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Sample records for kinase regulates migration

  1. DMPD: Regulation of phagocyte migration and recruitment by Src-family kinases. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 18385944 Regulation of phagocyte migration and recruitment by Src-family kinases. B...how Regulation of phagocyte migration and recruitment by Src-family kinases. PubmedID 18385944 Title Regulat...ion of phagocyte migration and recruitment by Src-family kinases. Authors Baruzzi

  2. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria); DeVaney, Trevor [Institute of Biophysics, Medical University of Graz (Austria); Zimmer, Andreas [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens University, Graz (Austria); Raynham, Tony; Ireson, Christopher [Cancer Research Technology Ltd, London (United Kingdom); Sattler, Wolfgang, E-mail: wolfgang.sattler@medunigraz.at [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria)

    2013-08-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun{sup S73} phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

  3. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea; DeVaney, Trevor; Zimmer, Andreas; Raynham, Tony; Ireson, Christopher; Sattler, Wolfgang

    2013-01-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun S73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

  4. p21-Activated kinase (PAK regulates cytoskeletal reorganization and directional migration in human neutrophils.

    Asako Itakura

    Full Text Available Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP, and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

  5. Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

    Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana; Pfeilschifter, Josef; Huwiler, Andrea

    2008-01-01

    Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1α and HIF-2α, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1α or HIF-2α by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression

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

    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...

  7. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

    Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

    2016-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

  8. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

    Matthew J Billard

    Full Text Available Triple negative breast cancer (TNBC is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3 is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

  9. CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

    Subramaniam, Venkateswaran; Vincent, Isabella R; Gardner, Helena; Chan, Emily; Dhamko, Helena; Jothy, Serge

    2007-10-01

    Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

  10. IκB kinase β regulates epithelium migration during corneal wound healing.

    Liang Chen

    2011-01-01

    Full Text Available The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt/tet-O-Cre/Ikkβ(F/F (Ikkβ(ΔCE/ΔCE mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F mice. Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing.

  11. Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

    Schofield, Alice V; Steel, Rohan; Bernard, Ora

    2012-12-21

    The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

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

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

    2015-12-18

    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.

  13. Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors.

    Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B

    2010-03-01

    Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.

  14. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

  15. Regulation of Autophagy by Kinases

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  16. Regulation of Autophagy by Kinases

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  17. Regulation of Autophagy by Kinases

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  18. Regulation of Autophagy by Kinases

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  19. Negative regulation of AMP-activated protein kinase (AMPK) activity by macrophage migration inhibitory factor (MIF) family members in non-small cell lung carcinomas.

    Brock, Stephanie E; Rendon, Beatriz E; Yaddanapudi, Kavitha; Mitchell, Robert A

    2012-11-02

    AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca(2+)/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.

  20. Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

    Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

    2017-01-01

    Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  1. Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

    Anne N Shemon

    2009-06-01

    Full Text Available Raf Kinase Inhibitory Protein (RKIP, also PEBP1, a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/- mouse embryonic fibroblasts (MEFs to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/- MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

  2. Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

    Shemon, Anne N; Eves, Eva M; Clark, Matthew C; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira; Koide, Shohei; Rosner, Marsha Rich

    2009-06-24

    Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

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

    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.

  4. Fisetin regulates astrocyte migration and proliferation in vitro

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-01-01

    Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro. PMID:28204814

  5. Gene regulation by MAP kinase cascades

    Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

    2009-01-01

    Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

  6. Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1).

    Zhu, Xiang-Yu; Liu, Ning; Liu, Wei; Song, Shao-Wei; Guo, Ke-Jian

    2012-04-01

    Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

  7. Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1

    Xiang-Yu Zhu

    2012-01-01

    Full Text Available Integrin-linked kinase (ILK is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1 cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

  8. Fisetin regulates astrocyte migration and proliferation in vitro.

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-04-01

    Fisetin (3,3',4',7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro.

  9. The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase

    Zhu, Zengrong; Bhat, Krishna Moorthi

    2011-01-01

    In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. We have previously shown that RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. Here, we show that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at al and fail to send out its axon projection. We report that Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, our data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. We also find that Abelson Tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. Our results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl. PMID:21726548

  10. Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

    Taro Mikami

    2015-01-01

    Full Text Available BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

  11. Insulin promotes cell migration by regulating PSA-NCAM

    Monzo, Hector J.; Coppieters, Natacha [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Park, Thomas I.H. [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Dieriks, Birger V.; Faull, Richard L.M. [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Dragunow, Mike [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Curtis, Maurice A., E-mail: m.curtis@auckland.ac.nz [Centre for Brain Research, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand); Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland (New Zealand)

    2017-06-01

    Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.

  12. Insulin promotes cell migration by regulating PSA-NCAM

    Monzo, Hector J.; Coppieters, Natacha; Park, Thomas I.H.; Dieriks, Birger V.; Faull, Richard L.M.; Dragunow, Mike; Curtis, Maurice A.

    2017-01-01

    Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.

  13. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion

    Nisha Durand

    2016-02-01

    Full Text Available The Protein Kinase D (PKD isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs and diacylglycerol (DAG. PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development.

  14. Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

    Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

    2016-09-01

    Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

    Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

    2009-03-24

    ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

  16. Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases

    Lesslie, D P; Summy, J M; Parikh, N U; Fan, F; Trevino, J G; Sawyer, T K; Metcalf, C A; Shakespeare, W C; Hicklin, D J; Ellis, L M; Gallick, G E

    2006-01-01

    Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process. PMID:16685275

  17. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

    2017-06-15

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  18. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-01-01

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  19. Src kinase regulation by phosphorylation and dephosphorylation

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  20. AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma.

    Namiki, Takeshi; Tanemura, Atsushi; Valencia, Julio C; Coelho, Sergio G; Passeron, Thierry; Kawaguchi, Masakazu; Vieira, Wilfred D; Ishikawa, Masashi; Nishijima, Wataru; Izumo, Toshiyuki; Kaneko, Yasuhiko; Katayama, Ichiro; Yamaguchi, Yuji; Yin, Lanlan; Polley, Eric C; Liu, Hongfang; Kawakami, Yutaka; Eishi, Yoshinobu; Takahashi, Eishi; Yokozeki, Hiroo; Hearing, Vincent J

    2011-04-19

    The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan-Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44-10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.

  1. ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion.

    Chen, Wei-Ta; Ebelt, Nancy D; Stracker, Travis H; Xhemalce, Blerta; Van Den Berg, Carla L; Miller, Kyle M

    2015-06-01

    Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression.

  2. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  3. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  4. Substrate Curvature Regulates Cell Migration -A Computational Study

    He, Xiuxiu; Jiang, Yi

    Cell migration in host microenvironment is essential to cancer etiology, progression and metastasis. Cellular processes of adhesion, cytoskeletal polymerization, contraction, and matrix remodeling act in concert to regulate cell migration, while local extracellular matrix architecture modulate these processes. In this work we study how stromal microenvironment with native and cell-derived curvature at micron-meter scale regulate cell motility pattern. We developed a 3D model of single cell migration on a curved substrate. Mathematical analysis of cell morphological adaption to the cell-substrate interface shows that cell migration on convex surfaces deforms more than on concave surfaces. Both analytical and simulation results show that curved surfaces regulate the cell motile force for cell's protruding front through force balance with focal adhesion and cell contraction. We also found that cell migration on concave substrates is more persistent. These results offer a novel biomechanical explanation to substrate curvature regulation of cell migration. NIH 1U01CA143069.

  5. Nonautonomous Regulation of Neuronal Migration by Insulin Signaling, DAF-16/FOXO, and PAK-1

    Lisa M. Kennedy

    2013-09-01

    Full Text Available Neuronal migration is essential for nervous system development in all organisms and is regulated in the nematode, C. elegans, by signaling pathways that are conserved in humans. Here, we demonstrate that the insulin/IGF-1-PI3K signaling pathway modulates the activity of the DAF-16/FOXO transcription factor to regulate the anterior migrations of the hermaphrodite-specific neurons (HSNs during embryogenesis of C. elegans. When signaling is reduced, DAF-16 is activated and promotes migration; conversely, when signaling is enhanced, DAF-16 is inactivated, and migration is inhibited. We show that DAF-16 acts nonautonomously in the hypodermis to promote HSN migration. Furthermore, we identify PAK-1, a p21-activated kinase, as a downstream mediator of insulin/IGF-1-DAF-16 signaling in the nonautonomous control of HSN migration. Because a FOXO-Pak1 pathway was recently shown to regulate mammalian neuronal polarity, our findings indicate that the roles of FOXO and Pak1 in neuronal migration are most likely conserved from C. elegans to higher organisms.

  6. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

    of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

  7. Regulators of Intestinal Epithelial Migration in Sepsis.

    Meng, Mei; Klingensmith, Nathan J; Liang, Zhe; Lyons, John D; Fay, Katherine T; Chen, Ching-Wen; Ford, Mandy L; Coopersmith, Craig M

    2018-02-08

    The gut is a continuously renewing organ, with cell proliferation, migration and death occurring rapidly under basal conditions. Since the impact of critical illness on cell movement from crypt base to villus tip is poorly understood, the purpose of this study was to determine how sepsis alters enterocyte migration. Wild type, transgenic and knockout mice were injected with 5-bromo-2'deoxyuridine (BrdU) to label cells in S phase before and after the onset of cecal ligation and puncture and were sacrificed at pre-determined endpoints to determine distance proliferating cells migrated up the crypt-villus unit. Enterocyte migration rate was decreased from 24-96 hours following sepsis. BrdU was not detectable on villi 6 days after sham laparotomy, meaning all cells had migrated the length of the gut and been exfoliated into its lumen. However, BrdU positive cells were detectable on villi 10 days after sepsis. Multiple components of gut integrity altered enterocyte migration. Sepsis decreased crypt proliferation, which further slowed enterocyte transit as mice injected with BrdU after the onset of sepsis (decreased proliferation) had slower migration than mice injected with BrdU prior to the onset of sepsis (normal proliferation). Decreasing intestinal apoptosis via gut-specific overexpression of Bcl-2 prevented sepsis-induced slowing of enterocyte migration. In contrast, worsened intestinal hyperpermeability by genetic deletion of JAM-A increased enterocyte migration. Sepsis therefore significantly slows enterocyte migration, and intestinal proliferation, apoptosis and permeability all affect migration time, which can potentially be targeted both genetically and pharmacologically.

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

    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

  9. Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

    Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

    2014-12-01

    Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

  10. SHARPIN Regulates Uropod Detachment in Migrating Lymphocytes

    Jeroen Pouwels

    2013-11-01

    Full Text Available SHARPIN-deficient mice display a multiorgan chronic inflammatory phenotype suggestive of altered leukocyte migration. We therefore studied the role of SHARPIN in lymphocyte adhesion, polarization, and migration. We found that SHARPIN localizes to the trailing edges (uropods of both mouse and human chemokine-activated lymphocytes migrating on intercellular adhesion molecule-1 (ICAM-1, which is one of the major endothelial ligands for migrating leukocytes. SHARPIN-deficient cells adhere better to ICAM-1 and show highly elongated tails when migrating. The increased tail lifetime in SHARPIN-deficient lymphocytes decreases the migration velocity. The adhesion, migration, and uropod defects in SHARPIN-deficient lymphocytes were rescued by reintroducing SHARPIN into the cells. Mechanistically, we show that SHARPIN interacts directly with lymphocyte-function-associated antigen-1 (LFA-1, a leukocyte counterreceptor for ICAM-1, and inhibits the expression of intermediate and high-affinity forms of LFA-1. Thus, SHARPIN controls lymphocyte migration by endogenously maintaining LFA-1 inactive to allow adjustable detachment of the uropods in polarized cells.

  11. Non-autonomous Regulation of Neuronal Migration by Insulin Signaling, DAF-16/FOXO and PAK-1

    Kennedy, Lisa M.; Pham, Steven C.D.L.; Grishok, Alla

    2013-01-01

    SUMMARY Neuronal migration is essential for nervous system development in all organisms and is regulated in the nematode, C. elegans, by signaling pathways that are conserved in humans. Here, we demonstrate that the Insulin/IGF-1-PI3K signaling pathway modulates the activity of the DAF-16/FOXO transcription factor to promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. When signaling is reduced, DAF-16 is activated and promotes migration, conversely, when signaling is enhanced, DAF-16 is inactivated and migration is inhibited. We show that DAF-16 acts non-autonomously in the hypodermis to promote HSN migration. Furthermore, we identify PAK-1, a p21-activated kinase, as a downstream mediator of Insulin/IGF-1-DAF-16 signaling in the non-autonomous control of HSN migration. As a FOXO-Pak1 pathway was recently shown to regulate mammalian neuronal polarity, our findings indicate that the roles of FOXO and Pak1 in neuronal migration are likely conserved from C. elegans to higher organisms. PMID:23994474

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

    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

  13. ASIC proteins regulate smooth muscle cell migration.

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

  14. Regulation of glucose metabolism in T cells; new insight into the role of Phosphoinositide 3-kinases

    David K Finlay

    2012-08-01

    Full Text Available Naïve T cells are relatively quiescent cells that only require energy to prevent atrophy and for survival and migration. However, in response to developmental or extrinsic cues T cells can engage in rapid growth and robust proliferation, produce of a range of effector molecules and migrate through peripheral tissues. To meet the significantly increased metabolic demands of these activities, T cells switch from primarily metabolizing glucose to carbon dioxide through oxidative phosphorylation to utilizing glycolysis to convert glucose to lactate (termed aerobic glycolysis. This metabolic switch allows glucose to be used as a source of carbon to generate biosynthetic precursors for the production of protein, DNA and phospholipids, and is crucial for T cells to meet metabolic demands. Phosphoinositide 3-kinases (PI3K are a family of inositol lipid kinases linked with a broad range of cellular functions in T lymphocytes that include cell growth, proliferation, metabolism, differentiation, survival and migration. Initial research described a critical role for PI3K signaling through Akt (also called Protein kinase B for the increased glucose uptake and glycolysis that accompanies T cell activation. This review article relates this original research with more recent data and discusses the evidence for and against a role for PI3K in regulating the metabolic switch to aerobic glycolysis in T cells.

  15. The F-BAR domain protein PACSIN2 associates with Rac1 and regulates cell spreading and migration

    de Kreuk, Bart-Jan; Nethe, Micha; Fernandez-Borja, Mar; Anthony, Eloise C.; Hensbergen, Paul J.; Deelder, Andre M.; Plomann, Markus; Hordijk, Peter L.

    2011-01-01

    The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins, such as the PAK kinases and the Scar and WAVE proteins. We previously identified a series of regulatory proteins that associate with Rac1 through its

  16. SOCS proteins in regulation of receptor tyrosine kinase signaling

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  17. Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

    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. Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells

    van Oosterwijk, J G; van Ruler, M A J H; Briaire-de Bruijn, I H; Herpers, B; Gelderblom, H; van de Water, B; Bovée, J V M G

    2013-01-01

    Background: Chondrosarcomas are malignant cartilage-forming tumours of bone. Because of their resistance to conventional chemotherapy and radiotherapy, currently no treatment strategies exist for unresectable and metastatic chondrosarcoma. Previously, PI3K/AKT/GSK3β and Src kinase pathways were shown to be activated in chondrosarcoma cell lines. Our aim was to investigate the role of these kinases in chemoresistance and migration in chondrosarcoma in relation to TP53 mutation status. Methods: We used five conventional and three dedifferentiated chondrosarcoma cell lines and investigated the effect of PI3K/AKT/GSK3β pathway inhibition (enzastaurin) and Src pathway inhibition (dasatinib) in chemoresistance using WST assay and live cell imaging with AnnexinV staining. Immunohistochemistry on tissue microarrays (TMAs) containing 157 cartilaginous tumours was performed for Src family members. Migration assays were performed with the RTCA xCelligence System. Results: Src inhibition was found to overcome chemoresistance, to induce apoptosis and to inhibit migration. Cell lines with TP53 mutations responded better to combination therapy than wild-type cell lines (P=0.002). Tissue microarray immunohistochemistry confirmed active Src (pSrc) signalling, with Fyn being most abundantly expressed (76.1%). Conclusion: These results strongly indicate Src family kinases, in particular Fyn, as a potential target for the treatment of inoperable and metastatic chondrosarcomas, and to sensitise for doxorubicin especially in the presence of TP53 mutations. PMID:23922104

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

    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

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

    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.

  1. FOREIGN EXPERIENCE OF STATE REGULATION OF MIGRATION PROCESSES

    Ekaterina Nikolaevna Tarasenko

    2018-05-01

    Full Text Available International migration of population has existed for centuries, as it has activated as a result of globalization. Share the non-economic causes of international migration (causes related to wars, political and religious persecution, the desire to explore new spaces, the desire for family reunification, natural disasters and economic problems (the search for a new job in the absence of the opportunity to find a job in their own country, the search for more paid or creative work, a higher quality of life. Recently, the main reason for migration is economic reasons, on the basis of which the popular migration corridors and the leading directions of migration of labor personnel are identified. Analyzed the main centers of attraction of migration, namely, the United States of America, Federal Republic of Germany and the Russian Federation. Noted that the means and methods of implementation of the State migration policy vary depending on the specific situation on the labor market. So, given the shortage of labor in some European countries, such as Germany, used methods of stimulating immigration. When there is a need to reduce the level of immigration, as in the case of the United States, government regulation sets barriers to a new influx of foreign workers. Revealed, the dynamics of migration primarily due to social phenomena. Adverse external conditions: the deterioration of the economic, environmental or political situation in the country of residence is becoming an important factor in the readiness of potential migrants for forced migration. However, migrants have different socio-economic characteristics, and so they choose the wrong country for migration that they will be closer to social and psychological features. However, migrants have different socio-economic characteristics, and so they choose the wrong country for migration that they will be closer to social and psychological features. The purpose is to study international experience of

  2. ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

    Grifoni, Samira C.; Jernigan, Nikki L.; Hamilton, Gina; Drummond, Heather A.

    2007-01-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated Epithelial Na+ Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration, however the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence indi...

  3. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

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

    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.

  5. Frank-ter Haar syndrome protein Tks4 regulates epidermal growth factor-dependent cell migration.

    Bögel, Gábor; Gujdár, Annamária; Geiszt, Miklós; Lányi, Árpád; Fekete, Anna; Sipeki, Szabolcs; Downward, Julian; Buday, László

    2012-09-07

    Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

  6. PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration.

    Bahm, Isabel; Barriga, Elias H; Frolov, Antonina; Theveneau, Eric; Frankel, Paul; Mayor, Roberto

    2017-07-01

    A fundamental property of neural crest (NC) migration is contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibians and zebrafish by controlling cell polarity in a cell contact-dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin, which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial-to-mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. Here, we show that platelet-derived growth factor receptor alpha (PDGFRα) and its ligand platelet-derived growth factor A (PDGF-A) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently, impairing CIL. Moreover, we identify phosphatidylinositol-3-kinase (PI3K)/AKT as a downstream effector of the PDGFRα cellular response during CIL. Our results lead us to propose PDGF-A/PDGFRα signalling as a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, we show that once NC cells have undergone EMT, the same PDGF-A/PDGFRα works as an NC chemoattractant, guiding their directional migration. © 2017. Published by The Company of Biologists Ltd.

  7. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-01-01

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

  8. The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

    Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John; Rhind, Nicholas

    2008-01-01

    Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or ...

  9. The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation.

    Parsons, Sean A; Mewburn, Jeffrey D; Truesdell, Peter; Greer, Peter A

    2007-12-01

    Fps/Fes and Fer comprise a distinct subfamily of cytoplasmic protein-tyrosine kinases, and have both been implicated in the regulation of innate immunity. Previous studies showed that Fps/Fes-knockout mice were hypersensitive to systemic lipopolysaccharide (LPS) challenge, and Fer-deficient mice displayed enhanced recruitment of leucocytes in response to localized LPS challenge. We show here for the first time, a role for Fps in the regulation of leucocyte recruitment to areas of inflammation. Using the cremaster muscle intravital microscopy model, we observed increased leucocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-knockout mice relative to wild-type animals subsequent to localized LPS challenge. There was also a decreased vessel wall shear rate in the post-capillary venules of LPS-challenged Fps/Fes-knockout mice, and an increase in neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry to quantify the expression of surface molecules, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex vivo with LPS. These observations provide important insights into the observed in vivo behaviour of leucocytes in LPS-challenged Fps/Fes-knockout mice and provide evidence that the Fps/Fes kinase plays an important role in the innate immune response.

  10. SOX15 regulates proliferation and migration of endometrial cancer cells.

    Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

    2017-10-31

    The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

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

    Crowe, David L; Ohannessian, Arthur

    2004-01-01

    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. 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. 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. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

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

    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.

  13. Angiogenin enhances cell migration by regulating stress fiber assembly and focal adhesion dynamics.

    Saisai Wei

    Full Text Available Angiogenin (ANG acts on both vascular endothelial cells and cancer cells, but the underlying mechanism remains elusive. In this study, we carried out a co-immunoprecipitation assay in HeLa cells and identified 14 potential ANG-interacting proteins. Among these proteins, β-actin, α-actinin 4, and non-muscle myosin heavy chain 9 are stress fiber components and involved in cytoskeleton organization and movement, which prompted us to investigate the mechanism of action of ANG in cell migration. Upon confirmation of the interactions between ANG and the three proteins, further studies revealed that ANG co-localized with β-actin and α-actinin 4 at the leading edge of migrating cells. Down-regulation of ANG resulted in fewer but thicker stress fibers with less dynamics, which was associated with the enlargements of focal adhesions. The focal adhesion kinase activity and cell migration capacity were significantly decreased in ANG-deficient cells. Taken together, our data demonstrated that the existence of ANG in the cytoplasm optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration. The finding that ANG promoted cancer cell migration might provide new clues for tumor metastasis research.

  14. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

    Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

    2017-11-09

    CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

  15. Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

    Wu, Weibin; Sun, Zhichao; Wu, Jingwen; Peng, Xiaomin; Gan, Huacheng; Zhang, Chunyi; Ji, Lingling; Xie, Jianhui; Zhu, Haiyan; Ren, Shifang

    2012-01-01

    c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. PMID:22238675

  16. A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing.

    Ghosh, Manik C; Makena, Patrudu S; Kennedy, Joseph; Teng, Bin; Luellen, Charlean; Sinclair, Scott E; Waters, Christopher M

    2017-05-19

    Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

  17. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  18. Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

    Margarida Ressurreição

    2014-06-01

    Full Text Available Protein kinases C (PKCs and extracellular signal-regulated kinases (ERKs are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.

