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)

Previous work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3--30 minutes of incubation (Sas el al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor =X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into "activated" and "non activated" groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This aggreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.
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PMID:Spectrophotometric determination of factor Xa generation in factor IX concentrates. 57 33

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

Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.
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PMID:Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. 171 82

We studied the mode of action of the low molecular weight heparin PK10169 and two of its constituent fractions: EMT 966 High Molecular Weight Fraction and EMT 967 Low Molecular Weight Fraction. EMT 966 like standard heparin, acts primarily on thrombin formed and not on prothrombinase (S type heparin). In contrast EMT 967 has no direct effect on thrombin. At high concentrations, it inhibits the prothrombinase complex (P type heparin). PK10169, that contains the two EMTs shows both activities: antithrombin and antiprothrombinase (mixed type heparin). The addition of increasing amounts of EMT 967 to a constant amount of EMT 966 does not influence the breakdown constant of endogenous thrombin which is determined by the concentration of EMT 966 only. This demonstrates the absence of competition for AT III between the two components of PK10169. In platelet rich plasma, EMT 966 inhibits and postpones thrombin generation more efficiently than unfractionated heparin, probably because it is less sensitive to neutralization by platelet components (platelet factor 4). Amounts of EMT 967 that hardly inhibit thrombin generation in platelet rich plasma enhance the effect of EMT 966 probably by neutralizing platelet factor 4.
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PMID:The mode of action of low molecular weight heparin preparation (PK10169) and two of its major components on thrombin generation in plasma. 254 77

We have isolated a cDNA encoding a novel human intracytoplasmic tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). In addition, we have cloned and characterized the murine homolog of the human RAFTK cDNA. Comparison of the deduced amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA clone, encoding a polypeptide of 1009 amino acids, has closest homology (48% identity, 65% similarity) to the focal adhesion kinase (pp125FAK). Comparison of the deduced amino acid sequences also indicates that RAFTK, like pp125FAK, lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. In addition, like pp125FAK, RAFTK contains a kinase domain flanked by large N-terminal (426 residues) and C-terminal (331 residues) domains, and the C-terminal region contains a predicted proline-rich stretch of residues. In fetal tissues, RAFTK expression was abundant in brain, and low levels were observed in lung and liver. In adult tissues, it was less restricted, indicating that RAFTK expression is developmentally up-regulated. Expression of RAFTK was also observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a approximately 123-kDa protein from the human megakaryocytic CMK cell line was immunoprecipitated. Treatment of the megakaryocytic CMK cells with thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK protein. The structural features of RAFTK suggest that it is a member of the focal adhesion kinase gene family and may participate in signal transduction in human megakaryocytes and brain as well as in other cell types.
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PMID:Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. 749 42

Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')2-anti-p62 in a way insensitive to elevation of cAMP. In contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive activation of c-Src and Syk was also observed in collagen but not thrombin-stimulated platelets. Moreover, either F(ab')2-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.
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PMID:Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI. 749 87

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

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.
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PMID:Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase. 753 75

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

We show the presence of the tyrosine kinase JAK2 in human platelets and demonstrate that it undergoes phosphorylation on tyrosine residues on challenge with the G protein receptor stimulus, thrombin, or the tyrosine phosphatase inhibitor, peroxovanadate. Thrombin-induced phosphorylation of JAK2 is inhibited by two structurally distinct inhibitors of tyrosine kinases, staurosporine and the tyrphostin ST271. The protein kinase C (PKC) inhibitor, Ro 31-8220, and intracellular Ca2+ chelator, BAPTA-AM, also inhibit thrombin-induced phosphorylation of JAK2, while the phorbol ester, phorbol dibutyrate (PDBu), and Ca2+ ionophore, A23187, induce tyrosine phosphorylation of JAK2. These results suggest that tyrosine phosphorylation of JAK2 stimulated by thrombin may be mediated downstream of phosphoinositide metabolism.
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PMID:Phosphorylation of JAK2 in thrombin-stimulated human platelets. 792 97


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