald; Canan, Stacie; Hawryluk, Natalie; Mortensen, Deborah; Khetani, Vikram; Zeldis, Jerome; Siekierka, John J; Rotella, David P

    2018-03-08

    Lymphatic filariasis infects over 120 million people worldwide and can lead to significant disfigurement and disease. Resistance is emerging with current treatments, and these therapies have dose limiting adverse events; consequently new targets are needed. One approach to achieve this goal is inhibition of parasitic protein kinases involved in circumventing host defense mechanisms. This report describes structure-activity relationships leading to the identification of a potent, orally bioavailable stress activated protein kinase inhibitor that may be used to investigate this hypothesis.

  19. Role of phosphatidylinositol 3-kinase activation on insulin action and its alteration in diabetic conditions.

    Science.gov (United States)

    Asano, Tomoichiro; Fujishiro, Midori; Kushiyama, Akifumi; Nakatsu, Yusuke; Yoneda, Masayasu; Kamata, Hideaki; Sakoda, Hideyuki

    2007-09-01

    Inositol phospholipids phosphorylated on D3-position of their inositol rings (3-phosphoinositides) are known to play important roles in various cellular events. Activation of PI (phosphatidylinositol) 3-kinase is essential for aspects of insulin-induced glucose metabolism, including translocation of GLUT4 to the cell surface and glycogen synthesis. The enzyme exists as a heterodimer containing a regulatory subunit and one of two widely-distributed isoforms of the p110 catalytic subunit: p110alpha or p110beta. Activation of PI 3-kinase and its downstream AKT has been demonstrated to be essential for almost all of the insulin-induced glucose and lipid metabolism such as glucose uptake, glycogen synthesis, suppression of glucose output and triglyceride synthesis as well as insulin-induced mitogenesis. Accumulated PI(3,4,5)P(3) activates several serine/threonine kinases containing a PH (pleckstrin homology) domain, including Akt, atypical PKCs, p70S6 kinase and GSK. In the obesity-induced insulin resistant condition, JNK and p70S6K are activated and phosphorylate IRS-proteins, which diminishes the insulin-induced tyrosine phosphorylation of IRS-proteins and thereby impairs the PI 3-kinase/AKT activations. Thus, the drugs which restore the impaired insulin-induced PI 3-kinase/AKT activation, for example, by suppressing JNK or p70S6K, PTEN or SHIP2, could be novel agents to treat diabetes mellitus.

  20. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    International Nuclear Information System (INIS)

    Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

    2008-01-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

  1. Activation of AMP-activated protein kinase rapidly suppresses multiple pro-inflammatory pathways in adipocytes including IL-1 receptor-associated kinase-4 phosphorylation

    DEFF Research Database (Denmark)

    Mancini, Sarah J; White, Anna D; Bijland, Silvia

    2017-01-01

    Inflammation of adipose tissue in obesity is associated with increased IL-1β, IL-6 and TNF-α secretion and proposed to contribute to insulin resistance. AMP-activated protein kinase (AMPK) regulates nutrient metabolism and is reported to have anti-inflammatory actions in adipose tissue, yet the m...

  2. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  3. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    Science.gov (United States)

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  5. Nuclear factor kappaB-mediated down-regulation of adhesion molecules: possible mechanism for inhibitory activity of bigelovin against inflammatory monocytes adhesion to endothelial cells.

    Science.gov (United States)

    Nam, Kung-Woo; Oh, Goo Taeg; Seo, Eun-Kyoung; Kim, Kyeong Ho; Koo, Uk; Lee, Sung-Jin; Mar, Woongchon

    2009-06-22

    The flowers of Inula britannica L. var. chinensis (Rupr.) Reg. (Compositae) are used in traditional medicine to treat asthma, chronic bronchitis, and acute pleurisy in China and Korea. However, the pharmacological actions of Inula britannica L. var. chinensis on endothelial cells and inflammatory monocytes are not clear. In this study, we investigated whether bigelovin, a sesquiterpene lactone isolated from the flowers of Inula britannica L. var. chinensis, inhibits monocyte adhesion and adhesion molecule expression in brain endothelial cells. We measured tumor necrosis factor-alpha (TNF-alpha)-enhanced Raw264.7 monocyte binding to brain endothelial cells and the levels of cell adhesion molecules, including vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-selectin (E-selectin) on the surface of brain endothelial cells. Bigelovin significantly inhibited these in a dose-dependent manner without affecting cell viability. Furthermore, bigelovin suppressed the nuclear factor kappaB (NF-kappaB) promoter-driven luciferase activity, NF-kappaB activation, and degradation of NF-kappaB inhibitor protein alpha (IkappaBalpha). These results indicate that bigelovin inhibits inflammatory monocyte adhesion to endothelial cells and the expression of VCAM-1, ICAM-1, and E-selectin by blocking IkappaBalpha degradation and NF-kappaB activation.

  6. The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

    2015-01-01

    Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...... activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular...... intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded...

  7. FAK kinase activity is required for the progression of c-Met/β-catenin-driven HCC

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Zaidi, Ali; Cotler, Scott J.; Breslin, Peter; Ding, Xianzhong; Kuo, Paul; Nishimura, Michael; Zhang, Jiwang; Qiu, Wei

    2016-01-01

    Background & Aims There is an urgent need to develop new and more effective therapeutic strategies and agents to treat hepatocellular carcinoma (HCC). We have recently found that deletion of Fak in hepatocytes before tumors form inhibits tumor development and prolongs survival of animals in a c-Met (MET)/β-catenin (CAT)-driven HCC mouse model. However, it has yet to be determined whether FAK expression in hepatocytes promotes MET/CAT-induced HCC progression after tumor initiation. In addition, it remains unclear whether FAK promotes HCC development through its kinase activity. Methods We generated hepatocyte-specific inducible Fak-deficient mice (Alb-creERT2; Fakflox/flox) to examine the role of FAK in HCC progression. We re-expressed wild-type and mutant FAK in Fak-deficient mice to determine FAK’s kinase activity in HCC development. We also examined the efficacy of a FAK kinase inhibitor PF-562271 on HCC inhibition. Results We found that deletion of Fak after tumors form significantly repressed MET/CAT-induced tumor progression. Ectopic FAK expression restored HCC formation in hepatocyte-specific Fak-deficient mice. However, overexpression of a FAK kinase-dead mutant led to reduced tumor load compared to mice which express wild-type FAK. Furthermore, PF-562271 significantly suppressed progression of MET/CAT-induced HCC. Conclusion Fak kinase activity is important for MET/CAT-induced HCC progression. Inhibiting FAK kinase activity provides a potential therapeutic strategy to treat HCC. PMID:27142958

  8. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  9. Tec family kinases: regulation of FcεRI-mediated mast-cell activation.

    Science.gov (United States)

    Ellmeier, Wilfried; Abramova, Anastasia; Schebesta, Alexandra

    2011-06-01

    Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell. © 2011 The Authors Journal compilation © 2011 FEBS.

  10. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  11. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    Science.gov (United States)

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-07-06

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (PMRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. CONCLUSIONS This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis.

  12. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    DEFF Research Database (Denmark)

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically......, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation. RESEARCH DESIGN...... caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P...

  13. Involvement of the mitogen-activated protein kinase kinase 2 in the induction of cell dissociation in pancreatic cancer.

    Science.gov (United States)

    Tan, Xiaodong; Egami, Hiroshi; Kamohara, Hidenobu; Ishikawa, Shinji; Kurizaki, Takashi; Yoshida, Naoya; Tamori, Yasuhiko; Takai, Eiji; Hirota, Masahiko; Ogawa, Michio

    2004-01-01

    In our previous investigation, mitogen-activated protein kinase kinase 2 (MEK2) was detected as a factor which was correlated to the potential of invasion-metastasis. In this study, the immunocytochemical, immunohistochemical and mRNA expressions of MEK2 were examined in pancreatic cancer cell lines and tissue samples, respectively. Constitutive expressions of MEK2 and phosphorylated MEK (p-MEK) were observed in PC-1.0 and ASPC-1 cells, which exhibited a growth pattern of single cells, whereas the relevant expressions were quite faint in PC-1 cells and CAPAN-2 cells, which exhibited a growth pattern of island-like clonies. Simultaneous inductions of MEK2 expressions and cell dissociation were observed after the treatment with a conditioned medium (CM) of PC-1.0 cells. The expression of MEK2 and p-MEK were reduced and the cell aggregation was found in PC-1.0 and ASPC-1 cells after U0126 (a MEK inhibitor) treatment. In vivo, both the MEK2 and p-MEK overexpressed in human pancreatic cancer tissues and p-MEK was found to be more strongly expressed in the invasive front than that in the center of tumor (Pcell dissociation. MEK2 activation is probably involved in the first step of the cascade in the invasion-metastasis of pancreatic cancer.

  14. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  15. Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning.

    Science.gov (United States)

    Farley, J; Auerbach, S

    Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.

  16. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    Directory of Open Access Journals (Sweden)

    Xing-Xing Shi

    Full Text Available Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1 and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR, and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01. Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05. In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain.

  17. Activation of p38 and Erk Mitogen-Activated Protein Kinases Signaling in Ocular Rosacea.

    Science.gov (United States)

    Wladis, Edward J; Swamy, Supraja; Herrmann, Alyssa; Yang, Jinhong; Carlson, J Andrew; Adam, Alejandro P

    2017-02-01

    Rosacea-related cutaneous inflammation is a common cause of ocular surface disease. Currently, there are no specific pharmacologic therapies to treat ocular rosacea. Here, we aimed at determining the differences in intracellular signaling activity in eyelid skin from patients with and without ocular rosacea. This was an observational, comparative case series including 21 patients undergoing lower lid ectropion surgery at one practice during 2013 and 2014 (18 patients with rosacea, 13 control patients), and 24 paraffin-embedded archival samples from Albany Medical Center, selected randomly (12 patients with rosacea, 12 control patients). Cutaneous biopsies resulting from elective lower lid ectropion surgery were analyzed by Proteome Profiler Human Phospho-Kinase Array, Western blot, and/or immunohistochemistry. Samples derived from ocular rosacea patients showed increased levels of phosphorylated (active) p38 and Erk kinases. Phosphoproteins were mainly localized to the epidermis of affected eyelids. This finding provides a novel potential therapeutic target for treatment of ocular rosacea and possibly other forms of rosacea. Further testing is required to determine if p38 and Erk activation have a causal role in ocular rosacea. The selective activation of keratinocytes in the affected skin suggests that topical pathway inhibition may be an effective treatment that will ultimately prevent ocular surface damage due to ocular rosacea.

  18. Adhesion molecules levels in blood correlate with MRI activity and clinical activity in multiple sclerosis

    International Nuclear Information System (INIS)

    Millers, A.; Enina, G.; Platkajis, A.; Metra, M.; Kukaine, R.

    2002-01-01

    Research into pathogenesis of multiple sclerosis (MS) has prompted efforts to identify immunological markers associated with disease activity. Adhesion molecules ICAM-1 and VCAM-1 are associated with inflammatory mediated blood-brain barrier (BBB) dysfunction. In this study investigates the correlation between blood level of circulating ICAM-1 and VCAM-1 and magnetic resonance imaging (MRI) activity in different clinical phases of patients with MS. We show that RRMS and SPMS patients in clinically active phase with Gd-enhancing lesions in CNS had higher blood levels of cICAM-1 and cVCAM-1 compared these parameters levers of RRMS patients in remission stage. These results suggest that cICAM-1 and cVCAM-1 is a sensitive indicator of disease activity associated with BBB inflammatory dysfunction. Elevated blood level of cICAM-1 more strongly correlated with clinical activity and BBB damage, than cVCAM-1 and that could be used as biological marker of disease activity. Circulating VCAM-1 as an early indicator of BBB disturbance, may also serve as marker of beneficial activity in relapses phase of MS course. (authors)

  19. Polyunsaturated fatty acids induce ovarian cancer cell death through ROS-dependent MAP kinase activation.

    Science.gov (United States)

    Tanaka, Aiko; Yamamoto, Akane; Murota, Kaeko; Tsujiuchi, Toshifumi; Iwamori, Masao; Fukushima, Nobuyuki

    2017-11-04

    Free fatty acids not only play a role in cell membrane construction and energy production but also exert diverse cellular effects through receptor and non-receptor mechanisms. Moreover, epidemiological and clinical studies have so far suggested that polyunsaturated fatty acids (PUFAs) could have health benefits and the advantage as therapeutic use in cancer treatment. However, the underlying mechanisms of PUFA-induced cellular effects remained to be cleared. Here, we examined the effects of ω-3 and ω-6 PUFAs on cell death in ovarian cancer cell lines. ω-3 PUFA, docosahexaenoic acid (DHA) and ω-6 PUFA, γ-linolenic acid (γ-LNA) induced cell death in KF28 cells at the levels of physiological concentrations, but not HAC2 cells. Pharmacological and biochemical analyses demonstrated that cell death induced by DHA and γ-LNA was correlated with activation of JNK and p38 MAP kinases, and further an upstream MAP kinase kinase, apoptosis signal-regulating kinase 1, which is stimulated by reactive oxygen species (ROS). Furthermore, an antioxidant vitamin E attenuated PUFA-induced cell death and MAP kinase activation. These findings indicate that PUFA-induced cell death involves ROS-dependent MAP kinase activation and is a cell type-specific action. A further study of the underlying mechanisms for ROS-dependent cell death induced by PUFAs will lead to the discovery of a new target for cancer therapy or diagnosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    International Nuclear Information System (INIS)

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G.

    2006-01-01

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites

  1. Activation of the ATR kinase by the RPA-binding protein ETAA1

    DEFF Research Database (Denmark)

    Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

    2016-01-01

    Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway......, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate...... that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress....