  19. Pyruvate kinase M2: a potential target for regulating inflammation

    Jose Carlos eAlves-Filho

    2016-04-01

    Full Text Available Pyruvate kinase (PK is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signalling pathways, affecting both the enzymatic activity of PKM2 as a pyruvate kinase and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for PKM2 as a therapeutic target in inflammatory and metabolic disorders.

  20. Role of integrin-linked kinase in vascular smooth muscle cells: Regulation by statins and angiotensin II

    Friedrich, Erik B.; Clever, Yvonne P.; Wassmann, Sven; Werner, Nikos; Boehm, Michael; Nickenig, Georg

    2006-01-01

    Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy

  1. Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

    Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

    2018-05-10

    The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue

  2. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  3. Protein kinase C signaling and cell cycle regulation

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  4. Tyrosine kinase chromosomal translocations mediate distinct and overlapping gene regulation events

    Kim, Hani; Gillis, Lisa C; Jarvis, Jordan D; Yang, Stuart; Huang, Kai; Der, Sandy; Barber, Dwayne L

    2011-01-01

    Leukemia is a heterogeneous disease commonly associated with recurrent chromosomal translocations that involve tyrosine kinases including BCR-ABL, TEL-PDGFRB and TEL-JAK2. Most studies on the activated tyrosine kinases have focused on proximal signaling events, but little is known about gene transcription regulated by these fusions. Oligonucleotide microarray was performed to compare mRNA changes attributable to BCR-ABL, TEL-PDGFRB and TEL-JAK2 after 1 week of activation of each fusion in Ba/F3 cell lines. Imatinib was used to control the activation of BCR-ABL and TEL-PDGFRB, and TEL-JAK2-mediated gene expression was examined 1 week after Ba/F3-TEL-JAK2 cells were switched to factor-independent conditions. Microarray analysis revealed between 800 to 2000 genes induced or suppressed by two-fold or greater by each tyrosine kinase, with a subset of these genes commonly induced or suppressed among the three fusions. Validation by Quantitative PCR confirmed that eight genes (Dok2, Mrvi1, Isg20, Id1, gp49b, Cxcl10, Scinderin, and collagen Vα1(Col5a1)) displayed an overlapping regulation among the three tested fusion proteins. Stat1 and Gbp1 were induced uniquely by TEL-PDGFRB. Our results suggest that BCR-ABL, TEL-PDGFRB and TEL-JAK2 regulate distinct and overlapping gene transcription profiles. Many of the genes identified are known to be involved in processes associated with leukemogenesis, including cell migration, proliferation and differentiation. This study offers the basis for further work that could lead to an understanding of the specificity of diseases caused by these three chromosomal translocations

  5. Involvement of JAK2 upstream of the PI 3-kinase in cell-cell adhesion regulation by gastrin

    Ferrand, Audrey; Kowalski-Chauvel, Aline; Bertrand, Claudine; Pradayrol, Lucien; Fourmy, Daniel; Dufresne, Marlene; Seva, Catherine

    2004-01-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway has been implicated in cell transformation and proliferation. Besides aberrant cell proliferation, loss of cell-cell adhesion during epithelial-mesenchymal transition (EMT) is an important event which occurs during development of epithelial cancers. However, the role of JAK-dependent pathways in this process is not known. We analyzed the involvement of these pathways in the regulation of E-cadherin-dependent cell-cell adhesion by gastrin, a mitogenic factor for gastrointestinal (GI) tract. We identified JAK2/STAT3 as a new pathway in gastrin signaling. We demonstrated that JAK2 functions as an upstream mediator of the phosphatidylinositol 3 (PI 3)-kinase activity in gastrin signaling. Indeed, we observed a coprecipitation of both kinases and an inhibition of gastrin-induced PI 3-kinase activation when JAK2 activity is blocked. We also demonstrated that loss of cell-cell adhesion and the increase in cell motility induced by gastrin required the activation of JAK2 and the PI 3-kinase. Indeed, the modifications in localization of adherens junctions proteins and the migration, observed in gastrin-stimulated cells, were reversed by inhibition of both kinases. These results described the involvement of JAK2 in the modulation of cell-cell adhesion in epithelial cells. They support a possible role of JAK2 in the epithelial-mesenchymal transition which occurs during malignant development

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

    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.

  7. Flotillins Regulate Focal Adhesions by Interacting with α-Actinin and by Influencing the Activation of Focal Adhesion Kinase

    Antje Banning

    2018-04-01

    Full Text Available Cell–matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell–matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation.

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

    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...

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

    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.

  10. Telocinobufagin inhibits the epithelial-mesenchymal transition of breast cancer cells through the phosphoinositide 3-kinase/protein kinase B/extracellular signal-regulated kinase/Snail signaling pathway.

    Gao, Yuxue; Shi, Lihong; Cao, Zhen; Zhu, Xuetao; Li, Feng; Wang, Ruyan; Xu, Jinyuan; Zhong, Jinyi; Zhang, Baogang; Lu, Shijun

    2018-05-01

    Telocinobufagin (TBG), an active ingredient of Venenumbufonis , exhibits an immunomodulatory activity. However, its antimetastatic activity in breast cancer remains unknown. The present study investigated whether TBG prevents breast cancer metastasis and evaluated its regulatory mechanism. TBG inhibited the migration and invasion of 4T1 breast cancer cells. Furthermore, TBG triggered the collapse of F-actin filaments in breast cancer. The epithelial-mesenchymal transition (EMT) markers, vimentin and fibronectin, were downregulated following TBG treatment. However, E-cadherin was upregulated following TBG treatment. Snail, a crucial transcriptional factor of EMT, was downregulated following TBG treatment. Signaling pathway markers, including phosphorylated protein kinase B (P-Akt), p-mechanistic target of rapamycin (mTOR) and p-extracellular signal-regulated kinase (ERK), were decreased following TBG treatment. The same results were obtained from in vivo experiments. In conclusion, in vitro and in vivo experiments reveal that TBG inhibited migration, invasion and EMT via the phosphoinositide 3-kinase (PI3K)/Akt/ERK/Snail signaling pathway in breast cancer.

  11. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. © 2014 John Wiley & Sons Ltd.

  12. Y-27632 Increases Sensitivity of PANC-1 Cells to EGCG in Regulating Cell Proliferation and Migration.

    Liu, Xing; Bi, Yongyi

    2016-10-03

    BACKGROUND The study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (-)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer. MATERIAL AND METHODS PANC-1 cells, maintained in Dulbecco's Modified Eagle's Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator-activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR). RESULTS EGCG (20-80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARa and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632. CONCLUSIONS Y-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARa mRNA and Caspase-3 mRNA.

  13. The Hsk1(Cdc7) replication kinase regulates origin efficiency.

    Patel, Prasanta K; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John; Rhind, Nicholas

    2008-12-01

    Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

  14. The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

    Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John

    2008-01-01

    Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency. PMID:18799612

  15. Neuronal migration is regulated by endogenous RNAi and chromatin-binding factor ZFP-1/AF10 in Caenorhabditis elegans.

    Kennedy, Lisa M; Grishok, Alla

    2014-05-01

    Endogenous short RNAs and the conserved plant homeodomain (PHD) zinc-finger protein ZFP-1/AF10 regulate overlapping sets of genes in Caenorhabditis elegans, which suggests that they control common biological pathways. We have shown recently that the RNAi factor RDE-4 and ZFP-1 negatively modulate transcription of the insulin/PI3 signaling-dependent kinase PDK-1 to promote C. elegans fitness. Moreover, we have demonstrated that the insulin/IGF-1-PI3K-signaling pathway regulates the activity of the DAF-16/FOXO transcription factor in the hypodermis to nonautonomously promote the anterior migrations of the hermaphrodite-specific neurons (HSNs) during embryogenesis of C. elegans. In this study, we implicate the PHD-containing isoform of ZFP-1 and endogenous RNAi in the regulation of HSN migration. ZFP-1 affects HSN migration in part through its negative effect on pdk-1 transcription and modulation of downstream DAF-16 activity. We also identify a novel role for ZFP-1 and RNAi pathway components, including RDE-4, in the regulation of HSN migration in parallel with DAF-16. Therefore, the coordinated activities of DAF-16, ZFP-1, and endogenous RNAi contribute to gene regulation during development to ensure proper neuronal positioning.

  16. Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

    Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

    2014-05-01

    Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H{sub 2}O{sub 2} in HL-1 mouse cardiac muscle cells

    Rao, F. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Deng, C.Y. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Zhang, Q.H.; Xue, Y.M. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Xiao, D.Z.; Kuang, S.J.; Lin, Q.X.; Shan, Z.X.; Liu, X.Y.; Zhu, J.N. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Yu, X.Y. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Wu, S.L. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China)

    2013-09-06

    Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H{sub 2}O{sub 2}), but not angiotensin II, stimulated MIF expression in HL-1 cells. H{sub 2}O{sub 2}-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H{sub 2}O{sub 2}-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

  18. Protein kinase Cδ signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

    Kharait, Sourabh; Dhir, Rajiv; Lauffenburger, Douglas; Wells, Alan

    2006-01-01

    Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cδ (PKCδ)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCδ is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCδ using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCδ and phosphorylated PKCδ protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCδ inhibition can limit migration and invasion of prostate cancer cells

  19. Csk Homologous Kinase, a Potential Regulator of CXCR4-mediated Breast Cancer Cell Metastasis

    2010-08-31

    SH2 ) and SH3 domains and lacks the consensus tyrosine phosphorylation and myristylation sites found in Src family kinases . CHK has been shown to...0350 TITLE: Csk Homologous Kinase , a Potential Regulator of CXCR4-mediated Breast Cancer Cell Metastasis PRINCIPAL INVESTIGATOR: Byeong-Chel...1 AUG 2009 - 31 JUL 2010 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-09-1-0350 Csk Homologous Kinase , a Potential Regulator

  20. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...osine kinases and the regulation of macrophage activation. PubmedID 14726496 Title Receptor tyrosine...rell PH, Morrison AC, Lutz MA. J Leukoc Biol. 2004 May;75(5):731-7. Epub 2004 Jan 14. (.png) (.svg) (.html) (.csml) Show Receptor tyr

  1. Glycogen synthase kinase-3 regulation of urinary concentrating ability.

    Rao, Reena

    2012-09-01

    Glycogen synthase kinase-3 (GSK3) is an enzyme that is gaining prominence as a critical signaling molecule in the epithelial cells of renal tubules. This review will focus on recent findings exploring the role of GSK3 in renal collecting ducts, especially its role in urine concentration involving vasopressin signaling. Recent studies using inhibition or tissue-specific gene deletion of GSK3 revealed the mechanism by which GSK3 regulates aquaporin 2 water channels via adenylate cyclase or the prostaglandin-E2 pathway. In other studies, postnatal treatment with lithium, an inhibitor of GSK3, increased cell proliferation and led to microcyst formation in rat kidneys. These studies suggest that loss of GSK3 activity could interfere with renal water transport at two levels. In the short term, it could disrupt vasopressin signaling in collecting duct cells and in the long term it could alter the structure of the collecting ducts, making them less responsive to the hydro-osmotic effects of vasopressin. Ongoing studies reveal the crucial role played by GSK3 in the regulation of vasopressin action in the renal collecting ducts and suggest a possible use of GSK3 inhibitors in disease conditions associated with disrupted vasopressin signaling.

  2. Glycogen synthase kinase-3 regulates inflammatory tolerance in astrocytes

    Beurel, Eléonore; Jope, Richard S.

    2010-01-01

    Inflammatory tolerance is the down-regulation of inflammation upon repeated stimuli, which is well-established to occur in peripheral immune cells. However, less is known about inflammatory tolerance in the brain although it may provide an important protective mechanism from detrimental consequences of prolonged inflammation, which appears to occur in many psychiatric and neurodegenerative conditions. Array analysis of 308 inflammatory molecules produced by mouse primary astrocytes after two sequential stimulations with lipopolysaccharide (LPS) distinguished three classes, tolerant, sensitized and unaltered groups. For many of these inflammatory molecules, inhibition of glycogen synthase kinase-3 (GSK3) increased tolerance and reduced sensitization. Focusing on LPS-tolerance in interleukin-6 (IL-6) production, we found that microglia exhibited a strong tolerance response that matched that of macrophages, whereas astrocytes exhibited only partial tolerance. The astrocyte semi-tolerance was found to be regulated by GSK3. GSK3 inhibitors or knocking down GSK3 levels promoted LPS-tolerance and astrocytes expressing constitutively active GSK3 did not develop LPS-tolerance. These findings identify the critical role of GSK3 in counteracting IL-6 inflammatory tolerance in cells of the CNS, supporting the therapeutic potential of GSK3 inhibitors to reduce neuroinflammation by promoting tolerance. PMID:20553816

  3. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

    Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

  4. VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration.

    Williams, B Blairanne; Cantrell, V Ashley; Mundell, Nathan A; Bennett, Andrea C; Quick, Rachel E; Jessen, Jason R

    2012-05-01

    Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes.

  5. Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells

    Bansal, Nitu; Mishra, Prasun J.; Stein, Mark; DiPaola, Robert S.; Bertino, Joseph R.

    2015-01-01

    Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl. PMID:26036314

  6. STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.

    Pankaj Goyal

    Full Text Available BACKGROUND: Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs, and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a leading to G(1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays. CONCLUSION/SIGNIFICANCE: The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear

  7. Cytoskeletal Regulation by AUTS2 in Neuronal Migration and Neuritogenesis

    Kei Hori

    2014-12-01

    Full Text Available Mutations in the Autism susceptibility candidate 2 gene (AUTS2, whose protein is believed to act in neuronal cell nuclei, have been associated with multiple psychiatric illnesses, including autism spectrum disorders, intellectual disability, and schizophrenia. Here we show that cytoplasmic AUTS2 is involved in the regulation of the cytoskeleton and neural development. Immunohistochemistry and fractionation studies show that AUTS2 localizes not only in nuclei, but also in the cytoplasm, including in the growth cones in the developing brain. AUTS2 activates Rac1 to induce lamellipodia but downregulates Cdc42 to suppress filopodia. Our loss-of-function and rescue experiments show that a cytoplasmic AUTS2-Rac1 pathway is involved in cortical neuronal migration and neuritogenesis in the developing brain. These findings suggest that cytoplasmic AUTS2 acts as a regulator of Rho family GTPases to contribute to brain development and give insight into the pathology of human psychiatric disorders with AUTS2 mutations.

  8. MicroRNA-200b Suppresses Arsenic-transformed Cell Migration by Targeting Protein Kinase Cα and Wnt5b-Protein Kinase Cα Positive Feedback Loop and Inhibiting Rac1 Activation*

    Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

    2014-01-01

    MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3′-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation. PMID:24841200

  9. MicroRNA-200b suppresses arsenic-transformed cell migration by targeting protein kinase Cα and Wnt5b-protein kinase Cα positive feedback loop and inhibiting Rac1 activation.

    Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

    2014-06-27

    MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3'-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation. © 2014 by The American Society for Biochemistry and Molecular

  10. Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

    Serrano, Isabel; Diez-Marques, Maria L.; Rodriguez-Puyol, Manuel [Department of Physiology, University of Alcala, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain); Herrero-Fresneda, Inmaculada [Nephrology Unit, IDIBELL, Hospital de Bellvitge, Barcelona (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Garcia del Moral, Raimundo [Department of Pathology, University of Granada (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Dedhar, Shoukat [Department of Integrative Oncology, BC Cancer Research Center, Vancouver, BC (Canada); Ruiz-Torres, Maria P., E-mail: mpiedad.ruiz@uah.es [Department of Physiology, University of Alcala, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain); Rodriguez-Puyol, Diego [Nephrology Unit, Hospital Universitario Principe de Asturias, Alcala de Henares, Madrid (Spain); Red de Investigacion Renal Cooperativa (RedinRen) (Spain); Instituto Reina Sofia de Investigacion Nefrologica (Spain)

    2012-11-15

    Integrin-linked kinase (ILK) is an intracellular effector of cell-matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing. -- Highlights: Black-Right-Pointing-Pointer ILK deletion results in decreased HGF expression and delayed scratch wound repair. Black-Right-Pointing-Pointer PI3K/ILK/AKT pathway signals through HGF to regulate wound healing. Black-Right-Pointing-Pointer An ILK-dependent increase in HGF expression is responsible for wound healing in vivo. Black-Right-Pointing-Pointer ILK-KO mice are used to confirm the requirement for ILK function in wound healing. Black-Right-Pointing-Pointer Human HGF treatment restores delayed wound closure in vitro and in vivo.

  11. ATR kinase regulates its attenuation via PPM1D phosphatase ...

    In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogationleads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition ofATR kinase activity specifically during replication stress recovery results in persistent ...

  12. Evolutionary adaptations of plant AGC kinases: from light signaling to cell polarity regulation

    Eike Hendrik Rademacher

    2012-11-01

    Full Text Available Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues internal stimuli can modify the activity or abundance of these proteins at the plasma membrane. One such regulatory mechanism is protein phosphorylation by membrane-associated kinases and phosphatases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the plant AGC kinases that most likely allow for sensing of external stimuli (i.e. light through controlled modification of kinase activity.Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the plasma membrane depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP2A phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth.

  13. Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration.

    Erika Costa de Alvarenga

    Full Text Available The angiotensin-I converting enzyme (ACE plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II. More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet.Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration.We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC, and experimental results in CHO cells, demonstrated that the β3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5 showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein.ACE activation regulates melanoma cell proliferation and migration.

  14. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    Fabian V Filipp

    2013-01-01

    Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor acti...

  15. Src protein-tyrosine kinase structure and regulation

    Roskoski, Robert

    2004-01-01

    Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

  16. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2007-01-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85α and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1

  17. Lipopolysaccharide induces the migration of human dental pulp cells by up-regulating miR-146a.

    Wang, Min-Ching; Hung, Pei-Shih; Tu, Hsi-Feng; Shih, Wen-Yu; Li, Wan-Chun; Chang, Kuo-Wei

    2012-12-01

    MicroRNAs are small noncoding RNAs that play crucial roles in regulating normal and pathologic functions. Bacterial lipopolysaccharide (LPS) is one of the key regulators of pulpal pathogenesis. This study investigated how LPS regulates microRNA expression and affects the phenotype of human dental pulp cells (DPCs). Primary DPCs were established and immortalized to achieve immortalized DPCs (I-DPCs). DPCs and I-DPCs were treated with LPS and examined to identify changes in microRNA expression, cell proliferation, and cell migration. Quantitative reverse-transcriptase polymerase chain reaction was used to detect changes in gene expression. Exogenous miR-146a expression was performed transfection with pre-mir-146a mimic. Knockdown of interleukin receptor-associated kinase (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) expression was performed by small interference oligonucleotide transfection. Western blot analysis was used to detect changes in the expression of the IRAK1 and TRAF6 proteins. The differentiation of DPCs was induced by osteogenic medium. I-DPCs had a higher level of human telomerase reverse transcriptase gene than the parental DPCs. Up-regulation of miR-146a expression and an increase in migration was induced by LPS treatment of DPCs and I-DPCs. Exogenous miR-146a expression increased the migration of DPCs and I-DPCs and down-regulated the expression of IRAK1 and TRAF6. Knockdown of IRAK1 and/or TRAF6 increased the migration of DPCs. The results suggested that LPS is able to increase the migration of DPCs by modulating the miR-146a-TRAF6/IRAK1 regulatory cascade. Copyright © 2012 American Association of Endodontists. All rights reserved.

  18. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2016-12-16

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae*

    Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M.

    2016-01-01

    In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1–77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1–77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. PMID:27834677

  20. Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells.

    Cho, Sun-Mi; Lee, Eun-Ok; Kim, Sung-Hoon; Lee, Hyo-Jeong

    2014-07-30

    The essential oil of Pinus koraiensis (EOPK) is biologically active compound obtained from the leaves of P. koraiensis. The goal of this study was to investigate the anti-cancer mechanism of EOPK in HCT116 colorectal cancer cells. HCT116 cell proliferation was assessed by conducting crystal violet and BrdU assays. To assess the effects of EOPK on cell migration, we performed a wound-healing assay. Further, the contribution of PAK1 to EOPK-induced AKT and extracellular signal-regulated kinase (ERK) suppression was assessed by siRNA-mediated PAK1 knockdown. Changes to the expression and phosphorylation of PAK1 and its effectors were determined by western blotting, and changes to the actin cytoskeleton were determined by performing an immunofluorescence assay. EOPK significantly decreased HCT116 cell proliferation and migration, and induced G1 arrest without affecting normal cells. Additionally, EOPK suppressed the expression of PAK1, and decreased ERK and AKT phosphorylation in HCT116 cells. Finally, EOPK suppressed β-catenin, cyclin D1, and CDK4/6 expression. Our studies indicate that EOPK significantly reduced proliferation and migration of colorectal cancer cells. Furthermore, EOPK suppressed PAK1 expression in a dose-dependent manner, and this suppression of PAK1 led to inhibition of ERK, AKT, and β-catenin activities. Our findings suggest that EOPK exerts its anticancer activity via the inhibition of PAK1 expression, suggesting it may be a potent chemotherapeutic agent for colorectal cancer.

  1. Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

    Stephanie Jamison

    Full Text Available Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK in response to endoplasmic reticulum (ER stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A. Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2, to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

  2. Protein kinase C signaling and cell cycle regulation

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  3. Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

    Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

    2017-10-01

    Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

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

    Ren-You Gan

    2014-09-01

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

  5. Upregulated STAT3 and RhoA signaling in colorectal cancer (CRC) regulate the invasion and migration of CRC cells.

    Zhang, G-Y; Yang, W-H; Chen, Z

    2016-05-01

    We aimed to reveal the expression and activation of signal transducers and activators of transcription 3 (STAT3) and RhoA/Rho-associated coiled-coil forming kinase 1 (ROCK1) signaling in CRC tissues, and to investigate the regulatory role of STAT3 and RhoA signaling in the invasion and migration of colorectal cancer cells. We examined the expression of STAT3, RhoA and ROCK1 in CRC tissues with real-time PCR and Western blotting methods. And then we examined the interaction between STAT3 and RhoA/ROCK1 signaling in CRC HT-29 cells with gain-of-function and loss-of-function strategies. In addition, we determined the regulation by STAT3 and RhoA/ROCK1 on the invasion and migration of CRC HT-29 cells. Our study demonstrated a significant upregulation of RhoA and ROCK1 expression and STAT3-Y705 phosphorylation in 32 CRC specimens, compared to the 17 normal CRC tissues. Further study demonstrated there was a coordination between STAT3 and RhoA/Rock signaling in the HT-29 cells. Moreover, STAT3 knockdown or RhoA knockdown significantly repressed the migration and invasion in HT-29 cells and vice versa. STAT3 and RhoA signaling regulate the invasion and migration of CRC cells, implying the orchestrated and oncogenic roles of STAT3 and RhoA/ROCK1 signaling in CRC.

