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)

Platelet integrin alphaIIbbeta3 must be activated via intracellular mechanisms before it binds soluble ligands, and it is thought to be activated at its extracellular site by surface-bound ligands. Integrin activation is associated with rearrangement of the cytoskeleton and phosphorylation of proteins that become localized in focal contacts. In these processes, the cytoplasmic tail of the beta-subunit plays a central role. We introduced peptides homologous to the E749ATSTFTN756 domain (E-N peptide) and the T755NITYRGT762 domain (T-T peptide) of beta3 in streptolysin O-permeabilized platelets and analyzed the initial interaction with soluble fibronectin, fibrinogen and PAC-1 after stimulation with thrombin. E-N peptide left the initial binding of fibronectin intact but interfered with stable receptor occupancy. E-N peptide also inhibited fibrinogen binding, thereby reducing the formation of large aggregates. Strikingly, E-N peptide did not disturb the binding of PAC-1, which is known to reflect activation of the integrin. E-N peptide also inhibited tyrosine phosphorylation of focal adhesion kinase, a response known to be dependent on alphaIIbbeta3. T-T peptide did not affect these processes. In a model for outside-in integrin activation, E-N peptide disrupted the binding of CHO cells expressing alphaIIbbeta3 to surface-bound ligand. Again, T-T peptide had no effect. We conclude that the E749ATSTFTN756 region of the beta3-tail stabilizes the binding of soluble and surface-bound ligand to integrin alphaIIbbeta3 via a mechanism that involves the phosphorylation of FAK.
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PMID:Involvement of the beta3 E749ATSTFTN756 region in stabilizing integrin alphaIIbbeta3-ligand interaction. 1452 7

The leflunomide metabolite analog alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) is a rationally-designed specific inhibitor of the TEC family protein tyrosine kinase, Bruton's tyrosine kinase (BTK) which plays an important role in platelet physiology by regulating the glycoprotein GPVI-FcRgamma-coupled collagen receptor signaling pathway. At low micromolar concentrations, LFM-A13 inhibited collagen-induced ultrastructural changes indicative of activation. LFM-A13 inhibited collagen (but not thrombin, TRAP-6, or ADP)-induced platelet aggregation in a concentration-dependent fashion with an IC50 value of 2.8 microM. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100 mg/kg. At nontoxic dose levels, LFM-A13 prolonged the tail bleeding times of mice and improved event-free survival in two mouse models of agonist-induced invariably fatal pulmonary thromboembolism. To our knowledge, LFM-A13 is the first anti-thrombotic agent which prevents platelet aggregation by inhibiting BTK.
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PMID:The anti-leukemic Bruton's tyrosine kinase inhibitor alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl) propenamide (LFM-A13) prevents fatal thromboembolism. 1456 61

Sphingosine-1 phosphate (S1P) and thrombin are agents with profound but divergent effects on vascular endothelial cell (EC) barrier properties. We have previously reported that S1P-induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G-protein-coupled receptor kinase-interacting proteins GIT1 and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) and site-specific FAK tyrosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S1P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S1P and thrombin induced distinct patterns of FAK site-specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src-specific inhibitor PP2 abolished S1P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter thrombin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phosphorylation. These observations highlight the differential mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.
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PMID:Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion remodeling: role of Src and GIT. 1465 86

Tissue factor (TF), apart from activating the extrinsic pathway of the blood coagulation, is a principal regulator of embryonic and oncogenic angiogenesis, inflammation, leukocyte reverse transmigration, and tumor progression. It has become clear that these events are mediated by intracellular signal transduction elicited by TF/factor VIIa (FVIIa) interaction, but the details of this signaling remain largely obscure. In this study, we show that FVIIa/TF-interaction produces STAT5 phosphorylation, STAT5 nuclear translocation and transactivation of a STAT5 reporter construct. FVIIa-dependent STAT5 activation was dependent on FVIIa proteolytic activity but not on generation of the downstream coagulation factors Xa and thrombin, nor on the TF cytoplasmic domain. FVIIa-induced STAT5 phosphorylation was dependent on functional G12/G13 class G proteins and Jak2 activity, but not Jak1 or Tyk2. Finally, we show that FVIIa leads to cell survival through a Jak2/STAT5-dependent production of the antiapoptotic STAT5 target Bcl(XL) as well as Jak2-dependent activation of the antiapoptotic protein PKB. In conclusion, our results show that FVIIa induces cell survival through STAT5-dependent Bcl(XL) production and Jak2-dependent activation of PKB. Finally, we demonstrated for the first time that TF/FVIIa-signal transduction is dependent on G12/G13 class G proteins.
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PMID:FVIIa:TF induces cell survival via G12/G13-dependent Jak/STAT activation and BclXL production. 1501 32