  2. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    Science.gov (United States)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  3. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    International Nuclear Information System (INIS)

    Miyata, Yoshiki; Sato, Takashi; Ito, Akira

    2005-01-01

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH 2 -terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation

  4. Adhesion rings surround invadopodia and promote maturation

    Directory of Open Access Journals (Sweden)

    Kevin M. Branch

    2012-06-01

    Invasion and metastasis are aggressive cancer phenotypes that are highly related to the ability of cancer cells to degrade extracellular matrix (ECM. At the cellular level, specialized actin-rich structures called invadopodia mediate focal matrix degradation by serving as exocytic sites for ECM-degrading proteinases. Adhesion signaling is likely to be a critical regulatory input to invadopodia, but the mechanism and location of such adhesion signaling events are poorly understood. Here, we report that adhesion rings surround invadopodia shortly after formation and correlate strongly with invadopodium activity on a cell-by-cell basis. By contrast, there was little correlation of focal adhesion number or size with cellular invadopodium activity. Prevention of adhesion ring formation by inhibition of RGD-binding integrins or knockdown (KD of integrin-linked kinase (ILK reduced the number of ECM-degrading invadopodia and reduced recruitment of IQGAP to invadopodium actin puncta. Furthermore, live cell imaging revealed that the rate of extracellular MT1-MMP accumulation at invadopodia was greatly reduced in both integrin-inhibited and ILK-KD cells. Conversely, KD of MT1-MMP reduced invadopodium activity and dynamics but not the number of adhesion-ringed invadopodia. These results suggest a model in which adhesion rings are recruited to invadopodia shortly after formation and promote invadopodium maturation by enhancing proteinase secretion. Since adhesion rings are a defining characteristic of podosomes, similar structures formed by normal cells, our data also suggest further similarities between invadopodia and podosomes.

  5. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Xiong Zhang

    2014-11-01

    Full Text Available Embryonic stem (ES cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent

  6. The Cytolytically Inactive Terminal Complement Complex Activates Endothelial Cells to Express Adhesion Molecules and Tissue Factor Procoagulant Activity

    Science.gov (United States)

    Tedesco, Francesco; Pausa, Mario; Nardon, Ermanno; Introna, Martino; Mantovani, Alberto; Dobrina, Aldo

    1997-01-01

    The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC. PMID:9151899

  7. PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation

    DEFF Research Database (Denmark)

    Dovas, Athanassios; Yoneda, Atsuko; Couchman, John R

    2006-01-01

    necessary for the formation and maintenance of stress fibres in primary rat embryo fibroblasts. Inhibition of PKCalpha activity through the use of specific pharmacological inhibitors, a dominant-negative construct, or siRNA downregulation of protein levels, attenuated focal adhesion formation...

  8. Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens.

    Science.gov (United States)

    Singh, Tejinder P; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K

    2017-01-01

    Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were able to adhere to Caco-2 cells. L. reuteri strains tested were able to inhibit and displace ( P strain L. reuteri LR6 showed the strongest adhesion and pathogen inhibition ability among the eight L. reuteri strains tested. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting the involvement of various mechanisms. The adhesion and antagonistic potential of the probiotic strains were significantly decreased upon exposure to 5 M LiCl, showing that surface molecules, proteinaceous in nature, are involved. The heat-killed forms of the probiotic L. reuteri strains also inhibited the attachment of selected pathogens to Caco-2 cells. In conclusion, in vitro assays showed that L. reuteri strains, as viable or heat-killed forms, are adherent to Caco-2 cells and are highly antagonistic to pathogens tested in which surface associated proteins play an important role.

  9. Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose

    International Nuclear Information System (INIS)

    Craven, P.A.; DeRubertis, F.R.

    1989-01-01

    Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded

  10. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    International Nuclear Information System (INIS)

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-01-01

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  11. Is tyrosine kinase activation involved in basophil histamine release in asthma due to western red cedar?

    Science.gov (United States)

    Frew, A; Chan, H; Salari, H; Chan-Yeung, M

    1998-02-01

    Occupational asthma due to western red cedar is associated with histamine release from basophils and mast cells on exposure to plicatic acid (PA), but the mechanisms underlying this response remain unclear. Specific kinase inhibitors were used to study the role of tyrosine and serine/threonine kinases in PA-induced histamine release from human basophils. Pretreatment with the tyrosine kinase inhibitor methyl 2,5-dihydroxy-cinnamate (MDHC) attenuated histamine release from basophils triggered by anti-IgE (29.8% inhibition; n = 15; P < 0.01) or grass pollen (48% inhibition; n = 6; P < 0.01). Inhibition was concentration-dependent and could be reversed by washing the cells in buffer, while the inactive stereoisomer of MDHC did not affect histamine release. In contrast, the protein kinase C inhibitor staurosporine did not affect histamine release by either anti-IgE or grass pollen. Pretreatment with MDHC partially inhibited PA-induced histamine release from basophils of 6/9 patients with red cedar asthma (25.4% vs 33.8%; P = NS). Staurosporine gave a similar level of inhibition of PA-induced histamine release (25.3% vs 33.8%; P = NS). Thus, signal transduction of the human basophil Fc epsilon RI appears to depend upon tyrosine kinase activation, but not on protein kinase C (serine/threonine kinase) activation. The lack of specific effect on plicatic acid-induced histamine release in basophils obtained from patients with occupational asthma due to western red cedar suggests that tyrosine kinases are not as important in this disease as in atopic asthma, and is consistent with the view that histamine release in red cedar asthma is largely IgE-independent.

  12. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...

  13. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J. (Merck)

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  14. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    Directory of Open Access Journals (Sweden)

    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  15. EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion

    NARCIS (Netherlands)

    Sikkema, Arend H.; den Dunnen, Wilfred F. A.; Hulleman, Esther; van Vuurden, Dannis G.; Garcia-Manero, Guillermo; Yang, Hui; Scherpen, Frank J. G.; Kampen, Kim R.; Hoving, Eelco W.; Kamps, Willem A.; Diks, Sander H.; Peppelenbosch, Maikel P.; de Bont, Eve Line S. J. M.

    Eph/ephrin signaling has bcen implicated in various types of key cancer-enhancing processes, like migration, proliferation, and angiogenesis. In medulloblastoma, invading tumor cells characteristically lead to early recurrence and a decreased prognosis. Based on kinase-activity profiling data

  16. Phosphorylation and activation of protamine kinase by two forms of a myelin basic protein kinase from extracts of bovine kidney cortex.

    Science.gov (United States)

    Reddy, S A; Guo, H; Tarun, S Z; Damuni, Z

    1993-07-15

    Two myelin basic protein kinases designated MBPK-1 and MBPK-2 were purified to apparent homogeneity from extracts of bovine kidney cortex. The purified preparations exhibited an apparent M(r) approximately 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 42,000 (MBPK-1) and 45,000 (MBPK-2) by gel permeation chromatography. Up to 0.4 and 1.8 mol of phosphoryl groups were incorporated per mol of MBPK-1 and MBPK-2, respectively, on threonines following incubation with ATP. Autophosphorylation, incubation with protein phosphatase 2A2 (PP2A2), CD45, or T-cell protein tyrosine phosphatase did not affect MBPK-1 activity. Autophosphorylation increased by about 3-fold MBPK-2 activity. This autophosphorylation and activation was reversed by PP2A2 but not by CD45 or T-cell protein tyrosine phosphatase. MBPK-1 and MBPK-2 displayed a positive reaction with an antibody to mitogen-activated protein kinase. Purified preparations of protamine kinase were activated by about 1.5-6-fold and, after inactivation with PP2A2, were reactivated by about 30% by MBPK-1 and MBPK-2. Activation and reactivation correlated with the incorporation, respectively, of 0.1-0.5 and 0.5 mol of phosphoryl groups/mol of the protamine kinase on serines. The results show that MBPK-1 and MBPK-2 are protamine kinase-activating kinases and suggest that MBPK-1 and MBPK-2 may be related to mitogen-activated protein kinase.

  17. Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease

    NARCIS (Netherlands)

    Hommes, Daan; van den Blink, Bernt; Plasse, Terry; Bartelsman, Joep; Xu, Cuiping; Macpherson, Bret; Tytgat, Guido; Peppelenbosch, Mailkel; van Deventer, Sander

    2002-01-01

    Background & Aims: We investigated if inhibition of mitogen-activated protein kinases (MAPKs) was beneficial in Crohn's disease. Methods: Inhibition of JNK and p38 MAPK activation with CNI-1493, a guanylhydrazone, was tested in vitro. Twelve patients with severe Crohn's disease (mean baseline, CDAI

  18. Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

    Science.gov (United States)

    Carmena, Mar; Lombardia, Miguel Ortiz; Ogawa, Hiromi; Earnshaw, William C

    2014-11-01

    Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

  19. New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

    Science.gov (United States)

    De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

    2015-01-01

    Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge

  20. Activation of a calcium-dependent protein kinase involved in the Azospirillum growth promotion in rice.

    Science.gov (United States)

    Ribaudo, Claudia M; Curá, José A; Cantore, María L

    2017-02-01

    Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca 2+ -dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca 2+ -dependent protein kinase in promotion of rice plants growth by A. brasilense.

  1. Janus kinase/signal transducer and activator of transcription pathways in spondyloarthritis.

    Science.gov (United States)

    Raychaudhuri, Smriti K; Raychaudhuri, Siba P

    2017-07-01

    Cytokines are major drivers of autoimmunity, and biologic agents targeting cytokines have revolutionized the treatment of immune-mediated diseases. Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway represents a group of several intracellular molecules with a key role in signal pathways activated by growth factors and cytokines. These kinase proteins are associated with the signaling process of multiple key cytokines, which regulates various T-cell subpopulations and their effector cytokines. Development of novel drugs to inhibit this kinase cascade is an emerging field in clinical immunology. Thus, it is essential to have insights about the regulatory role of the JAK-STAT cytokine signaling in relation to autoimmune diseases and its applications in spondyloarthritis. JAK-STAT kinase signaling proteins have been extensively studied in rheumatoid arthritis. Initial observations suggest that JAK-STAT kinase signaling cascade regulates activation and proliferation of the IL17 effector memory T cells and thus has a potential role in the pathogenesis of psoriasis, psoriatic arthritis and ankylosing spondylitis. Here, we provide an overview of the clinical rheumatologists about the significance of JAK-STAT signaling system in rheumatic diseases and introduce the potential application of JAK and STAT inhibitors in spondyloarthritis.

  2. Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase

    NARCIS (Netherlands)

    van den Blink, B.; Branger, J.; Weijer, S.; Deventer, S. H.; van der Poll, T.; Peppelenbosch, M. P.

    2001-01-01

    All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to

  3. Mururins A-C, three new lignoids from Brosimum acutifolium and their protein kinase inhibitory activity.

    Science.gov (United States)

    Takashima, Junko; Asano, Shoichi; Ohsaki, Ayumi

    2002-07-01

    Two new flavonolignans, mururins A and B ( 1 and 2), and a new lignan, mururin C ( 3), were isolated from the bark of Brosimum acutifolium Huber together with three known lignans. Their structures were elucidated by spectroscopic means and chemical modifications. They were tested for protein kinase A (PKA) and protein kinase C (PKC) inhibitory activity. Mururin A showed 3 % and 63 % inhibition to PKA and PKC, respectively, at 20 microM. Mururin B showed 58 % and 38 % inhibition, respectively. Mururin C did not have significant activity.

  4. Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators.

    Science.gov (United States)

    Guigas, Bruno; Sakamoto, Kei; Taleux, Nellie; Reyna, Sara M; Musi, Nicolas; Viollet, Benoit; Hue, Louis

    2009-01-01

    5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.

  5. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  6. Nitric Oxide Binds to and Modulates the Activity of a Pollen Specific Arabidopsis Diacylglycerol Kinase

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  7. Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase

    International Nuclear Information System (INIS)

    Huang, C.S.; Ma, W.Y.; Ryan, C.A.; Dong, Z.G.

    1997-01-01

    Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation

  8. BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival.

    Science.gov (United States)

    Jacque, Emilie; Schweighoffer, Edina; Tybulewicz, Victor L J; Ley, Steven C

    2015-06-01

    B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers. © 2015 Jacque et al.

  9. Active p21-Activated Kinase 1 Rescues MCF10A Breast Epithelial Cells from Undergoing Anoikis

    Directory of Open Access Journals (Sweden)

    Raymond E. Menard

    2005-07-01

    Full Text Available The protein kinase, PAKi, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAKi in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADPribose polymerase (PARP, after more than 8 hours of detachment. Endogenous Akt, PAKi, BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAKi or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Cooverexpression of active PAKi with dominant-negative Akt, or of active Akt with dominant-negative PAKi, still suppresses anoikis. Thus, Akt and PAKi enhance survival through pathways that are at least partially independent. PAKi-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAKi increased the cleavage of the upstream caspase 9, while constitutively active PAKi inhibited caspase 9 activation. These results support a role for activated PAKi in the suppression of anoikis in MCF10A epithelial cells.

  10. Active p21-activated kinase 1 rescues MCF10A breast epithelial cells from undergoing anoikis.

    Science.gov (United States)

    Menard, Raymond E; Jovanovski, Andrew P; Mattingly, Raymond R

    2005-07-01

    The protein kinase, PAK1, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAK1 in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), after more than 8 hours of detachment. Endogenous Akt, PAK1, and BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAK1 or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Co-overexpression of active PAK1 with dominant-negative Akt, or of active Akt with dominant-negative PAK1, still suppresses anoikis. Thus, Akt and PAK1 enhance survival through pathways that are at least partially independent. PAK1-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAK1 increased the cleavage of the upstream caspase 9, while constitutively active PAK1 inhibited caspase 9 activation. These results support a role for activated PAK1 in the suppression of anoikis in MCF10A epithelial cells.

  11. Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kim, Tae Sung; Shin, Yern-Hyerk; Lee, Hye-Mi; Kim, Jin Kyung; Choe, Jin Ho; Jang, Ji-Chan; Um, Soohyun; Jin, Hyo Sun; Komatsu, Masaaki; Cha, Guang-Ho; Chae, Han-Jung; Oh, Dong-Chan; Jo, Eun-Kyeong

    2017-06-13

    The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. In this study, we present a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster-Mycobacterium marinum infection model, we showed that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. We further showed that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.

  12. Regulation of AMP-activated protein kinase by natural and synthetic activators

    Directory of Open Access Journals (Sweden)

    David Grahame Hardie

    2016-01-01

    Full Text Available The AMP-activated protein kinase (AMPK is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.

  13. MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

    DEFF Research Database (Denmark)

    Skov, S; Odum, Niels; Claesson, M H

    1995-01-01

    We have studied the biochemical signal pathway leading to a rise in intracellular free calcium concentration ([Ca2+]i) following cross-linking of MHC class I (MHC-I) molecules on human T leukemic Jurkat cells. Evidence is presented that MHC-I signaling is dependent on tyrosine kinase activity......). Collectively, these results indicate that the MHC-I signaling pathway is linked to activation of tyrosine kinase(s) in Jurkat cells....