  6. Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy

    Michael eMutlak

    2015-07-01

    Full Text Available Cardiac hypertrophy results from increased mechanical load on the heart and through the actions of local and systemic neuro-humoral factors, cytokines and growth factors. These mechanical and neuroendocrine effectors act through stretch, G protein-coupled receptors and tyrosine kinases to induce the activation of a myriad of intracellular signaling pathways including the extracellular signal-regulated kinases 1/2 (ERK1/2. Since most stimuli that provoke myocardial hypertrophy also elicit an acute phosphorylation of the threonine-glutamate-tyrosine (TEY motif within the activation loops of ERK1 and ERK2 kinases, resulting in their activation, ERKs have long been considered promotors of cardiac hypertrophy. Several mouse models were generated in order to directly understand the causal role of ERK1/2 activation in the heart. These models include direct manipulation of ERK1/2 such as overexpression, mutagenesis or knockout models, manipulations of upstream kinases such as MEK1 and manipulations of the phosphatases that depohosphorylate ERK1/2 such as DUSP6. The emerging understanding from these studies, as will be discussed here, is more complex than originally considered. While there is little doubt that ERK1/2 activation or the lack of it modulates the hypertrophic process or the type of hypertrophy that develops, it appears that not all ERK1/2 activation events are the same. While much has been learned, some questions remain regarding the exact role of ERK1/2 in the heart, the upstream events that result in ERK1/2 activation and the downstream effector in hypertrophy.

  7. Regulation of hematopoietic cell function by protein tyrosine kinase-encoding oncogenes, a review

    Punt, C. J.

    1992-01-01

    Tyrosine phosphorylation of proteins by protein tyrosine kinases (PTKs) is an important mechanism in the regulation of various cellular processes such as proliferation, differentiation, and transformation. Accumulating data implicate PTKs as essential intermediates in the transduction of

  8. Resveratrol blocks interleukin-18-EMMPRIN cross-regulation and smooth muscle cell migration

    Venkatesan, Balachandar; Valente, Anthony J.; Reddy, Venkatapuram Seenu; Siwik, Deborah A.; Chandrasekar, Bysani

    2009-01-01

    Vascular smooth muscle cell (SMC) migration is an important mechanism in atherogenesis and postangioplasty arterial remodeling. Previously, we demonstrated that the proinflammatory cytokine interleukin (IL)-18 is a potent inducer of SMC migration. Since extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates ECM degradation and facilitates cell migration, we investigated whether IL-18 and EMMPRIN regulate each other's expression, whether their cross talk induces SMC migration, and...

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

    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......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

  10. AGC kinases, mechanisms of regulation ‎and innovative drug development.

    Leroux, Alejandro E; Schulze, Jörg O; Biondi, Ricardo M

    2018-02-01

    The group of AGC kinases consists of 63 evolutionarily related serine/threonine protein kinases comprising PDK1, PKB/Akt, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, and YANK, while two other families, Aurora and PLK, are the most closely related to the group. Eight of these families are physiologically activated downstream of growth factor signalling, while other AGC kinases are downstream effectors of a wide range of signals. The different AGC kinase families share aspects of their mechanisms of inhibition and activation. In the present review, we update the knowledge of the mechanisms of regulation of different AGC kinases. The conformation of the catalytic domain of many AGC kinases is regulated allosterically through the modulation of the conformation of a regulatory site on the small lobe of the kinase domain, the PIF-pocket. The PIF-pocket acts like an ON-OFF switch in AGC kinases with different modes of regulation, i.e. PDK1, PKB/Akt, LATS and Aurora kinases. In this review, we make emphasis on how the knowledge of the molecular mechanisms of regulation can guide the discovery and development of small allosteric modulators. Molecular probes stabilizing the PIF-pocket in the active conformation are activators, while compounds stabilizing the disrupted site are allosteric inhibitors. One challenge for the rational development of allosteric modulators is the lack of complete structural information of the inhibited forms of full-length AGC kinases. On the other hand, we suggest that the available information derived from molecular biology and biochemical studies can already guide screening strategies for the identification of innovative mode of action molecular probes and the development of selective allosteric drugs for the treatment of human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction

    Frödin, M; Gammeltoft, S

    1999-01-01

    ), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK......-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c...

  12. Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-10-01

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

  13. Lack of Csk-mediated negative regulation in a unicellular SRC kinase.

    Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, Iñaki; Miller, W Todd

    2012-10-16

    Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.

  14. Fisetin regulates astrocyte migration and proliferation in vitro

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-01-01

    Fisetin (3,3?,4?,7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte m...

  15. Tomato thymidine kinase is subject to inefficient TTP feedback regulation

    Larsen, Nicolai Balle; Munch-Petersen, Birgitte; Piskur, Jure

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. Transduction with toTK1 has been found to effi...

  16. Mib1 contributes to persistent directional cell migration by regulating the Ctnnd1-Rac1 pathway.

    Mizoguchi, Takamasa; Ikeda, Shoko; Watanabe, Saori; Sugawara, Michiko; Itoh, Motoyuki

    2017-10-31

    Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1 ta52b mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1 ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway. Published under the PNAS license.

  17. Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

    Morgan-Fisher, Marie; Couchman, John R; Yoneda, Atsuko

    2013-01-01

    The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II......, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2....

  18. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

  19. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2012-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  20. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  1. The State Regulation of External Labor Migration: the Experience of the EU, France, Germany and the USA

    Андрей Георгиевич Иванов

    2009-09-01

    Full Text Available The given article is devoted to the actual problem of the state regulation of external labor migration. It's based on examples of developed western countries. The author makes a conclusion about the importance of the state regulation of migration processes, warning that narrow understanding of migration policy as restriction of external migration flows is insufficient nowadays.

  2. Tyrosine kinase ETK/BMX is up-regulated in bladder cancer and predicts poor prognosis in patients with cystectomy.

    Shengjie Guo

    2011-03-01

    Full Text Available Deregulation of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors. In this report, we demonstrated that ETK expression is progressively increased during bladder cancer progression. We found that down-regulation of ETK in bladder cancer cells attenuated STAT3 and AKT activity whereas exogenous overexpression of ETK had opposite effects, suggesting that deregulation of ETK may attribute to the elevated activity of STAT3 and AKT frequently detected in bladder cancer. The survival, migration and invasion of bladder cancer cells were significantly compromised when ETK expression was knocked down by a specific shRNA. In addition, we showed that ETK localizes to mitochondria in bladder cancer cells through interacting with Bcl-XL and regulating ROS production and drug sensitivity. Therefore, ETK may play an important role in regulating survival, migration and invasion by modulating multiple signaling pathways in bladder cancer cells. Immunohistochemistry analysis on tissue microarrays containing 619 human bladder tissue samples shows that ETK is significantly upregulated during bladder cancer development and progression and ETK expression level predicts the survival rate of patients with cystectomy. Taken together, our results suggest that ETK may potentially serve as a new drug target for bladder cancer treatment as well as a biomarker which could be used to identify patients with higher mortality risk, who may be benefited from therapeutics targeting ETK activity.

  3. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

  4. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-11-01

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2506-2512, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  5. Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

    2018-03-01

    Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

    Petersen, M.; Brodersen, P.; Naested, H.

    2000-01-01

    Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) revels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern...... of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression....

  7. Structural aspects of protein kinase ASK1 regulation

    Obšil, Tomáš; Obšilová, Veronika

    2017-01-01

    Roč. 66, 1 Dec (2017), s. 31-36 ISSN 2212-4926 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ASK1 kinase * apoptosis * thioredoxin * 14-3-3 protein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  8. Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

    Andersen, Martin Nybo; Skibsbye, Lasse; Saljic, Arnela

    2018-01-01

    around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane...... targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid......-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further...

  9. The spinal muscular atrophy with pontocerebellar hypoplasia gene VRK1 regulates neuronal migration through an amyloid-β precursor protein-dependent mechanism.

    Vinograd-Byk, Hadar; Sapir, Tamar; Cantarero, Lara; Lazo, Pedro A; Zeligson, Sharon; Lev, Dorit; Lerman-Sagie, Tally; Renbaum, Paul; Reiner, Orly; Levy-Lahad, Ephrat

    2015-01-21

    Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH) is an infantile SMA variant with additional manifestations, particularly severe microcephaly. We previously identified a nonsense mutation in Vaccinia-related kinase 1 (VRK1), R358X, as a cause of SMA-PCH. VRK1-R358X is a rare founder mutation in Ashkenazi Jews, and additional mutations in patients of different origins have recently been identified. VRK1 is a nuclear serine/threonine protein kinase known to play multiple roles in cellular proliferation, cell cycle regulation, and carcinogenesis. However, VRK1 was not known to have neuronal functions before its identification as a gene mutated in SMA-PCH. Here we show that VRK1-R358X homozygosity results in lack of VRK1 protein, and demonstrate a role for VRK1 in neuronal migration and neuronal stem cell proliferation. Using shRNA in utero electroporation in mice, we show that Vrk1 knockdown significantly impairs cortical neuronal migration, and affects the cell cycle of neuronal progenitors. Expression of wild-type human VRK1 rescues both proliferation and migration phenotypes. However, kinase-dead human VRK1 rescues only the migration impairment, suggesting the role of VRK1 in neuronal migration is partly noncatalytic. Furthermore, we found that VRK1 deficiency in human and mouse leads to downregulation of amyloid-β precursor protein (APP), a known neuronal migration gene. APP overexpression rescues the phenotype caused by Vrk1 knockdown, suggesting that VRK1 affects neuronal migration through an APP-dependent mechanism. Copyright © 2015 the authors 0270-6474/15/350936-08$15.00/0.

  10. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  11. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  12. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  13. AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

    2013-01-01

    Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

  14. Csk Homologous Kinase, a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis

    2011-08-01

    is a non-receptor tyrosine kinase and a second member of the Csk family. Like Csk, CHK has Src homology 2 ( SH2 ) and SH3 domains and lacks the...MSCV-retroviral vectors encoding either wild-type CHK or kinase -dead CHK or wild type SH2 domain or SH2 -R147A or SH2 -G129A. All these constructs were... Kinase , a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis Byeong-Chel Lee The University of Pittsburgh Pittsburgh, PA 15213

  15. Odorant Receptor 51E2 Agonist β-ionone Regulates RPE Cell Migration and Proliferation

    Nikolina Jovancevic

    2017-11-01

    Full Text Available The odorant receptor 51E2 (OR51E2, which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand β-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders.

  16. CRF2 signaling is a novel regulator of cellular adhesion and migration in colorectal cancer cells.

    Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Lainé, Michèle; Cristina, Nadine; Vachez, Yvan; Scoazec, Jean-Yves; Bonaz, Bruno; Jacquier-Sarlin, Muriel

    2013-01-01

    Stress has been proposed to be a tumor promoting factor through the secretion of specific neuromediators, such as Urocortin2 and 3 (Ucn2/3), however its role in colorectal cancer (CRC) remains elusive. We observed that Ucn2/3 and their receptor the Corticotropin Releasing Factor receptor 2 (CRF2) were up-regulated in high grade and poorly differentiated CRC. This suggests a role for CRF2 in the loss of cellular organization and tumor progression. Using HT-29 and SW620 cells, two CRC cell lines differing in their abilities to perform cell-cell contacts, we found that CRF2 signals through Src/ERK pathway to induce the alteration of cell-cell junctions and the shuttle of p120ctn and Kaiso in the nucleus. In HT-29 cells, this signaling pathway also leads to the remodeling of cell adhesion by i) the phosphorylation of Focal Adhesion Kinase and ii) a modification of actin cytoskeleton and focal adhesion complexes. These events stimulate cell migration and invasion. In conclusion, our findings indicate that CRF2 signaling controls cellular organization and may promote metastatic potential of human CRC cells through an epithelial-mesenchymal transition like process. This contributes to the comprehension of the tumor-promoting effects of stress molecules and designates Ucn2/3-CRF2 tandem as a target to prevent CRC progression and aggressiveness.

  17. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

    Boon Siang Nicholas Tan

    Full Text Available Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL cells from mouse embryonic stem (mES cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

  18. Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

    Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

    2016-01-01

    Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

  19. Overexpression of microRNA-375 impedes platelet-derived growth factor-induced proliferation and migration of human fetal airway smooth muscle cells by targeting Janus kinase 2.

    Ji, Yamei; Yang, Xin; Su, Huixia

    2018-02-01

    The abnormal proliferation and migration of airway smooth muscle (ASM) cells play a critical role in airway remodeling during the development of asthma. MicroRNAs (miRNAs) have emerged as critical regulators of ASM cell proliferation and migration in airway remodeling. In this study, we aimed to investigate the potential role of miR-375 in the regulation of platelet-derived growth factor (PDGF)-induced fetal ASM cell proliferation and migration. Our results showed that miR-375 expression was significantly decreased in fetal ASM cells that were treated with PDGF. Functional data showed that overexpression of miR-375 inhibited the proliferation and migration of fetal ASM cells, whereas inhibition of miR-375 enhanced the proliferation and migration of fetal ASM cells. The results of bioinformatics analysis and a dual-luciferase reporter assay showed that miR-375 binds directly to the 3'-untranslated region of Janus kinase 2 (JAK2). Further data confirmed that miR-375 negatively regulates the expression of JAK2 in fetal ASM cells. Moreover, miR-375 also impeded the PDGF-induced activation of signal transducer and activator of transcription 3 (STAT3) in fetal ASM cells. However, restoration of JAK2 expression partially reversed the inhibitory effect of miR-375 on fetal ASM cell proliferation and migration. Overall, our results demonstrate that miR-375 inhibits fetal ASM cell proliferation and migration by targeting JAK2/STAT3 signaling. Our study provides a potential therapeutic target for the development of novel treatment strategies for pediatric asthma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5

    Zhang, Duanwu; Tomisato, Wataru; Su, Lijing; Sun, Lei; Choi, Jin Huk; Zhang, Zhao; Wang, Kuan-wen; Zhan, Xiaoming; Choi, Mihwa; Li, Xiaohong; Tang, Miao; Castro-Perez, Jose M.; Hildebrand, Sara; Murray, Anne R.; Moresco, Eva Marie Y.

    2017-01-01

    We discovered a previously unrecognized regulator of cholesterol biosynthesis, glycerol kinase 5 (GK5), which functions exclusively in the skin independently of cholesterol regulation in other tissues. GK5 negatively regulates the processing and nuclear localization of sterol regulatory element binding proteins, transcription factors that control expression of virtually all cholesterol synthesis enzymes. Excessive amounts of cholesterol, triglycerides, and ceramides were found in the skin of ...

  1. E-cadherin Is Critical for Collective Sheet Migration and Is Regulated by the Chemokine CXCL12 Protein During Restitution*

    Hwang, Soonyean; Zimmerman, Noah P.; Agle, Kimberle A.; Turner, Jerrold R.; Kumar, Suresh N.; Dwinell, Michael B.

    2012-01-01

    Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2BBE and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-β1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-β1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution. PMID:22549778

  2. Focal Adhesion Kinase functions as an Akt downstream target in migration of colorectal cancer cells

    Turečková, Jolana; Vojtěchová, Martina; Krausová, Michaela; Šloncová, Eva; Kořínek, Vladimír

    2009-01-01

    Roč. 2, č. 4 (2009), s. 281-290 ISSN 1936-5233 R&D Projects: GA ČR GA204/06/1658; GA MŠk 2B06077 Institutional research plan: CEZ:AV0Z50520514 Keywords : FAK * migration * invasion Subject RIV: EB - Genetics ; Molecular Biology

  3. Regulation of cell cycle checkpoint kinase WEE1 by miR-195 in malignant melanoma.

    Bhattacharya, A; Schmitz, U; Wolkenhauer, O; Schönherr, M; Raatz, Y; Kunz, M

    2013-06-27

    WEE1 kinase has been described as a major gate keeper at the G2 cell cycle checkpoint and to be involved in tumour progression in different malignant tumours. Here we analysed the expression levels of WEE1 in a series of melanoma patient samples and melanoma cell lines using immunoblotting, quantitative real-time PCR and immunohistochemistry. WEE1 expression was significantly downregulated in patient samples of metastatic origin as compared with primary melanomas and in melanoma cell lines of high aggressiveness as compared with cell lines of low aggressiveness. Moreover, there was an inverse correlation between the expression of WEE1 and WEE1-targeting microRNA miR-195. Further analyses showed that transfection of melanoma cell lines with miR-195 indeed reduced WEE1 mRNA and protein expression in these cells. Reporter gene analysis confirmed direct targeting of the WEE1 3' untranslated region (3'UTR) by miR-195. Overexpression of miR-195 in SK-Mel-28 melanoma cells was accompanied by WEE1 reduction and significantly reduced stress-induced G2-M cell cycle arrest, which could be restored by stable overexpression of WEE1. Moreover, miR-195 overexpression and WEE1 knockdown, respectively, increased melanoma cell proliferation. miR-195 overexpression also enhanced migration and invasiveness of melanoma cells. Taken together, the present study shows that WEE1 expression in malignant melanoma is directly regulated by miR-195. miR-195-mediated downregulation of WEE1 in metastatic lesions may help to overcome cell cycle arrest under stress conditions in the local tissue microenvironment to allow unrestricted growth of tumour cells.

  4. Y-27632 Increases Sensitivity of PANC-1 Cells to Epigallocatechin Gallate (EGCG) in Regulating Cell Proliferation and Migration

    Liu, Xing; Bi, Yongyi

    2016-01-01

    Background The study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (−)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer. Material/Methods PANC-1 cells, maintained in Dulbecco’s Modified Eagle’s Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator–activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR). Results EGCG (20–80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARα and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632. Conclusions Y-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARα mRNA and Caspase-3 mRNA. PMID:27694793

  5. Notch1-Dll4 signaling and mechanical force regulate leader cell formation during collective cell migration

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D.; Wong, Pak Kin

    2015-01-01

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct “leader” phenotype with characteristic morphology and motility. However, the factors driving leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here, we use single cell gene expression analysis and computational modeling to show that leader cell identity is dynamically regulated by Dll4 sign...

  6. Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

    Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

    2002-01-01

    Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

  7. Differential regulation of microtubule severing by APC underlies distinct patterns of projection neuron and interneuron migration

    Eom, Tae-Yeon; Stanco, Amelia; Guo, Jiami; Wilkins, Gary; Deslauriers, Danielle; Yan, Jessica; Monckton, Chase; Blair, Josh; Oon, Eesim; Perez, Abby; Salas, Eduardo; Oh, Adrianna; Ghukasyan, Vladimir; Snider, William D.; Rubenstein, John L. R.; Anton, E. S.

    2014-01-01

    Coordinated migration of distinct classes of neurons to appropriate positions leads to the formation of functional neuronal circuitry in the cerebral cortex. Two major classes of cortical neurons, interneurons and projection neurons, utilize distinctly different modes (radial vs. tangential) and routes of migration to arrive at their final positions in the cerebral cortex. Here, we show that adenomatous polyposis coli (APC) modulates microtubule (MT) severing in interneurons to facilitate tangential mode of interneuron migration, but not the glial-guided, radial migration of projection neurons. APC regulates the stability and activity of the MT severing protein p60-katanin in interneurons to promote the rapid remodeling of neuronal processes necessary for interneuron migration. These findings reveal how severing and restructuring of MTs facilitate distinct modes of neuronal migration necessary for laminar organization of neurons in the developing cerebral cortex. PMID:25535916

  8. A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

    Fuller, Stephen J; McGuffin, Liam J; Marshall, Andrew K; Giraldo, Alejandro; Pikkarainen, Sampsa; Clerk, Angela; Sugden, Peter

    2012-01-01

    The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily thro...

  9. Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

    Rieder Gabriele

    2011-11-01

    Full Text Available Abstract Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS and the effector protein cytotoxin-associated gene A (CagA of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells.

  10. Casein kinase 1-Like 3 is required for abscisic acid regulation of ...

    Casein kinase 1-Like 3 is required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. M Wang, D Yu, X Guo, X Li, J Zhang, L Zhao, H Chang, S Hu, C Zhang, J Shi, X Liu ...

  11. Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

    Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

  12. The MAP kinase substrate MKS1 is a regulator of plant defense responses

    Andreasson, E.; Jenkins, T.; Brodersen, P.

    2005-01-01

    Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used...

  13. Regulation of the vertebrate cell cycle by the cdc2 protein kinase

    Draetta, G.; Brizuela, L.; Moran, B.; Beach, D.

    1988-01-01

    A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. The authors have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. They find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G 1 but displays maximal enzymatic activity during mitotic metaphase. These observations indicate that in a wide variety of vertebrate cells, the cdc2 protein kinase is involved in regulating mitosis. The authors' approach taken toward study of the cdc2 protein kinase highlights the possibilities that now exist for combining the advantages of ascomycete genetics with the cell-free systems of Xenopus and the biochemical advantages of tissue culture cells to investigate fundamental problems of the cell cycle

  14. SCFβ-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration

    Tron, Adriana E.; Wang, Zhiwei; Sun, Liankun; Inuzuka, Hiroyuki; Wei, Wenyi

    2013-01-01

    Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers. PMID:24318128

  15. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima

    2015-01-01

    -intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given...

  16. Kinase-Mediated Regulation of P4-ATPases

    Frøsig, Merethe Mørch

    designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

  17. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

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

    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.

  19. Regulation of glycogen synthase kinase-3 during bipolar mania treatment.

    Li, Xiaohong; Liu, Min; Cai, Zhuoji; Wang, Gang; Li, Xiaohua

    2010-11-01

    Bipolar disorder is a debilitating psychiatric illness presenting with recurrent mania and depression. The pathophysiology of bipolar disorder is poorly understood, and molecular targets in the treatment of bipolar disorder remain to be identified. Preclinical studies have suggested that glycogen synthase kinase-3 (GSK3) is a potential therapeutic target in bipolar disorder, but evidence of abnormal GSK3 in human bipolar disorder and its response to treatment is still lacking. This study was conducted in acutely ill type I bipolar disorder subjects who were hospitalized for a manic episode. The protein level and the inhibitory serine phosphorylation of GSK3 in peripheral blood mononuclear cells of bipolar manic and healthy control subjects were compared, and the response of GSK3 to antimanic treatment was evaluated. The levels of GSK3α and GSK3β in this group of bipolar manic subjects were higher than healthy controls. Symptom improvement during an eight-week antimanic treatment with lithium, valproate, and atypical antipsychotics was accompanied by a significant increase in the inhibitory serine phosphorylation of GSK3, but not the total level of GSK3, whereas concomitant electroconvulsive therapy treatment during a manic episode appeared to dampen the response of GSK3 to pharmacological treatment. Results of this study suggest that GSK3 can be modified during the treatment of bipolar mania. This finding in human bipolar disorder is in agreement with preclinical data suggesting that inhibition of GSK3 by increasing serine phosphorylation is a response of GSK3 to psychotropics used in bipolar disorder, supporting the notion that GSK3 is a promising molecular target in the pharmacological treatment of bipolar disorder. © 2010 John Wiley and Sons A/S.

