We report that expressing interfering mutants of the small Ras-related GTPase Rac, using either recombinant vaccinia virus or stable DNA transfection, eliminates epidermal growth factor-induced Ca2+ ...signaling, without affecting Ca2+ mobilization or influx from G protein-coupled receptors. Platelet-derived growth factor-dependent Ca2+ influx, however, is only partly sensitive to dominant negative Rac proteins. Thus, whereas epidermal growth factor-induced Ca2+ influx is completely mediated by Rac proteins, platelet-derived growth factor-induced Ca2+ influx involves Rac-dependent and -independent signaling pathways.
Association of the p21ras guanine nucleotide exchange factor mSOS with tyrosine-phosphorylated Shc has been implicated in the activation of p21ras. In addition, after growth factor stimulation mSOS ...becomes phosphorylated as indicated by the appearance of a form of mSOS with reduced electrophoretic mobility. This phosphorylation is delayed with respect to Shc-Grb2-mSOS complex formation and activation of p21ras. To investigate the role of mSOS phosphorylation in further detail we have investigated the effect of phosphorylation on mSOS complex formation and p21ras activation. We found that Shc is associated with the unphosphorylated, faster migrating form of mSOS. Furthermore, although there is a correlation between the amount of complexes formed and the activation of p21ras, there is no such a correlation between mSOS phosphorylation and p21ras activation. In addition, inhibition of mSOS phosphorylation did not affect complex formation of mSOS with tyrosine phosphorylated Shc. Also, induction of mSOS phosphorylation prior to complex formation did not affect EGF-induced association of mSOS with Shc significantly, and Shc still associated predominantly with the faster migrating form of mSOS. From these results we conclude that the unphosphorylated form of mSOS is associated with Shc and that perhaps a phosphorylation-dephosphorylation step is part of the mSOS activation-inactivation cycle.
Several tyrosine-phosphorylated proteins have been identified that associate with p120GAP, the GTPase activating protein of p21ras. In keratinocytes, calcium induced the tyrosine phosphorylation of a ...65 kDa p120GAP-associated protein (p65Ca). This protein did not comigrate with two previously reported p120GAP-associated proteins, i.e. a 68 kDa protein from src-transformed cells (p68) and an insulin-induced protein of 60 kDa (p60(2C4)). P65Ca was neither recognized by poly(U)-sepharose, which efficiently precipitates p68, nor did it crossreact with antibodies against p68. In addition, a monoclonal antibody directed to p60(2C4) did not recognize p65Ca. From these results we conclude that p65Ca is different from p68 and p60(2C4) and thus, a novel p120GAP-associated protein. Since calcium has an important, tyrosine kinase dependent, role in the differentiation of keratinocytes, phosphorylation of p65Ca may be important for this differentiation process. However, surprisingly, calcium induced the phosphorylation of a similar-sized p120GAP-associated 65 kDa protein in fibroblast cell lines.
p21ras GAP is phosphorylated on tyrosine residues and associates with 62 kDa and 190 kDa tyrosine phosphorylated proteins in v-src-transformed fibroblasts. We were interested in identifying the ...tyrosine kinase responsible for phosphorylation of GAP and the two associated proteins. Here, we report that GAP-immunoprecipitates from v-src transformed cells contain a tyrosine kinase activity that phosphorylates GAP, p62 and p190. Tryptic peptide analysis indicated that the sites phosphorylated in vitro and in vivo are indistinguishable, suggesting that the precipitated kinase could be responsible for tyrosine phosphorylation of GAP in vivo. The GAP-associated kinase activity might be due to v-src itself, because pp60v-src is able to associate with GAP in vitro and GAP can be phosphorylated by pp60v-src immunecomplexes.
Expression of p21
rasAsn-17
, a dominant negative mutant of p21
ras
that blocks p21
ras
activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin ...and platelet-derived growth factor in rat-1 cells A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992. Here we report that expression of p21
rasAsn-17
does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21
ras
in these cells, this indicates that EGF induces a p21
ras
-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21
ras
-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21
ras
and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21
ras
activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.
She proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have ...studied the role of She in insulin- and EGF-induced activation of p21
ras
in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21
ras
. Analysis of She immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in She tyrosine phosphorylation and (ii) She antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of She and the presence of Grb2 and mSOS in She immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, She could be detected as well. Furthermore, She immune complexes contained guanine nucleotide exchange activity toward p21
ras
in vitro. From these results, we conclude that after insulin and EGF treatment, She associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21
ras
In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with She was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-mSOS with She may be an important event in insulin-induced, mSOS-mediated activation of p21
ras
.
A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21
ras
. This accumulation could be caused either by an increase in ...guanine nucleotide exchange on p21
ras
or by a decrease in the GTPase activity of p21
ras
. To investigate whether insulin and EGF affect nucleotide exchange on p21
ras
, we measured binding of α-
32
PGTP to p21
ras
in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21
ras
. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21
ras
, p21
ras
(Asn-17). Activation of p21
ras
by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21
ras
(Asn-17). In addition, the enhanced nucleotide binding to p21
ras
in response to insulin and EGF in permeabilized cells was blocked upon expression of p21
ras
(Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21
ras
.
We have used two approaches to identify possible substrates of the insulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), which is ...reported to be specific for the insulin-induced signal transduction route, to augment tyrosine phosphorylation. Second, we used src homology 2 (SH2) domains fused to glutathione S-transferase as high affinity binding agents for tyrosine-phosphorylated proteins. Using the SH2 domain-containing region of p120 GTPase-activating protein and growth factor-bound protein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in insulin- plus PAO-treated NIH3T3 cells overexpressing the IR. This M(r) 70,000 protein, which migrated as a doublet on SDS-polyacrylamide gels, efficiently bound to polyuridylic acid-Sepharose but is distinct from similar-size RNA-binding proteins such as p62 (sam68) and heterogeneous nuclear ribonucleoproteins I, K, L, and M. In addition, it differs from other M(r) 70,000 tyrosine-phosphorylated proteins, such as SH2-containing tyrosine phosphatase, raf1, and paxillin. Tyrosine phosphorylation of this protein was hardly observed after epidermal growth factor treatment. This suggests that the M(r) 70,000 protein is a novel and specific substrate for the IR kinase or an insulin-induced tyrosine kinase. The requirement for PAO to identify this tyrosine phosphorylation indicates a high turnover rate of the tyrosine phosphate.
PDF