  14. Protein-tyrosine kinase activity profiling in knock down zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Simone Lemeer

    Full Text Available BACKGROUND: Protein-tyrosine kinases (PTKs regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. METHODOLOGY: We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. RESULTS AND CONCLUSION: Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile

  15. Plant toxin β-ODAP activates integrin β1 and focal adhesion: A critical pathway to cause neurolathyrism.

    Science.gov (United States)

    Tan, Rui-Yue; Xing, Geng-Yan; Zhou, Guang-Ming; Li, Feng-Min; Hu, Wen-Tao; Lambein, Fernand; Xiong, Jun-Lan; Zhang, Sheng-Xiang; Kong, Hai-Yan; Zhu, Hao; Li, Zhi-Xiao; Xiong, You-Cai

    2017-01-17

    Neurolathyrism is a unique neurodegeneration disease caused by β-N-oxalyl-L-α, β- diaminopropionic (β-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, β-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca 2+ from mitochondria to cellular matrix. Increased Ca 2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of β1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

  16. Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress

    Science.gov (United States)

    Zagórska, Anna; Pozo-Guisado, Eulalia; Boudeau, Jérôme; Vitari, Alberto C.; Rafiqi, Fatema H.; Thastrup, Jacob; Deak, Maria; Campbell, David G.; Morrice, Nick A.; Prescott, Alan R.; Alessi, Dario R.

    2007-01-01

    Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress. PMID:17190791

  17. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    Science.gov (United States)

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2013-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α-AMP-activated protein kinase phosphorylation in mice sacrificed by cervical dislocation. Sacrifice of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation and similar levels of phosphorylation were observed compared to mice sacrificed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation, but phosphorylation was higher in mice sacrificed by cervical dislocation compared to mice sacrificed by focused microwave irradiation. These results demonstrate that α-AMP-activated protein kinase phosphorylation is dependent on method of sacrifice and tissue preparation and that α-AMP-activated protein kinase phosphorylation can increase in a manner that does not reflect biological alterations. PMID:18088373

  18. Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetables.

    Science.gov (United States)

    Feng, Junchang; Liu, Pilong; Yang, Xin; Zhao, Xin

    2015-12-01

    The purpose of this study was to select strains of lactic acid bacteria (LAB) by their in vitro adhesive and immunomodulatory properties for potential use as probiotics. In this study, 16 randomly selected LAB strains from fermented vegetables (sauerkraut, bean and cabbage) were first screened for their tolerance to acid, bile salts, pepsin and pancreatin, bacterial inhibitory activities and abilities to adherence to Caco-2 cells. Then, 4 strains with the highest adhesion abilities were selected for further studies of their immunomodulatory properties and inhibitory effects against Salmonella adhesion and invasion to Caco-2 cells in vitro. The results showed that these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of six tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of tumor necrosis factor-α and interleukin-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection.

  19. LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

    DEFF Research Database (Denmark)

    John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

    2010-01-01

    The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identifie...... for new therapeutic drugs against leishmaniasis....

  20. Involvement of the mitogen-activated protein (MAP kinase signalling pathway in host cell invasion by Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Robert-Gangneux F.

    2000-06-01

    Full Text Available Little is known about signalling in Toxoplasma gondii, but it is likely that protein kinases might play a key role in the parasite proliferation, differentiation and probably invasion. We previously characterized Mitogen-Activated Protein (MAP kinases in T. gondii lysates. In this study, cultured cells were tested for their susceptibility to Toxoplasma gondii infection after tachyzoite pretreatment with drugs interfering with AMP kinase activation pathways. Protein kinases inhibitors, i.e. genistein, R031-8220 and PD098059, reduced tachyzoite infectivity by 38 ± 4.5 %, 85.5 ± 9 % and 56 ± 10 %, respectively. Conversely, protein kinases activators, i.e. bombesin and PMA, markedly increased infectivity (by 202 ± 37 % and 258 ± 14 %, respectively. These results suggest that signalling pathways involving PKC and AAAP kinases play a role in host cell invasion by Toxoplasma.

  1. Activation and translocation of p38 mitogen-activated protein kinase after stimulation of monocytes with contact sensitizers.

    Science.gov (United States)

    Brand, Pia; Plochmann, Sibylle; Valk, Elke; Zahn, Sabine; Saloga, Joachim; Knop, Jürgen; Becker, Detlef

    2002-07-01

    Recently we described the induction of tyrosine phosphorylation by contact sensitizers as an early molecular event during the activation of antigen- presenting cells. In this study, the role of the p38 mitogen-activated protein kinase for the activation of human monocytes after exposure to four structurally unrelated contact sensitizers was analyzed in comparison with the irritant benzalkonium chloride and an inductor of oxidative stress (H2O2) using immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay techniques. Bio chemical analysis revealed a translocation of p38 from the cytoplasm to the detergent-resistant cell fraction only upon stimulation with contact sensitizers. The activity of p38 was studied by quantification of its phosphorylated active form with a specific antibody and by kinase assay. Although all stimulants used in this study led to the activation of p38, a translocation to the detergent-resistant fraction as well phosphorylation of the mitogen-activated protein kinase dependent transcription factor Elk-1 was induced only by contact sensitizers. Evidence for a functional relevance of mitogen-activated protein kinase activation was provided by measurement of the hapten-induced production of the proinflammatory cytokine interleukin-1beta. Its release was inhibited by blocking p38-mediated signaling using the imidazole compounds SB203580 and SB202190. These data show that contact sensitizers are strong activators of the p38 mitogen-activated protein kinase. Although activation of this stress-associated pathway has been reported for many other stimuli, a unique translocation of p38 from the cytoplasm to the detergent-resistant fraction seems to be a specific event during hapten-induced activation of antigen-presenting cells.

  2. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  3. Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase.

    Science.gov (United States)

    Yamamoto, Naohiro; Otsuka, Takanobu; Kuroyanagi, Gen; Kondo, Akira; Kainuma, Shingo; Nakakami, Akira; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Tokuda, Haruhiko

    2015-01-01

    Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. ADHESIVES WITH DIFFERENT PHS: EFFECT ON THE MTBS OF CHEMICALLY ACTIVATED AND LIGHT-ACTIVATED COMPOSITES TO HUMAN DENTIN

    Science.gov (United States)

    Mallmann, André; de Melo, Renata Marques; Estrela, Verbênia; Pelogia, Fernanda; Campos, Laura; Bottino, Marco Antonio; Valandro, Luiz Felipe

    2007-01-01

    Purpose: To evaluate the bond strength between human dentin and composites, using two light-activated single-bottle total-etch adhesive systems with different pHs combined with chemically activated and light-activated-composites. The tested hypothesis was that the dentin bond strength is not influenced by an adhesive system of low pH, combined with chemically activated or light-activated composites. Material and Method: Flat dentin surfaces of twenty-eight human third molars were allocated in 4 groups (n=7), depending on the adhesive system: (One Step Plus-OS and Prime & Bond NT-PB) and composite (light-activated Filtek Z-100 [Z100] and chemically activated Bisfil 2B [B2B]). Each adhesive system was applied on acid-etched dentin and then one of the composites was added to form a 5 mm-high resin block. The specimens were stored in tap water (37°C/24 h) and sectioned into two axes, x and y. This was done with a diamond disk under coolant irrigation to obtain beams with a cross-section area of approximately 0.8 mm2. Each specimen was then attached to a custom-made device and submitted to the microtensile test (1 mm.min−1). Data were analyzed using two-way ANOVA and Tukey’s tests (p<0.05). Results: The anticipated hypothesis was not confirmed (p<0.0001). The bond strengths (MPa) were not statistically different between the two adhesive systems when light-activated composite was used (OS+Z100 = 24.7±7.1ª; PB+Z100 = 23.8±5.7ª). However, with use of the chemically activated composite (B2B), PB (7.8±3.6b MPa) showed significantly lower dentin bond strengths than OS (32.2±7.6ª). Conclusion: The low pH of the adhesive system can affect the bond of chemically activated composite to dentin. On the other hand, under the present conditions, the low pH did not seem to affect the bond of light-activated composites to dentin significantly. PMID:19089142

  5. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

    OpenAIRE

    Chouinard, Nadine; Valerie, Kristoffer; Rouabhia, Mahmoud; Huot, Jacques

    2002-01-01

    Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity ...

  6. 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.

  7. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    Science.gov (United States)

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.

  8. Structures of the inactive and active states of RIP2 kinase inform on the mechanism of activation.

    Directory of Open Access Journals (Sweden)

    Erika Pellegrini

    Full Text Available Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.

  9. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    DEFF Research Database (Denmark)

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...

  10. Fueling the engine: induction of AMP-activated protein kinase in trout skeletal muscle by swimming

    NARCIS (Netherlands)

    Magnoni, L.J.; Palstra, A.P.; Planas, J.V.

    2014-01-01

    AMP-activated protein kinase (AMPK) is well known to be induced by exercise and to mediate important metabolic changes in the skeletal muscle of mammals. Despite the physiological importance of exercise as a modulator of energy use by locomotory muscle, the regulation of this enzyme by swimming has

  11. Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation.

    Science.gov (United States)

    Tisdale, Ellen J; Shisheva, Assia; Artalejo, Cristina R

    2014-06-01

    Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration. Copyright © 2014. Published by Elsevier Inc.

  12. Protein kinase C activity is a protective modifier of Purkinje neuron degeneration in cerebellar ataxia

    NARCIS (Netherlands)

    Chopra, Ravi; Wasserman, Aaron H; Pulst, Stefan M; De Zeeuw, Chris I; Shakkottai, Vikram G

    2018-01-01

    Among the many types of neurons expressing protein kinase C (PKC) enzymes, cerebellar Purkinje neurons are particularly reliant on appropriate PKC activity for maintaining homeostasis. The importance of PKC enzymes in Purkinje neuron health is apparent as mutations in PRKCG (encoding PKCγ) cause

  13. Immunohistochemical analysis of receptor tyrosine kinase signal transduction activity in chordoma.

    Science.gov (United States)

    Fasig, J H; Dupont, W D; LaFleur, B J; Olson, S J; Cates, J M M

    2008-02-01

    Currently, there are no effective chemotherapeutic protocols for chordoma. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumours may respond to kinase inhibitor therapy. However, RTK signalling activity has not been extensively investigated in chordoma. A tissue microarray containing 21 cases of chordoma was analysed for expression of a number of proteins involved in signal transduction from RTKs by immunohistochemistry. Platelet-derived growth factor receptor-beta, epidermal growth factor receptor (EGFR), KIT and HER2 were detected in 100%, 67%, 33% and 0% of cases, respectively. Platelet-derived growth factor receptor-beta staining was of moderate-to-strong intensity in 20 of 21 cases. In contrast, KIT immunoreactivity was weak and focal in each of the seven positive cases. Total EGFR staining was variable; weak staining for phosphorylated EGFR was detected in nine cases. Phosphorylated isoforms of p44/42 mitogen-activated protein kinase, Akt and STAT3, indicative of tyrosine kinase activity, were detected in 86%, 76% and 67% of cases, respectively. Chordomas commonly express RTKs and activated signal transduction molecules. Although there were no statistically significant correlations between the expression of any of the markers studied and disease-free survival or tumour location, the results nonetheless indicate that chordomas may respond to RTK inhibitors or modulators of other downstream signalling molecules.

  14. Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

    Directory of Open Access Journals (Sweden)

    Linghui Kong

    Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

  15. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

  16. Role of 5'AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Wojtaszewski, Jørgen F. P.

    2008-01-01

    5'AMP-activated protein kinase (AMPK) is recognized as an important intracellular energy sensor, shutting down energy-consuming processes and turning on energy-generating processes. Discovery of target proteins of AMPK has dramatically increased in the past 10 years. Historically, AMPK was first...

  17. Expression of adhesion and activation molecules on lymphocytes during open-heart surgery with cardiopulmonary bypass

    DEFF Research Database (Denmark)

    Toft, P; Tønnesen, Else Kirstine; Zülow, I

    1997-01-01

    -heart surgery and eight with abdominal surgery were studied. The adhesion molecules CD11a/CD18 (LFA-1_, CD11c/CD18 and CD44 and the activation molecules CD25, CD69, CD71 and MHCII were measured, using monoclonal antibodies and flow cytometry. Lymphocytopenia was observed during CPB and for some hours after both...... open-heart and abdominal surgery. The proportion of CD11a/CD18-positive lymphocytes rose from 67.6 +/- 8% to 86.4 +/- 3% after aortic declamping (p ... was associated with increased expression of the adhesion molecule CD11a/CD18 on lymphocytes, while the expression of activation molecules on lymphocytes was unchanged....

  18. A conserved cysteine motif is critical for rice ceramide kinase activity and function.

    Directory of Open Access Journals (Sweden)

    Fang-Cheng Bi

    Full Text Available Ceramide kinase (CERK is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare and investigate the effects of ceramides on rice cell viability.OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.

  19. SI113, a Specific Inhibitor of the Sgk1 Kinase Activity that Counteracts Cancer Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Lucia D''Antona

    2015-03-01

    Full Text Available Background/Aims: Published observations on serum and glucocorticoid regulated kinase 1 (Sgk1 knockout murine models and Sgk1-specific RNA silencing in the RKO human colon carcinoma cell line point to this kinase as a central player in colon carcinogenesis and in resistance to taxanes. Methods: By in vitro kinase activity inhibition assays, cell cycle and viability analysis in human cancer model systems, we describe the biologic effects of a recently identified kinase inhibitor, SI113, characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold, that shows specificity for Sgk1. Results: SI113 was able to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. In RKO cells, this kinase inhibitor blocked insulin-dependent phosphorylation of the Sgk1 substrate Mdm2, the main regulator of p53 protein stability, and induced necrosis and apoptosis when used as a single agent. Finally, SI113 potentiated the effects of paclitaxel on cell viability. Conclusion: Since SI113 appears to be effective in inducing cell death in RKO cells, potentiating paclitaxel sensitivity, we believe that this new molecule could be efficiently employed, alone or in combination with paclitaxel, in colon cancer chemotherapy.

  20. 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 ...

  1. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  2. Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

    Science.gov (United States)

    Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

    2009-06-20

    The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

  3. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  4. Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

    Directory of Open Access Journals (Sweden)

    Abhisheka Bansal

    2016-12-01

    Full Text Available We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs. Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1. The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2, a specific inhibitor of protein kinase G (PKG. These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes.

  5. Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells.