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

    Elizabeth M Chislock

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

  1. Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala.

    Lamprecht, R; Margulies, D S; Farb, C R; Hou, M; Johnson, L R; LeDoux, J E

    2006-01-01

    Learning and memory depend on signaling molecules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the training stimuli were presented in a non-associative manner. Anatomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically implicated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nucleus of the amygdala. When ML-7 was applied without associative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the circuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

  2. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Munk, Stephanie; Sigurðsson, Jón Otti; Xiao, Zhenyu

    2017-01-01

    The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essentia....... They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage....

  3. URG11 Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion

    Bin Pan

    2018-01-01

    Full Text Available Upregulated gene 11 (URG11, a new gene upregulated by hepatitis B virus X protein, is involved in the development and progression of several tumors, including liver, stomach, lung, and colon cancers. However, the role of URG11 in prostate cancer remains yet to be elucidated. By determined expression in human prostate cancer tissues, URG11 was found significantly upregulated and positively correlated with the severity of prostate cancer, compared with that in benign prostatic hyperplasia tissues. Further, the mRNA and protein levels of URG11 were significantly upregulated in human prostate cancer cell lines (DU145, PC3, and LNCaP, compared with human prostate epithelial cell line (RWPE-1. Moreover, by the application of siRNA against URG11, the proliferation, migration, and invasion of prostate cancer cells were markedly inhibited. Genetic knockdown of URG11 also induced cell cycle arrest at G1/S phase, induced apoptosis, and decreased the expression level of β-catenin in prostate cancer cells. Overexpression of URG11 promoted the expression of β-catenin, the growth, the migration, and invasion ability of prostate cancer cells. Taken together, this study reveals that URG11 is critical for the proliferation, migration, and invasion in prostate cancer cells, providing the evidence of URG11 to be a novel potential therapeutic target of prostate cancer.

  4. Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.

    Guerrero-Cazares, Hugo; Lavell, Emily; Chen, Linda; Schiapparelli, Paula; Lara-Velazquez, Montserrat; Capilla-Gonzalez, Vivian; Clements, Anna Christina; Drummond, Gabrielle; Noiman, Liron; Thaler, Katrina; Burke, Anne; Quiñones-Hinojosa, Alfredo

    2017-07-01

    Human neural progenitor cell (NPC) migration within the subventricular zone (SVZ) of the lateral ganglionic eminence is an active process throughout early brain development. The migration of human NPCs from the SVZ to the olfactory bulb during fetal stages resembles what occurs in adult rodents. As the human brain develops during infancy, this migratory stream is drastically reduced in cell number and becomes barely evident in adults. The mechanisms regulating human NPC migration are unknown. The Slit-Robo signaling pathway has been defined as a chemorepulsive cue involved in axon guidance and neuroblast migration in rodents. Slit and Robo proteins expressed in the rodent brain help guide neuroblast migration from the SVZ through the rostral migratory stream to the olfactory bulb. Here, we present the first study on the role that Slit and Robo proteins play in human-derived fetal neural progenitor cell migration (hfNPC). We describe that Robo1 and Robo2 isoforms are expressed in the human fetal SVZ. Furthermore, we demonstrate that Slit2 is able to induce a chemorepellent effect on the migration of hfNPCs derived from the human fetal SVZ. In addition, when Robo1 expression is inhibited, hfNPCs are unable to migrate to the olfactory bulb of mice when injected in the anterior SVZ. Our findings indicate that the migration of human NPCs from the SVZ is partially regulated by the Slit-Robo axis. This pathway could be regulated to direct the migration of NPCs in human endogenous neural cell therapy. Stem Cells 2017;35:1860-1865. © 2017 AlphaMed Press.

  5. GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-06-01

    Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  7. RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation.

    Wu, Xiangbing; Walker, Chandler L; Lu, Qingbo; Wu, Wei; Eddelman, Daniel B; Parish, Jonathan M; Xu, Xiao-Ming

    2017-11-01

    Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A 2 (cPLA 2 ), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA 2 . Inhibition of RhoA, Rho kinase and cPLA 2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA 2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA 2 . The immunofluorescence staining showed that ROCK 1 or ROCK 2 , two isoforms of Rho kinase, was co-localized with cPLA 2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK 1 or ROCK 2 bonded directly with cPLA 2 and phospho-cPLA 2 . When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA 2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA 2 activation.

  8. Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer.

    Michie, Alison M; McCaig, Alison M; Nakagawa, Rinako; Vukovic, Milica

    2010-01-01

    Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

  9. Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

    Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

    2014-01-31

    Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

  10. STRATEGIC PRIORITIES FOR THE INTERNAL MIGRATION PROCESSES REGULATION IN UKRAINE

    Olga Balueva

    2018-01-01

    Full Text Available As the result of the annexation of the Crimea and Donbass military conflict, there was an emergence of forced migration. It ought to be emphasized that today issues related to addressing problems of internally displaced persons (IDPs, are relevant and require an integrated approach. The increase in the number of IDPs in the country raises the issue of identifying the strategic priorities of the state policy. The purpose of this research is to formulate strategic priorities of the state policy of support for IDPs’. The subject of the study is theoretical and methodological principles of the state policy to support IDPs. The research methodology includes a set of scientific research methods that provide identifying the strategic priorities of the state policy for IDPs’ support, developing effective mechanisms for these priorities achievement, and conceptual vision of solving IDPs’ problems. In the course of investigations, the following methods are used: analysis while studying the main problems of internally displaced persons; logical synthesis and synthesis while drawing conclusions. Results. Scientific novelty of the article. In the article, the conceptual foundations of the IDPs support have been formed, an attention has been focused on the feasibility of using a strategic approach, which involves the formation and implementation of a set of strategically oriented measures aimed at solving the most acute issues and matters related to internal forced migration, directing the IDPs potential and internally displaced businesses to strengthen local communities and social and economic development of the resettlement territories. The main stages and conceptual foundations for the Strategy formation on the IDPs support have been proposed. The strategy is based on an integrated approach to addressing IDPs and internally displaced businesses, taking into account the interests of host communities. Practical implications. The result of the Strategy

  11. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry

  12. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs Effects on AMP-Activated Protein Kinase (AMPK Regulation of Chicken Sperm Functions.

    Thi Mong Diep Nguyen

    Full Text Available Sperm require high levels of energy to ensure motility and acrosome reaction (AR accomplishment. The AMP-activated protein kinase (AMPK has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+/calmodulin-dependent protein kinase kinases (CaMKKs mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+, or of CaMKKs inhibitor (STO-609. Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β, CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+. Our results show for the first time the presence of CaMKKs (α and β and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+ entry in sperm through the Ca(2+/CaM/CaMKKs/CaMKI pathway. The Ca(2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2

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

    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-regulated

  14. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Negative regulation of MAP kinase signaling in Drosophila by Ptp61F/PTP1B.

    Tchankouo-Nguetcheu, Stéphane; Udinotti, Mario; Durand, Marjorie; Meng, Tzu-Ching; Taouis, Mohammed; Rabinow, Leonard

    2014-10-01

    PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, we generated a new allele of Ptp61F as well as employed both RNA interference and overexpression alleles. Our results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, we establish negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, our results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.

  16. Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara

    2013-01-01

    Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, 183KKRKRKCLLL192 (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1. PMID:24043306

  17. Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

    Karacosta, Loukia G; Kuroski, Laura A; Hofmann, Wilma A; Azabdaftari, Gissou; Mastri, Michalis; Gocher, Angela M; Dai, Shuhang; Hoste, Allen J; Edelman, Arthur M

    2016-02-15

    Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells. The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting. CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non

  18. MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

    Yang, H; Raizada, M K

    1998-09-01

    Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons

  19. Protein kinase C regulates human pluripotent stem cell self-renewal.

    Masaki Kinehara

    Full Text Available The self-renewal of human pluripotent stem (hPS cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2 appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC, GF109203X (GFX, increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β, suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2 synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK, PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though h

  20. Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

    Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.

    2013-01-01

    Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long

  1. SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

    Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

    2018-05-01

    SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

  2. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  3. Crosstalk between intracellular and extracellular signals regulating interneuron production migration and integration into the cortex

    Elise ePeyre

    2015-04-01

    Full Text Available During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: 1/ Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; 2/ Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; 3/ Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex.

  4. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex.

    Peyre, Elise; Silva, Carla G; Nguyen, Laurent

    2015-01-01

    During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: (1) Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; (2) Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; (3) Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex.

  5. Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

    Song, Jiho; Shawky, Joseph H; Kim, YongTae; Hazar, Melis; LeDuc, Philip R; Sitti, Metin; Davidson, Lance A

    2015-07-01

    Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in tr...

  7. IKKα regulates human keratinocyte migration through surveillance of the redox environment.

    Lisse, Thomas S; Rieger, Sandra

    2017-03-01

    Although the functions of H 2 O 2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H 2 O 2 -dependent wound repair mechanisms. Scratch wounding led to H 2 O 2 production in two or three cell layers at the wound margin within ∼30 min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H 2 O 2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H 2 O 2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H 2 O 2 -dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK ), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells. © 2017. Published by The Company of Biologists Ltd.

  8. The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

    Li, Zhiru; Dugan, Aisling S; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R

    2009-09-17

    The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

  9. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H.; Hickson, Gilles R.

    2014-01-01

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks. PMID:25332165

  10. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  11. Low Expression of DYRK2 (Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2 Correlates with Poor Prognosis in Colorectal Cancer.

    Haiyan Yan

    Full Text Available Dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2 is a member of dual-specificity kinase family, which could phosphorylate both Ser/Thr and Tyr substrates. The role of DYRK2 in human cancer remains controversial. For example, overexpression of DYRK2 predicts a better survival in human non-small cell lung cancer. In contrast, amplification of DYRK2 gene occurs in esophageal/lung adenocarcinoma, implying the role of DYRK2 as a potential oncogene. However, its clinical role in colorectal cancer (CRC has not been explored. In this study, we analyzed the expression of DYRK2 from Oncomine database and found that DYRK2 level is lower in primary or metastatic CRC compared to adjacent normal colon tissue or non-metastatic CRC, respectively, in 6 colorectal carcinoma data sets. The correlation between DYRK2 expression and clinical outcome in 181 CRC patients was also investigated by real-time PCR and IHC. DYRK2 expression was significantly down-regulated in colorectal cancer tissues compared with adjacent non-tumorous tissues. Functional studies confirmed that DYRK2 inhibited cell invasion and migration in both HCT116 and SW480 cells and functioned as a tumor suppressor in CRC cells. Furthermore, the lower DYRK2 levels were correlated with tumor sites (P = 0.023, advanced clinical stages (P = 0.006 and shorter survival in the advanced clinical stages. Univariate and multivariate analyses indicated that DYRK2 expression was an independent prognostic factor (P < 0.001. Taking all, we concluded that DYRK2 a novel prognostic biomarker of human colorectal cancer.

  12. Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells.

    Roman J Krawetz

    Full Text Available The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK play pivotal roles in cell migration, apoptosis (membrane blebbing, cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a 'ground' state after the inhibition had been removed.Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the

  13. CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

    Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

    2017-06-15

    Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  14. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

  15. [Regulation of microRNA-199a on adhesion, migration and invasion ability of human endometrial stromal cells].

    Dai, Lan; Gu, Li-ying; Zhu, Jie; Shi, Jun; Wang, Yao; Ji, Fang; Di, Wen

    2011-11-01

    To study the regulation of microRNA 199a (miR-199a) on adhesion, migration and invasion ability of human eutopic endometrial stromal cells (ESC) from patients with endometriosis. ESC were transfected with miR-199a mimics or negative control (NC) RNA by lipofectamine 2000. The adhesion, migration and invasion ability of ESC were detected by cell adhesion assay, scratch assay, cell migration assay and matrigel invasion assay, respectively. Luciferase reporter assay was used to evaluate whether IKKβ was the target gene of miR-199a. The expression of ikappa B kinase beta (IKKβ), inhibitory kappa B alpha (IκB-α), phospho-IκB-α(p-IκB-α) and nuclear factor-kappa B (NF-κB) protein were measured by western blot. (1) Adhesion potential: the adhesion inhibitory rates were (14 ± 4)% in miR-199a group and 0 in control group, which showed significant difference (P scratch assay, ESC transfected with miR-199a exhibited a lower scratch closure rate than that of controls. In migration and invasion assays, the migration and invasion ability of miR-199a group were significantly decreased compared with those of NC group [130 ± 31 vs. 247 ± 36 (P < 0.01); 63 ± 15 vs. 133 ± 17 (P < 0.01), respectively]. (3) The luciferase activity of miR-199a group was significantly lowered than that of control group [0.160 ± 0.006 vs. 0.383 ± 0.083 (P < 0.01)]. The protein levels of IKKβ, p-IκB-α, IκB-α and NF-κB of 0.350 ± 0.195, 0.443 ± 0.076, 1.970 ± 0.486 and 0.454 ± 0.147 in miR-199a group were significantly different compared with the NC group in which the protein levels were set at 1.000 (P < 0.01). miR-199a can inhibit the adhesion, migration and invasion of the ESC. IKKβ is the target gene of miR-199a in ESC. One of the mechanisms of the inhibition effect is probably that miR-199a inhibits the activation of NF-κB signaling pathway by targeting IKKβ gene.

  16. Impact, regulation and health policy implications of physician migration in OECD countries

    Simoens Steven

    2004-07-01

    Full Text Available Abstract Background In the face of rising demand for medical services due to ageing populations, physician migration flows are increasingly affecting the supply of physicians in Organisation for Economic Co-operation and development (OECD countries. This paper offers an integrated perspective on the impact of physician migration on home and host countries and discusses international regulation and policy approaches governing physician migration. Methods Information about migration flows, international regulation and policies governing physician migration were derived from two questionnaires sent to OECD countries, a secondary analysis of EUROSTAT Labour Force Surveys, a literature review and official policy documents of OECD countries. Results OECD countries increasingly perceive immigration of foreign physicians as a way of sustaining their physician workforce. As a result, countries have entered into international agreements regulating physician migration, although their success has been limited due to the imposition of licensing requirements and the protection of vested interests by domestic physicians. OECD countries have therefore adopted specific policies designed to stimulate the immigration of foreign physicians, whilst minimising its negative impact on the home country. Measures promoting immigration have included international recruitment campaigns, less strict immigration requirements and arrangements that foster shared learning between health care systems. Policies restricting the societal costs of physician emigration from developing countries such as good practice guidelines and taxes on host countries have not yet produced their expected effect or in some cases have not been established at all. Conclusions Although OECD countries generally favour long-term policies of national self-sufficiency to sustain their physician workforce, such policies usually co-exist with short-term or medium-term policies to attract foreign physicians

  17. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. © 2015 Hughes, Oudin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration.

    Grifoni, Samira C; McKey, Susan E; Drummond, Heather A

    2008-05-01

    Recent studies suggest members of the degenerin (DEG)/epithelial Na(+) channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes.

  19. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myobla...

  20. Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

    Burden, Steven; Lee, Jennifer

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm ...

  1. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

    Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

  2. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  3. Focal adhesion kinase regulates neuronal growth, synaptic plasticity and hippocampus-dependent spatial learning and memory.

    Monje, Francisco J; Kim, Eun-Jung; Pollak, Daniela D; Cabatic, Maureen; Li, Lin; Baston, Arthur; Lubec, Gert

    2012-01-01

    The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory. Copyright © 2011 S. Karger AG, Basel.

  4. KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

    Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

    2002-05-15

    K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

  5. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  6. Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1.

    Komaravolu, Ravi K; Adam, Christian; Moonen, Jan-Renier A J; Harmsen, Martin C; Goebeler, Matthias; Schmidt, Marc

    2015-01-01

    The MEK5/Erk5 pathway mediates beneficial effects of laminar flow, a major physiological factor preventing vascular dysfunction. Forced Erk5 activation induces a protective phenotype in endothelial cell (EC) that is associated with a dramatically decreased migration capacity of those cells. Transcriptional profiling identified the Krüppel-like transcription factors KLF2 and KLF4 as central mediators of Erk5-dependent gene expression. However, their downstream role regarding migration is unclear and relevant secondary effectors remain elusive. Here, we further investigated the mechanism underlying Erk5-dependent migration arrest in ECs. Our experiments reveal KLF2-dependent loss of the pro-migratory Rac/Cdc42 mediator, p21-activated kinase 1 (PAK1), as an important mechanism of Erk5-induced migration inhibition. We show that endothelial Erk5 activation by expression of a constitutively active MEK5 mutant, by statin treatment, or by application of laminar shear stress strongly decreased PAK1 mRNA and protein expression. Knockdown of KLF2 but not of KLF4 prevented Erk5-mediated PAK1 mRNA inhibition, revealing KLF2 as a novel PAK1 repressor in ECs. Importantly, both PAK1 re-expression and KLF2 knockdown restored the migration capacity of Erk5-activated ECs underscoring their functional relevance downstream of Erk5. Our data provide first evidence for existence of a previously unknown Erk5/KLF2/PAK1 axis, which may limit undesired cell migration in unperturbed endothelium and lower its sensitivity for migratory cues that promote vascular diseases including atherosclerosis. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  7. Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes

    Lippert Dustin ND

    2012-04-01

    Full Text Available Abstract Background Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration. Results Microscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1. Conclusions The present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated

  8. The master Greatwall kinase, a critical regulator of mitosis and meiosis.

    Vigneron, Suzanne; Robert, Perle; Hached, Khaled; Sundermann, Lena; Charrasse, Sophie; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2016-01-01

    Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.

  9. SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

    Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

    2005-10-28

    SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

  10. Regulation of peripheral inflammation by spinal p38 MAP kinase in rats.

    David L Boyle

    2006-09-01

    Full Text Available Somatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.Selective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3 gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha in the nervous system because IT etanercept (a TNF inhibitor given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.These data suggest that peripheral inflammation is sensed by the central nervous system (CNS, which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.

  11. SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

    Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

    2011-02-04

    SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

  12. Markets, citizenship and rights: state regulation of labour migration in Malaysia and Spain

    Garcés-Mascareñas, B.

    2010-01-01

    The State regulation of labour migration seems to be confronted with a double dilemma. First, while markets require a policy of open borders to provide as many migrant workers as demanded, citizenship seems to require some degree of closure to the outside. Second, while the exclusive character of

  13. NDRG2 inhibits hepatocellular carcinoma adhesion, migration and invasion by regulating CD24 expression

    Zheng, Jin; Guo, Hang; Tao, Yurong; Xue, Yan; Jiang, Ning; Yao, Libo; Liu, Wenchao; Li, Yan; Yang, Jiandong; Liu, Qiang; Shi, Ming; Zhang, Rui; Shi, Hengjun; Ren, Qinyou; Ma, Ji

    2011-01-01

    The prognosis of most hepatocellular carcinoma (HCC) patients is poor due to the high metastatic rate of the disease. Understanding the molecular mechanisms underlying HCC metastasis is extremely urgent. The role of CD24 and NDRG2 (N-myc downstream-regulated gene 2), a candidate tumor suppressor gene, has not yet been explored in HCC. The mRNA and protein expression of CD24 and NDRG2 was analyzed in MHCC97H, Huh7 and L-02 cells. Changes in cell adhesion, migration and invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. The expression pattern of NDRG2 and CD24 in HCC tissues and the relationship between NDRG2 and HCC clinical features was analyzed by immunohistochemical and western blotting analysis. NDRG2 expression was negatively correlated with malignancy in HCC. NDRG2 exerted anti-tumor activity by regulating CD24, a molecule that mediates cell-cell interaction, tumor proliferation and adhesion. NDRG2 up-regulation decreased CD24 expression and cell adhesion, migration and invasion. By contrast, NDRG2 down-regulation enhanced CD24 expression and cell adhesion, migration and invasion. Immunohistochemical analysis of 50 human HCC clinical specimens showed a strong correlation between NDRG2 down-regulation and CD24 overexpression (P = 0.04). In addition, increased frequency of NDRG2 down-regulation was observed in patients with elevated AFP serum level (P = 0.006), late TNM stage (P = 0.009), poor differentiation grade (P = 0.002), tumor invasion (P = 0.004) and recurrence (P = 0.024). Our findings indicate that NDRG2 and CD24 regulate HCC adhesion, migration and invasion. The expression level of NDRG2 is closely related to the clinical features of HCC. Thus, NDRG2 plays an important physiological role in HCC metastasis

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

    Lee A Borthwick

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

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

    Tewari, Rita; Straschil, Ursula; Bateman, Alex; Bö hme, Ulrike; Cherevach, Inna; Gong, Peng; Pain, Arnab; Billker, Oliver

    2010-01-01

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

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

    Tewari, Rita

    2010-10-21

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

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

    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.

  18. Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory.

    Nasrollahi, Samila; Walter, Christopher; Loza, Andrew J; Schimizzi, Gregory V; Longmore, Gregory D; Pathak, Amit

    2017-11-01

    During morphogenesis and cancer metastasis, grouped cells migrate through tissues of dissimilar stiffness. Although the influence of matrix stiffness on cellular mechanosensitivity and motility are well-recognized, it remains unknown whether these matrix-dependent cellular features persist after cells move to a new microenvironment. Here, we interrogate whether priming of epithelial cells by a given matrix stiffness influences their future collective migration on a different matrix - a property we refer to as the 'mechanical memory' of migratory cells. To prime cells on a defined matrix and track their collective migration onto an adjoining secondary matrix of dissimilar stiffness, we develop a modular polyacrylamide substrate through step-by-step polymerization of different PA compositions. We report that epithelial cells primed on a stiff matrix migrate faster, display higher actomyosin expression, form larger focal adhesions, and retain nuclear YAP even after arriving onto a soft secondary matrix, as compared to their control behavior on a homogeneously soft matrix. Priming on a soft ECM causes a reverse effect. The depletion of YAP dramatically reduces this memory-dependent migration. Our results present a previously unidentified regulation of mechanosensitive collective cell migration by past matrix stiffness, in which mechanical memory depends on YAP activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. R-Ras regulates migration through an interaction with filamin A in melanoma cells.

    Joanna E Gawecka

    2010-06-01

    Full Text Available Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin beta1, beta2 and beta7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaDelta3 abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaDelta3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.

  1. Arabidopsis MAP Kinase 4 regulates gene expression via transcription factor release in the nucleus

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus

    2008-01-01

    kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from...... MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further...... supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation....

