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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of human platelets causes a dramatic increase in phosphorylation of various proteins at tyrosine residues. The abundance of protein tyrosine kinases of the src-family in platelets, particularly pp60c-src, suggests an important role of these kinases in response to stimulation events. We have shown that pp60c-src is activated on agonist-induced platelet stimulation with respect to its substrate affinity. This was accompanied by phosphorylation of pp60c-src at Ser-12, a residue which is phosphorylated by PKC. Inhibition of PKC with a specific inhibitor, Ro-31-8220, suppressed thrombin-induced translocation of pp60c-src to the cytoskeleton. On the basis of our data, we suggest that the cytoskeletal association of pp60c-src is dependent on phosphorylation of pp60c-src at Ser-12 by PKC. Phosphorylation at Ser-12 in the membrane-binding domain might be the signal that displaces pp60c-src from the plasma membrane and, accompanied with the increased substrate affinity, facilitates phosphorylation of putative substrates.
Cell Mol Biol (Noisy-le-grand) 1994 Jul
PMID:The protein tyrosine kinase pp60c-src is activated upon platelet stimulation. 752 17

Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of IGF1, insulin, PDGF, EGF and FGFs on phosphatidylinositol-3 kinase. IGF1, PDGF and EGF which stimulate cell proliferation of astroglial cells, increased phosphatidylinositol-3 kinase activity immunoprecipitated with anti-phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation of astroglial cells, have no effect on phosphatidylinositol-3 kinase activity. Addition of 1 nM PDGF, 10 nM IGF1 or 100 nM EGF to the culture medium rapidly stimulated phosphatidylinositol-3 kinase activity which declined slowly after 2 min. The stimulation of phosphatidylinositol-3 kinase increased with growth factor concentration. The maximum increase in phosphatidylinositol-3 kinase activity occurred with 50 nM IGF1, 1 nM PDGF or 100 nM EGF. Since insulin was active only at high concentration (1 microM), its effect was probably mediated through IGF1 receptors and not through insulin receptors. Treatment with IGF1-plus-EGF, had an additive effect on PI(3)-kinase activity, PDGF-plus-IGF1 did not. IGF1 and PDGF, to a lesser degree, also increased the phosphatidylinositol-3 kinase activity associated with pp60c-src protein. Immunoblots performed with an antibody directed against the p85-subunit of the phosphatidylinositol-3 kinase confirmed that IGF1 increased the number of phosphatidylinositol-3 kinase molecules associated with phosphotyrosine-containing proteins or with c-src protein. Each growth factor affects in a different manner the association of phosphatidylinositol-3 kinase with phosphotyrosine-containing proteins and with c-src protein and thus probably modulates intracellular signals downstream of phosphatidylinositol-3 kinase in astroglial cells.
Cell Mol Biol (Noisy-le-grand) 1994 Jul
PMID:Growth factor-regulated phosphatidylinositol-3-kinase in astrocytes. Involvement of pp60c-src. 752 18

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.
Mol Cell Biol 1995 Feb
PMID:Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. 752 76

Overexpression of pp60c-src in mouse fibroblasts potentiates both agonist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttrell, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400]. In reconstitution experiments in vitro, phosphorylation of Gs alpha by immune-complexed pp60c-src resulted in enhanced rates of receptor-mediated guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding and GTP hydrolysis [Hausdorff, Pitcher, Luttrell, Linder, Kurose, Parsons, Caron and Lefkowitz (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5720-5724]. These results suggest that one mechanism by which pp60c-src affects signalling through the beta-adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of Gs alpha might affect its function, we subjected phosphorylated, recombinant Gs alpha to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edman degradation to determine the cycle numbers at which radiolabelled phosphotyrosine was released. Candidate peptides that contained Tyr residues at the corresponding positions were synthesized, phosphorylated in vitro by pp60c-src, and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact Gs alpha. We report here that Gs alpha is phosphorylated on two residues by pp60c-src, namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP [Johnson, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cell Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-terminus, within a region of Gs alpha important for receptor interaction [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Nature (London) 334, 712-715]. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains.
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PMID:Identification of the in vitro phosphorylation sites on Gs alpha mediated by pp60c-src. 753 Apr 45

pp60c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443-23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependent activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+ cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+ cells, but the effect was smaller in PTPase+ cells. In an in vitro assay, hormone-stimulated pp60c-src autophosphorylation from PTPase+ cells was decreased 64 +/- 22%, and substrate phosphorylation by pp60c-src was reduced 54 +/- 16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, an in vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition, in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was an in vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.
Mol Cell Biochem 1994 Oct 26
PMID:Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity. 753 75

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.
Mol Cell Biol 1995 Sep
PMID:The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src. 754 35

The decision to enter the cell division cycle is governed by the interplay between growth activators and growth inhibitors. The retinoblastoma protein (RB) is an example of a growth inhibitor whose main function appears to be the binding and inactivation of key cell cycle activators. One target of RB is a proto-oncoprotein, the c-Abl tyrosine kinase. RB binds to the ATP-binding lobe in the kinase domain and inhibits the nuclear pool of c-Abl in quiescent and G1 cells. Phosphorylation of RB at G1/S releases c-Abl, leading to the activation of this nuclear tyrosine kinase. In this report, we describe the construction of a mutant Abl, replacing the ATP-binding lobe of c-Abl with that of c-Src. The mutant protein AS2 is active as a tyrosine kinase and can phosphorylate Abl substrates, such as the C-terminal repeated domain of RNA polymerase II. AS2, however, does not bind to RB, and its activity is not inhibited by RB. As a result, the nuclear pool of AS2 is no longer cell cycle regulated. Excess AS2, but not its kinase-defective counterpart, can overcome RB-induced growth arrest in Saos-2 cells. Interestingly, wild-type c-Abl, in both its kinase-active and -inactive forms, can also overcome RB. Furthermore, overexpression of a kinase-defective c-Abl in rodent fibroblasts accelerates the transition from quiescence to S phase and cooperates with c-Myc to induce transformation. These effects, however, do not occur with the kinase-defective form of AS2. Thus, the growth-stimulating function of the kinase-defective c-Abl is dependent on the binding and the abrogation of RB function. That RB function can be abolished by the overproduction of one of its binding proteins is consistent with the hypothesis that RB induces cell cycle arrest by acting as a "molecular matchmaker" to assemble protein complexes. Exclusive engagement of RB by one of its many targets is incompatible with the biological function of this growth suppressor protein.
Mol Cell Biol 1995 Oct
PMID:Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms. 756 6

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.
Mol Cell Biol 1993 Mar
PMID:Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis. 768 96

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.
Mol Cell Biol 1993 Mar
PMID:Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets. 768 Jan

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.
Mol Cell Biol 1993 Apr
PMID:The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src. 768 Nov 47


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