    Science.gov (United States)

    Aoki, Koichi; Yokosuka, Akihito; Mimaki, Yoshihiro; Fukunaga, Kohji; Yamakuni, Tohru

    2013-01-01

    Ras, a small G-protein, physiologically directs cell proliferation and cell cycle via regulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Dysregulation of Ras/MEK/ERK signaling has been reported to cause tumorigenesis and gliomas. Nobiletin, a citrus flavonoid, has been shown to have anti-tumor cells action. However, it remains elusive whether nobiletin could affect Ras activity. In this study, we provide the first evidence that nobiletin suppresses the proliferation by inhibiting Ras activity in C6 glioma cells, a rat glioma cell line. First, Ras pull-down assay showed that nobiletin inhibits Ras activity in a concentration-dependent manner in C6 cells. Second, farnesyltransferase inhibitor I, a Ras inhibitor, and U0126, a MEK inhibitor, induced an inhibition of the cell proliferation in C6 cells, while the cell proliferation was inhibited by nobiletin as well. Third, western blotting revealed that nobiletin showed inhibitory effects on MEK and ERK phopsphorylation levels in a concentration-dependent manner. Finally, such an inhibitory effect on the level of ERK phosphorylation by nobiletin was appreciably prevented by Gö6976, a selective inhibitor of conventional protein kinase Cs (PKCs) showing Ca(2+)-sensitivity, while GF109203X, a general inhibitor for PKCs, and BAPTA, a cell-permeable Ca(2+) chelator, to a lesser extent, suppressed a reduction of the phosphorylation. These findings suggest that the proliferation of C6 cells is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the cell proliferation by inhibiting Ras activity and MEK/ERK signaling cascade probably via a Ca(2+)-sensitive PKC-dependent mechanism. Thus, the natural compound has potential to be a therapeutic agent for glioma.

  6. Creatine supplementation: effects on blood creatine kinase activity responses to resistance exercise and creatine kinase activity measurement

    Directory of Open Access Journals (Sweden)

    Marco Machado

    2009-12-01

    Full Text Available The purpose of this study was to determine the effects of creatine supplementation and exercise on the integrity of muscle fiber, as well as the effect of the supplementation on the creatine kinase (CK assay measurement. Forty-nine sedentary individuals participated in a double-blind study and were divided into two groups: C (n=26 received 4x5-day packages of 0.6 g.kg-1 of body weight contained 50% of creatine + 50% of dextrose, and P (n=23 received packages containing only dextrose. On the first day the groups performed a 1RM test for bench press, seated row, leg extension, leg curl and leg press. On D7 they received the supplements. On the fourteenth day, they performed a training session of five exercises, each in three sets of ten repetitions at 75% of 1RM. Blood was collected before (D14 and after the exercise session (D15. Differing levels of blood creatine were tested to determine the influence on the assay measurements of CK. ANOVA and Tukey's post-hoc tests were used to compare groups and different times of study protocol (PO objetivo do presente estudo foi determinar o efeito da suplementação de creatina e do exercício na integridade das fibras musculares e, também, o efeito da suplementação na técnica de mensuração da atividade da creatina kinase (CK. Quarenta e nove sedentários participaram de um estudo duplo-cego e foram divididos em dois grupos: C (n=26 que receberam 4x5 dias embalagens com 0,6 g.kg-1 de massa corporal com 50% de creatina + 50% de dextrose, e P (n=23 que receberam embalagens contendo apenas dextrose. No primeiro dia, eles realizaram o teste de 1RM para os exercícios supino reto, remada sentada, cadeira extensora, mesa flexora, e leg press. No D7 receberam os suplementos. No décimo quarto dia eles realizaram uma sessão de treinos com os cinco exercícios, cada um com 3x10 repetições a 75% de 1RM. Sangue foi coletado antes (D14 e depois da sessão de exercícios (D15. Diferentes concentrações de

  7. Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment

    Directory of Open Access Journals (Sweden)

    Bruno Aquino

    2017-05-01

    Full Text Available Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK, have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK, the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1 is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists.

  8. Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

    Science.gov (United States)

    González, R; Carvajal, N; Morán, A

    1984-01-01

    In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

  9. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    International Nuclear Information System (INIS)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-01-01

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

  10. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  11. 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.

  12. Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

    LENUS (Irish Health Repository)

    McEneaney, Victoria

    2010-01-01

    Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1\\/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1\\/2-dependent. Aldosterone induced the rapid activation of ERK1\\/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1\\/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1\\/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1\\/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1\\/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1\\/2 was inhibited in cells suppressed in the expression of PKD1.

  13. Structural insights into the architecture and allostery of full-length AMP-activated protein kinase.

    Science.gov (United States)

    Zhu, Li; Chen, Lei; Zhou, Xiao-Ming; Zhang, Yuan-Yuan; Zhang, Yi-Jiong; Zhao, Jing; Ji, Shang-Rong; Wu, Jia-Wei; Wu, Yi

    2011-04-13

    AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy homeostasis. However, the molecular architecture of the intact complex and the allostery associated with the adenosine binding-induced regulation of kinase activity remain unclear. Here, we determine the three-dimensional reconstruction and subunit organization of the full-length rat AMPK (α1β1γ1) through single-particle electron-microscopy. By comparing the structures of AMPK in ATP- and AMP-bound states, we are able to visualize the sequential conformational changes underlying kinase activation that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. These results not only make substantial revision to the current model of AMPK assembly, but also highlight a central role of the linker sequence of the α subunit in mediating the allostery of AMPK. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase.

    Science.gov (United States)

    Pazos, Elena; Vázquez, M Eugenio

    2014-02-01

    Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide-based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide-based luminescent peptide sensors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Loss of ATM kinase activity leads to embryonic lethality in mice.

    Science.gov (United States)

    Daniel, Jeremy A; Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V; You, Zhongsheng; Paull, Tanya T; Sleckman, Barry P; Feigenbaum, Lionel; Nussenzweig, André

    2012-08-06

    Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

  16. Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase.

    OpenAIRE

    Tang, Xiuwen; Wang, Lijun; Proud, Christopher G; Downes, C Peter

    2003-01-01

    In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammali...

  17. Acetylcorynoline impairs the maturation of mouse bone marrow-derived dendritic cells via suppression of IκB kinase and mitogen-activated protein kinase activities.

    Directory of Open Access Journals (Sweden)

    Ru-Huei Fu

    Full Text Available BACKGROUND: Dendritic cells (DCs are major modulators in the immune system. One active field of research is the manipulation of DCs as pharmacological targets to screen novel biological modifiers for the treatment of inflammatory and autoimmune disorders. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. We assessed the capability of acetylcorynoline to regulate lipopolysaccharide (LPS-stimulated activation of mouse bone marrow-derived DCs. METHODOLOGY/PRINCIPAL FINDINGS: Our experimental data showed that treatment with up to 20 µM acetylcorynoline does not cause cytotoxicity in cells. Acetylcorynoline significantly inhibited the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also decreased by acetylcorynoline, and the endocytic capacity of LPS-stimulated DCs was restored by acetylcorynoline. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was blocked by acetylcorynoline, and the migratory ability of LPS-stimulated DCs was reduced by acetylcorynoline. Moreover, acetylcorynoline significantly inhibits LPS-induced activation of IκB kinase and mitogen-activated protein kinase. Importantly, administration of acetylcorynoline significantly attenuates 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity. CONCLUSIONS/SIGNIFICANCE: Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of DC maturation and function.

  18. In vivo effects of vanadium in diabetic rats are independent of changes in PI-3 kinase activity in skeletal muscle.

    Science.gov (United States)

    Mohammad, A; Bhanot, S; McNeill, J H

    2001-07-01

    The PI-3 kinase signalling pathway is an important pathway in mediating the glucoregulatory effects of insulin and skeletal muscle (SKM) is the major tissue involved in glucose utilization. In diabetes this pathway is impaired, either due to lack of insulin as in Type I diabetes, or due to insulin resistance as in Type 2 diabetes. Bis(maltolato)-oxovanadium IV (BMOV), an insulin mimetic/enhancing agent, produces a marked glucose lowering effect in models of both types of diabetes. Some in vitro studies have shown that phosphatidylinositol 3 kinase (PI-3 kinase) activity is enhanced by vanadium. In the present study we looked at changes in PI-3 kinase expression and activity in SKM from STZ-diabetic and fa/fa Zucker rats treated with BMOV for 3 weeks. Although BMOV treatment completely normalized glucose levels in STZ-diabetic rats, no effect was observed on basal or insulin-stimulated PI-3 kinase activity. In fatty Zucker rats, activation of PI-3 kinase activity after insulin injection was impaired as compared to age matched lean controls, but BMOV again did not affect the activity. These results suggest that although PI-3 kinase is an important signalling factor in glucose utilization, vanadium treatment does not reduce hyperglycemia through activation of SKM PI-3 kinase in vivo.

  19. Activation loop dynamics determine the different catalytic efficiencies of B cell- and T cell-specific tec kinases.

    Science.gov (United States)

    Joseph, Raji E; Kleino, Iivari; Wales, Thomas E; Xie, Qian; Fulton, D Bruce; Engen, John R; Berg, Leslie J; Andreotti, Amy H

    2013-08-27

    Itk (interleukin-2-inducible T cell kinase) and Btk (Bruton's tyrosine kinase) are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of 6 of the 619 amino acid residues of Itk with the corresponding residues of Btk (and vice versa) was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties, depending on the cellular context. We suggest that the weaker catalytic activities of T cell-specific kinases serve to regulate cellular activation and prevent aberrant immune responses.

  20. The Oncogenic Lung Cancer Fusion Kinase CD74-ROS Activates a Novel Invasiveness Pathway through E-Syt1 Phosphorylation

    OpenAIRE

    Johnson, Hannah; White, Forest M.; Jun, Hyun Jung; Bronson, Roderick T.; de Feraudy, Sebastien; Charest, Alain

    2012-01-01

    Patients with lung cancer often present with metastatic disease and therefore have a very poor prognosis. The recent discovery of several novel ROS receptor tyrosine kinase molecular alterations in non-small-cell lung cancer (NSCLC) presents a therapeutic opportunity for the development of new targeted treatment strategies. Here, we report that the NSCLC-derived fusion CD74-ROS, which accounts for 30% of all ROS fusion kinases in NSCLC, is an active and oncogenic tyrosine kinase. We found tha...

  1. Dual inhibition of MET and SRC kinase activity as a combined targeting strategy for colon cancer.

    Science.gov (United States)

    Song, Na; Qu, Xiujuan; Liu, Shizhou; Zhang, Simeng; Liu, Jing; Qu, Jinglei; Zheng, Huachuan; Liu, Yunpeng; Che, Xiaofang

    2017-08-01

    Hepatocyte growth factor (HGF)/MET signaling is implicated in the development of colorectal cancer (CRC) and possesses therapeutic value for various types of cancer. However, inhibition of MET alone has been demonstrated to have limited efficacy. The present study examined the combined inhibition of MET and SRC kinase activity in colon cancer cells. Furthermore, the role of the HGF/MET pathway in ligand-dependent and -independent activation was demonstrated. The single inhibition of MET by knockdown small interfering RNA or inhibitor indicated a limited anti-viability effects without inhibiting the basal phosphorylation levels of SRC, protein kinase B (AKT) or extracellular signal-regulated kinase (ERK). In view of the strong association between MET and SRC identified by direct regulation, growth factor-induced MET activation was suppressed by pretreatment with the SRC inhibitor, dasatinib, and downstream phosphorylation of AKT and ERK partially decreased, which suggested that SRC activation was essential for ligand-dependent and -independent activation of MET. Considering that both the activation of MET and SRC was required in ligand-dependent and -independent MET activation, the antitumor effect of concurrent inhibition of MET and SRC was examined, and it was demonstrated that combination treatment exerted increased viability inhibition and apoptosis enhancement in mutant and wild type RAS colon cancer cells. Therefore, combinational inhibition of MET and SRC may be a promising strategy for the treatment of CRC.

  2. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  3. Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.

    Science.gov (United States)

    Liu, Yin; Belkina, Natalya V; Graham, Caroline; Shaw, Stephen

    2006-04-28

    Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

  4. Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

    OpenAIRE

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2006-01-01

    Abstract Background AMP-activated protein kinase (AMPK) has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data ar...

  5. Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca2+/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain.

    Science.gov (United States)

    Kratimenos, Panagiotis; Koutroulis, Ioannis; Jain, Amit; Malaeb, Shadi; Delivoria-Papadopoulos, Maria

    2018-01-01

    Hypoxia-ischemia (HI) results in increased activation of Ca2+/calmodulin kinase IV (CaM kinase IV) mediated by Src kinase. Therapeutic hypothermia ameliorates neuronal injury in the newborn. Inhibition of Src kinase concurrently with hypothermia further attenuates the hypoxia-induced increased activation of CaM kinase IV compared with hypothermia alone. Ventilated piglets were exposed to HI, received saline or a selective Src kinase inhibitor (PP2), and were cooled to 33°C. Neuropathology, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and CaM kinase IV activity were determined. The neuropathology mean score (mean ± SD) was 0.4 ± 0.43 in normoxia-normothermia (p Src kinase inhibitor were comparable in the levels of ATP and PCr, indicating that they were similar in their degree of energy failure prior to treatments. Hypothermia or Src kinase inhibitor (PP2) did not restore the ATP and PCr levels. Hypothermia and Src kinase inhibition attenuated apoptotic cell death and improved neuropathology after hypoxia. The combination of short-duration hypothermia with Src kinase inhibition following hypoxia further attenuates the increased activation of CaM kinase IV compared to hypothermia alone in the newborn swine brain. © 2017 S. Karger AG, Basel.

  6. Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity

    Directory of Open Access Journals (Sweden)

    Wynn Michelle L

    2011-10-01

    Full Text Available Abstract Background It has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone. Results We used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator. Conclusions A proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective

  7. MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism.

    Directory of Open Access Journals (Sweden)

    Laura P Bernard

    Full Text Available The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs, are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer's disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA, through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.

  8. [Effects of polydatin on learning and memory and Cdk5 kinase activity in the hippocampus of rats with chronic alcoholism].