  2. Polo kinase Cdc5 is a central regulator of meiosis I

    Attner, Michelle A.; Miller, Matthew P.; Ee, Ly-sha; Elkin, Sheryl K.; Amon, Angelika

    2013-01-01

    During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5’s central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern. PMID:23918381

  3. Fission yeast shelterin regulates DNA polymerases and Rad3(ATR kinase to limit telomere extension.

    Ya-Ting Chang

    2013-11-01

    Full Text Available Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established Rad3(ATR/Tel1(ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 as the critical post-translational modification for telomerase recruitment to telomeres. Furthermore, shelterin subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. Thus, we investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1(TERT, DNA polymerases, Replication Protein A (RPA complex, Rad3(ATR-Rad26(ATRIP checkpoint kinase complex, Tel1(ATM kinase, shelterin subunits (Tpz1, Ccq1 and Poz1 and Stn1. We further investigated how telomere shortening, caused by trt1Δ or catalytically dead Trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. These analyses established that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε and lagging (Polα strand DNA polymerases at telomeres to modulate Rad3(ATR association, Ccq1 Thr93 phosphorylation and telomerase recruitment.

  4. MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability.

    Zhao, Chunzhao; Wang, Pengcheng; Si, Tong; Hsu, Chuan-Chih; Wang, Lu; Zayed, Omar; Yu, Zheping; Zhu, Yingfang; Dong, Juan; Tao, W Andy; Zhu, Jian-Kang

    2017-12-04

    Mitogen-activated protein kinase cascades are important signaling modules that convert environmental stimuli into cellular responses. We show that MPK3, MPK4, and MPK6 are rapidly activated after cold treatment. The mpk3 and mpk6 mutants display increased expression of CBF genes and enhanced freezing tolerance, whereas constitutive activation of the MKK4/5-MPK3/6 cascade in plants causes reduced expression of CBF genes and hypersensitivity to freezing, suggesting that the MKK4/5-MPK3/6 cascade negatively regulates the cold response. MPK3 and MPK6 can phosphorylate ICE1, a basic-helix-loop-helix transcription factor that regulates the expression of CBF genes, and the phosphorylation promotes the degradation of ICE1. Interestingly, the MEKK1-MKK2-MPK4 pathway constitutively suppresses MPK3 and MPK6 activities and has a positive role in the cold response. Furthermore, the MAPKKK YDA and two calcium/calmodulin-regulated receptor-like kinases, CRLK1 and CRLK2, negatively modulate the cold activation of MPK3/6. Our results uncover important roles of MAPK cascades in the regulation of plant cold response. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. C. elegans serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

    Cohen Stephen

    2005-04-01

    Full Text Available Background In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. Results kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. Conclusion kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFβ outputs, such as tail morphogenesis.

  6. Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

    Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

    2018-06-01

    Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

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

    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)

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

    Mierke, Claudia Tanja

    2013-12-01

    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.

  9. p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals

    Epifano, Carolina; Megias, Diego; Perez-Moreno, Mirna

    2014-01-01

    The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic...... migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell...... motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results...

  10. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro

    Varley, Claire; Hill, Gemma; Pellegrin, Stephanie; Shaw, Nicola J.; Selby, Peter J.; Trejdosiewicz, Ludwik K.; Southgate, Jennifer

    2005-01-01

    Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-α, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator

  11. Ras-Induced and Extracellular Signal-Regulated Kinase 1 and 2 Phosphorylation-Dependent Isomerization of Protein Tyrosine Phosphatase (PTP)-PEST by PIN1 Promotes FAK Dephosphorylation by PTP-PEST ▿

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X.; Lu, Zhimin

    2011-01-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression. PMID:21876001

  12. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P.

    2005-01-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  13. Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility*

    Ayoub, Emily; Hall, Anita; Scott, Adam M.; Chagnon, Mélanie J.; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L.

    2013-01-01

    PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis. PMID:23897807

  14. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

  15. Activated Fps/Fes tyrosine kinase regulates erythroid differentiation and survival.

    Sangrar, Waheed; Gao, Yan; Bates, Barbara; Zirngibl, Ralph; Greer, Peter A

    2004-10-01

    A substantial body of evidence implicates the cytoplasmic protein tyrosine kinase Fps/Fes in regulation of myeloid differentiation and survival. In this study we wished to determine if Fps/Fes also plays a role in the regulation of erythropoiesis. Mice tissue-specifically expressing a "gain-of-function" mutant fps/fes transgene (fps(MF)) encoding an activated variant of Fps/Fes (MFps), were used to explore the in vivo biological role of Fps/Fes. Erythropoiesis in these mice was assessed by hematological analysis, lineage marker analysis, bone-marrow colony assays, and biochemical approaches. fps(MF) mice displayed reductions in peripheral red cell counts. However, there was an accumulation of immature erythroid precursors, which displayed increased survival. Fps/Fes and the related Fer kinase were both detected in early erythroid progenitors/blasts and in mature red cells. Fps/Fes was also activated in response to erythropoietin (EPO) and stem cell factor (SCF), two critical factors in erythroid development. In addition, increased Stat5A/B activation and reduced Erk1/2 phosphorylation was observed in fps(MF) primary erythroid cells in response to EPO or SCF, respectively. These data support a role for Fps/Fes in regulating the survival and differentiation of erythroid cells through modulation of Stat5A/B and Erk kinase pathways induced by EPO and SCF. The increased numbers and survival of erythroid progenitors from fps(MF) mice, and their differential responsiveness to SCF and EPO, implicates Fps/Fes in the commitment of multilineage progenitors to the erythroid lineage. The anemic phenotype in fps(MF) mice suggests that downregulation of Fps/Fes activity might be required for terminal erythroid differentiation.

  16. Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex▿

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R.; Elbing, Karin; Schmidt, Martin C.

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation. PMID:19897735

  17. Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

    Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

    2010-01-01

    Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

  18. Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora A Kinase Expression

    Xuesong Liu

    2008-08-01

    Full Text Available Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654, a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

  19. Regulation of mouse brain glycogen synthase kinase-3 by atypical antipsychotics.

    Li, Xiaohua; Rosborough, Kelley M; Friedman, Ari B; Zhu, Wawa; Roth, Kevin A

    2007-02-01

    Glycogen synthase kinase-3 (GSK3) has been recognized as an important enzyme that modulates many aspects of neuronal function. Accumulating evidence implicates abnormal activity of GSK3 in mood disorders and schizophrenia, and GSK3 is a potential protein kinase target for psychotropics used in these disorders. We previously reported that serotonin, a major neurotransmitter involved in mood disorders, regulates GSK3 by acutely increasing its N-terminal serine phosphorylation. The present study was undertaken to further determine if atypical antipsychotics, which have therapeutic effects in both mood disorders and schizophrenia, can regulate phospho-Ser-GSK3 and inhibit its activity. The results showed that acute treatment of mice with risperidone rapidly increased the level of brain phospho-Ser-GSK3 in the cortex, hippocampus, striatum, and cerebellum in a dose-dependent manner. Regulation of phospho-Ser-GSK3 was a shared effect among several atypical antipsychotics, including olanzapine, clozapine, quetiapine, and ziprasidone. In addition, combination treatment of mice with risperidone and a monoamine reuptake inhibitor antidepressant imipramine or fluoxetine elicited larger increases in brain phospho-Ser-GSK3 than each agent alone. Taken together, these results provide new information suggesting that atypical antipsychotics, in addition to mood stabilizers and antidepressants, can inhibit the activity of GSK3. These findings may support the pharmacological mechanisms of atypical antipsychotics in the treatment of mood disorders.

  20. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans.

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-21

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.

  1. Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

    Ho, Ernest; Dagnino, Lina

    2012-01-01

    Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594

  2. Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

    Ho, Ernest; Dagnino, Lina

    2012-02-01

    Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

  3. Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants

    Louise Tzung-Harn Hsieh

    2017-03-01

    Full Text Available In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1 in a TLR4- and Toll/interleukin (IL-1R domain-containing adaptor inducing interferon (IFN-β (TRIF-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

  4. Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5

    Zhang, Duanwu; Tomisato, Wataru; Su, Lijing; Sun, Lei; Choi, Jin Huk; Zhang, Zhao; Wang, Kuan-wen; Zhan, Xiaoming; Choi, Mihwa; Li, Xiaohong; Tang, Miao; Castro-Perez, Jose M.; Hildebrand, Sara; Murray, Anne R.; Moresco, Eva Marie Y.; Beutler, Bruce

    2017-01-01

    The recessive N-ethyl-N-nitrosourea–induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body. PMID:28607088

  5. A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

    Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

    2001-12-15

    Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

  6. Syk Tyrosine Kinase Acts as a Pancreatic Adenocarcinoma Tumor Suppressor by Regulating Cellular Growth and Invasion

    Layton, Tracy; Stalens, Cristel; Gunderson, Felizza; Goodison, Steve; Silletti, Steve

    2009-01-01

    We have identified the nonreceptor tyrosine kinase syk as a marker of differentiation/tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Syk expression is lost in poorly differentiated PDAC cells in vitro and in situ, and stable reexpression of syk in endogenously syk-negative Panc1 (Panc1/syk) cells retarded their growth in vitro and in vivo and reduced anchorage-independent growth in vitro. Panc1/syk cells exhibited a more differentiated morphology and down-regulated cyclin D1, ak...

  7. Glycogen synthase kinaseregulates urine concentrating mechanism in mice

    Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R.; Kakade, Vijayakumar; Yu, Alan S. L.; Howard, Christiana; Rao, Reena

    2015-01-01

    In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine outp...

  8. FK506-binding protein 10 (FKBP10) regulates lung fibroblast migration via collagen VI synthesis.

    Knüppel, Larissa; Heinzelmann, Katharina; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

    2018-04-19

    In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration. Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays. FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration. These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.

  9. Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4.

    Barhanpurkar-Naik, Amruta; Mhaske, Suhas T; Pote, Satish T; Singh, Kanupriya; Wani, Mohan R

    2017-07-14

    subcutaneously implanted matrigel-releasing-SDF-1α in immunocompromised mice. The present study demonstrates for the first time that IL-3 has an important role in enhancing the migration of human MSCs through regulation of the CXCR4/SDF-1α axis. These findings suggest a potential role of IL-3 in improving the efficacy of MSCs in regenerative cell therapy.

  10. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-01-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  11. Rac1 Regulates the Proliferation, Adhesion, Migration, and Differentiation of MDPC-23 Cells.

    Ren, Jing; Liang, Guobin; Gong, Li; Guo, Bing; Jiang, Hongwei

    2017-04-01

    Stem cells are responsible for replacing damaged pulp tissue; therefore, promoting their survival and inducing their adhesion to dentin are vital. As a member of the Rho family of guanosine triphosphatases, Rac1 is an important regulator of osteoblast functions. However, little is known about its role in regenerative endodontic procedures. The current study examined the role of Rac1 in the proliferation, migration, and odontoblastic differentiation of MDPC-23 cells. MDPC-23 cells were transfected with small interfering RNA to knock down Rac1 expression, and then their proliferation, migration, adhesion, and odontoblastic differentiation were examined in vitro. MDPC-23 cells transfected with si-Rac1 exhibited the increased expression of several key odontogenic protein markers, including Dmp1, Dspp, Runx2, and alkaline phosphatase, as well as decreased proliferation and migration in vitro. The results suggest that Rac1 might regulate nuclear factor kappa B signaling in MDPC-23 cells. Rac1 may have vital roles in the proliferation, migration, adhesion, and odontoblastic differentiation of MDPC-23 cells. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  12. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  13. Reduction of Fibrogenesis by Selective Delivery of a Rho Kinase Inhibitor to Hepatic Stellate Cells in Mice

    van Beuge, M. M.; Prakash, J.; Lacombe, M.; Gosens, R.; Post, E.; Reker-Smit, C.; Beljaars, L.; Poelstra, K.

    One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide

  14. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  15. Duodenal mucosal protein kinase C-δ regulates glucose production in rats.

    Kokorovic, Andrea; Cheung, Grace W C; Breen, Danna M; Chari, Madhu; Lam, Carol K L; Lam, Tony K T

    2011-11-01

    Activation of protein kinase C (PKC) enzymes in liver and brain alters hepatic glucose metabolism, but little is known about their role in glucose regulation in the gastrointestinal tract. We investigated whether activation of PKC-δ in the duodenum is sufficient and necessary for duodenal nutrient sensing and regulates hepatic glucose production through a neuronal network in rats. In rats, we inhibited duodenal PKC and evaluated whether nutrient-sensing mechanisms, activated by refeeding, have disruptions in glucose regulation. We then performed gain- and loss-of-function pharmacologic and molecular experiments to target duodenal PKC-δ; we evaluated the impact on glucose production regulation during the pancreatic clamping, while basal levels of insulin were maintained. PKC-δ was detected in the mucosal layer of the duodenum; intraduodenal infusion of PKC inhibitors disrupted glucose homeostasis during refeeding, indicating that duodenal activation of PKC-δ is necessary and sufficient to regulate glucose homeostasis. Intraduodenal infusion of the PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) specifically activated duodenal mucosal PKC-δ and a gut-brain-liver neuronal pathway to reduce glucose production. Molecular and pharmacologic inhibition of duodenal mucosal PKC-δ negated the ability of duodenal OAG and lipids to reduce glucose production. In the duodenal mucosa, PKC-δ regulates glucose homeostasis. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  16. ER-α36 mediates estrogen-stimulated MAPK/ERK activation and regulates migration, invasion, proliferation in cervical cancer cells

    Sun, Qing; Liang, Ying; Zhang, Tianli; Wang, Kun; Yang, Xingsheng

    2017-01-01

    Objective: Estrogen receptor alpha 36 (ER-α36), a truncated variant of ER-α, is different from other nuclear receptors of the ER-α family. Previous findings indicate that ER-α36 might be involved in cell growth, proliferation, and differentiation in carcinomas and primarily mediates non-genomic estrogen signaling. However, studies on ER-α36 and cervical cancer are rare. This study aimed to detect the expression of ER-α36 in cervical cancer; the role of ER-α36 in 17-β-estradiol (E2)-induced invasion, migration and proliferation of cervical cancer; and their probable molecular mechanisms. Methods: Immunohistochemistry and immunofluorescence were used to determine the location of ER-α36 in cervical cancer tissues and cervical cell lines. CaSki and HeLa cell lines were transfected with lentiviruses to establish stable cell lines with knockdown and overexpression of ER-α36. Wound healing assay, transwell invasion assay, and EdU incorporation proliferation assay were performed to evaluate the migration, invasion, and proliferation ability. The phosphorylation levels of mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling molecules were examined with western blot analysis. Results: ER-α36 expression was detected in both cervical cell lines and cervical cancer tissues. Downregulation of ER-α36 significantly inhibited cell invasion, migration, and proliferation. Moreover, upregulation of ER-α36 increased the invasion, migration, and proliferation ability of CaSki and HeLa cell lines. ER-α36 mediates estrogen-stimulated MAPK/ERK activation. Conclusion: ER-α36 is localized on the plasma membrane and cytoplasm in both cervical cancer tissues and cell lines. ER-α36 mediates estrogen-stimulated MAPK/ERK activation and regulates migration, invasion, proliferation in cervical cancer cells. - Highlights: • ER-α36 is expressed on both cervical cell lines and cervical cancer tissues. • ER-α36 mediates estrogen

  17. The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

    Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

    2010-06-01

    Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

  18. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    Serena A D'Souza

    2016-04-01

    Full Text Available The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

  19. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    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

  20. An epidermal microRNA regulates neuronal migration through control of the cellular glycosylation state

    Pedersen, Mikael Egebjerg; Snieckute, Goda; Kagias, Konstantinos

    2013-01-01

    An appropriate balance in glycosylation of proteoglycans is crucial for their ability to regulate animal development. Here, we report that the Caenorhabditis elegans microRNA mir-79, an ortholog of mammalian miR-9, controls sugar-chain homeostasis by targeting two proteins in the proteoglycan bio...... that impinges on a LON-2/glypican pathway and disrupts neuronal migration. Our results identify a regulatory axis controlled by a conserved microRNA that maintains proteoglycan homeostasis in cells....

  1. Markets, citizenship and rights: state regulation of labour migration in Malaysia and Spain

    Garcés-Mascareñas, B.

    2010-01-01

    The State regulation of labour migration seems to be confronted with a double dilemma. First, while markets require a policy of open borders to provide as many migrant workers as demanded, citizenship seems to require some degree of closure to the outside. Second, while the exclusive character of citizenship demands closed membership, civil and human rights seem to undermine the State capacity to exclude foreigners once in the country. The present thesis analyses this trilemma between markets...

  2. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number...

  3. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

    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.

  4. Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival.

    McDonald, Paul C; Oloumi, Arusha; Mills, Julia; Dobreva, Iveta; Maidan, Mykola; Gray, Virginia; Wederell, Elizabeth D; Bally, Marcel B; Foster, Leonard J; Dedhar, Shoukat

    2008-03-15

    An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH(2)- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser(473) phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser(473)P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser(473)P-Akt in the ILK complex. Expression of the NH(2)-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser(473) phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival.

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

    Gary A. Koretzky

    2013-03-01

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

  6. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length.

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-12-12

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2 +/- mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation.

  7. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  8. WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

    Inmaculada Banon-Rodriguez

    Full Text Available The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs or G-protein coupled receptors (GPCR regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein family. WIP (WASP Interacting Protein is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast and amoeboid (B lymphocytes motility.

  9. Selective Expression of an Endogenous Inhibitor of FAK Regulates Proliferation and Migration of Vascular Smooth Muscle Cells

    Taylor, Joan M.; Mack, Christopher P.; Nolan, Kate; Regan, Christopher P.; Owens, Gary K.; Parsons, J. Thomas

    2001-01-01

    Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538–540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [3H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals. PMID:11238893

  10. MIIP, a cytoskeleton regulator that blocks cell migration and invasion, delays mitosis, and suppresses tumorogenesis.

    Wang, Yingmei; Wen, Jing; Zhang, Wei

    2011-02-01

    The migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). Recent studies have shown that MIIP not only modulates IGFBP2 but also regulates microtubule by binding to and inhibiting HDAC6, a class 2 histone deacetylase that deacetylates α-tubulin, heat-shock protein 90 (HSP90), and cortactin. In addition, MIIP also regulates the mitosis checkpoint, another microtubule-associated process. The location of the MIIP gene in chromosomal region 1p36, a commonly deleted region in a broad spectrum of human cancers, and the observation that MIIP attenuates tumorigenesis in a mouse model suggest that it functions as a tumor-suppressor gene. This review summarizes the recent progress in characterizing this novel protein, which regulates cell migration and mitosis, two processes that rely on the highly coordinated dynamics of the microtubule and cytoskeleton systems.

  11. Retinoic Acid Differentially Regulates the Migration of Innate Lymphoid Cell Subsets to the Gut.

    Kim, Myung H; Taparowsky, Elizabeth J; Kim, Chang H

    2015-07-21

    Distinct groups of innate lymphoid cells (ILCs) such as ILC1, ILC2, and ILC3 populate the intestine, but how these ILCs develop tissue tropism for this organ is unclear. We report that prior to migration to the intestine ILCs first undergo a "switch" in their expression of homing receptors from lymphoid to gut homing receptors. This process is regulated by mucosal dendritic cells and the gut-specific tissue factor retinoic acid (RA). This change in homing receptors is required for long-term population and effector function of ILCs in the intestine. Only ILC1 and ILC3, but not ILC2, undergo the RA-dependent homing receptor switch in gut-associated lymphoid tissues. In contrast, ILC2 acquire gut homing receptors in a largely RA-independent manner during their development in the bone marrow and can migrate directly to the intestine. Thus, distinct programs regulate the migration of ILC subsets to the intestine for regulation of innate immunity. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The Hog1p kinase regulates Aft1p transcription factor to control iron accumulation.

    Martins, Telma S; Pereira, Clara; Canadell, David; Vilaça, Rita; Teixeira, Vítor; Moradas-Ferreira, Pedro; de Nadal, Eulàlia; Posas, Francesc; Costa, Vítor

    2018-01-01

    Iron acquisition systems have to be tightly regulated to assure a continuous supply of iron, since it is essential for survival, but simultaneously to prevent iron overload that is toxic to the cells. In budding yeast, the low‑iron sensing transcription factor Aft1p is a master regulator of the iron regulon. Our previous work revealed that bioactive sphingolipids modulate iron homeostasis as yeast cells lacking the sphingomyelinase Isc1p exhibit an upregulation of the iron regulon. In this study, we show that Isc1p impacts on iron accumulation and localization. Notably, Aft1p is activated in isc1Δ cells due to a decrease in its phosphorylation and an increase in its nuclear levels. Consistently, the expression of a phosphomimetic version of Aft1p-S210/S224 that favours its nuclear export abolished iron accumulation in isc1Δ cells. Notably, the Hog1p kinase, homologue of mammalian p38, interacts with and directly phosphorylates Aft1p at residues S210 and S224. However, Hog1p-Aft1p interaction decreases in isc1Δ cells, which likely contributes to Aft1p dephosphorylation and consequently to Aft1p activation and iron overload in isc1Δ cells. These results suggest that alterations in sphingolipid composition in isc1Δ cells may impact on iron homeostasis by disturbing the regulation of Aft1p by Hog1p. To our knowledge, Hog1p is the first kinase reported to directly regulate Aft1p, impacting on iron homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. MicroRNA-218 inhibits cell invasion and migration of pancreatic cancer via regulating ROBO1.

    He, Hang; Hao, Si-Jie; Yao, Lie; Yang, Feng; Di, Yang; Li, Ji; Jiang, Yong-Jian; Jin, Chen; Fu, De-Liang

    2014-10-01

    miRNA-218 is a highlighted tumor suppressor and its underlying role in tumor progression is still unknown. Here, we restored the expression of miRNA-218 in pancreatic cancer to clarify the function and potent downstream pathway of miRNA-218. The expressions of both miRNA-218 and its potent target gene ROBO1 were revealed by RT-PCR and western blotting analysis. Transfection of miRNA-218 precursor mimics and luciferase assay were performed to elucidate the regulation mechanism between miRNA-218 and ROBO1. Cells, stably expressing miRNA-218 followed by forced expression of mutant ROBO1, were established through co-transfections of both lentivirus vector and plasmid vector. The cell migration and invasion abilities were evaluated by migration assay and invasion assay respectively. An increased expression of ROBO1 was revealed in cell BxPC-3-LN compared with cell BxPC-3. Elevated expression of miRNA-218 would suppress the expression of ROBO1 via complementary binding to a specific region within 3'UTR of ROBO1 mRNA (sites 971-978) in pancreatic cancer cells. Stably restoring the expression of miRNA-218 in pancreatic cancer significantly downregulated the expression of ROBO1 and effectively inhibited cell migration and invasion. Forced expression of mutant ROBO1 could reverse the repression effects of miRNA-218 on cell migration and invasion. Consequently, miRNA-218 acted as a tumor suppressor in pancreatic cancer by inhibiting cell invasion and migration. ROBO1 was a functional target of miRNA-218's downstream pathway involving in cell invasion and migration of pancreatic cancer.

