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)

Ca(2+) is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs). A role for intracellular Ca(2+) in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 and by Ca(2+)/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF; however, there is no Ca(2+)-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca(2+)-promoted Ras inactivator), a Ca(2+)-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca(2+). Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca(2+) regulates the GAP by a fast C2 domain-dependent translocation mechanism.
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PMID:CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway. 1144 76

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.
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PMID:The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly. 1205 76

The small G protein RAP1 and the kinase B-RAF have been proposed to link elevations of cAMP to activation of ERK/mitogen-activated protein (MAP) kinase. In order to delineate signaling pathways that link receptor-generated cAMP to the activation of MAP kinase, the human A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, was heterologously expressed in Chinese hamster ovary cells (referred as CHO-A(2A) cells). In CHO-A(2A) cells, the stimulation of the A(2A)-receptor resulted in an activation of RAP1 and formation of RAP1-B-RAF complexes. However, overexpression of a RAP1 GTPase-activating protein (RAP1GAP), which efficiently clamped cellular RAP1 in the inactive GDP-bound form, did not affect A(2A)-agonist-mediated MAP kinase stimulation. In contrast, the inhibitor of protein kinase A H89 efficiently suppressed A(2A)-agonist-mediated MAP kinase stimulation. Neither dynamin-dependent receptor internalization nor receptor-promoted shedding of matrix-bound growth factors accounted for A(2A)-receptor-dependent MAP kinase activation. PP1, an inhibitor of SRC family kinases, blunted both the A(2A)-receptor- and the forskolin-induced MAP kinase stimulation (IC(50) = 50 nm); this was also seen in PC12 cells, which express the A(2A)-receptor endogenously, and in NIH3T3 fibroblasts, in which cAMP causes MAP kinase stimulation. In the corresponding murine fibroblast cell line SYF, which lacks the ubiquitously expressed SRC family kinases SRC, YES, and FYN, forskolin barely stimulated MAP kinase; this reduction was reversed in cells in which c-SRC had been reintroduced. These findings show that activation of MAP kinase by cAMP requires a SRC family kinase that lies downstream of protein kinase A. A role for RAP1, as documented for the beta(2)-adrenergic receptor, is apparently contingent on receptor endocytosis.
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PMID:MAP kinase stimulation by cAMP does not require RAP1 but SRC family kinases. 1208 90

The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (approximately 150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including betaPIX, focal adhesion kinase, liprin-alpha, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, betaPIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and betaPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.
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PMID:The GIT family of proteins forms multimers and associates with the presynaptic cytomatrix protein Piccolo. 1247 61

Leupaxin is a cytoskeleton adaptor protein that was first identified in human macrophages and was found to share homology with the focal adhesion protein, paxillin. Leupaxin possesses several protein-binding domains that have been implicated in targeting proteins such as focal adhesion kinase (pp125FAK) to focal adhesions. Leupaxin can be detected in monocytes and osteoclasts, both cells of hematopoietic origin. We have identified leupaxin to be a component of the osteoclast podosomal signaling complex. We have found that leupaxin in murine osteoclasts is associated with both PYK2 and pp125FAK in the osteoclast. Treatment of osteoclasts with TNF-alpha and soluble osteopontin were found to stimulate tyrosine phosphorylation of both leupaxin and leupaxin-associated PYK2. Leupaxin was found to co-immunoprecipitate with the protein tyrosine phosphatase PTP-PEST. The cellular distribution of leupaxin, PYK2, and protein tyrosine phosphorylation-PEST co-localized at or near the osteoclast podosomal complex. Leupaxin was also found to associate with the ARF-GTPase-activating protein, paxillin kinase linker p95PKL, thereby providing a link to regulators of cytoskeletal dynamics in the osteoclast. Overexpression of leupaxin by transduction into osteoclasts evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of leupaxin expression in the osteoclast led to a decrease in resorptive capacity. Our data suggest that leupaxin may be a critical nucleating component of the osteoclast podosomal signaling complex.
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PMID:Leupaxin is a critical adaptor protein in the adhesion zone of the osteoclast. 1267 28

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.
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PMID:The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1. 1277 Nov 46

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

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i), Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.
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PMID:CXC chemokine ligand 12-induced focal adhesion kinase activation and segregation into membrane domains is modulated by regulator of G protein signaling 1 in pro-B cells. 1572 64

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

Protein kinase B (PKB or Akt) plays an essential role in the actions of insulin, cytokines, and growth factors, although the substrates for PKB that are relevant to many of its actions require identification. In this study, we have reported the identification of p122RhoGAP, a GTPase-activating protein selective for RhoA and rodent homologue of the tumor suppressor deleted in liver cancer (DLC1) as a novel insulin-stimulated phosphoprotein in primary rat adipocytes. We have demonstrated that Ser-322 is phosphorylated upon insulin stimulation of intact cells and that this site is directly phosphorylated in vitro by PKB and ribosomal S6 kinase, members of the AGC (protein kinases A, G, and C) family of insulin-stimulated protein kinases. Furthermore, expression of constitutively active mutants of PKB or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) stimulates Ser-322 phosphorylation in intact cells, demonstrating that activation of the PKB or MEK pathway is sufficient for Ser-322 phosphorylation in vivo. Indeed, in primary adipocytes, insulin-stimulated Ser-322 phosphorylation was almost exclusively regulated by the phosphatidylinositol 3-kinase/PKB pathway, whereas in immortalized cells, insulin-stimulated phosphorylation was predominantly regulated by the MEK/extracellular signal-regulated kinase/ribosomal S6 kinase pathway, with the phosphatidylinositol 3-kinase/PKB pathway playing a minor role. These results demonstrate that p122RhoGAP Ser-322 acts as an integrator of signal transduction in a manner dependent on the cellular context.
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PMID:Identification of p122RhoGAP (deleted in liver cancer-1) Serine 322 as a substrate for protein kinase B and ribosomal S6 kinase in insulin-stimulated cells. 1633 27


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