    Science.gov (United States)

    Li, Xin-juan; Zhang, Yan; Xu, Chun-yang; Li, Shuang; Du, Ai-lin; Zhang, Li-bin; Zhang, Rui-ling

    2015-03-01

    To observe the effects of polydatin on learning and memory and cyclin-dependent kinase 5 (Cdk5) kinase activity in the hippocampus of rats with chronic alcoholism. Forty rats were randomly divided into 4 groups: control group, chronic alcoholism group, low and high polydatin group. The rat chronic alcoholism model was established by ethanol 3.0 g/(kg · d) (intragastric administration). The abstinence scoring was used to evaluate the rats withdrawal symptoms; cognitive function was measured by Morris water maze experiment; Cdk5 protein expression in the hippocampus was detected by immunofluorescence; Cdk5 kinase activity in the hippocampus was detected by liquid scintillation counting method. The abstinence score, escape latency, Cdk5 kinase activity in chronic alcoholism group rats were significantly higher than those of control group (P alcoholism group (P alcoholism group( P alcoholism group were significantly increased compared with control group (P alcoholism group ( P alcoholism damage may interrelate with regulation of Cdk5 kinase activity.

  9. Structure and Ubiquitination-Dependent Activation of TANK-Binding Kinase 1

    Directory of Open Access Journals (Sweden)

    Daqi Tu

    2013-03-01

    Full Text Available Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1 induces type I interferon expression and modulates nuclear factor κB (NF-κB signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ∊ (IKK∊ but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

  10. Structure and ubiquitination-dependent activation of TANK-binding kinase 1.

    Science.gov (United States)

    Tu, Daqi; Zhu, Zehua; Zhou, Alicia Y; Yun, Cai-hong; Lee, Kyung-Eun; Toms, Angela V; Li, Yiqun; Dunn, Gavin P; Chan, Edmond; Thai, Tran; Yang, Shenghong; Ficarro, Scott B; Marto, Jarrod A; Jeon, Hyesung; Hahn, William C; Barbie, David A; Eck, Michael J

    2013-03-28

    Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  11. HYDROGEN PEROXIDE PRODUCTION ACTIVITY AND ADHESIVE PROPERTIES OF AEROCOCCI, ISOLATED IN WOMEN

    Directory of Open Access Journals (Sweden)

    Stepanskyi D.O.

    2017-06-01

    Full Text Available Introduction. Antagonistic activity of probiotic microorganisms against other species of bacteria is an important mechanism of their ecology and it is widely used in practice. This activity is inherent in many heme-deficient bacteria, which include aerococci, and can be composed of several components: the production of organic acids, antibiotics, lysozyme, hydrogen peroxide and others. Ability to produce hydrogen peroxide under aerobic conditions and in a state of relative anaerobiosis was established in aerococci. They were divided into strong and weak producers, depending on the amount of peroxides. Lack of data about peroxide-productive ability of aerococci, isolated from the lower genital tract of women, as well as a proven mechanism of hydrogen peroxide excretion in the oxidation of lactic acid, led to need in studying the aerococci hydrogen peroxide production level, to create autobacterial drugs, based on aerococci symbiont strains for sanitation of birth canal. Colonization resistance of the vaginal mucous and normal microflora value depends largely on the degree of adhesion of microbial cells to the mucosal surface. Along with numerous studies of lactobacilli adhesive properties to the vaginal epithelium, there are no data on the adsorption capacity of aerococci to the vaginal epithelial cells. Material and methods. 18 aerococci resident strains and 1 museum strain were explored in total. Presence and quantity of autosymbiont aerococci content in different parts of the birth tract (cervical canal, vagina, external genitalia skin (EGS and perineum was studied in 44 healthy women. Isolation and identification of aerococci from the women body was conducted by the method, taking into account growth on selective indicator medium, growth and biochemical activity in environments with selenium and tellurium salts, lactate oxidase and superoxide dismutase activity. Hydrogen peroxide was determined by iodometric method. Hydrogen peroxide

  12. Preventing Pseudomonas aeruginosa and Chromobacterium violaceum infections by anti-adhesion-active components of edible seeds

    Directory of Open Access Journals (Sweden)

    Rachmaninov Ofra

    2012-02-01

    Full Text Available Abstract Background Pseudomonas aeruginosa adhesion to animal/human cells for infection establishment involves adhesive proteins, including its galactose- and fucose-binding lectins PA-IL (LecA and PA-IIL (LecB. The lectin binding to the target-cell receptors may be blocked by compatible glycans that compete with those of the receptors, functioning as anti-adhesion glycodecoys. The anti-adhesion treatment is of the utmost importance for abrogating devastating antibiotic-resistant P. aeruginosa infections in immunodeficient and cystic fibrosis (CF patients. This strategy functions in nature in protecting embryos and neonates. We have shown that PA-IL, PA-IIL, and also CV-IIL (a PA-IIL homolog produced in the related pathogen Chromobacterium violaceum are highly useful for revealing natural glycodecoys that surround embryos in diverse avian eggs and are supplied to neonates in milks and royal jelly. In the present study, these lectins were used as probes to search for seed embryo-protecting glycodecoys. Methods The lectin-blocking glycodecoy activities were shown by the hemagglutination-inhibition test. Lectin-binding glycoproteins were detected by Western blotting with peroxidase-labeled lectins. Results The present work reports the finding - by using PA-IL, PA-IIL, and CV-IIL - of rich glycodecoy activities of low ( 10 kDa compounds (including glycoproteins in extracts of cashew, cocoa, coffee, pumpkin, and tomato seeds, resembling those of avian egg whites, mammal milks, and royal jelly. Conclusions Edible seed extracts possess lectin-blocking glycodecoys that might protect their embryos from infections and also might be useful for hampering human and animal infections.

  13. Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

    Science.gov (United States)

    Lim, M A; Selak, M A; Xiang, Z; Krainc, D; Neve, R L; Kraemer, B C; Watts, J L; Kalb, R G

    2012-01-18

    A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

  14. Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

    Directory of Open Access Journals (Sweden)

    Shepherd Trevor G

    2010-02-01

    Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

  15. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

    Directory of Open Access Journals (Sweden)

    Nasar Virk

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

  16. Casein kinase II activity in the brain of an insect, Acheta domesticus: characterization and hormonal regulation.

    Science.gov (United States)

    Degrelle, F; Renucci, M; Charpin, P; Tirard, A

    1997-01-01

    This study documented casein kinase II (CK II) activity in Acheta domesticus brain using specific antibodies and its regulation by polyamines. In control animals a transient decrease in CK II activity at day 3 after imaginal moult was observed in the brain but not in the fat body. If deprived of ecdysone by ovariectomy a different pattern was observed, with CK II activity being significantly higher on days 3 and 4 after emergence. After ecdysone injection in ovariectomized females, CK II activity decreased to levels similar to those in controls. The implications of ecdysone regulation of brain CK II activity are discussed.

  17. Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

    Directory of Open Access Journals (Sweden)

    Silvia Volpe

    Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

  18. Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development.

    Science.gov (United States)

    Krishnamurthy, Vishnu V; Turgeon, Aurora J; Khamo, John S; Mondal, Payel; Sharum, Savanna R; Mei, Wenyan; Yang, Jing; Zhang, Kai

    2017-06-15

    Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

  19. Insulin deficiency results in reversible protein kinase A activation and tau phosphorylation.

    Science.gov (United States)

    van der Harg, Judith M; Eggels, Leslie; Bangel, Fabian N; Ruigrok, Silvie R; Zwart, Rob; Hoozemans, Jeroen J M; la Fleur, Susanne E; Scheper, Wiep

    2017-07-01

    Alzheimer's disease (AD) is a highly prevalent multifactorial disease for which Diabetes Mellitus (DM) is a risk factor. Abnormal phosphorylation and aggregation of tau is a key hallmark of AD. In animal models, DM induces or exacerbates the phosphorylation of tau, suggesting that DM may influence the risk at AD by directly facilitating tau pathology. Previously we reported that tau phosphorylation induced in response to metabolic stress is reversible. Since identification and understanding of early players in tau pathology is pivotal for therapeutic intervention, we here investigated the mechanism underlying tau phosphorylation in the diabetic brain and its potential for reversibility. To model DM we used streptozotocin-treatment to induce insulin deficiency in rats. Insulin depletion leads to increased tau phosphorylation in the brain and we investigated the activation status of known tau kinases and phosphatases in this model. We identified protein kinase A (PKA) as a tau kinase activated by DM in the brain. The potential relevance of this signaling pathway to AD pathogenesis is indicated by the increased level of active PKA in temporal cortex of early stage AD patients. Our data indicate that activation of PKA and tau phosphorylation are associated with insulin deficiency per se, rather than the downstream energy deprivation. In vitro studies confirm that insulin deficiency results in PKA activation and tau phosphorylation. Strikingly, both active PKA and induced tau phosphorylation are reversed upon insulin treatment in the steptozotocin animal model. Our data identify insulin deficiency as a direct trigger that induces the activity of the tau kinase PKA and results in tau phosphorylation. The reversibility upon insulin treatment underscores the potential of insulin as an early disease-modifying intervention in AD and other tauopathies. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. PGD2 stimulates osteoprotegerin synthesis via AMP-activated protein kinase in osteoblasts: Regulation of ERK and SAPK/JNK.

    Science.gov (United States)

    Kainuma, Shingo; Tokuda, Haruhiko; Kuroyanagi, Gen; Yamamoto, Naohiro; Ohguchi, Reou; Fujita, Kazuhiko; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2015-10-01

    AMP-activated protein kinase (AMPK), a key enzyme sensing cellular energy metabolism, is currently known to regulate multiple metabolic pathways. Osteoprotegerin plays a pivotal role in the regulation of bone metabolism by inhibiting osteoclast activation. We have previously reported that prostaglandin D2 (PGD2) stimulates the synthesis of osteoprotegerin through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. On the basis of these findings, we herein investigated the implication of AMPK in PGD2-stimulated osteoprotegerin synthesis in these cells. PGD2 induced the phosphorylation of AMPKα (Thr-172) and AMPKβ (Ser-108), and the phosphorylation of acetyl-coenzyme A carboxylase, a direct AMPK substrate. Compound C, an AMPK inhibitor, which suppressed the phosphorylation of acetyl-coenzyme A carboxylase, significantly attenuated both the release and the mRNA levels of osteoprotegerin stimulated by PGD2. The PGD2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK but not p38 MAP kinase were markedly inhibited by compound C. These results strongly suggest that AMPK regulates the PGD2-stimulated osteoprotegerin synthesis at a point upstream of p44/p42 MAP kinase and SAPK/JNK in osteoblasts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. The Interaction of Src Kinase with beta 3 Integrin Tails : A Potential Therapeutic Target in Thrombosis and Cancer

    NARCIS (Netherlands)

    Huveneers, Stephan; Danen, Erik H. J.

    2010-01-01

    Activation of Src family kinases is an important event downstream of integrin adhesion signaling in many cell types. A particularly intriguing connection between an integrin and a Src family kinase was first discovered in platelets, where the selective direct interaction of alpha IIb beta 3

  2. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J.

    2006-01-01

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  3. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    DEFF Research Database (Denmark)

    Massip, L; Garand, C; Labbé, A

    2010-01-01

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we...... show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...

  4. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells.

    Science.gov (United States)

    Diakos, Christos; Prieschl, Eva E; Säemann, Marcus D; Böhmig, Georg A; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J

    2006-10-20

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-alpha transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling.

  5. Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation.

    Science.gov (United States)

    Zemans, Rachel L; Arndt, Patrick G

    2009-01-01

    The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH(2) terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta expression are dependent on Tec kinase activity.

  6. Shuttle active thermal control system development testing. Volume 7: Improved radiator coating adhesive tests

    Science.gov (United States)

    Reed, M. W.

    1973-01-01

    Silver/Teflon thermal control coatings have been tested on a modular radiator system projected for use on the space shuttle. Seven candidate adhesives have been evaluated in a thermal vacuum test on radiator panels similar to the anticipated flight hardware configuration. Several classes of adhesives based on polyester, silicone, and urethane resin systems were tested. These included contact adhesives, heat cured adhesives, heat and pressure cured adhesives, pressure sensitive adhesives, and two part paint on or spray on adhesives. The coatings attached with four of the adhesives, two silicones and two urethanes, had no changes develop during the thermal vacuum test. The two silicone adhesives, both of which were applied to the silver/Teflon as transfer laminates to form a tape, offered the most promise based on application process and thermal performance. Each of the successful silicone adhesives required a heat and pressure cure to adhere during the cryogenic temperature excursion of the thermal-vacuum test.

  7. Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases.

    Science.gov (United States)

    Ojansivu, M; Hyväri, L; Kellomäki, M; Hupa, L; Vanhatupa, S; Miettinen, S

    2018-01-30

    Bioactive glasses (BaGs) are widely utilised in bone tissue engineering (TE) but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs) on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-%)) and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2) in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP) and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene). Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK)-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK) inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

  8. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

    International Nuclear Information System (INIS)

    Ryu, S.H.; Lee, S.Y.; Lee, K.Y.; Rhee, S.G.

    1987-01-01

    Inositol 1,4,5-triphosphate (Ins-1,4,5-P 3 ) is an important second-messenger molecule that mobilizes Ca 2+ from intracellular stores in response to the occupancy of receptor by various Ca 2+ -mobilizing agonists. The fate of Ins-1,4,5-P 3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P 3 to Ins-1,3,4,5-P 4 , whereas the latter forms Ins-1,4-P 2 . Recent studies suggest that Ins-1,3,4,5-P 4 might modulate the entry of Ca 2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca 2+ /calmodulin complex. Therefore, they propose that Ca 2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P 3 forms a complex with calmodulin, and that the Ca 2+ /calmodulin complex stimulates the conversion of Ins-1,4,5-P 3 , and intracellular Ca 2+ mobilizer, to Ins-1,3,4,5-P 4 , an extracellular Ca 2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3- 32 P]Ins-1,3,4,5-P 4 and [γ- 32 P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P 3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg 2+ > > Mn 2+ > Ca 2+ ) for the 3-kinase

  9. Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone

    NARCIS (Netherlands)

    Sharma, P.; Stagge, S.; Bekker, M.; Bettenbrock, K.; Hellingwerf, K.

    2013-01-01

    Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase.

  10. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Title Macrophage-stimu

  11. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    Science.gov (United States)

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  12. cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2009-09-01

    The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

  13. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  14. Action of mercurials on activity of partially purified soluble protein kinase C from mice brain

    International Nuclear Information System (INIS)

    Inoue, Y.; Saijoh, K.; Sumino, K.