  14. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    Yunta, Cristina

    2011-11-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  15. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    Yunta, Cristina; Martí nez-Ripoll, Martí n; Zhu, Jian-Kang; Albert, Armando

    2011-01-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  16. Interleukin 20 regulates dendritic cell migration and expression of co-stimulatory molecules

    Bech, Rikke; Jalilian, Babak; Agger, Ralf

    2016-01-01

    BACKGROUND: Psoriasis is an inflammatory disease characterized by leukocyte skin infiltration. Interestingly, recent works suggest that the migration of dendritic cells (DCs) is abnormal in psoriatic skin. DCs have significant role in regulating the function of T lymphocytes, at least in part...... influenced by the local environment of cytokines. In psoriatic skin lesions the expression of IL-20 is highly up-regulated. It is unclear if this cytokine has any influence on DCs. METHODS: Here, we investigated the influence of IL-20 in monocyte-derived dendritic cell (MDDCs) in vitro. This work addressed...

  17. Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

    Petrvalská, Olivia; Košek, Dalibor; Kukačka, Zdeněk; Tošner, Z.; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

    2016-01-01

    Roč. 291, č. 39 (2016), s. 20753-20765 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 ; RVO:61388971 Keywords : 14-3-3 protein * apoptosis signal-regulating kinase 1 (ASK1) * fluorescence * nuclear magnetic resonance (NMR) * protein cross-linking * small-angle x-ray scattering (SAXS) Subject RIV: CE - Biochemistry Impact factor: 4.125, year: 2016

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

    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

  19. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  20. Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes

    Balboula, A. Z.; Nguyen, A. L.; Gentilello, A. S.; Quartuccio, S. M.; Drutovič, Dávid; Šolc, Petr; Schindler, K.

    2016-01-01

    Roč. 129, č. 19 (2016), s. 3648-3660 ISSN 0021-9533 R&D Projects: GA MŠk(CZ) LO1609 Institutional support: RVO:67985904 Keywords : haspin * aurora kinase * spindle * MTOC * oocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.431, year: 2016

  1. DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth

    DiAntonio Aaron

    2007-08-01

    Full Text Available Abstract Background The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK. To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. Results We identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth. Conclusion The F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.

  2. p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

    Sudo, Tatsuhiko; Kawai, Kayoko; Matsuzaki, Hiroshi; Osada, Hiroyuki

    2005-01-01

    One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38α is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38α, we utilized newly established mouse fibroblast cell lines originated from a p38α null mouse, namely, a parental cell line without p38α gene locus, knockout of p38α (KOP), Zeosin-resistant (ZKOP), revertant of p38α (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38α. The loss of MAPKAPK2 expression accompanied by the defect of p38α is confirmed in an embryonic extract prepared from p38α null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in

  3. Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

    Yu, Hongchi; Gao, Min; Ma, Yunlong; Wang, Lijuan; Shen, Yang; Liu, Xiaoheng

    2018-05-01

    angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti‑angiogenic therapy. In the present study, a small‑molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

  4. Taurine Inhibits K+-Cl− Cotransporter KCC2 to Regulate Embryonic Cl− Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway*

    Inoue, Koichi; Furukawa, Tomonori; Kumada, Tatsuro; Yamada, Junko; Wang, Tianying; Inoue, Rieko; Fukuda, Atsuo

    2012-01-01

    GABA inhibits mature neurons and conversely excites immature neurons due to lower K+-Cl− cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl− homeostasis, which is required for normal brain development. PMID:22544747

  5. Regulation of Glioma Cell Migration by Seri ne-Phosphorylated P3111

    Wendy S. McDonough

    2005-09-01

    Full Text Available P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59 near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation, induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311, reduced glioma cell migration. Coimmunoprecipitation coupled with matrixassisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of β1 integrin function using TACβ1A, a dominant-negative inhibitor of β1 integrin signaling, suggesting that P311 acts downstream of β1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Raci GTPase; small interfering RNA-mediated depletion of Raci suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility, invasion through the reorganization of actin cytoskeleton at the cell periphery.

  6. The Syk kinase SmTK4 of Schistosoma mansoni is involved in the regulation of spermatogenesis and oogenesis.

    Svenja Beckmann

    2010-02-01

    Full Text Available The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6 acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.

  7. Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

    Aquino-Martínez, Rubén; Angelo, Alcira P; Pujol, Francesc Ventura

    2017-11-16

    Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca 2+ -containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO 4 ) on MSC migration. In addition, to evaluate the influence of CaSO 4 on MSC differentiation and the potential molecular mechanisms involved. A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO 4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO 4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO 4 treatment was also evaluated by qPCR. CaSO 4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO 4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO 4 -containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO 4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO 4 effects on MSC migration. Specific CaSO 4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO 4 regulates BMP-2-induced MSC migration by differentially activating the PI3

  8. Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration

    Rubén Aquino-Martínez

    2017-11-01

    Full Text Available Abstract Background Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4 on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. Methods A circular calvarial bone defect (5 mm diameter was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. Results CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. Conclusions Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host’s undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced

  9. The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

    Chih-Wei Chou

    Full Text Available BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio. Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a or matrix metalloproteinase 2 (mmp2, two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through

  10. Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1 Regulation.

    Wei Shen

    Full Text Available As one of the most commonly used eukaryotic recombinant protein expression systems, P. pastoris relies heavily on the AOX1 promoter (PAOX1, which is strongly induced by methanol but strictly repressed by glycerol and glucose. However, the complicated signaling pathways involved in PAOX1 regulation when supplemented with different carbon sources are poorly understood. Here we constructed a kinase deletion library in P. pastoris and identified 27 mutants which showed peculiar phenotypes in cell growth or PAOX1 regulation. We analyzed both annotations and possible functions of these 27 targets, and then focused on the MAP kinase Hog1. In order to locate its potential downstream components, we performed the phosphoproteome analysis on glycerol cultured WT and Δhog1 strains and identified 157 differentially phosphorylated proteins. Our results identified important kinases involved in P. pastoris cell growth and PAOX1 regulation, which could serve as valuable targets for further mechanistic studies.

  11. A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

    Nanjareddy, Kalpana; Blanco, Lourdes; Arthikala, Manoj-Kumar; Alvarado-Affantranger, Xóchitl; Quinto, Carmen; Sánchez, Federico; Lara, Miguel

    2016-11-01

    The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean. © 2016 American Society of Plant Biologists. All Rights Reserved.

  12. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  13. Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

    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

  14. Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

    Andrade, Luiza Freire de; Mourão, Marina de Moraes; Geraldo, Juliana Assis; Coelho, Fernanda Sales; Silva, Larissa Lopes; Neves, Renata Heisler; Volpini, Angela; Machado-Silva, José Roberto; Araujo, Neusa; Nacif-Pimenta, Rafael; Caffrey, Conor R; Oliveira, Guilherme

    2014-06-01

    Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade. We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

  15. Functional Role of Cyclin-Dependent Kinase 5 in the Regulation of Melanogenesis and Epidermal Structure.

    Dong, Changsheng; Yang, Shanshan; Fan, Ruiwen; Ji, Kaiyuan; Zhang, Junzhen; Liu, Xuexian; Hu, Shuaipeng; Xie, Jianshan; Liu, Yu; Gao, Wenjun; Wang, Haidong; Yao, Jianbo; Smith, George W; Herrid, Muren

    2017-10-23

    The mammalian integumentary system plays important roles in body homeostasis, and dysfunction of melanogenesis or epidermal development may lead to a variety of skin diseases, including melanoma. Skin pigmentation in humans and coat color in fleece-producing animals are regulated by many genes. Among them, microphthalmia-associated transcription factor (MITF) and paired-box 3 (PAX3) are at the top of the cascade and regulate activities of many important melanogenic enzymes. Here, we report for the first time that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of MITF and PAX3. Cdk5 knockdown in mice causes a lightened coat color, a polarized distribution of melanin and hyperproliferation of basal keratinocytes. Reduced expression of Keratin 10 (K10) resulting from Cdk5 knockdown may be responsible for an abnormal epidermal structure. In contrast, overexpression of Cdk5 in sheep (Ovis aries) only produces brown patches on a white background, with no other observable abnormalities. Collectively, our findings show that Cdk5 has an important functional role in the regulation of melanin production and transportation and in normal development of the integumentary system.

  16. Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis.

    Hill, Jennifer W; Xu, Yong; Preitner, Frederic; Fukuda, Makota; Cho, You-Ree; Luo, Ji; Balthasar, Nina; Coppari, Roberto; Cantley, Lewis C; Kahn, Barbara B; Zhao, Jean J; Elmquist, Joel K

    2009-11-01

    Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

  17. Nano-food packaging: an overview of market, migration research, and safety regulations.

    Bumbudsanpharoke, Nattinee; Ko, Seonghyuk

    2015-05-01

    Recently, food packages produced with nanoparticles, "nano-food packaging," have become more available in the current market. However, although the use of nanomaterials is increasing in food packaging applications, concern over toxicity affects consumer perceptions and acceptance. Quite a number of commercialized forms of nano-food packaging are coated or composited product with inorganic materials, for example, nanosilver and nanoclay as representative examples. Several studies have shown the possibility of nanomaterial migration from packaging or containers to foodstuff. The debate is still ongoing among researchers about the extent of migration and whether it is negligible and safe. Government agencies and stakeholders must hurry to determine use limitations and release conclusive legislation and regulations as soon as possible since nano-food packaging may have great impacts on human health. This paper aims to review the availability of nano-food packaging in the current market, report case studies on nanomaterial migration, and present the current status of safety regulations and management of nano-food packaging in leading countries across regions. This review should enable governments and researchers to develop further nanomaterial risk assessment studies. © 2015 Institute of Food Technologists®

  18. SAD-B kinase regulates pre-synaptic vesicular dynamics at hippocampal Schaffer collateral synapses and affects contextual fear memory.

    Watabe, Ayako M; Nagase, Masashi; Hagiwara, Akari; Hida, Yamato; Tsuji, Megumi; Ochiai, Toshitaka; Kato, Fusao; Ohtsuka, Toshihisa

    2016-01-01

    Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. © 2015 International

  19. Regulation of p73 by Hck through kinase-dependent and independent mechanisms

    Radha Vegesna

    2007-05-01

    Full Text Available Abstract Background p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73. Results Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP, a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation. Conclusion We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73

  20. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  1. Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells.

    María José Olivares

    Full Text Available The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

  2. CENPI is overexpressed in colorectal cancer and regulates cell migration and invasion.

    Ding, Na; Li, Rongxin; Shi, Wenhao; He, Cui

    2018-06-21

    Centromere protein I (CENPI),an important member of centromere protein family, has been suggest to serve as a oncogene in breast cancer, but the clinical significance and biological function of CENPI in colorectal cancer (CRC) is still unclear. In our results, we found CENPI was overexpressed in CRC tissues and cells, and associated with clinical stage, tumor depth, lymph node metastasis, distant metastasis and differentiation in CRC patients. However, there was no significant association between CENPI protein expression and overall survival time in colon cancer patients and rectal cancer patients through analyzing TCGA survival data. Moreover, CENPI mRNA and protein were increased in metastatic lymph nodes compared with primary CRC tissues. Down-regulation of CENPI expression suppresses CRC cell migration, invasion and epithelial mesenchymal transition process. In conclusion, CENPI is overexpressed in CRC and functions as oncogene in modulating CRC cell migration, invasion and EMT process. Copyright © 2018. Published by Elsevier B.V.

  3. Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

    Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

    2016-06-01

    The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. The Drosophila PNG kinase complex regulates the translation of cyclin B.

    Vardy, Leah; Orr-Weaver, Terry L

    2007-01-01

    The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.

  5. Protein kinase C-dependent regulation of connexin43 gap junctions and hemichannels

    Alstrøm, Jette Skov; Stroemlund, Line Waring; Nielsen, Morten Schak

    2015-01-01

    Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix...... and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current...... knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results....

  6. A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

    Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

    2013-01-01

    The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

  7. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  8. Src-family kinases negatively regulate NFAT signaling in resting human T cells.

    Alan Baer

    Full Text Available T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.

  9. The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

    Cole Johnson

    2014-03-01

    Full Text Available The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans.

  10. [Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

    Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

    2016-10-01

    Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

  11. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Hyung Soon; Kang, Gum-Yong; Bang, Joo Young [Center for Biomedical Mass Spectrometry, Diatech Korea Co., Ltd., Seoul (Korea, Republic of); Cho, Eun-Jung [National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence and Technology, Seoul National University, Seoul (Korea, Republic of)

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  12. Agonist-induced down-regulation of endogenous protein kinase c α through an endolysosomal mechanism.

    Lum, Michelle A; Pundt, Krista E; Paluch, Benjamin E; Black, Adrian R; Black, Jennifer D

    2013-05-03

    Protein kinase C (PKC) isozymes undergo down-regulation upon sustained stimulation. Previous studies have pointed to the existence of both proteasome-dependent and -independent pathways of PKCα processing. Here we demonstrate that these down-regulation pathways are engaged in different subcellular compartments; proteasomal degradation occurs mainly at the plasma membrane, whereas non-proteasomal processing occurs in the perinuclear region. Using cholesterol depletion, pharmacological inhibitors, RNA interference, and dominant-negative mutants, we define the mechanisms involved in perinuclear accumulation of PKCα and identify the non-proteasomal mechanism mediating its degradation. We show that intracellular accumulation of PKCα involves at least two clathrin-independent, cholesterol/lipid raft-mediated pathways that do not require ubiquitination of the protein; one is dynamin-dependent and likely involves caveolae, whereas the other is dynamin- and small GTPase-independent. Internalized PKCα traffics through endosomes and is delivered to the lysosome for degradation. Supportive evidence includes (a) detection of the enzyme in EEA1-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes and (b) inhibition of its down-regulation by lysosome-disrupting agents and leupeptin. Only limited dephosphorylation of PKCα occurs during trafficking, with fully mature enzyme being the main target for lysosomal degradation. These studies define a novel and widespread mechanism of desensitization of PKCα signaling that involves endocytic trafficking and lysosome-mediated degradation of the mature, fully phosphorylated protein.

  13. Computational Modelling of the Metabolic States Regulated by the Kinase Akt

    Ettore eMosca

    2012-11-01

    Full Text Available Signal transduction pathways and gene regulation determine a major reorganization of metabolic activities in order to support cell proliferation. Protein Kinase B (PKB, also known as Akt, participates in the PI3K/Akt/mTOR pathway, a master regulator of aerobic glycolysis and cellular biosynthesis, two activities shown by both normal and cancer proliferating cells. Not surprisingly considering its relevance for cellular metabolism, Akt/PKB is often found hyperactive in cancer cells. In the last decade, many efforts have been made to improve the understanding of the control of glucose metabolism and the identification of a therapeutic window between proliferating cancer cells and proliferating normal cells. In this context, we have modelled the link between the PI3K/Akt/mTOR pathway, glycolysis, lactic acid production and nucleotide biosynthesis. We used a computational model in order to compare two metabolic states generated by the specific variation of the metabolic fluxes regulated by the activity of the PI3K/Akt/mTOR pathway. One of the two states represented the metabolism of a growing cancer cell characterised by aerobic glycolysis and cellular biosynthesis, while the other state represented the same metabolic network with a reduced glycolytic rate and a higher mitochondrial pyruvate metabolism, as reported in literature in relation to the activity of the PI3K/Akt/mTOR. Some steps that link glycolysis and pentose phosphate pathway revealed their importance for controlling the dynamics of cancer glucose metabolism.

  14. Protein Kinases C-Mediated Regulations of Drug Transporter Activity, Localization and Expression

    Abdullah Mayati

    2017-04-01

    Full Text Available Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug–drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs in transporter regulations are summarized and discussed. Both solute carrier (SLC and ATP-binding cassette (ABC drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug–drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.

  15. Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

    Enugutti, Balaji; Kirchhelle, Charlotte; Oelschner, Maxi; Torres Ruiz, Ramón Angel; Schliebner, Ivo; Leister, Dario; Schneitz, Kay

    2012-09-11

    The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity.

  16. Cxcl8b and Cxcr2 Regulate Neutrophil Migration through Bloodstream in Zebrafish

    Constanza Zuñiga-Traslaviña

    2017-01-01

    Full Text Available Neutrophils play an essential role during an inflammatory response, which is dependent on their rapid recruitment from the bone marrow to the vasculature. However, there is no information about the molecular signals that regulate neutrophil entry to circulation during an inflammatory process in humans. This is mainly due to the lack of a suitable model of study that contains similar set of molecules and that allows in vivo analyses. In this study, we used the zebrafish to assess the role of Cxcl8a, Cxcl8b, and Cxcr2 in neutrophil migration to blood circulation after injury. Using Tg(BACmpx:GFPi114 transgenic embryos and two damage models (severe and mild, we developed in vivo lack of function assays. We found that the transcription levels of cxcl8a, cxcl8b, and cxcr2 were upregulated in the severe damage model. In contrast, only cxcr2 and cxcl8a mRNA levels were increased during mild damage. After knocking down Cxcl8a, neutrophil quantity decreased at the injury site, while Cxcl8b decreased neutrophils in circulation. When inhibiting Cxcr2, we observed a decrease in neutrophil entry to the bloodstream. In conclusion, we identified different functions for both Cxcl8 paralogues, being the Cxcl8b/Cxcr2 axis that regulates neutrophil entry to the bloodstream, while Cxcl8a/Cxcr2 regulates the migration to the affected area.

  17. Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

    Dobisova, Tereza; Hrdinova, Vendula; Cuesta, Candela; Michlickova, Sarka; Urbankova, Ivana; Hejatkova, Romana; Zadnikova, Petra; Pernisova, Marketa; Benkova, Eva; Hejatko, Jan

    2017-05-01

    In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL ( LPH ) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 ( CKI1 ), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 ( HY1 ) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph / hy1 - 7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  18. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    Anne-Sophie eLeprince

    2015-01-01

    Full Text Available Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signalling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K, VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1, a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.

  19. Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

    Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

    2006-08-15

    Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

  20. Down-regulation of serum/glucocorticoid regulated kinase 1 in colorectal tumours is largely independent of promoter hypermethylation.

    Francesca Lessi

    2010-11-01

    Full Text Available We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1 is down-regulated in colorectal cancers (CRC with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples.We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963 which affects methylation of the corresponding CpG.Our results show that even though partial methylation of the promoter region of SGK1 is present, this does not account for the different expression levels seen between normal and tumour tissue.

  1. Sphingosine 1-Phosphate (S1P) Receptors 1 and 2 Coordinately Induce Mesenchymal Cell Migration through S1P Activation of Complementary Kinase Pathways*

    Quint, Patrick; Ruan, Ming; Pederson, Larry; Kassem, Moustapha; Westendorf, Jennifer J.; Khosla, Sundeep; Oursler, Merry Jo

    2013-01-01

    Normal bone turnover requires tight coupling of bone resorption and bone formation to preserve bone quantity and structure. With aging and during several pathological conditions, this coupling breaks down, leading to either net bone loss or excess bone formation. To preserve or restore normal bone metabolism, it is crucial to determine the mechanisms by which osteoclasts and osteoblast precursors interact and contribute to coupling. We showed that osteoclasts produce the chemokine sphingosine 1-phosphate (S1P), which stimulates osteoblast migration. Thus, osteoclast-derived S1P may recruit osteoblasts to sites of bone resorption as an initial step in replacing lost bone. In this study we investigated the mechanisms by which S1P stimulates mesenchymal (skeletal) cell chemotaxis. S1P treatment of mesenchymal (skeletal) cells activated RhoA GTPase, but this small G protein did not contribute to migration. Rather, two S1P receptors, S1PR1 and S1PR2, coordinately promoted migration through activation of the JAK/STAT3 and FAK/PI3K/AKT signaling pathways, respectively. These data demonstrate that the chemokine S1P couples bone formation to bone resorption through activation of kinase signaling pathways. PMID:23300082

  2. Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

    Pabst, A M; Walter, C; Grassmann, L; Weyhrauch, M; Brüllmann, D D; Ziebart, T; Scheller, H; Lehmann, K M

    2014-05-01

    The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each materials at all points of measurement (between -36 % and -71 %; p each ceramic materials could be investigated. This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

  3. Retinoic acid differentially regulates the migration of innate lymphoid cell subsets to the gut

    Kim, Myung H.; Taparowsky, Elizabeth J.; Kim, Chang H.

    2015-01-01

    Distinct groups of innate lymphoid cells (ILCs) such as ILC1, ILC2 and ILC3 populate the intestine, but how these ILCs develop tissue tropism for this organ is unclear. We report that prior to migration to the intestine ILCs first undergo a `switch' in their expression of homing receptors from lymphoid to gut homing receptors. This process is regulated by mucosal dendritic cells and the gut-specific tissue factor retinoic acid (RA). This change in homing receptors is required for long-term po...

  4. Proneural Transcription Factors Regulate Different Steps of Cortical Neuron Migration through Rnd-Mediated Inhibition of RhoA Signaling

    Pacary, Emilie; Heng, Julian; Azzarelli, Roberta; Riou, Philippe; Castro, Diogo; Lebel-Potter, Mélanie; Parras, Carlos; Bell, Donald M.; Ridley, Anne J.; Parsons, Maddy; Guillemot, François

    2011-01-01

    Summary Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program. PMID:21435554

  5. Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

    Asai, Jun; Hirakawa, Satoshi; Sakabe, Jun-ichi; Kishida, Tsunao; Wada, Makoto; Nakamura, Naomi; Takenaka, Hideya; Mazda, Osam; Urano, Tetsumei; Suzuki-Inoue, Katsue; Tokura, Yoshiki; Katoh, Norito

    2016-01-01

    Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  6. Regulation of vernal migration in Gambel's white-crowned sparrows: Role of thyroxine and triiodothyronine.

