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 principal substrate of protealytic enzymes is fibrinogen. Thrombin severs four ARG-GLY bonds in the alpha A and beta B chains of its molecule, on the side of the terminal-N. It thus liberates two fibrinopeptides A and B, and leads to the formation of fibrin. Plasmin, by contrast, acts upon the fibrinogen molecule first by hydrolysis of the alpha and beta chains liberating the X fragment and three peptides A, B and C. It continues on the alpha, beta and gamma chains of fragment X, leading to the appearance of fragments Y and D. Fragment Y is in turn hydrolysed into a second fragment D and fragment E. The initial (X and Y) or terminal (D and E) fibrinogen breakdown products each possess their own anticoagulant properties together with immunological properties which may be used in their estimation.
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PMID:[Action of proteolytic enzymes on fibrinogen]. 3 Nov 11

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.
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PMID:Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. 138 45

FAK is a focal adhesion kinase that is phosphorylated on tyrosine in activated platelets. Induction of FAK phosphorylation requires both fibrinogen binding to integrin alpha IIb beta 3 and post-occupancy events during agonist-induced platelet aggregation or platelet spreading on a fibrinogen matrix. To identify the signaling pathways necessary for tyrosine phosphorylation of FAK, we have examined the conditions that stimulate or inhibit this phosphorylation in platelets in which fibrinogen binding to alpha IIb beta 3 and platelet aggregation were induced directly with an anti-beta 3 Fab fragment (anti-LIBS6). Apyrase was added to prevent effects of the endogenous platelet agonist, ADP. Under these conditions, neither fibrinogen binding nor primary platelet aggregation was sufficient to induce FAK phosphorylation, suggesting that a second "costimulatory" event was required. Indeed, when epinephrine was added with fibrinogen and anti-LIBS6, large platelet aggregates formed and FAK phosphorylation occurred. This response was prevented by blockade of cyclooxygenase with indomethacin or thromboxane A2 receptors with SQ 30,741. A stable thromboxane A2 analogue (U46619) could substitute for epinephrine as the costimulus. Epinephrine costimulation of FAK phosphorylation was also prevented by chelation of intracellular Ca2+ with BAPTA or selective inhibition of protein kinase C (PKC) with bisindolylmaleimide, indicating that Ca2+ and PKC are necessary for FAK phosphorylation under these conditions. Epinephrine also promoted FAK phosphorylation and adhesive spreading of apyrase-treated platelets on a fibrinogen matrix. Cytochalasin D, an inhibitor of actin polymerization, blocked FAK phosphorylation under all these conditions. Thus, tyrosine phosphorylation of FAK in platelets requires coordinated signaling through occupied integrin and agonist receptors. These separate pathways may converge to increase free Ca2+ and activate PKC and thus promote the cytoskeletal reorganization required for activation of FAK.
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PMID:Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. 751 81

Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.
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PMID:Structurally distinct disintegrins contortrostatin and multisquamatin differentially regulate platelet tyrosine phosphorylation. 752 Sep 9

Integrins bind extracellular matrix and transduce signals mediating cell adhesion, spreading, and migration. It is unclear how these distinct responses follow from a common event: integrin clustering. We examined the relationship between integrin-mediated signals and the integrin's activation state using a cell line expressing alpha IIb beta 3 (Clone B) and a panel of monoclonal antibodies against this integrin. Non-activating antibodies used to cluster alpha IIb beta 3 stimulated focal adhesion kinase (FAK) phosphorylation, regardless of affinity, subunit specificity, or ligand-blocking phenotype. Coated on plastic, these antibodies supported cell adhesion, spreading, and FAK phosphorylation. In contrast, clustering of alpha IIb beta 3 induced with activating antibodies, or binding of soluble fibrinogen to antibody-activated alpha IIb beta 3, did not induce FAK phosphorylation. Thus, clustering of alpha IIb beta 3 on Clone B does not necessarily result in FAK phosphorylation. Coated on plastic, activating antibodies supported cell adhesion, but not spreading or FAK phosphorylation. Therefore, it appears the resting, not the active form of alpha IIb beta 3, induces cell spreading and FAK phosphorylation in Clone B. These data indicate that "inside-out" signals may alter not only the binding specificity of an integrin, but the "outside-in" biochemical signals that integrin initiates as well. This activation state-linked signaling represents a novel mechanism, which may explain how diverse cellular responses are induced by integrin-matrix interactions.
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PMID:The activation state of the integrin alpha IIb beta 3 affects outside-in signals leading to cell spreading and focal adhesion kinase phosphorylation. 754 96