    1988-01-01

    The enzymatic activity of soluble protein kinase C from mice brain was inhibited by mercuric chloride (II) (HgCl 2 ) and organic mercurials, i.e. methyl mercury, phenyl mercury and p-chloromercuribenzoic acid (PCMB). The IC50 was 0.08 μM for HgCl 2 and about 1 μM for organic mercurials. Sulfhydryl blocking reagents such as 5.5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) were less potent but nevertheless inhibited the enzymic activity of protein kinase C. The Hill coefficients of HgCl 2 , DTNB and NEM were close to unity whereas the values for organic mercurials were 1.3 to 1.5. The inhibition was of a non-competitive type with respect to Hl histone. 3 H-PDBu binding activity was also inhibited by all of the reagents in a non-competitive manner. Mercurials apparently bind to sulfhydryl groups of protein kinase C to inhibit the enzymatic activity. (author)

  15. TGF-βRI kinase activity mediates Emdogain-stimulated in vitro osteoclastogenesis.

    Science.gov (United States)

    Gruber, Reinhard; Roos, Gilles; Caballé-Serrano, Jordi; Miron, Rick; Bosshardt, Dieter D; Sculean, Anton

    2014-07-01

    Emdogain, containing an extract of fetal porcine enamel matrix proteins, is a potent stimulator of in vitro osteoclastogenesis. The underlying molecular mechanisms are, however, unclear. Here, we have addressed the role of transforming growth factor-beta receptor type 1 (TGF-βRI) kinase activity on osteoclastogenesis in murine bone marrow cultures. Inhibition of TGF-βRI kinase activity with SB431542 abolished the effect of Emdogain on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand or tumor necrosis factor-alpha. SB431542 also suppressed the Emdogain-mediated increase of OSCAR, a co-stimulatory protein, and dendritic cell-specific transmembrane protein and Atp6v0d2, the latter two being involved in cell fusion. Similar to transforming growth factor-beta1 (TGF-β), Emdogain could not compensate for the inhibition of IL-4 and IFNγ on osteoclast formation. When using the murine macrophage cell line RAW246.7, SB431542 and the smad-3 inhibitor SIS3 blocked Emdogain-stimulated expression of the transcription factor NFATc1. Taken together, the data suggest that TGF-βRI kinase activity is necessary to mediate in vitro effects of Emdogain on osteoclastogenesis. Based on these in vitro data, we can speculate that at least part of the clinical effects of Emdogain on osteoclastogenesis is mediated via TGF-β signaling.

  16. Interaction between Salt-inducible Kinase 2 and Protein Phosphatase 2A Regulates the Activity of Calcium/Calmodulin-dependent Protein Kinase I and Protein Phosphatase Methylesterase-1*

    Science.gov (United States)

    Lee, Chia-Wei; Yang, Fu-Chia; Chang, Hsin-Yun; Chou, Hanyi; Tan, Bertrand Chin-Ming; Lee, Sheng-Chung

    2014-01-01

    Salt-inducible kinase 2 (SIK2) is the only AMP-activated kinase (AMPK) family member known to interact with protein phosphatase 2 (PP2A). However, the functional aspects of this complex are largely unknown. Here we report that the SIK2·PP2A complex preserves both kinase and phosphatase activities. In this capacity, SIK2 attenuates the association of the PP2A repressor, the protein phosphatase methylesterase-1 (PME-1), thus preserving the methylation status of the PP2A catalytic subunit. Furthermore, the SIK2·PP2A holoenzyme complex dephosphorylates and inactivates Ca2+/calmodulin-dependent protein kinase I (CaMKI), an upstream kinase for phosphorylating PME-1/Ser15. The functionally antagonistic SIK2·PP2A and CaMKI and PME-1 networks thus constitute a negative feedback loop that modulates the phosphatase activity of PP2A. Depletion of SIK2 led to disruption of the SIK2·PP2A complex, activation of CaMKI, and downstream effects, including phosphorylation of HDAC5/Ser259, sequestration of HDAC5 in the cytoplasm, and activation of myocyte-specific enhancer factor 2C (MEF2C)-mediated gene expression. These results suggest that the SIK2·PP2A complex functions in the regulation of MEF2C-dependent transcription. Furthermore, this study suggests that the tightly linked regulatory loop comprised of the SIK2·PP2A and CaMKI and PME-1 networks may function in fine-tuning cell proliferation and stress response. PMID:24841198

  17. Platelet Activation and Thrombus Formation over IgG Immune Complexes Requires Integrin αIIbβ3 and Lyn Kinase.

    Directory of Open Access Journals (Sweden)

    Huiying Zhi

    Full Text Available IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.

  18. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

    Science.gov (United States)

    Rewitz, Kim F; Yamanaka, Naoki; Gilbert, Lawrence I; O'Connor, Michael B

    2009-12-04

    Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal-regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation of the Torso/ERK pathway.

  19. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear...... and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  20. DMPD: Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-signalingcascades during intracellular Toxoplasma gondii infection. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15361242 Manipulation of mitogen-activated protein kinase/nuclear factor-kappaB-sig...mmunol Rev. 2004 Oct;201:191-205. (.png) (.svg) (.html) (.csml) Show Manipulation of mitogen-activated protein kinase/nuclear... gondii infection. PubmedID 15361242 Title Manipulation of mitogen-activated protein kinase/nuclear factor-k

  1. Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase.

    Science.gov (United States)

    Fu, Xiaoyong; Creighton, Chad J; Biswal, Nrusingh C; Kumar, Vijetha; Shea, Martin; Herrera, Sabrina; Contreras, Alejandro; Gutierrez, Carolina; Wang, Tao; Nanda, Sarmistha; Giuliano, Mario; Morrison, Gladys; Nardone, Agostina; Karlin, Kristen L; Westbrook, Thomas F; Heiser, Laura M; Anur, Pavana; Spellman, Paul; Guichard, Sylvie M; Smith, Paul D; Davies, Barry R; Klinowska, Teresa; Lee, Adrian V; Mills, Gordon B; Rimawi, Mothaffar F; Hilsenbeck, Susan G; Gray, Joe W; Joshi, Amit; Osborne, C Kent; Schiff, Rachel

    2014-09-11

    Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance. Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy. Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression. Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

  2. Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2017-12-01

    Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

  3. Targeting Mitogen-activated Protein Kinase-activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts to Lead Small Molecule Inhibitors to Clinical Trials

    Science.gov (United States)

    Fiore, Mario; Forli, Stefano; Manetti, Fabrizio

    2015-01-01

    The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway, but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases, to prevent tumor metastasis, and to increase tumor sensitivity to chemotherapeutics. PMID:26502061

  4. The non-receptor tyrosine kinase Tec controls assembly and activity of the noncanonical caspase-8 inflammasome.

    Science.gov (United States)

    Zwolanek, Florian; Riedelberger, Michael; Stolz, Valentina; Jenull, Sabrina; Istel, Fabian; Köprülü, Afitap Derya; Ellmeier, Wilfried; Kuchler, Karl

    2014-12-01

    Tec family kinases are intracellular non-receptor tyrosine kinases implicated in numerous functions, including T cell and B cell regulation. However, a role in microbial pathogenesis has not been described. Here, we identified Tec kinase as a novel key mediator of the inflammatory immune response in macrophages invaded by the human fungal pathogen C. albicans. Tec is required for both activation and assembly of the noncanonical caspase-8, but not of the caspase-1 inflammasome, during infections with fungal but not bacterial pathogens, triggering the antifungal response through IL-1β. Furthermore, we identify dectin-1 as the pathogen recognition receptor being required for Syk-dependent Tec activation. Hence, Tec is a novel innate-specific inflammatory kinase, whose genetic ablation or inhibition by small molecule drugs strongly protects mice from fungal sepsis. These data demonstrate a therapeutic potential for Tec kinase inhibition to combat invasive microbial infections by attenuating the host inflammatory response.

  5. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    Directory of Open Access Journals (Sweden)

    Kumar N Alagramam

    2014-01-01

    Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  6. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

    Science.gov (United States)

    Alagramam, Kumar N; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H-C; Davis, Rickie R

    2014-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

  7. Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress.

    Science.gov (United States)

    Dutilleul, Christelle; Chavarria, Heidy; Rézé, Nathalie; Sotta, Bruno; Baudouin, Emmanuel; Guillas, Isabelle

    2015-12-01

    Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress. © 2015 John Wiley & Sons Ltd.

  8. Ret receptor tyrosine kinase activates extracellular signal-regulated kinase 2 in SK-N-MC cells

    NARCIS (Netherlands)

    van Weering, D. H.; Medema, J. P.; van Puijenbroek, A.; Burgering, B. M.; Baas, P. D.; Bos, J. L.

    1995-01-01

    Ret is a receptor tyrosine kinase predominantly expressed in tissue derived from the neuroectoderm and is involved in multiple endocrine neoplasia type 2A and 2B, familiar medullary thyroid carcinoma, and Hirschsprung's disease. The ligand for the receptor is still unknown. Previously, using a human

  9. Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms.

    NARCIS (Netherlands)

    Herpen, R.E.M.A. van; Tjeertes, J.V.; Mulders, S.A.M.; Oude Ophuis, R.J.A.; Wieringa, B.; Wansink, D.G.

    2006-01-01

    The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein

  10. Pharmaceutically inhibiting polo-like kinase 1 exerts a broad anti-tumour activity in retinoblastoma cell lines.

    Science.gov (United States)

    Schwermer, Melanie; Dreesmann, Sabine; Eggert, Angelika; Althoff, Kristina; Steenpass, Laura; Schramm, Alexander; Schulte, Johannes H; Temming, Petra

    2017-04-01

    Retinoblastoma is the most common malignant cancer of the eye in children. Although metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level polo-like kinase 1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. Polo-like kinase 1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. Polo-like kinase 1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here, we assessed its efficacy against retinoblastoma cell lines. Expression of polo-like kinase 1 was determined in a panel of retinoblastoma cell lines by polymerase chain reaction and western blot analysis. We analysed viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT assay), proliferation (5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay), cell cycle progression (propidium iodid staining) and apoptosis (cell death enzyme-linked immunosorbent assay) in three retinoblastoma cell lines after treatment with two adenosine triphosphate-competitive polo-like kinase 1 inhibitors, BI6727 or GSK461364. Activation of polo-like kinase 1 downstream signalling components including TP53 were assessed. Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity and induced both cell cycle arrest and apoptosis. Polo-like kinase 1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either polo-like kinase 1 inhibitor upregulated cyclin B1 and increased activated cyclin-dependent kinase 1 (phosphorylated at Y15) in retinoblastoma cell lines. These preclinical data indicate that polo-like kinase 1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasized retinoblastoma in early phase clinical

  11. [Comparative study of antimicrobial activity of "Biodentin" and "Rootdent" cements and "Futurabond NR" adhesive].

    Science.gov (United States)

    Shamkhalov, G S; Ivanova, E V; Dmitrieva, N A; Akhmedova, Z R

    2013-01-01

    Bacterial contamination under fillings proved to be the main reason for pulp inflammation thus emphasizing the importance of antibacterial properties of restorative materials. The aim of the current study was to assess antibacterial properties of "Biodentine" (Septodont), "Rootdent" (TehnoDent) and adhesive "Futurabond НР" (Voco). Two lines of experiments were carried out using cements water solutions and firm tablet-like samples (made by means of special pattern). All the examined materials showed antibacterial activity against E. coli, S. aureus, C. albiсans, Str. faecalis. The results confirm the analyzed materials to be a useful tool for deep caries lesions treatment.

  12. The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.

    Science.gov (United States)

    Guerrero-Mendiola, Carlos; García-Trejo, José J; Encalada, Rusely; Saavedra, Emma; Ramírez-Silva, Leticia

    2017-01-01

    In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may "mimic" the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.

  13. Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

    DEFF Research Database (Denmark)

    Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.

    2005-01-01

    In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...

  14. Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

    Science.gov (United States)

    Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

    2015-01-01

    Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

  15. A STUDY ON THE USE OF GRAPHENE PEEK COMPOSITES AS HIGH TEMPERATURE ADHESIVES: MECHANICAL PROPERTIES AND MICROWAVE ACTIVATION

    Science.gov (United States)

    2017-11-03

    Technical Report ARWSB-TR-18001 A STUDY ON THE USE OF GRAPHENE-PEEK COMPOSITES AS HIGH TEMPERATURE ADHESIVES: MECHANICAL PROPERTIES...AND SUBTITLE A STUDY ON THE USE OF GRAPHENE-PEEK COMPOSITES AS HIGH TEMPERATURE ADHESIVES: MECHANICAL PROPERTIES AND MICROWAVE ACTIVATION 5a...is a widely used engineering polymer that is especially suitable for high - temperature applications. Graphene is a two-dimensional form of carbon

  16. Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality

    Science.gov (United States)

    Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; Brouhard, Gary J.

    2014-01-01

    Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates. PMID:24498282

  17. Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

    Science.gov (United States)

    Noujaim, Michael; Bechstedt, Susanne; Wieczorek, Michal; Brouhard, Gary J

    2014-01-01

    Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a "reduction in dimensionality." We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

  18. Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

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    Michael Noujaim

    Full Text Available Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC, a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a "reduction in dimensionality." We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

  19. Highly biological active antibiofilm, anticancer and osteoblast adhesion efficacy from MWCNT/PPy/Pd nanocomposite

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    Murugesan, Balaji; Sonamuthu, Jegatheeswaran; Samayanan, Selvam; Arumugam, Sangili; Mahalingam, Sundrarajan

    2018-03-01

    Multifunctional biologically active materials have approached for antibiofilm, anticancer and osteoblast adhesion activities with significant biomedical applications, owing to this MWCNT modified with polypyrrole (PPy) matrix with the incorporation of palladium nanoparticles (NPs). The synthesized composite displays a tube-shaped morphology with highly dispersed crystalline Pd NPs, which are established through XRD, SEM, TEM and SAED studies. The pyridinic-N(∼402.7), pyrrolic sbnd N (∼400.8) peak in XPS spectra evidenced the interaction of PPy with Pd and MWCNT. Polymer stretching frequencies in FTIR and Raman spectroscopy proves successful formation of PPy and the Pd-N (1609 cm-1) interaction. In the stability aspect, it is up to 58.73% mass withstood at 800 °C in TGA analysis. The composite exhibits an efficient Anti-biofilm against a set of bacterial stain with planktonic cell growth. In vitro cytotoxicity of Vero and HeLa cell line assess the composites toxicity and anticancer activity up to 100 μg. The outcome of cell adhesions showed that human osteosarcoma cells (HOS) can adhere and to develop on the MWCNT/PPy/Pd composites. Furthermore, the proliferation of cells on MWCNT/PPy/Pd composites was also proved the biocompatibility of the composites against HOS cells. These results suggest that Pd-doped MWCNT/PPy composites are promising materials for biomedical applications.