    Pérez, Jonathan H; Furlow, J David; Wingfield, John C; Ramenofsky, Marilyn

    2016-08-01

    Appropriate timing of migratory behavior is critical for migrant species. For many temperate zone birds in the spring, lengthening photoperiod is the initial cue leading to morphological, physiological and behavior changes that are necessary for vernal migration and breeding. Strong evidence has emerged in recent years linking thyroid hormone signaling to the photoinduction of breeding in birds while more limited information suggest a potential role in the regulation of vernal migration in photoperiodic songbirds. Here we investigate the development and expression of the vernal migratory life history stage in captive Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) in a hypothyroidic state, induced by chemical inhibition of thyroid hormone production. To explore possible variations in the effects of the two thyroid hormones, triiodothyronine and thyroxine, we subsequently performed a thyroid inhibition coupled with replacement therapy. We found that chemical inhibition of thyroid hormones resulted in complete abolishment of mass gain, fattening, and muscle hypertrophy associated with migratory preparation as well as resulting in failure to display nocturnal restlessness behavior. Replacement of thyroxine rescued all of these elements to near control levels while triiodothyronine replacement displayed partial or delayed rescue. Our findings support thyroid hormones as being necessary for the expression of changes in morphology and physiology associated with migration as well as migratory behavior itself. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

    Fairley, Jennifer A.; Mitchell, Louise E.; Berg, Tracy; Kenneth, Niall S.; von Schubert, Conrad; Sillje, Herman H. W.; Medema, Rene H.; Nigg, Erich A.; White, Robert J.

    2012-01-01

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase Ill (pol Ill) through direct binding and phosphorylation of transcription factor Brit During interphase, Plk1 promotes

  8. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  9. Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating

    Kristensen, Anders Skov; Jenkins, Meagan A; Banke, Tue G

    2011-01-01

    The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductanc...

  10. Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

    Usaite, Renata; Jewett, Michael Christopher; Soberano de Oliveira, Ana Paula

    2009-01-01

    Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite...

  11. Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

    Qian-Shi Zhang

    Full Text Available Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1, also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5, after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1, the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93, or the downstream target, c-Jun N-terminal kinase (SP600125 also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580 had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

  12. Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration

    Zhang, Qian-Shi; Kurpad, Deepa S.; Mahoney, My G.; Steinbeck, Marla J.

    2017-01-01

    Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration. PMID:29045420

  13. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...

  14. An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration.

    Kutys, Matthew L; Yamada, Kenneth M

    2014-09-01

    Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

  15. Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

    Wang, Hong; Brautigan, David L

    2006-11-01

    Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

  16. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    Larsen, Claus Morten; Døssing, M G; Papa, S

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  17. Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

    Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

    2017-01-08

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

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

    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.

  20. Microtubule affinity-regulating kinases are potential druggable targets for Alzheimer's disease.

    Annadurai, Narendran; Agrawal, Khushboo; Džubák, Petr; Hajdúch, Marián; Das, Viswanath

    2017-11-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects normal functions of the brain. Currently, AD is one of the leading causes of death in developed countries and the only one of the top ten diseases without a means to prevent, cure, or significantly slow down its progression. Therefore, newer therapeutic concepts are urgently needed to improve survival and the quality of life of AD patients. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neurons. However, their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy. Despite the relevance of MARKs in AD pathogenesis, only a few small molecules are known to have anti-MARK activity and not much has been done to progress these compounds into therapeutic candidates. But given the diverse role of MARKs, the specificity of novel inhibitors is imperative for their successful translation from bench to bedside. In this regard, a recent co-crystal structure of MARK4 in association with a pyrazolopyrimidine-based inhibitor offers a potential scaffold for the development of more specific MARK inhibitors. In this manuscript, we review the biological role of MARKs in health and disease, and draw attention to the largely unexplored area of MARK inhibitors for AD.

  1. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    Lee, Jennifer K; Hallock, Peter T

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2+/− mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation. PMID:29231808

  2. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    John Paul Christianson

    2014-10-01

    Full Text Available Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed behavioral immunization. Recent data implicate neuroplasticity within the ventromedial prefrontal cortex (mPFC as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and one week later to uncontrollable tailshocks, followed 24h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDAR and the extracellular signal-regulated kinase (ERK cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL or infralimbic (IL mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent plasticity within the PL region is required for behavioral immunization, a learned form of stressor resistance.

  3. Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.

    Chamorro, David; Alarcón, Lourdes; Ponce, Arturo; Tapia, Rocio; González-Aguilar, Héctor; Robles-Flores, Martha; Mejía-Castillo, Teresa; Segovia, José; Bandala, Yamir; Juaristi, Eusebio; González-Mariscal, Lorenza

    2009-09-01

    Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

  4. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  5. Transcriptional regulation of the protein kinase a subunits in Saccharomyces cerevisiae during fermentative growth.

    Galello, Fiorella; Pautasso, Constanza; Reca, Sol; Cañonero, Luciana; Portela, Paula; Moreno, Silvia; Rossi, Silvia

    2017-12-01

    Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favourite carbon source of Saccharomyces cerevisiae, which prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signalling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

    Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

    2011-01-01

    For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

  7. Turning behaviors of T cells climbing up ramp-like structures are regulated by myosin light chain kinase activity and lamellipodia formation.

    Song, Kwang Hoon; Lee, Jaehyun; Jung, Hong-Ryul; Park, HyoungJun; Doh, Junsang

    2017-09-14

    T cells navigate diverse microenvironments to perform immune responses. Micro-scale topographical structures within the tissues, which may inherently exist in normal tissues or may be formed by inflammation or injury, can influence T cell migration, but how T cell migration is affected by such topographical structures have not been investigated. In this study, we fabricated ramp-like structures with a 5 μm height and various slopes, and observed T cells climbing up the ramp-like structures. T cells encountering the ramp-like structures exhibited MLC accumulation near head-tail junctions contacting the ramp-like structures, and made turns to the direction perpendicular to the ramp-like structures. Pharmacological study revealed that lamellipodia formation mediated by arp2/3 and contractility regulated by myosin light chain kinase (MLCK) were responsible for the intriguing turning behavior of T cells climbing the ramp-like structures. Arp2/3 or MLCK inhibition substantially reduced probability of T cells climbing sharp-edged ramp-like structures, indicating intriguing turning behavior of T cells mediated by lamellipodia formation and MLCK activity may be important for T cells to access inflamed or injured tissues with abrupt topographical changes.

  8. Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells.

    Jiang, Shan; Zhang, Dongxin; Huang, Hong; Lei, Yonghong; Han, Yan; Han, Weidong

    2017-01-01

    Background . The aim of this study was to assess the effects of low concentrations of H 2 O 2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods . HUVECs were cultured and stimulated with different concentrations of H 2 O 2 . Flow cytometric analysis was used to select an optimal concentration of H 2 O 2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results . We found that low concentrations of H 2 O 2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H 2 O 2 . Enhanced ERK5 activity significantly amplified the proangiogenic effects of H 2 O 2 ; in contrast, ERK5 knock-down abrogated the effects of H 2 O 2 . Conclusions . Our results confirmed that low concentrations of H 2 O 2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.

  9. Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9

    Susann Pohlers

    2017-01-01

    Full Text Available Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases—metalloenzymes that have been found in all domains of life—enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2. Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata. Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi.

  10. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

    2016-08-01

    -Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. © FASEB.

  11. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

    Zheng, Yanhua; Lu, Zhimin

    2013-01-01

    Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

  12. Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro

    Nieuw Amerongen, G.P. van; Koolwijk, P.; Versteilen, A.; Hinsbergh, V.W.M. van

    2003-01-01

    Objective - Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced

  13. Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

    Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-01-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

  14. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-03-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

  15. Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.

    Ito, Daisuke; Yogosawa, Satomi; Mimoto, Rei; Hirooka, Shinichi; Horiuchi, Takashi; Eto, Ken; Yanaga, Katsuhiko; Yoshida, Kiyotsugu

    2017-08-01

    Colorectal cancer is a common cancer and a leading cause of cancer-related death worldwide. The liver is a dominant metastatic site for patients with colorectal cancer. Molecular mechanisms that allow colorectal cancer cells to form liver metastases are largely unknown. Activation of epithelial-mesenchymal transition is the key step for metastasis of cancer cells. We recently reported that dual-specificity tyrosine-regulated kinase 2 (DYRK2) controls epithelial-mesenchymal transition in breast cancer and ovarian serous adenocarcinoma. The aim of this study is to clarify whether DYRK2 regulates liver metastases of colorectal cancer. We show that the ability of cell invasion and migration was abrogated in DYRK2-overexpressing cells. In an in vivo xenograft model, liver metastatic lesions were markedly diminished by ectopic expression of DYRK2. Furthermore, we found that patients whose liver metastases expressed low DYRK2 levels had significantly worse overall and disease-free survival. Given the findings that DYRK2 regulates cancer cell metastasis, we concluded that the expression status of DYRK2 could be a predictive marker for liver metastases of colorectal cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  16. Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes.

    Duong, Michelle; Yu, Xuejiao; Teng, Beina; Schroder, Patricia; Haller, Hermann; Eschenburg, Susanne; Schiffer, Mario

    2017-07-21

    Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of β-catenin, which promotes its degradation. β-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with β-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower β-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, β-catenin showed a reversed subcellular localization pattern: Although β-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates β-catenin expression and stabilization in a glycogen synthase kinase 3β-independent manner. Further, β-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the β-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in β-catenin that are required for full β-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and β-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of β-catenin levels and define PKCϵ as an important β-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. HOG MAP kinase regulation of alternariol biosynthesis in Alternaria alternata is important for substrate colonization.

    Graf, Eva; Schmidt-Heydt, Markus; Geisen, Rolf

    2012-07-16

    Strains of the genus Alternaria are ubiquitously present and frequently found on fruits, vegetables and cereals. One of the most commonly found species from this genus is A. alternata which is able to produce the mycotoxin alternariol among others. To date only limited knowledge is available about the regulation of the biosynthesis of alternariol, especially under conditions relevant to food. Tomatoes are a typical substrate of A. alternata and have a high water activity. On the other hand cereals with moderate water activity are also frequently colonized by A. alternata. In the current analysis it was demonstrated that even minor changes in the osmotic status of the substrate affect the alternariol biosynthesis of strains from vegetables resulting in nearly complete inhibition. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade) which is a MAP kinase transduction pathway. The phosphorylation status of the A. alternata HOG (AaHOG) was determined. Various concentrations of NaCl induce the phosphorylation of AaHOG in a concentration, time and strain dependent manner. A strain with a genetically inactivated aahog gene was no longer able to produce alternariol indicating that the activity of the aahog gene is required for alternariol biosynthesis. Further experiments revealed that the biosynthesis of alternariol is important for the fungus to colonize tomato tissue. The tight water activity dependent regulation of alternariol biosynthesis ensures alternariol biosynthesis at conditions which indicate an optimal colonization substrate for the fungus. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  19. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

    2013-01-01

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma

  20. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C. However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM, a constituent of HDL, was affected by dihydrotestosterone (DHT. Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC, phorbol-12-myristate-13-acetate (PMA, blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the

  1. Sphingosine kinase-1 is central to androgen-regulated prostate cancer growth and survival.

    Audrey Dayon

    Full Text Available BACKGROUND: Sphingosine kinase-1 (SphK1 is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. METHODOLOGY/PRINCIPAL FINDINGS: Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway--by negatively impacting SphK1 activity--could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. CONCLUSIONS/SIGNIFICANCE: We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a

  2. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  3. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-01-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

  4. IL-27 regulates the adherence, proliferation, and migration of MSCs and enhances their regulatory effects on Th1 and Th2 subset generations.

    Xu, Fenghuang; Yi, Junzhu; Wang, Zhuoya; Hu, Yejia; Han, Chunlei; Xue, Qun; Zhang, Xueguang; Luan, Xiying

    2017-08-01

    Interleukin 27 (IL-27) regulates T cell function and is involved in inflammation. It has been reported that human placenta-derived mesenchymal stromal cells (hPMSCs) can inhibit T cell responses and attenuate inflammation reactions. However, it is unclear whether IL-27 can regulate hPMSC function. Here, we examined the effects of IL-27 upon adherence, migration, and proliferation as well as the immunomodulatory effects of hPMSCs. The results show that IL-27 receptor α chain (IL-27Rα) is expressed in hPMSCs. IL-27 at 30 ng/ml inhibited hPMSC adherence and proliferation, while the migration of hPMSCs was promoted with IL-27 at doses of 20 or 30 ng/ml, as determined with use of real-time cell analysis (RTCA). Moreover, IL-27 promoted regulatory effects of hPMSCs through enhancing Th2 and suppressing Th1 subset generation from activated T cells in human peripheral blood. IL-27 also enhanced the ability of hPMSCs to secrete IL-10 from CD4 + T cells through increased expression levels of the programmed death ligand 1 (PDL1) in hPMSCs via the Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway. In conclusion, IL-27 has significant modulatory effects on adherence, proliferation, and migration of hPMSCs. IL-27 increased PDL1 expression levels in hPMSCs via the JAK/STAT1 pathway, which then enhanced the regulatory effects of hPMSCs upon Th1 and Th2 cell generations and IL-10 secretion from CD4 + T cells.

  5. Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury

    Huiying Liu

    2016-12-01

    Full Text Available Background Acute lung injury and acute respiratory distress syndrome (ALI/ARDS is a severe clinical syndrome with mortality rate as high as 30–40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs exhibit make it possible to regulate the homeostatic control of S1P. Methods By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. Results It was found that the down-regulation of TNF-α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. Discussions The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1

  6. Regulation of S1P receptors and sphingosine kinases expression in acute pulmonary endothelial cell injury.

    Liu, Huiying; Zhang, Zili; Li, Puyuan; Yuan, Xin; Zheng, Jing; Liu, Jinwen; Bai, Changqing; Niu, Wenkai

    2016-01-01

    Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a severe clinical syndrome with mortality rate as high as 30-40%. There is no treatment yet to improve pulmonary endothelial barrier function in patients with severe pulmonary edema. Developing therapies to protect endothelial barrier integrity and stabilizing gas exchange is getting more and more attention. Sphingosine-1-phosphate (S1P) is able to enhance the resistance of endothelial cell barrier. S1P at physiological concentrations plays an important role in maintaining endothelial barrier function. Proliferation, regeneration and anti-inflammatory activity that mesenchymal stem cells (MSCs) exhibit make it possible to regulate the homeostatic control of S1P. By building a pulmonary endothelial cell model of acute injury, we investigated the regulation of S1P receptors and sphingosine kinases expression by MSCs during the treatment of acute lung injury using RT-PCR, and investigated the HPAECs Micro-electronics impedance using Real Time Cellular Analysis. It was found that the down-regulation of TNF- α expression was more significant when MSC was used in combination with S1P. The combination effection mainly worked on S1PR2, S1PR3 and SphK2. The results show that when MSCs were used in combination with S1P, the selectivity of S1P receptors was increased and the homeostatic control of S1P concentration was improved through regulation of expression of S1P metabolic enzymes. The study found that, as a potential treatment, MSCs could work on multiple S1P related genes simultaneously. When it was used in combination with S1P, the expression regulation result of related genes was not simply the superposition of each other, but more significant outcome was obtained. This study establishes the experimental basis for further exploring the efficacy of improving endothelial barrier function in acute lung injury, using MSCs in combination with S1P and their possible synergistic mechanism.

  7. Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

    Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

    2017-01-01

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

  8. Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions.

    Groveman, Bradley R; Xue, Sheng; Marin, Vedrana; Xu, Jindong; Ali, Mohammad K; Bienkiewicz, Ewa A; Yu, Xian-Min

    2011-02-01

    Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS.

  9. The Essential WalK Histidine Kinase and WalR Regulator Differentially Mediate Autolysis of Staphylococcus aureus RN4220.

    Zheng, Li; Yan, Meiying; Fan, Frank; Ji, Yinduo

    2015-06-01

    The two-component regulatory system, WalR/WalK is necessary for growth of different gram-positive bacteria, including Staphylococcus aureus . In present study, we confirmed the essentiality of both the histidine kinase protein WalK and the response regulator WalR for growth using S. aureus RN4220 strain and demonstrated that the histidine kinase protein WalK and the response regulator WalR function differently in regulation of staphylococcal autolysis. The down-regulation of walR expression effectively inhibited Triton X-100-induced lysis and had a weak impact on bacterial tolerance to penicillin induced cell lysis. In contrast, the down-regulation of walK expression had no influence on either Triton X-100- or penicillin-caused autolysis. Moreover, we determined the effect of WalR and WalK on bacterial hydrolase activity using a zymogram analysis. The results showed that the cell lysate of down-regulated walR expression mutant displayed several bands of decreased cell wall hydrolytic activities; however, the down-regulation of WalK had no dramatic impact on the hydrolytic activities. Furthermore, we examined the impact of WalR on the transcription of cidA associated with staphylococcal autolysis, and the results showed that the down-regulation of WalR led to decreased transcription of cidA in the log phase of growth. Taken together, the above results suggest that the essential WalR response regulator and the essential WalK histidine kinase might differently control bacterial lysis in RN4220 strain.

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

    Arsheed Hussain Sheikh

    2016-02-01

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

  11. Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5'-Phosphosulfate Kinase from Cyanobacteria to Plants.

    Herrmann, Jonathan; Nathin, David; Lee, Soon Goo; Sun, Tony; Jez, Joseph M

    2015-10-09

    In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

    Lishi Xie

    Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

  13. Glucose-induced serum- and glucocorticoid-regulated kinase activation in oncofetal fibronectin expression

    Khan, Zia A.; Barbin, Yousef P.; Farhangkhoee, Hana; Beier, Norbert; Scholz, Wolfgang; Chakrabarti, Subrata

    2005-01-01

    Preferential expression of oncofetal extra domain-B fibronectin (EDB + FN), a proposed angiogenic marker, has been shown in proliferative diabetic retinopathy. High levels of glucose also increase EDB + FN expression in endothelial cells (ECs) via transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). The present study was aimed at elucidating the role of serum- and glucocorticoid-regulated kinase (SGK-1) in glucose-induced EDB + FN expression. Using human macro- and microvascular ECs, we show that high levels of glucose, TGF-β1, and ET-1 increase the EDB + FN expression via SGK-1 alteration at the mRNA, protein, and activity levels. Inhibition of TGF-β1 and ET-1 prevented glucose-induced SGK-1 activation and the EDB + FN expression. Furthermore, using siRNA-mediated SGK-1 gene silencing, we show that glucose-induced EDB + FN expression can be completely prevented. These findings provide first evidence of glucose-induced SGK-1 activation in altered EDB + FN expression and provide novel avenues for therapeutic modalities

  14. Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

    Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

    2012-10-01

    Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

  15. P21-activated kinase 2 (PAK2) regulates glucose uptake and insulin sensitivity in neuronal cells.

    Varshney, Pallavi; Dey, Chinmoy Sankar

    2016-07-05

    P21-activated kinases (PAKs) are recently reported as important players of insulin signaling and glucose homeostasis in tissues like muscle, pancreas and liver. However, their role in neuronal insulin signaling is still unknown. Present study reports the involvement of PAK2 in neuronal insulin signaling, glucose uptake and insulin resistance. Irrespective of insulin sensitivity, insulin stimulation decreased PAK2 activity. PAK2 downregulation displayed marked enhancement of GLUT4 translocation with increase in glucose uptake whereas PAK2 over-expression showed its reduction. Treatment with Akti-1/2 and wortmannin suggested that Akt and PI3K are mediators of insulin effect on PAK2 and glucose uptake. Rac1 inhibition demonstrated decreased PAK2 activity while inhibition of PP2A resulted in increased PAK2 activity, with corresponding changes in glucose uptake. Taken together, present study demonstrates an inhibitory role of insulin signaling (via PI3K-Akt) and PP2A on PAK2 activity and establishes PAK2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-01-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses.

  17. Expression of phosphorylated extracellular signal-regulated kinase in rat kidneys exposed to high +Gz

    Hyun-Soo Kim

    2012-11-01

    Full Text Available Exposure to high gravitational acceleration forces acting along the body axis from the head to the feet (+Gz severely reduces blood flow to the visceral organs, including the kidneys. Extracellular signal-regulated kinase (ERK figures predominantly in mediating kidney cell responses to a wide variety of stress-related stimuli. Though previous studies have shown the activation of ERK in some experimental models, the regulation of ERK associated with +Gz exposure has not yet been investigated. The aim of this study was to examine the effect of high +Gz exposure on ERK activation in the kidneys. Using a small animal centrifuge, eight male Sprague-Dawley rats were exposed to +10Gz or +13Gz three times for 3 minutes each. The bilateral kidneys were obtained from each rat, and the expression levels of phosphorylated ERK (p-ERK were evaluated using immunohistochemistry. In the control group, the collecting duct epithelium displayed faint cytoplasmic staining with no nuclear staining of p-ERK. By contrast, rats exposed to +10Gz showed strong nuclear staining intensity for p-ERK. In the renal papilla, the epithelial cells of collecting ducts and thin segments of the loop of Henle exhibited strong nuclear immunoreactivity for p-ERK. Rats exposed to +13Gz also showed the same staining intensity and distribution of p-ERK expression as that of rats exposed to +10Gz. This study is the first to describe +Gz exposure-induced alteration in the expression of p-ERK in the kidneys. Our finding suggests that high +Gz exposure leads to the activation of ERK in the renal papilla.

  18. Effects of butyltin exposures on MAP kinase dependent transcription regulators in human natural killer cells

    Person, Rachel J.; Whalen, Margaret M.

    2010-01-01

    Natural Killer (NK) cells are a major immune defense mechanism against cancer development and viral infection. The butyltins (BTs), tributyltin (TBT) and dibutyltin (DBT) have been widely used in industrial and other applications and significantly contaminate the environment. Both TBT and DBT have been detected in human blood. These compounds inhibit the lytic and binding function of human NK cells and thus could increase the incidence of cancer and viral infections. Butyltin (BT)-induced loss of NK function is accompanied by activation of mitogen activated protein kinases (MAPKs) and decreases in expression of cell-surface and cytolytic proteins. MAPKs activate components of the transcription regulator AP-1 and activate the transcription regulator Elk-1. Based on the fact that BTs activate MAPKs and alter protein expression, the current study examined the effect of BT exposures on the levels and phosphorylation states of the components of AP-1 and the phosphorylation state of Elk-1. Exposure to 300 nM TBT for 10 min increased the phosphorylation of c-Jun in NK cells. 1 h exposures to 300 nM and 200 nM TBT increased the phosphorylation and overall level of c-Jun. During a 300 nM treatment with TBT for 1 h the binding activity of AP-1 was significantly decreased. There were no significant alterations of AP-1 components or of Elk-1 with DBT exposures. Thus, it appears that TBT-induced alterations on phosphorylation, total levels and binding activity of c-Jun might contribute to, but are not fully responsible for, TBT-induced alterations of NK protein expression. PMID:20370538

  19. Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii*

    Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H.; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D.

    2014-01-01

    In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

  20. [Mifepristone inhibites the migration of endometrial cancer cells through regulating H19 methylation].