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.
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PMID:GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions. 1501 33

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.
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PMID:The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin. 1507 Sep

Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000. J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokine-induced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI-P97 and WHI-P154) had little effect on thrombin-induced CIS expression. In contrast, cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA(2), cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N-acetyl-cysteine [NAC] and trolox) reduced thrombin-induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE(2) and LTB(4) generated by COX and LO, mediate CIS expression. Since interferon-gamma (IFN-gamma)-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS-transfected cells compared to control vector-transfected cells, CIS could have anti-inflammatory activity. These data suggest that thrombin-stimulation of ROS and prostaglandin and leukotriene production via the cPLA(2), COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.
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PMID:Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase, and lipoxygenase. 1537 59

In a previous report we have presented evidence that thrombin interacts with alpha(v)beta(3) integrin in endothelial cells at the molecular and cellular level. This interaction was shown to be of functional significance in vitro and in vivo and contributed to activation of angiogenesis by thrombin. In the present study, we have used a synthetic thrombin peptide, TP508, which represents residues 183 to 200 of human thrombin. This peptide lacks the catalytic site of thrombin but contains the thrombin RGD sequence. Immobilized (surface-coated) TP508 peptide, like thrombin, supported alpha(v)beta(3) integrin-dependent endothelial cell attachment and haptotactic migration. These effects were specific (a scrambled TP508 peptide was without effect), and dosedependent. The RGD sequence was essential since a modified TP508 peptide, which contained RAD sequence instead of RGD, was inactive. Immobilized TP508 peptide stimulated phosphorylation of mitogen-activated protein kinases and focal adhesion kinase, the signal transduction pathways characteristic for integrin activation. On the other hand, TP508 peptide, when in solution, did not mimic other thrombin-promoted angiogenic effects, such as that of activation gelatinase A, upregulation of expression of vascular endothelial growth factor receptor mRNA or prostacyclin PGI(2) release in endothelial cells. On the contrary, soluble TP508 acted as an antagonist for the aforementioned effects of thrombin. TP508 peptide inhibited these thrombin-induced effects through a RGD and alpha(v)beta(3)-related mechanism. The antagonism with thrombin or thrombin receptor activating peptide was specific and involved at least in part mitogen-activated protein kinases activation. These results point to the importance of RGD sequence of thrombin in mediating effects on endothelial cells and angiogenesis.
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PMID:On the mode of action of thrombin-induced angiogenesis: thrombin peptide, TP508, mediates effects in endothelial cells via alphavbeta3 integrin. 1546 17

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by beta(1)-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125(FAK) (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin alpha(2)beta(1) receptor on collagen production, prolidase activity, expression of prolidase, beta(1)-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK(1) and ERK(2)) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK(1) and ERK(2). An opposite phenomenon was observed in fibroblasts treated with 0.1IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve beta(1)-integrin-dependent signaling.
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PMID:Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in beta1-integrin-mediated signaling. 1566 71

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins can form lateral associations with integrins and may act as 'organizers' of multimolecular networks that modulate integrinmediated signaling, cell morphology, motility and migration. In resting platelets, CD63 is present on the membranes of dense granules and lysosomes but relocates to the plasma membrane following platelet activation and exocytosis where it associates with the platelet integrin alphaIIBbeta3-CD9 complex and with the actin cytoskeleton in an alphaIIBbeta 3-dependent manner. D545, a monoclonal antibody directed at the second extracellular loop of CD63,was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation. Using immunofluorescence microscopy and confocal imaging, we have demonstrated that D545 does not alter adhesion of platelets to immobilized fibrinogen, but instead platelet spreading. In the presence of buffer or non-specific mouse IgG, activated platelets showed fully spread morphology, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. D545 also inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate alphaIIBbeta3-dependent cytoskeletal reorganization. To identify signaling enzymes associated with CD63 that could affect this pathway, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase which, on the basis of enzymatic properties(stimulated by nonionic detergents, inhibited by adenosine), is consistent with PI 4-kinase type II. The CD63-PI 4-kinase complex was not activation-dependent as the constituents were co-purified from both resting and activated platelets. The linkage of CD63 with PI 4-kinase may result in the recruitment of this signaling enzyme to specific membrane locations in the platelet where it influences phosphoinositide-dependent signaling and platelet spreading.
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PMID:CD63 modulates spreading and tyrosine phosphorylation of platelets on immobilized fibrinogen. 1571 48


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