  20. LIM domains regulate protein kinase C activity: a novel molecular function.

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    Maturana, Andrés D; Nakagawa, Noritaka; Yoshimoto, Nobuo; Tatematsu, Kenji; Hoshijima, Masahiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2011-05-01

    Enigma homolog protein 1 (ENH1) acts as a scaffold that selectively associates protein kinases and transcription factors with cytoskeletal elements. ENH1 comprises an N-terminal PDZ domain and three C-terminal LIM domains. Through the LIM domains ENH1 interacts with the N-terminal region of protein kinase C βI (PKCβI). Here, we show that when ENH1 is co-expressed, PKCβI is translocated from the cytoplasm to the plasma membrane in the absence of any other stimulation. Moreover expression of ENH1 markedly increases PKCβI activity in the absence of PKC activators. A similar activation of PKCβI was observed with co-expression of Cypher1 or Enigma, but not other LIM proteins. The region including the three LIM domains of ENH1 (residues 415-591) appears to be sufficient for this PKCβI activation. Finally, interaction with ENH1 also increases the activity of PKCα and PKCγ, whereas it reduces PKCζ activity. These findings provide strong evidence that ENH1 activates conventional PKCs by directly binding through its LIM domains. Thus, LIM domains have a novel molecular function: the regulation of PKC activities in a PKC isoform-specific manner. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity

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    Hayeß Katrin

    2005-08-01

    Full Text Available Abstract Background Creatine kinase (CK links phosphocreatine, an energy storage system, to cellular ATPases. CK activity serves as a temporal and spatial buffer for ATP content, particularly in fast-twitch skeletal muscles. The extraocular muscles are notoriously fast and active, suggesting the need for efficient ATP buffering. This study tested the hypotheses that (1 CK isoform expression and activity in rat extraocular muscles would be higher, and (2 the resistance of these muscles to fatigue would depend on CK activity. Results We found that mRNA and protein levels for cytosolic and mitochondrial CK isoforms were lower in the extraocular muscles than in extensor digitorum longus (EDL. Total CK activity was correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a fraction of CK activity is found, were downregulated in the extraocular muscles as was shown by immunocytochemistry and western blotting. CK inhibition significantly accelerated the development of fatigue in EDL muscle bundles, but had no major effect on the extraocular muscles. Searching for alternative ATP buffers that could compensate for the relative lack of CK in extraocular muscles, we determined that mRNAs for two adenylate kinase (AK isoforms were expressed at higher levels in these muscles. Total AK activity was similar in EDL and extraocular muscles. Conclusion These data indicate that the characteristic fatigue resistance of the extraocular muscles does not depend on CK activity.

  2. Metformin activates type I interferon signaling against HCV via activation of adenosine monophosphate-activated protein kinase.

    Science.gov (United States)

    Tsai, Wei-Lun; Chang, Tsung-Hsien; Sun, Wei-Chi; Chan, Hoi-Hung; Wu, Chun-Ching; Hsu, Ping-I; Cheng, Jin-Shiung; Yu, Ming-Lung

    2017-11-03

    Activation of the type I interferon (IFN) signaling pathway is essential for the eradication of hepatitis C virus (HCV). Metformin can activate adenosine monophosphate-activated protein kinase (AMPK) to reduce insulin resistance. Cross talks between AMPK and IFN signaling remain unclear. To understand the influence of metformin on the type I IFN signaling pathway and HCV infection, the full-length HCV replicon OR6 cells and the infectious HCV clones JFH1 were used to assess the anti-HCV effect of the insulin sensitizers, metformin and pioglitazone. Immunofluorescence staining and the immunoblotting of HCV viral protein demonstrated that metformin, but not pioglitazone, inhibited HCV replication in OR-6 and JFH-1-infected Huh 7.5.1 cells. Immunoblotting data showed that metformin activated the phosphorylation of STAT-1 and STAT-2 in OR-6 and JFH-1 infected Huh 7.5.1 cells. Metformin enhanced the phosphorylation of AMPK, and the metformin-activated IFN signaling was down-regulated by AMPK inhibitor. After treatment of AMPK inhibitor, the level of HCV core protein decreased by metformin can be rescued. In conclusion, metformin activates type I interferon signaling and inhibits the replication of HCV via activation of AMPK.

  3. Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

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    Chen Hairu

    2010-10-01

    Full Text Available Abstract Background Activated leukocyte cell adhesion molecule (ALCAM is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. Results A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. Conclusion Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

  4. The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

    Science.gov (United States)

    Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

    2017-04-24

    Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon

  5. Structure of protein O-mannose kinase reveals a unique active site architecture.

    Science.gov (United States)

    Zhu, Qinyu; Venzke, David; Walimbe, Ameya S; Anderson, Mary E; Fu, Qiuyu; Kinch, Lisa N; Wang, Wei; Chen, Xing; Grishin, Nick V; Huang, Niu; Yu, Liping; Dixon, Jack E; Campbell, Kevin P; Xiao, Junyu

    2016-11-23

    The 'pseudokinase' SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg 2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome.

  6. Infralimbic cortex Rho-kinase inhibition causes antidepressant-like activity in rats.

    Science.gov (United States)

    Inan, Salim Yalcin; Soner, Burak Cem; Sahin, Ayse Saide

    2015-03-03

    Depression is one of the most common psychiatric disorders in the world; however, its mechanisms remain unclear. Recently, a new signal-transduction pathway, namely Rho/Rho-kinase signalling, has been suggested to be involved in diverse cellular events in the central nervous system; such as epilepsy, anxiety-related behaviors, regulation of dendritic and axonal morphology, antinociception, subarachnoid haemorrhage, spinal cord injury and amyotrophic lateral sclerosis. However there is no evidence showing the involvement of Rho-kinase pathway in depression. In addition, the infralimbic cortex, rodent equivalent to subgenual cingulate cortex has been shown to be responsible for emotional responses. Thus, in the present study, intracranial guide cannulae were stereotaxically implanted bilaterally into the infralimbic cortex, and the effects of repeated microinjections of a Rho-kinase (ROCK) inhibitor Y-27632 (10 nmol) were investigated in rats. Y-27632 significantly decreased immobility time and increased swimming and climbing behaviors when compared to fluoxetine (10 μg) and saline groups in the forced swim test. In addition, Y-27632 treatment did not affect spontaneous locomotor activity and forelimb use in the open-field and cylinder tests respectively; but it enhanced limb placing accuracy in the ladder rung walking test. Our results suggest that Y-27632 could be a potentially active antidepressant agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis.

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    Abdallah, Abbas M; Zhou, Xin; Kim, Christine; Shah, Kushani K; Hogden, Christopher; Schoenherr, Jessica A; Clemens, James C; Chang, Henry C

    2013-06-15

    Deregulation of the non-receptor tyrosine kinase ACK1 (Activated Cdc42-associated kinase) correlates with poor prognosis in cancers and has been implicated in promoting metastasis. To further understand its in vivo function, we have characterized the developmental defects of a null mutation in Drosophila Ack, which bears a high degree of sequence similarity to mammalian ACK1 but lacks a CRIB domain. We show that Ack, while not essential for viability, is critical for sperm formation. This function depends on Ack tyrosine kinase activity and is required cell autonomously in differentiating male germ cells at or after the spermatocyte stage. Ack associates predominantly with endocytic clathrin sites in spermatocytes, but disruption of Ack function has no apparent effect on clathrin localization and receptor-mediated internalization of Boss (Bride of sevenless) protein in eye discs. Instead, Ack is required for the subcellular distribution of Dock (dreadlocks), the Drosophila homolog of the SH2- and SH3-containing adaptor protein Nck. Moreover, Dock forms a complex with Ack, and the localization of Dock in male germ cells depends on its SH2 domain. Together, our results suggest that Ack-dependent tyrosine phosphorylation recruits Dock to promote sperm differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Insulin resistance in uremia: Insulin receptor kinase activity in liver and muscle from chronic uremic rats

    International Nuclear Information System (INIS)

    Cecchin, F.; Ittoop, O.; Sinha, M.K.; Caro, J.F.

    1988-01-01

    The authors have studied the structure and function of the partially purified insulin receptors from liver and skeletal muscle in a rat model of severe chronic uremia. 125 I-insulin binding was higher in the liver from uremic rats when compared with ad libitum- and pair-fed controls. Furthermore, the ability of insulin to stimulate the autophosphorylation of the β-subunit and insulin receptor kinase activity using Glu 80 , Tyr 20 as exogenous phosphoacceptor was increased in the liver of the uremic animals. The structural characteristics of the receptors, as determined by electrophoretic mobilities of affinity labeled α-subunit and the phosphorylated β-subunit, were normal in uremia. 125 I-insulin binding and insulin receptor kinase activity were similar in the skeletal muscle from uremic and pair- and ad libitum-fed animals. Thus the data are supportive of the hypothesis that in liver and muscle of chronic uremic rats, insulin resistance is due to a defect(s) distal to the insulin receptor kinase

  9. Effects of p38 mitogen-activated protein kinase on lung ischemia-reperfusion injury in diabetic rats.

    Science.gov (United States)

    Song, Linlin; Li, Di; Wang, Juan; Meng, Chao; Cui, Xiaoguang

    2017-08-01

    Lung ischemia-reperfusion injury (LIRI) is a pathologic process that is observed in several clinical conditions, and p38 mitogen-activated protein kinase (MAPK) is involved. Diabetes mellitus (DM) results in an increased incidence of ischemia-induced organ damage. The aims of this study were to examine the effects of DM on LIRI in a rat model of DM and to explore the possible mechanisms in relation to the p38 MAPK pathway. Forty rats were randomly divided into the following five groups (n = 8 each): a control + sham group, a control + IR group (CIR), a DM + sham group, a DM + IR group (DIR), and a DM + IR + SB203580 group. The control and streptozotocin-induced diabetic rats underwent a sham operation or left hilum occlusion for 90 min followed by reperfusion for 4 h. SB203580 was used to inhibit the p38 MAPK pathway. The pulmonary oxygenation index, inflammatory cytokines in the serum, lung edema, histopathology, oxidant stress, apoptosis, and phosphorylated/total-p38 MAPK protein levels were measured. The DIR group displayed greater concentrations of tumor necrosis factor-α, interleukin-6, and intercellular adhesion molecule-1 and increases in the wet weight-to-dry weight ratio, lung injury scores, malondialdehyde levels, and cellular apoptosis, and these effects were accompanied by lower pulmonary oxygenation compared with the CIR group (P < 0.05). In the DIR group, the expression levels of p38 MAPK protein were significantly upregulated compared with those of the CIR group. Additionally, all of these alterations were attenuated in the DM + IR + SB203580 group compared with the DIR group. Diabetes exacerbates LIRI by activating the p38 MAPK pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata

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    Kuang-Ren Chung

    2013-06-01

    Full Text Available Mitogen-activated protein kinase (MAPK- mediated signaling pathways have been known to have important functions in eukaryotic organisms. The mechanisms by which the filamentous fungus Alternaria alternata senses and responds to environmental signals have begun to be elucidated. Available data indicate that A. alternata utilizes the Fus3, Hog1 and Slt2 MAPK-mediated signaling pathways, either separately or in a cooperative manner, for conidia formation, resistance to oxidative and osmotic stress, and pathogenesis to citrus. This review provides an overview of our current knowledge of MAPK signaling pathways, in conjunction with the two-component histidine kinase and the Skn7 response regulator, in the tangerine pathotype of A. alternata.

  11. Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

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    Yanyan Yang

    2014-01-01

    Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

  12. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

    Science.gov (United States)

    Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David

    2012-01-01

    A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702

  13. p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease.

    Science.gov (United States)

    Waetzig, Georg H; Seegert, Dirk; Rosenstiel, Philip; Nikolaus, Susanna; Schreiber, Stefan

    2002-05-15

    Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.

  14. Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

    Science.gov (United States)

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

  15. Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

    Directory of Open Access Journals (Sweden)

    Peter Schierack

    Full Text Available We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2 and their probiotic activity against infection by enteropathogenic E. coli (EPEC. 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

  16. Sestrins Inhibit mTORC1 Kinase Activation through the GATOR Complex

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    Anita Parmigiani

    2014-11-01

    Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition.

  17. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

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    Bae, Sung Jun [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Ni, Lisheng [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Osinski, Adam [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Tomchick, Diana R. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Brautigam, Chad A. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States; Luo, Xuelian [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

    2017-10-24

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

  18. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

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    Kara R Barber

    2017-02-01

    Full Text Available Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.

  19. Inhibition of the TEF/TEAD transcription factor activity by nuclear calcium and distinct kinase pathways.

    Science.gov (United States)

    Thompson, M; Andrade, V A; Andrade, S J; Pusl, T; Ortega, J M; Goes, A M; Leite, M F

    2003-02-07

    Transcription enhancer factor (TEF/TEAD) is a family of four transcription factors that share a common TEA-DNA binding domain and are involved in similar cellular functions, such as cell differentiation and proliferation. All adult tissues express at least one of the four TEAD genes, so this family of transcription factors may be of widespread importance, yet little is known about their regulation. Here we examine the factors that regulate TEAD activity in CHO cells. RT-PCR indicated the presence of TEAD-1, TEAD-3, and both isoforms of TEAD-4, but not TEAD-2. Quantitative measurements showed that TEAD-4 is most abundant, followed by TEAD-3, then TEAD-1. We examined the relative effects of nuclear and cytosolic Ca(2+) on TEAD activity, since TEAD proteins are localized to the nucleus and since free Ca(2+) within the nucleus selectively regulates transcription in some systems. Chelation of nuclear but not cytosolic Ca(2+) increased TEAD activity two times above control. Inhibition of mitogen-activated protein kinase (MAPK) also increased TEAD activity, while cAMP decreased TEAD activity, and protein kinase C had no effect. Together, these results show that nuclear Ca(2+), MAPK, and cAMP each negatively regulate the activity of the TEAD transcription factor.

  20. Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

    DEFF Research Database (Denmark)

    Gesslein, Bodil; Håkansson, Gisela; Carpio, Ronald

    2010-01-01

    The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion....

  1. Negative regulation of active zone assembly by a newly identified SR protein kinase.

    Science.gov (United States)

    Johnson, Ervin L; Fetter, Richard D; Davis, Graeme W

    2009-09-01

    Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  2. Negative regulation of active zone assembly by a newly identified SR protein kinase.