Activation of human platelets by the peptide YFLLRNP has been shown to induce shape change but not secretion, Ca2+ mobilization, or pleckstrin phosphorylation (Rasmussen, U.B., Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J.P., and Pavirani, A. (1993) J. Biol. Chem. 268, 14322-14328). YFLLRNP was added to washed human platelets that had been pretreated with EGTA at 37 degrees C or preincubated with the fibrinogen receptor antagonist RGDS to preclude the activation of the integrin alpha IIb beta 3 (fibrinogen receptor). YFLLRNP induced shape change and stimulated the tyrosine phosphorylation of proteins of 62, 68, and 130 kDa within 7 s. Tyrosine phosphorylation of these proteins reached maximum levels (2-3-fold) 15-30 s after addition of YFLLRNP and decreased subsequently. The chelation of intracellular Ca2+ by BAPTA-AM decreased basal tyrosine protein phosphorylation but did not inhibit the increase of tyrosine phosphorylation of P62, P68, and P130 or the shape change induced by YFLLRNP. Preincubation of platelets with the tyrosine kinase inhibitors genistein or tyrphostin A23 completely inhibited platelet shape change and protein tyrosine phosphorylation induced by YFLLRNP. The inactive structural analogs daidzein and tyrphostin A1 were barely inhibitory. P62, P68, and P130, which exhibited increased tyrosine phosphorylation upon stimulation with YFLLRNP, were found in the cytoskeleton. P130 was not identical to vinculin or the focal adhesion kinase pp125FAK. The results indicate that stimulation of G-protein-coupled thrombin receptors rapidly induces protein tyrosine kinase activation through a Ca(2+)- and integrin-independent mechanism. Protein tyrosine kinase activation and tyrosine phosphorylation of novel protein substrates seem to play an essential role in the induction of platelet shape change.
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PMID:Platelet shape change induced by thrombin receptor activation. Rapid stimulation of tyrosine phosphorylation of novel protein substrates through an integrin- and Ca(2+)-independent mechanism. 783 59

Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.
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PMID:Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. 792 40

Platelet adhesion to immobilized fibrinogen stimulates the induction of tyrosine phosphorylation of multiple proteins. However, platelet spreading and tyrosine phosphorylation of three proteins, the focal adhesion kinase pp125FAK and proteins of 101 and 105 kD (pp101 and pp105), require a second adenosine diphosphate (ADP)-dependent costimulatory event. In this study we show that protein kinase C (PKC) inhibitors prevented the induction of tyrosine phosphorylation of pp125FAK, pp101 and pp105, and abolished spreading. These inhibitory effects were not observed after treatment of the platelets with the intracellular Ca2+ chelator BAPTA-AM. This suggested that in platelets, PKC regulates spreading and related protein tyrosine phosphorylation. In addition, the inhibitory effects of apyrase, an ADP scavenger, on spreading and tyrosine phosphorylation of pp125FAK, pp101, and pp105, were not observed in the presence of phorbol 12-myristate 13-acetate (PMA). These data implied that in fibrinogen-adherent platelets integrin ligation and an agonist receptor occupancy are required for the functional association of PKC and the alpha IIb beta 3-mediated signaling pathways. Taken together these results show that PKC plays a central role in the transduction of intracellular signals downstream from alpha IIb beta 3 that regulate spreading and pp125FAK phosphorylation.
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PMID:Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen. 854 37

The acute phase proteins (APPs) have been empirically defined as those whose plasma concentration changes following inflammatory reaction. Those proteins whose concentrations increase are referred to as positive APP, while those whose levels decline are termed negative APP. In man, positive APP are: alpha 1 acid glycoprotein, alpha 1 protease inhibitor, alpha 1 antichymotrypsin, haptoglobin, ceruloplasmin, fibrinogen, C-reactive protein, serum amyloid A. Great variability in the APP response between different species is observed. The principal functions of APP, result from the interaction of these proteins with ligands of various origins which give "protein-ligands" complexes. These complexes are cleared by the RES or by the hepatocyte. The results are protease inhibition, neutralization of toxic molecules such as hemoglobin or the superoxide anion, clearance of cell membranes and chromatin. The drop of the plasma concentration of negative APP during an inflammatory reaction carries a rise of free ligands (fatty acids, hormones, vitamins, trace elements). IL6 has been recognized as the principal regulator of most APP genes. The response of the hepatic cell to IL6 is characterized by the enhanced production of type 2 or IL6 specific APPs. The biochemical process of signal transduction is IL6--JAK2--APRF The set of APP genes regulated by IL1 type cytokines (type 1 APPs) is distinct from that regulated by IL6 type cytokine. IL1 and TNF alpha mediated stimulation of type 1 APP genes is synergistically enhanced by IL6 type cytokines. The biochemical process of signal transduction is IL1, IL6--Ras--MAP kinase--NFIL6 The targeted inflammatory proteic profile including the assay of C-reactive protein, haptoglobin and alpha 1 acid glycoprotein produces a "biological tool" to the clinician in order to manage an inflammatory response. IL6, a proteic marker for the future, connected with CRP, will be assayed during early inflammatory reaction.
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PMID:[Acute-phase proteins in inflammation]. 856 70

Platelets express a single low affinity receptor for immunoglobulin, FcgammaRII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show that immobilized IgG is also a potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to IgG was blocked by the FcgammaRII receptor-specific monoclonal antibody, IV. 3. Pretreatment of the platelets with cytochalasin D to inhibit actin polymerization similarly prevented cell binding to IgG having no effect on platelet binding to fibrinogen. Platelet adhesion to IgG also led to the induction of tyrosine phosphorylation of multiple proteins including pp125(FAK) and p72(SYK). These proteins were also tyrosine-phosphorylated in alphaIIbbeta3-deficient IgG-adherent platelets from patients with Glanzmann's thrombasthenia. These data demonstrate that FcgammaRII mediates pp125(FAK) phosphorylation and platelet adhesion to IgG independent of the integrin alphaIIbbeta3. Treatment of the platelets with bisindolylmaleimide to inhibit protein kinase C prevented phosphorylation of pp125(FAK) as well as several other proteins, but not p72(SYK) phosphorylation. This study establishes that the FcgammaRII receptor mediates pp125(FAK) phosphorylation via protein kinase C.
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PMID:The FcgammaRII receptor triggers pp125FAK phosphorylation in platelets. 866 17


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