Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytokine interleukin-1beta (IL-1beta) is critical to the formation of an astrocytic scar after CNS injury, but the mechanisms by which it induces a reactive phenotype remain unresolved. Here, we show that IL-1beta regulates the phenotype of astrocytes via deactivation of the Rho GTPase-Rho kinase (ROCK) pathway, which governs cellular morphology and migration via effects on F-actin and its interactions with focal adhesions, nonmuscle myosin, and microvillar adapter proteins of the ezrin-radixin-moesin (ERM) family. We found that IL-1beta induced cortical reorganization of F-actin and dephosphorylation of focal adhesion kinase, myosin light chain 2, and myosin phosphatase targeting subunit 1 in primary human astrocytes, and that all of these effects were mimicked by Rho-ROCK pathway blockade. We also found that IL-1beta conversely potentiated ERM phosphorylation, and that this effect was mediated via a Rho-ROCK-independent mechanism. Next, we used a rhotekin pulldown assay to confirm directly that IL-1beta deactivates Rho, and further demonstrated that a constitutively active Rho construct rescued astrocytes from developing an IL-1beta-induced reactive phenotype. These data implicate cytokine regulation of the Rho-ROCK pathway in the generation of a reactive astrogliosis, and we suggest that interventions targeted at this level may facilitate manipulation of the glial scar in inflammatory disorders of the human CNS.
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PMID:Interleukin-1beta induces a reactive astroglial phenotype via deactivation of the Rho GTPase-Rock axis. 1502 78

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) envelope glycoprotein gB possesses an RGD motif, interacts with alpha 3 beta 1 integrin, and uses it as one of the entry receptors. HHV-8 induces the integrin-dependent focal adhesion kinase (FAK), a critical step in the outside-in signaling pathways necessary for the subsequent phosphorylation of other cellular kinases, cytoskeletal rearrangements, and other functions. As an initial step toward deciphering the role of HHV-8 gB-integrin interaction in infection, signal pathways induced by gB were examined. A truncated form of gB without the transmembrane and carboxyl domains (gB Delta TM), a gB Delta TM mutant form (gB Delta TM-RGA) with an RGD-to-RGA mutation, and inhibitors of cellular kinases were used. HHV-8 gB Delta TM, but not gB Delta TM-RGA, induced FAK phosphorylation in target cells, which was in part dependent on the presence of alpha 3 beta 1 integrin. FAK was critical for the subsequent phosphorylation of Src by gB Delta TM, and Src induction was essential for the phosphorylation of phosphatidylinositol 3-kinase (PI-3K). HHV-8 gB Delta TM-induced PI-3K was essential for the induction of RhoA and Cdc42 Rho GTPases that was accompanied by the cytoskeletal rearrangements. These gB-induced morphological changes were inhibited by the PI-3K inhibitors. Ezrin, one of the essential elements required to cross-link the actin cytoskeleton with the plasma membrane and to induce the morphological changes, was induced by the Rho GTPases. Inhibition of cellular tyrosine kinases by the brief treatment of cells with 4',5,7-trihydroxyisoflavone (genistein) blocked the entry of HHV-8 into target cells. These findings suggest that, independently of other viral glycoproteins and via its RGD motif, HHV-8 gB induces integrin-dependent pre-existing FAK-Src-PI-3K-Rho GTPase kinases. Since these signal pathways play vital roles in host cell endocytosis and movement of particulate materials in the cytoplasm, the early stages of HHV-8 gB interaction with host cells may provide a very conducive environment for the successful infection of target cells.
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PMID:Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 envelope glycoprotein gB induces the integrin-dependent focal adhesion kinase-Src-phosphatidylinositol 3-kinase-rho GTPase signal pathways and cytoskeletal rearrangements. 1504 36

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.
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PMID:The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation. 1514 25

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5'-(beta,gamma-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.
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PMID:Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1. 1530 21

Chronic rejection is the major limitation to long-term allograft survival. HLA class I signaling pathways have been implicated in this process because ligation of class I molecules by anti-HLA antibodies (Ab) initiates intracellular signals in smooth muscle cells (SMC) and endothelial cells (EC) that synergize with growth factor receptors to elicit cell survival and proliferation. Anti-HLA Ab mediate cell proliferation and survival through a focal adhesion kinase dependent pathway that requires the integrity of the actin cytoskeleton. In this study, we investigated the role of Rho and Rho-kinase (ROK) in class I signal transduction. We show that class I ligation results in activation of Rho and increased stress fiber formation. In addition, inhibitors of Rho GTPase and ROK block HLA class I-mediated tyrosyl phosphorylation of paxillin and FAK, central elements of the focal adhesion signaling complex. These results suggest that HLA class I-induced signaling in EC is dependent on Rho GTPase and ROK.
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PMID:HLA class I signal transduction is dependent on Rho GTPase and ROK. 1535 23

