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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)

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, alpha IIb beta 3. To determine whether either of the cytoplasmic tails of alpha IIb beta 3 regulates FAK phosphorylation, CHO cells were stably transfected with alpha IIb beta 3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type alpha IIb beta 3 or alpha IIb beta 3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue alpha IIb tail adhered to and spread on fibrinogen or on an anti-alpha IIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue beta 3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phosphorylation was observed if 46 residues were deleted from the beta 3 tail. None of these beta 3 truncation mutants spread on the anti-alpha IIb antibody. When cells with wild-type alpha IIb beta 3 or truncated beta 3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two alpha IIb truncation mutants for up to 90 minutes in suspension. This persistent phosphorylation was not due to occupancy of alpha IIb beta 3 by adhesive ligands because it was also observed with an alpha IIb tail truncation mutant that contained an additional mutation in the extracellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the beta 3 cytoplasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the alpha IIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.
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PMID:Integrin signaling: roles for the cytoplasmic tails of alpha IIb beta 3 in the tyrosine phosphorylation of pp125FAK. 871 88

Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.
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PMID:Endogenous ADP prevents PGE1-induced tyrosine dephosphorylation of focal adhesion kinase in thrombin-activated platelets. 897 12

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules.
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PMID:Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes. 897 35

Fibronectin has been shown to stimulate tyrosine phosphorylation of a number of proteins in the 115-125 kDa range and facilitate degranulation by alloantigen-specific cytotoxic T lymphocyte (CTL) clones in response to substimulatory amounts of anti-CD3 or anti-T cell receptor (TCR). The current study was initiated to further characterize integrin expression and usage by these CTL clones. We demonstrate that vitronectin and fibrinogen, but not laminin or collagen, are also able to both facilitate degranulation in the presence of substimulatory anti-CD3 and stimulate tyrosine phosphorylation of these 115-125-kDa proteins, with a 115-kDa protein being the most prominently phosphorylated. These results implicate the expression and usage of the vitronectin receptor, alpha beta3 integrin, by these CTL clones. We demonstrate by both flow cytometry and immunoprecipitation that CTL clones do in fact express beta3 integrin. Immobilized antibody to beta3 stimulates the phosphorylation of the 115-125-kDa proteins, suggesting that engagement of beta3 transmits the same signal into these cells as fibronectin or vitronectin. The fibronectin and vitronectin-induced phosphorylation as well as adhesion to either fibronectin or vitronectin can be significantly inhibited with antibodies to beta3 integrins. Finally, we are able to immunoprecipitate 115-kDa proteins with antiserum to focal adhesion kinase and a related kinase, called PYK-2, that becomes phosphorylated in response to vitronectin or immobilized anti-beta3. Taken together, these results demonstrate that CTL express and use beta3-integrins as signaling molecules which can augment TCR-mediated stimulation.
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PMID:Cytotoxic T lymphocytes express a beta3 integrin which can induce the phosphorylation of focal adhesion kinase and the related PYK-2. 902 36

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.
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PMID:Tyrosine phosphorylation of the novel protein-tyrosine kinase RAFTK during an early phase of platelet activation by an integrin glycoprotein IIb-IIIa-independent mechanism. 909 53

Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1), the most abundant protein of platelet alpha granules. Although it associates with alphaIIbbeta3, IAP has no known function in platelets. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin alphaIIbbeta3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase. Furthermore, 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK. The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity column of 4N1K peptide. Both alphaIIb and beta3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK with the IAP-alphaIIbbeta3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of alphaIIbbeta3 whose unique properties result in its localization to the platelet surface and the fibrin clot.
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PMID:Thrombspondin acts via integrin-associated protein to activate the platelet integrin alphaIIbbeta3. 916 39

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.
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PMID:Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation. 926 1

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.
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PMID:Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants. 927 7

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.
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PMID:Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex. 935 72

The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin alphaIIbbeta3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3, 4-bisphosphate (PtdIns(3,4)P2), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). It has been assumed that PtdIns(3,4)P2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P2 is formed after directly activating alphaIIbbeta3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P3, but to transient formation of PtdIns3P and generation of PtdIns(3,4)P2, the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P3. Integrin-activated generation of the second messenger PtdIns(3,4)P2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.
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PMID:A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets. 941 38


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