    Directory of Open Access Journals (Sweden)

    Ervin L Johnson

    2009-09-01

    Full Text Available Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila or ribbon (vertebrates are believed to facilitate vesicle movement to the active zone (AZ of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D. This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  3. H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication.

    Science.gov (United States)

    Botting, Carolyn; Lu, Xu; Triezenberg, Steven J

    2016-01-27

    Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.

  4. PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2014-04-01

    Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

  5. Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade

    Directory of Open Access Journals (Sweden)

    Alessi Dario R

    2003-09-01

    Full Text Available Abstract Background The AMP-activated protein kinase (AMPK cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. Results We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1, but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. Conclusions These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

  6. DMPD: A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase pathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15809659 A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase pathways...csml) Show A pervasive role of ubiquitin conjugation in activation and termination ofIkappaB kinase pathways... and termination ofIkappaB kinase pathways. Authors Krappmann D, Scheidereit C. Publication EMBO Rep. 2005 A

  7. Calcium phosphate coating containing silver shows high antibacterial activity and low cytotoxicity and inhibits bacterial adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Ando, Yoshiki, E-mail: andoy@jmmc.jp [Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Research Department, Japan Medical Materials Corporation, Uemura Nissei Bldg.9F 3-3-31 Miyahara, Yodogawa-ku, Osaka 532-0003 (Japan); Miyamoto, Hiroshi [Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Noda, Iwao; Sakurai, Nobuko [Research Department, Japan Medical Materials Corporation, Uemura Nissei Bldg.9F 3-3-31 Miyahara, Yodogawa-ku, Osaka 532-0003 (Japan); Akiyama, Tomonori [Division of Microbiology, Department of Pathology and Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan); Yonekura, Yutaka; Shimazaki, Takafumi; Miyazaki, Masaki; Mawatari, Masaaki; Hotokebuchi, Takao [Department of Orthopaedic Surgery, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501 (Japan)

    2010-01-01

    Surgical site infection is one of the serious complications of orthopedic implants. In order to reduce the incidence of implant-associated infections, we developed a novel coating technology of calcium phosphate (CP) containing silver (Ag), designated Ag-CP coating, using a thermal spraying technique. In this study, we evaluated the antibacterial efficacy and biological safety of this coating. In vitro antibacterial activity tests showed that the growths of Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) are completely suppressed on Ag-CP coating. In vitro bacterial adherence tests revealed that the number of adherent bacteria on the surface of this coating is significantly less (p < 0.02) than that on the surface of the CP coating. Moreover, the Ag-CP coating completely inhibits MRSA adhesion [<10 colony-forming units (CFU)] when 10{sup 2} CFU MRSA is inoculated. On the other hand, V79 Chinese hamster lung cells were found to grow on the Ag-CP coating as well as on the CP coating in a cytotoxicity test. These results indicate that the Ag-CP coating on the surface of orthopedic implants exhibits antibacterial activity and inhibits bacterial adhesion without cytotoxicity.

  8. Molecular Mechanism of Distinct Salt-Dependent Enzyme Activity of Two Halophilic Nucleoside Diphosphate Kinases

    OpenAIRE

    Yamamura, Akihiro; Ichimura, Takefumi; Kamekura, Masahiro; Mizuki, Toru; Usami, Ron; Makino, Tsukasa; Ohtsuka, Jun; Miyazono, Ken-ichi; Okai, Masahiko; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    Nucleoside diphosphate kinases from haloarchaea Haloarcula quadrata (NDK-q) and H. sinaiiensis (NDK-s) are identical except for one out of 154 residues, i.e., Arg31 in NDK-q and Cys31 in NDK-s. However, the salt-dependent activity profiles of NDK-q and NDK-s are quite different: the optimal NaCl concentrations of NDK-q and NDK-s are 1 M and 2 M, respectively. We analyzed the relationships of the secondary, tertiary, and quaternary structures and NDK activity of these NDKs at various salt conc...

  9. The ABCD's of 5'-adenosine monophosphate-activated protein kinase and adrenoleukodystrophy.

    Science.gov (United States)

    Weidling, Ian; Swerdlow, Russell H

    2016-07-01

    This Editorial highlights a study by Singh and coworkers in the current issue of Journal of Neurochemistry, in which the authors present additional evidence that AMPKα1 is reduced in X-linked adrenoleukodystrophy (X-ALD). They make a case for increasing AMPKα1 activity for therapeutic purposes in this disease, and indicate how this goal may be achieved. Read the highlighted article 'Metformin-induced mitochondrial function and ABCD2 up regulation in X-linked adrenoleukodystrophy involves AMP activated protein kinase' on page 86. © 2016 International Society for Neurochemistry.

  10. Effects of denture adhesive on the retention of milled and heat-activated maxillary denture bases: A clinical study.

    Science.gov (United States)

    AlRumaih, Hamad S; AlHelal, Abdulaziz; Baba, Nadim Z; Goodacre, Charles J; Al-Qahtani, Ali

    2018-03-15

    Clinical studies have identified advantages of digital complete denture technology including patient satisfaction, improved mastication, increased retention, and technique efficiency. However, studies that focusing on the effect of denture adhesive on the retention of milled and heat-activated denture bases are lacking. The purpose of this clinical study was to evaluate the effectiveness of denture adhesive on the retention of milled and heat-activated denture bases. Twenty participants with complete maxillary edentulism were selected for this study (11 men and 9 women). Definitive impressions were obtained and scanned (iSeries impression scanner; Dental Wings). Digital data were sent to Global Dental Science for the fabrication of computer-aided design and computer-aided manufacturing (CAD-CAM) milled denture bases (MB condition). The physical impressions were poured in stone to produce casts for the fabrication of heat-activated acrylic resin denture bases (HB condition). A portable clinical motorized test stand and advance digital force gauge were modified to measure the amount of denture base retention in newtons. The denture bases were seated over the edentulous maxillary ridge and pulled 3 times vertically at 10-minute intervals without denture adhesive (MB and HB control conditions) and with denture adhesive (MBA and HBA test conditions). For statistical analysis, a repeated-measures ANOVA was performed (α=.05). The control MB condition had significantly higher retention values compared with all other conditions (Pbases. No significant differences were found with or without the use of denture adhesive among heat-activated denture bases (P>.05). Significantly higher retention values were recorded with milled denture bases than heat-activated resin bases without the use of denture adhesive. However, denture adhesive did negatively affect the retention of milled complete dentures. Copyright © 2017 Editorial Council for the Journal of Prosthetic Dentistry

  11. Nitrate Activation of Cytosolic Protein Kinases Diverts Photosynthetic Carbon from Sucrose to Amino Acid Biosynthesis

    Science.gov (United States)

    Champigny, Marie-Louise; Foyer, Christine

    1992-01-01

    The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis. PMID:16653003

  12. Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

    Directory of Open Access Journals (Sweden)

    Dennis J Templeton

    2010-11-01

    Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

  13. Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

    Science.gov (United States)

    Izrael-Tomasevic, Anita; Chalouni, Cécile; Pan, Borlan; Goldsmith, Joshua; Schoen, Alia P.; Quiñones, Gabriel A.; Kelly, Ryan; Lill, Jennie R.; Sandoval, Wendy; Costa, Mike; Polakis, Paul; Arnott, David; Rubinfeld, Bonnee; Ernst, James A.

    2014-01-01

    Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases. PMID:25029443

  14. Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

    Directory of Open Access Journals (Sweden)

    Travis W Bainbridge

    Full Text Available Receptor tyrosine kinase-like orphan receptors (ROR 1 and 2 are atypical members of the receptor tyrosine kinase (RTK family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

  15. Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

    International Nuclear Information System (INIS)

    Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

    2003-01-01

    We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

  16. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  17. Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.

    Science.gov (United States)

    Hallaq, Haifa; Wang, Dao W; Kunic, Jennifer D; George, Alfred L; Wells, K Sam; Murray, Katherine T

    2012-02-01

    Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

  18. Anomalous constitutive Src kinase activity promotes B lymphoma survival and growth

    Directory of Open Access Journals (Sweden)

    Robertson Darrell A

    2009-12-01

    Full Text Available Abstract Background Previously we have shown that B cell receptor (BCR expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK, ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs like Lyn are known to be needed for the phosphorylation of BCR co-receptors, Ig-α and Ig-β, we hypothesized that one or more SFKs will be constitutively activated in B lymphoma cells and may be necessary for B lymphoma growth. Results Src kinase activity was found to be constitutively high in many murine and human B lymphoma cell lines and primary lymphoma samples. The specific pharmacological inhibitors of SFKs, PP1 and PP2 inhibited the proliferation of a number of both murine and human B lymphomas in a dose-dependent manner. Importantly, dasatinib (BMS-354825, an oral dual BCR-ABL and SFK specific inhibitor inhibited the growth of B lymphomas in the nanomolar range in vitro and strongly inhibited a mouse lymphoma growth in vivo. Among the SFKs, Lyn is predominantly phosphorylated and Lyn-specific small interfering RNA inhibited the growth of B lymphomas, supporting an important role for Lyn in B lymphoma growth. Suppression of SFK activity blocks BCR mediated signaling pathways. PMA or CpG can partially reverse the growth inhibition induced by SFK inhibition. Although blocking SFK activity inhibited the growth of a number of B lymphomas, some lymphomas such as SudHL-4, SudHL-6, OCI-Ly3 and OCI-Ly10 are more resistant due to an increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Conclusions These studies further support our concept that BCR signaling pathways are important for the continued growth of established B lymphoma cells. Some of the intermediates in this

  19. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  20. Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells

    Science.gov (United States)

    Hao, Feng; Wu, Daniel Dongwei; Xu, Xuemin

    2012-01-01

    Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis. PMID:23001835

  1. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

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    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  2. PI-3 kinase activity is required for epithelial-mesenchymal transformation during palate fusion.

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    Kang, Pei; Svoboda, Kathy K H

    2002-11-01

    Epithelial-mesenchymal transformation (EMT) is the primary mechanism for the disappearance of medial edge epithelia (MEE) during palate fusion. This phenotype transition is highly regulated by growth factors, extracellular matrix, cell surface receptors, and a variety of intracellular signaling. Phosphatidylinositol-3 (PI-3) kinase regulates cytoskeleton reorganization, cell migration, and transforming growth factor (TGF) beta-regulated EMT. Therefore, we investigated the role of PI-3 kinase in EMT during palatal fusion in vitro. Palatal shelves from embryonic (E) 13.5 day mouse embryos were collected and cultured for up to 72 hr. A specific PI-3 kinase inhibitor, LY294002, was added to the medium at concentrations of 100 etaM, 1 microM, and 10 microM. The fate of midline epithelia was traced by carboxyfluorescence labeling and analyzed by confocal microscopy. Harvested tissues were also processed for immunohistochemical analysis of a specific marker for basal lamina (laminin). Palatal fusion stages were scored on a scale of 1 to 5, with 1 equal to complete nonfusion and 5 equal to complete fusion. The mean fusion score (MFS) was calculated for each treatment group. Palatal shelves fused after 72 hr of culture in control and 100 etaM LY294002 inhibitor-treated groups, with MFS of 4.67 and 4.5, respectively. Laminin was absent in the midline and epithelia transformed into mesenchyme. However, when cultured palates were treated with 1 and 10 microM LY294002, MEE persisted in the midline and the basal lamina remained intact after 72 hr. The MFS was significantly less in the 1 and 10 microM LY294002-treated tissues at 2.08 and 1.33, respectively. Our results demonstrate that EMT during palatal fusion in vitro is dependent on PI-3 kinase activity. Copyright 2002 Wiley-Liss, Inc.

  3. Antibacterial and EGFR-Tyrosine Kinase Inhibitory Activities of Polyhydroxylated Xanthones from Garcinia succifolia

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    Susawat Duangsrisai

    2014-11-01

    Full Text Available Chemical investigation of the methanol extract of the wood of Garcinia succifolia Kurz (Clusiaceae led to the isolation of 1,5-dihydroxyxanthone (1, 1,7-dihydroxyxanthone (2, 1,3,7-trihydroxyxanthone (3, 1,5,6-trihydroxyxanthone (4, 1,6,7-trihydroxyxanthone (5, and 1,3,6,7-tetrahydroxyxanthone (6. All of the isolated xanthones were evaluated for their antibacterial activity against bacterial reference strains, two Gram-positive (Staphylococcus aureus ATTC 25923, Bacillus subtillis ATCC 6633 and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and environmental drug-resistant isolates (S. aureus B1, Enteroccoccus faecalis W1, and E. coli G1, as well as for their epidermal growth factor receptor (EGFR of tyrosine kinase inhibitory activity. Only 1,5,6-trihydroxy-(4, 1,6,7-trihydroxy-(5, and 1,3,6,7-tetrahydroxyxanthones (6 exhibited antibacterial activity against Gram-positive bacteria, however none was active against vancomycin-resistant E. faecalis. Additionally, 1,7-dihydroxyxanthone (2 showed synergism with oxacillin, but not with ampicillin. On the other hand, only 1,5-dihydroxyxanthone (1 and 1,7-dihydroxyxanthone (2 were found to exhibit the EGFR-tyrosine kinase inhibitory activity, with IC50 values of 90.34 and 223 nM, respectively.

  4. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

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    Lin Yu-Hsin

    2010-04-01

    Full Text Available Abstract Photodynamic therapy (PDT has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK and mitogen-activated protein kinase (MAPK signaling cascades are activated following 5-aminolevulinic acid (ALA-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.

  5. Dibenzoylmethane exerts metabolic activity through regulation of AMP-activated protein kinase (AMPK-mediated glucose uptake and adipogenesis pathways.

    Directory of Open Access Journals (Sweden)

    Nami Kim

    Full Text Available Dibenzoylmethane (DBM has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor. DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK, which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4 was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC, the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS, was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.

  6. Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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    Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

    2018-04-04

    Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

  7. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

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    Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

    2017-08-18

    Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Crystal-induced neutrophil activation: X. Proinflammatory role of the tyrosine kinase Tec.

    Science.gov (United States)

    Popa-Nita, Oana; Marois, Louis; Paré, Guillaume; Naccache, Paul H

    2008-06-01

    Monosodium urate monohydrate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathogenesis of gouty arthritis. Release of the crystals into the joint cavity promotes an acute inflammation characterized by massive infiltration of neutrophils, which leads to tissue damage. Th