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 recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.
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PMID:Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. 805 85

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction and neoplastic transformation. These mechanisms are regulated by the activities of both protein-tyrosine kinases and protein-tyrosine phosphatases. Recent studies have identified a novel protein-tyrosine phosphatase, termed Syp, that is widely expressed in various tissues. Syp encodes a cytoplasmic phosphatase that contains two Src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules to activated tyrosine kinases, experiments were performed to determine whether Syp might form specific complexes with p210bcr-abl, a fusion protein believed to be involved in the pathogenesis of chronic myelogenous leukemia and, thus, possibly alter or mediate p210bcr-abl tyrosine kinase activity. We found that Syp was highly and constitutively tyrosine phosphorylated in three different murine cell lines transfected with a p210bcr-abl expression vector. Furthermore, p210bcr-abl, Syp, and Grb2 formed stable complexes in BCR-ABL-expressing cells. Complex formation between p210bcr-abl and Syp was mediated in vitro by the NH2-terminal SH2 domain of Syp. Last, p210bcr-abl tyrosine kinase was effectively dephosphorylated by Syp in vitro. These results suggest an interaction between Syp and BCR-ABL protein, which might play a role in cellular transformation of BCR-ABL.
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PMID:SH2-containing phosphotyrosine phosphatase Syp is a target of p210bcr-abl tyrosine kinase. 819 76

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.
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PMID:Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling. 891 Jun 7

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alphaIIbbeta3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin alphaIIbbeta3) and signaling enzymes (pp60c-src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin alphaIIbbeta3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alphaIIbbeta3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alphaIIbbeta3, PI 3-kinase, FAK, pp60(c-)src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alphaIIbbeta3 in vWf-induced signal transduction.
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PMID:Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction. 926 16

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
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PMID:Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. 944 80

Tyrosine phosphorylation of focal adhesion-associated proteins may be involved in the regulation of the cytoskeleton and in the control of signals for growth and survival. The focal adhesion kinase (FAK) functions in regulating tyrosine phosphorylation of several of these proteins, including paxillin, tensin, and p130(cas). Protein- tyrosine phosphatases, the counterparts of protein-tyrosine kinases, also presumably regulate phosphorylation of these proteins. We have tested the hypothesis that FAK intimately associates with a protein-tyrosine phosphatase. Protein-tyrosine phosphatase activity associated with the recombinant C-terminal domain of FAK in vitro and could be coimmunoprecipitated with both FAK and paxillin from lysates of chicken embryo cells. However, the interaction with FAK appeared to be indirect and mediated via paxillin. The protein-tyrosine phosphatase was subsequently identified as protein-tyrosine phosphatase-PEST, a nonreceptor protein-tyrosine phosphatase. The C-terminal noncatalytic domain of protein-tyrosine phosphatase-PEST directly bound to paxillin in vitro. The association of both a protein-tyrosine kinase and a protein-tyrosine phosphatase with paxillin suggests that paxillin may play a critical role in the regulation of the phosphotyrosine content of proteins in focal adhesions.
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PMID:Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin. 949 81

SIRPs (signal-regulatory proteins) are a family of transmembrane glycoproteins that were identified by their association with the Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2 in response to insulin. Here we examine whether SIRPalpha and SHP-2 are signaling molecules for the receptors for growth hormone (GH), leukemia inhibitory factor (LIF), or interferon-gamma (IFNgamma), cytokine receptor superfamily members that bind to and activate Janus kinase 2 (JAK2). In 3T3-F442A fibroblasts, GH rapidly stimulates tyrosyl phosphorylation of both SIRPalpha and SHP-2 and enhances association of SHP-2 with SIRPalpha. Consistent with JAK2 binding and phosphorylating SIRPalpha in response to GH, co-expression of SIRPalpha and JAK2 in COS cells results in tyrosyl phosphorylation of SIRPalpha and JAK2 association with SIRPalpha. LIF does not stimulate tyrosyl phosphorylation of SIRPalpha but stimulates greater tyrosyl phosphorylation of SHP-2 than GH. Additionally, LIF enhances association of SHP-2 with the gp130 subunit of the LIF receptor signaling complex. IFNgamma, which stimulates JAK2 to a greater extent than LIF, is ineffective at stimulating tyrosyl phosphorylation of SIRPalpha or SHP-2. These results suggest that SIRPalpha is a signaling molecule for GH but not for LIF or IFNgamma. Differential phosphorylation of SIRPalpha and SHP-2 may contribute to the distinct physiological effects of these ligands.
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PMID:Growth hormone regulation of SIRP and SHP-2 tyrosyl phosphorylation and association. 950 23

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.
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PMID:Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew. 958 52

We have studied the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J cells. The secretagogue bombesin, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), and the protein-tyrosine phosphatase inhibitor pervanadate induced tyrosine phosphorylation of different proteins, including paxillin and p125(FAK), which was reduced or blocked by the tyrosine kinase inhibitors genistein and tyrphostin B56, respectively. Both PMA and pervanadate continuously increased amylase secretion with a similar time course, reaching the level of bombesin-induced amylase release after 60 min. Their effects were not additive and could be inhibited by preincubation of AR4-2J cells with genistein or tyrphostin B56, respectively. Inhibition of protein kinase C with Ro 31-8220 nearly abolished the effects of PMA, but had no effect on either pervanadate-induced protein tyrosine phosphorylation or amylase secretion. An increase in cytosolic free Ca2+ concentration by thapsigargin or A23187 caused a rapid increase in amylase release within the initial 5 min. In the presence of PMA or pervanadate, amylase secretion was further stimulated to levels comparable to those induced by bombesin after 30 min of stimulation. Inhibition of PMA-induced amylase secretion by Ro 31-8220 was less at elevated cytosolic free Ca2+ concentrations than without Ca2+. Furthermore, an increase in cytosolic free Ca2+ concentration had no effect on protein tyrosine phosphorylation in either the absence or presence of PMA or pervanadate. We therefore conclude that in the cascade of events that lead to bombesin-induced protein secretion from AR4-2J cells, protein tyrosine phosphorylation occurs downstream of protein kinase C activation. A further step in secretion that is Ca2+-dependent occurs distal to protein tyrosine phosphorylation.
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PMID:Pervanadate stimulates amylase release and protein tyrosine phosphorylation of paxillin and p125(FAK) in differentiated AR4-2J pancreatic acinar cells. 963

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.
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PMID:Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion. 982 58


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