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-gamma-induced inflammatory responses in rat astrocytes. We revealed that IFN-gamma rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-gamma-responsive transcriptional responses. Compared with levels in control cells, IFN-gamma-induced IFN-gamma-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-gamma-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-gamma-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-gammaRalpha subunit and IFN-gamma-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-gammaRalpha and augment the interaction of IFN-gammaR with either STAT1 or STAT3 in response to IFN-gamma. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-gamma-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-gamma-responsive inflammatory signaling in rat astrocytes.
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PMID:Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes. 1549 21

Many leukemic oncogenes form as a consequence of gene fusions or mutation that result in the activation or overexpression of a tyrosine kinase. To identify commonalities and differences in the action of two such kinases, breakpoint cluster region (BCR)/ABL and TEL/PDGFRbeta, two-dimensional gel electrophoresis was employed to characterize their effects on the proteome. While both oncogenes affected expression of specific proteins, few common effects were observed. A number of proteins whose expression is altered by BCR/ABL, including gelsolin and stathmin, are related to cytoskeletal function whereas no such changes were seen in TEL/PDGFRbeta-transfected cells. Treatment of cells with the kinase inhibitor STI571 for 4-h reversed changes in expression of some of these cytoskeletal proteins. Correspondingly, BCR/ABL-transfected cells were less responsive to chemotactic and chemokinetic stimuli than non-transfected cells and TEL/PDGFRbeta-transfected Ba/F3 cells. Decreased motile response was reversed by a 16-h treatment with STI571. A phosphoprotein-specific gel stain was used to identify TEL/PDGFRbeta and BCR/ABL-mediated changes in the phosphoproteome. These included changes on Crkl, Ras-GAP-binding protein 1, and for BCR/ABL, cytoskeletal proteins such as tubulin, and Nedd5. Decreased phosphorylation of Rho-GTPase dissociation inhibitor (Rho GDI) was also observed in BCR/ABL-transfected cells. This results in the activation of the Rho pathway, and treatment of cells with Y27632, an inhibitor of Rho kinase, inhibited DNA synthesis in BCR/ABL-transfected Ba/F3 cells but not TEL/PDGFRbeta-expressing cells. Expression of a dominant-negative RhoA inhibited both DNA synthesis and transwell migration, demonstrating the significance of this pathway in BCR/ABL-mediated transformation.
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PMID:Global effects of BCR/ABL and TEL/PDGFRbeta expression on the proteome and phosphoproteome: identification of the Rho pathway as a target of BCR/ABL. 1556 70

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.
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PMID:Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling. 1563 62

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.
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PMID:Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin. 1580 46

Our previous studies demonstrated that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of both growth factor- and integrin-dependent sustained ERK MAPK activation, cyclin D1 expression, and cell cycle progression. We now report that MIF promotes the activation of the canonical ERK MAPK cascade and cyclin D1 expression by stimulating the activity of the Rho GTPase and downstream signaling to stress fiber formation. Rho-dependent stress fiber accumulation promotes the sustained activation of ERK and subsequent cyclin D1 expression during G(1)-S phase cell cycle progression. This pathway is reported to be dependent upon myosin light chain (MLC) kinase, integrin clustering, and subsequent activation of focal adhesion kinase, leading to sustained MAPK activity. Our studies reveal that recombinant MIF induces cyclin D1 expression in a Rho-, Rho kinase-, MLC kinase-, and ERK-dependent manner in asynchronous NIH 3T3 fibroblasts. Moreover, MIF(-/-) murine embryonic fibroblasts display aberrant cyclin D1 expression that is linked to defective Rho activity, stress fiber formation, and MLC phosphorylation. These results suggest that MIF is an integral autocrine mediator of Rho GTPase-dependent signaling events and provide mechanistic insight into how MIF regulates proliferative, migratory, and oncogenic processes.
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PMID:Rho GTPase-dependent signaling is required for macrophage migration inhibitory factor-mediated expression of cyclin D1. 1584 May 82


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