    Lu, Z Z; Yan, L; Zhang, H; Li, M J; Zhang, X H; Zhao, X X

    2016-06-23

    To investigate the effect and mechanism of mifepristone on the migration of human endometrial carcinoma cells. A human endometrial carcinoma cell line, Ishikawa cells, was cultured in vitro and treated with mifepristone at different concentrations. Wound healing assay was applied to detect the migration of Ishikawa cells. RT-PCR and methylation-specific PCR (MSP) were used to detect the levels of H19 mRNA and its DNA methylation. Western-blot was used to detect the expressions of HMGA2 and epithelial to mesenchymal transition (EMT) related proteins. When treated with different concentrations of mifepristone for 48 hours, the width of scratch of the the control group, the 5 mg/L and the 10 mg/L mifepristone treatment groups were (4.18±0.07)mm, (4.68±0.07)mm, and(4.99±0.07)mm, respectively (Pendometrial carcinoma cells partially through methylation-induced of transcriptional inhibition of H19, which results in the down-regulation of HMGA2 and vimentin and upregulation of E-cadherin.

  1. BAG3 regulates cell proliferation, migration, and invasion in human colorectal cancer.

    Shi, Huiyong; Xu, Haidong; Li, Zengjun; Zhen, Yanan; Wang, Bin; Huo, Shoujun; Xiao, Ruixue; Xu, Zhongfa

    2016-04-01

    Bcl2-associated athanogene 3 (BAG3) has been reported to be elevated in various tumors. However, it is unclear whether BAG3 has a functional role in the initiation and progression of colorectal cancer (CRC). Here, we collected CRC samples and cell lines to validate the pathway by using gene and protein assays. RT-PCR showed that the expression of BAG3 mRNA in CRC tissues was obviously higher than that in non-tumor tissues (p BAG3 was found in most CRC tissues and strongly correlated with TNM stage (p = 0.001), differentiation (p = 0.003), and metastasis (p = 0.010). Low expression of BAG3 in HCT-8 significantly reduced cellular proliferation, migration, and invasion. The analysis of in vitro cell showed that HCT-8 cells were exposed to si-BAG3, and its growth was inhibited depending on modulation of cell cycle G1/S checkpoints and cell cycle regulators, involving cyclin D1, cyclin A2, and cyclin B1. Furthermore, suppression of the epithelial-mesenchymal transition (EMT) by si-BAG3 is linked to the decreased expression of E-cadherin and the increased expression of N-cadherin, vimentin, and MMP9. In conclusion, in the present study, we demonstrated that BAG3 overexpression plays a critical role in cell proliferation, migration, and invasion of colorectal cancer. Our data suggests targeted inhibition of BAG3 may be useful for patients with CRC.

  2. RasGRP3 regulates the migration of glioma cells via interaction with Arp3

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

    2015-01-01

    Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

  3. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  4. State and legal regulation of refugees’ and asylum seekers’ migration in EU as institutional basis of management of international migrations

    V.V. Ievdokymov

    2017-12-01

    Full Text Available According to the analysis of World Bank data on the dynamics of migration indicators in Europe and Fragile State Index data the authors determine the groups of EU countries in geo-economic risk, which forms the pushing factors in the refugee flow formation. The geographic structure of refugees’ migratory flows to the EU is analyzed and the migration corridor dynamics is determined, in particular, it is found out that the overwhelming majority of refugees arrive to the EU through the Central and Eastern Mediterranean channels. In addition, It is determined that strengthening military and political instability in Africa and the Middle East, the tight policies and institutional mechanisms for crossing the EU's borders cause the increased flows of illegal migrants entering the EU in dangerous ways, in particular through the Mediterranean Sea, where a significant number of such persons, especially women and children, die. In addition, EU countries are structured by their migration policies (views on immigration, using of quantitative restrictions on immigration including highly skilled workers. As a result of the legal document study, the authors establish the system of documents identifying the institutional frameworks for the refugees’ and asylum seekers’ migratory flow management.

  5. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  6. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  7. NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin

    Paula Schiapparelli

    2017-07-01

    Full Text Available Glioblastoma (GBM is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1 is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD. To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.

  8. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

  9. Regulation of turkey myogenic satellite cell migration by MicroRNAs miR-128 and miR-24.

    Velleman, S G; Harding, R L

    2017-06-01

    Myogenic satellite cells are an adult stem cell responsible for all post-hatch muscle growth in poultry. As a stem cell population, satellite cells are highly heterogeneous, but the origin of this heterogeneity remains unclear. Heterogeneity is, in part, regulated by gene expression. One method of endogenous gene regulation that may contribute to heterogeneity is microRNAs (miRNAs). Two miRNAs previously shown to regulate poultry myogenic satellite cell proliferation and differentiation, miR-128 and miR-24, were studied to determine if they also affected satellite cell migration. Satellite cell migration is an essential step for both proliferation and differentiation. During proliferation, satellite cells will migrate and align to form new myofibers or donate their nuclei to existing myofibers leading to muscle fiber hypertrophy or regeneration. Transient transfection of miRNA specific mimics to each miRNA reduced migration of satellite cells following a cell culture scratch at 72 h of proliferation when the cultures were 90 to 100% confluent. However, only the migration in cells transfected with miR-24 mimics at 24 and 30 h following the scratch was significantly reduced (P ≤ 0.05) to around 70% of the distance migrated by controls. Alternately, transfection with inhibitors specific to miR-128 or miR-24 significantly (P ≤ 0.05) increased migration between 147 and 252% compared to their controls between 24 and 48 h following the scratch. These data demonstrate that miR-128 and miR-24 play a role in myogenic satellite cell migration, which will impact muscle development and growth. © 2016 Poultry Science Association Inc.

  10. Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

    Ni Zhu

    2018-03-01

    Full Text Available Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI, a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1, a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a

  11. Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

    Lu, D; Yang, H; Raizada, M K

    1996-12-01

    Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

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

    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.

  13. GAR22β regulates cell migration, sperm motility, and axoneme structure.

    Gamper, Ivonne; Fleck, David; Barlin, Meltem; Spehr, Marc; El Sayad, Sara; Kleine, Henning; Maxeiner, Sebastian; Schalla, Carmen; Aydin, Gülcan; Hoss, Mareike; Litchfield, David W; Lüscher, Bernhard; Zenke, Martin; Sechi, Antonio

    2016-01-15

    Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22β(-/-) cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β(-/-) cells reduced cell motility and focal adhesion turnover. GAR22β-actin interaction was stronger than GAR22β-microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β-EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes. © 2016 Gamper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases

    Nkosi Thokozani C

    2011-07-01

    Full Text Available Abstract Background The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the

  15. Glycogen synthase kinaseregulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-01-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 β (GSK3β) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3β has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3β (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3β delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3β is required for the activation of NFAT during wound repair

  16. Ionizing Radiation Induces Cellular Senescence of Articular Chondrocytes via Negative Regulation of SIRT1 by p38 Kinase

    Hong, Eun Hee; Hwang, Sang Gu [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    Senescent cells exhibit irreversible growth arrest, large flat morphology, and up-regulated senescence-associated {beta}-galactosidase activity at pH 6.0. Several conditions, including oncogenic stress, oxidative stress, and DNA damage are associated with cellular senescence. Massive acute DNA double-strand breaks occurring as a result of mechanical and chemical stress can be repaired, but some DNA damage persists, eventually triggering premature senescence. Since ionizing radiation directly induces DBS, it is possible that cellular senescence is activated under these conditions. The biological events in chondrocytes following irradiation are poorly understood, and limited information is available on the molecular signal transduction mechanisms of cellular senescence at present. In this study, we identify SIRT1 as a target molecule of p38 kinase and demonstrate that the interactions between p38 kinase and SIRT1 protein play an important role in the regulation of cellular senescence in response to IR.

  17. GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

    Massimilano Scolz

    Full Text Available The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

  18. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    Guo, Kai, E-mail: gk161@163.com [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Department of Respiration, 161th Hospital, PLA, Wuhan 430015 (China); Jin, Faguang, E-mail: jinfag@fmmu.edu.cn [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  19. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    Guo, Kai; Jin, Faguang

    2015-01-01

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells

  20. The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1

    Dong Sook Lee

    2017-10-01

    Full Text Available Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36 and overexpressing (CRK36OE plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity.

  1. Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

    Kyle K. Biggar

    2015-04-01

    Full Text Available Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK family members to primate torpor were compared in six organs of control (aroused versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs, c-jun NH2-terminal kinases (JNKs, MAPK kinase (MEK, and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

  2. Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

    Atoui, A.; Bao, D.; Kaur, N.; Grayburn, W.S.; Calvo, A.M.

    2008-01-01

    The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

  3. RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

    Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

    2017-08-01

    Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

  4. ERK-dependent and -independent pathways trigger human neural progenitor cell migration

    Moors, Michaela; Cline, Jason E.; Abel, Josef; Fritsche, Ellen

    2007-01-01

    Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways

  5. Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

    Go Sakai

    2017-11-01

    Full Text Available Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β stimulates the synthesis of vascular endothelial growth factor (VEGF through the activation of p38 mitogen-activated protein (MAP kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70 is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

  6. Theobromine, the primary methylxanthine found in Theobroma cacao, prevents malignant glioblastoma proliferation by negatively regulating phosphodiesterase-4, extracellular signal-regulated kinase, Akt/mammalian target of rapamycin kinase, and nuclear factor-kappa B.

    Sugimoto, Naotoshi; Miwa, Shinji; Hitomi, Yoshiaki; Nakamura, Hiroyuki; Tsuchiya, Hiroyuki; Yachie, Akihiro

    2014-01-01

    Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that methylxanthines, including caffeine and theophylline, have antitumor and antiinflammatory effects, which are in part mediated by their inhibition of phosphodiesterase (PDE). A member of the PDE family, PDE4, is widely expressed in and promotes the growth of glioblastoma, the most common type of brain tumor. The purpose of this study was to determine whether theobromine could exert growth inhibitory effects on U87-MG, a cell line derived from human malignant glioma. We show that theobromine treatment elevates intracellular cAMP levels and increases the activity of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, whereas it attenuates p44/42 extracellular signal-regulated kinase activity and the Akt/mammalian target of rapamycin kinase and nuclear factor-kappa B signal pathways. It also inhibits cell proliferation. These results suggest that foods and beverages containing cocoa bean extracts, including theobromine, might be extremely effective in preventing human glioblastoma.

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

    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...

  8. Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

    Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

    2016-10-01

    Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

  9. RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.

    Edwards, Holly; Xie, Chengzhi; LaFiura, Katherine M; Dombkowski, Alan A; Buck, Steven A; Boerner, Julie L; Taub, Jeffrey W; Matherly, Larry H; Ge, Yubin

    2009-09-24

    RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

  10. The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

    Jennifer Pöhlmann

    2014-09-01

    Full Text Available Microtubules (MTs are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

  11. Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration.

    Somogyi, Kálmán; Rørth, Pernille

    2004-07-01

    Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. We report that this migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and present evidence that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration.

  12. Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase, extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun N-terminal kinase downregulation in obese rats.

    Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal

    2015-02-01

    Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.

  13. The Tec kinase ITK regulates thymic expansion, emigration, and maturation of γδ NKT cells.

    Yin, Catherine C; Cho, Ok Hyun; Sylvia, Katelyn E; Narayan, Kavitha; Prince, Amanda L; Evans, John W; Kang, Joonsoo; Berg, Leslie J

    2013-03-15

    The Tec family tyrosine kinase, Itk, regulates signaling downstream of the TCR. The absence of Itk in CD4(+) T cells results in impaired Th2 responses along with defects in maturation, cytokine production, and survival of iNKT cells. Paradoxically, Itk(-/-) mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vγ1.1(+)Vδ6.3(+) subset of γδ T cells, known as γδ NKT cells. Comparisons between γδ NKT cells and αβ iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic γδ NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant γδ NKT cells plus a heterogeneous population of Vγ1.1(+)Vδ6.3(+) T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk(-/-) γδ NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally downregulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of γδ NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation.

  14. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

    Emily P Thi

    Full Text Available Of the various phosphatidylinositol 3- kinases (PI3Ks, only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5P3; however, p110α and PI(3,4,5P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP and homotypic vacuole fusion and protein sorting (HOPs components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

  16. ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase).

    Andrade, Erika C; Musante, Veronica; Horiuchi, Atsuko; Matsuzaki, Hideo; Brody, A Harrison; Wu, Terence; Greengard, Paul; Taylor, Jane R; Nairn, Angus C

    2017-03-08

    ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We

  17. Focal adhesion kinase-mediated activation of glycogen synthase kinaseregulates IL-33 receptor internalization and IL-33 signaling.

    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.

  18. β-Catenin–regulated myeloid cell adhesion and migration determine wound healing

    Amini-Nik, Saeid; Cambridge, Elizabeth; Yu, Winston; Guo, Anne; Whetstone, Heather; Nadesan, Puviindran; Poon, Raymond; Hinz, Boris; Alman, Benjamin A.

    2014-01-01

    A β-catenin/T cell factor–dependent transcriptional program is critical during cutaneous wound repair for the regulation of scar size; however, the relative contribution of β-catenin activity and function in specific cell types in the granulation tissue during the healing process is unknown. Here, cell lineage tracing revealed that cells in which β-catenin is transcriptionally active express a gene profile that is characteristic of the myeloid lineage. Mice harboring a macrophage-specific deletion of the gene encoding β-catenin exhibited insufficient skin wound healing due to macrophage-specific defects in migration, adhesion to fibroblasts, and ability to produce TGF-β1. In irradiated mice, only macrophages expressing β-catenin were able to rescue wound-healing deficiency. Evaluation of scar tissue collected from patients with hypertrophic and normal scars revealed a correlation between the number of macrophages within the wound, β-catenin levels, and cellularity. Our data indicate that β-catenin regulates myeloid cell motility and adhesion and that β-catenin–mediated macrophage motility contributes to the number of mesenchymal cells and ultimate scar size following cutaneous injury. PMID:24837430

  19. Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.

    Choi, Yun-Hee; McNally, Brian T; Igarashi, Peter

    2013-07-01

    Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

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

    Los, Alrik Pieter

    2007-01-01

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

  1. The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals.

    Su, Y C; Maurel-Zaffran, C; Treisman, J E; Skolnik, E Y

    2000-07-01

    We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct

  2. Adenovirus Protein E4-ORF1 activation of PI3 kinase reveals differential regulation of downstream effector pathways in adipocytes

    Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K.; McGraw, Timothy E.

    2016-01-01

    Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but...

  3. The Amoebal MAP Kinase Response to Legionella pneumophila Is Regulated by DupA

    Li, Zhiru; Dugan, Aisling S.; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R.

    2009-01-01

    The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type...

  4. Extracellular signal-regulated kinase activation is required for consolidation and reconsolidation of memory at an early stage of ontogenesis.

    Languille, Solène; Davis, Sabrina; Richer, Paulette; Alcacer, Cristina; Laroche, Serge; Hars, Bernard

    2009-11-01

    The ability to form long-term memories exists very early during ontogeny; however, the properties of early memory processes, brain structures involved and underlying cellular mechanisms are poorly defined. Here, we examine the role of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase/ERK signaling cascade, which is crucial for adult memory, in the consolidation and reconsolidation of an early memory using a conditioned taste aversion paradigm in 3-day-old rat pups. We show that intraperitoneal injection of SL327, the upstream mitogen-activated protein kinase kinase inhibitor, impairs both consolidation and reconsolidation of early memory, leaving short-term memory after acquisition and after reactivation intact. The amnesic effect of SL327 diminishes with increasing delays after acquisition and reactivation. Biochemical analyses revealed ERK hyperphosphorylation in the amygdala but not the hippocampus following acquisition, suggesting functional activation of the amygdala as early as post-natal day 3, although there was no clear evidence for amygdalar ERK activation after reactivation. These results indicate that, despite an immature brain, the basic properties of memory and at least some of the molecular mechanisms and brain structures implicated in aversion memory share a number of similarities with the adult and emerge very early during ontogeny.

  5. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1

    Hagit Bar-Yosef

    2018-02-01

    Full Text Available The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  6. A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

    Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A; Kornitzer, Daniel

    2018-01-01

    The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

  7. AMP-Activated Protein Kinase (AMPK) Regulates Energy Metabolism through Modulating Thermogenesis in Adipose Tissue

    Wu, Lingyan; Zhang, Lina; Li, Bohan; Jiang, Haowen; Duan, Yanan; Xie, Zhifu; Shuai, Lin; Li, Jia; Li, Jingya

    2018-01-01

    Obesity occurs when excess energy accumulates in white adipose tissue (WAT), whereas brown adipose tissue (BAT), which is specialized in dissipating energy through thermogenesis, potently counteracts obesity. White adipocytes can be converted to thermogenic “brown-like” cells (beige cells; WAT browning) under various stimuli, such as cold exposure. AMP-activated protein kinase (AMPK) is a crucial energy sensor that regulates energy metabolism in multiple tissues. However, the role of AMPK in adipose tissue function, especially in the WAT browning process, is not fully understood. To illuminate the effect of adipocyte AMPK on energy metabolism, we generated Adiponectin-Cre-driven adipose tissue-specific AMPK α1/α2 KO mice (AKO). These AKO mice were cold intolerant and their inguinal WAT displayed impaired mitochondrial integrity and biogenesis, and reduced expression of thermogenic markers upon cold exposure. High-fat-diet (HFD)-fed AKO mice exhibited increased adiposity and exacerbated hepatic steatosis and fibrosis and impaired glucose tolerance and insulin sensitivity. Meanwhile, energy expenditure and oxygen consumption were markedly decreased in the AKO mice both in basal conditions and after stimulation with a β3-adrenergic receptor agonist, CL 316,243. In contrast, we found that in HFD-fed obese mouse model, chronic AMPK activation by A-769662 protected against obesity and related metabolic dysfunction. A-769662 alleviated HFD-induced glucose intolerance and reduced body weight gain and WAT expansion. Notably, A-769662 increased energy expenditure and cold tolerance in HFD-fed mice. A-769662 treatment also induced the browning process in the inguinal fat depot of HFD-fed mice. Likewise, A-769662 enhanced thermogenesis in differentiated inguinal stromal vascular fraction (SVF) cells via AMPK signaling pathway. In summary, a lack of adipocyte AMPKα induced thermogenic impairment and obesity in response to cold and nutrient-overload, respectively

  8. Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

    Christian Schmidt-Lauber

    Full Text Available BACKGROUND: Tyrosine kinase inhibitors (TKIs are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF. Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein. CONCLUSION/SIGNIFICANCE: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important

  9. Altered CXCR3 isoform expression regulates prostate cancer cell migration and invasion

    Wu Qian

    2012-01-01

    Full Text Available Abstract Background Carcinoma cells must circumvent the normally suppressive signals to disseminate. While often considered 'stop' signals for adherent cells, CXCR3-binding chemokines have recently been correlated positively with cancer progression though the molecular basis remains unclear. Results Here, we examined the expression and function of two CXCR3 variants in human prostate cancer biopsies and cell lines. Globally, both CXCR3 mRNA and protein were elevated in localized and metastatic human cancer biopsies compared to normal. Additionally, CXCR3A mRNA level was upregulated while CXCR3B mRNA was downregulated in these prostate cancer specimens. In contrast to normal prostate epithelial cells (RWPE-1, CXCR3A was up to half the receptor in the invasive and metastatic DU-145 and PC-3 prostate cancer cells, but not in the localized LNCaP cells. Instead of inhibiting cell migration as in RWPE-1 cells, the CXCR3 ligands CXCL4/PF4 and CXCL10/IP10 promoted cell motility and invasiveness in both DU-145 and PC-3 cells via PLCβ3 and μ-calpain activation. CXCR3-mediated diminution of cell motility in RWPE-1 cells is likely a result of cAMP upregulation and m-calpain inhibition via CXCR3B signal transduction. Interestingly, overexpression of CXCR3B in DU-145 cells decreased cell movement and invasion. Conclusion These data suggest that the aberrant expression of CXCR3A and down-regulation of CXCR3B may switch a progression "stop" to a "go" signal to promote prostate tumor metastasis via stimulating cell migration and invasion.

  10. Regulation of EphA4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function

    Egea, Joaquim; Nissen, Ulla Vig; Dufour, Audrey

    2005-01-01

    Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also...... requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4 EE), whose kinase is constitutively activated in the absence of ephrinB ligands......, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4EE, including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation...

  11. Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis.

    Sanja Coso

    Full Text Available BACKGROUND: Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF family is a major regulator of lymphatic endothelial cell (LEC function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. METHODS AND RESULTS: Here we delineate the VEGF-C/VEGF receptor (VEGFR-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCγ1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. CONCLUSIONS: Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

  12. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  13. Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation.

    Hara, Masatoshi; Petrova, Boryana; Orr-Weaver, Terry L

    2017-05-30

    The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity we show here to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition.

  14. The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway.

    Kim, Hye-Sook; Willett, Jonathan W; Jain-Gupta, Neeta; Fiebig, Aretha; Crosson, Sean

    2014-11-01

    In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long-term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin-binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long-term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N-terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology. © 2014 John Wiley & Sons Ltd.

  15. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    Mili Jeon

    2012-04-01

    The respiratory (tracheal system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs, Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr tyrosine kinase (TK. Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  16. Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

    Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

    2013-06-01

    In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

  17. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    Jin, Yonghui; Kato, Tomohisa; Furu, Moritoshi; Nasu, Akira; Kajita, Yoichiro; Mitsui, Hiroto; Ueda, Michiko; Aoyama, Tomoki; Nakayama, Tomitaka; Nakamura, Takashi; Toguchida, Junya

    2010-01-01

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO 2 ) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO 2 ) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  18. The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

    Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

    2009-02-01

    The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

  19. A sensor kinase recognizing the cell-cell signal BDSF (cis-2-dodecenoic acid) regulates virulence in Burkholderia cenocepacia

    McCarthy, Y.; Yang, Liang; Twomey, K.B.

    2010-01-01

    Xanthomonas campestris. The mechanism of perception of this signal and the range of functions regulated in B. cenocepacia are, however, unknown. A screen for transposon mutants unable to respond to exogenous signal identified BCAM0227 as a potential BDSF sensor. BCAM0227 is a histidine sensor kinase...... with an input domain unrelated to that of RpfC, the DSF sensor found in xanthomonads. Transcriptome profiling established the scope of the BDSF regulon and demonstrated that the sensor controls expression of a subset of these genes. A chimeric sensor kinase in which the input domain of BCAM0227 replaced...... the input domain of RpfC was active in BDSF signal perception when expressed in X. campestris. Mutation of BCAM0227 gave rise to reduced cytotoxicity to Chinese hamster ovary cells and reduced virulence to Wax moth larvae and in the agar-bead mouse model of pulmonary infection. The findings identify BCAM...

  20. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.