Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation.

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

The phosphotyrosine interaction domain of Shc binds an LXNPXY motif on the epidermal growth factor receptor.

Shc is an SH2 domain protein that is tyrosine phosphorylated in cells stimulated with a variety of growth factors and cytokines. Once phosphorylated, Shc binds the Grb2-Sos complex, leading to Ras activation. Shc can interact with tyrosine-phosphorylated proteins by binding to phosphotyrosine in the context of an NPXpY motif, where pY is a phosphotyrosine. This is an unusual binding site for an SH2 domain protein whose binding specificity is usually controlled by residues carboxy terminal, not amino terminal, to the phosphotyrosine. Recently we identified a second region in Shc, named the phosphotyrosine interaction (PI) domain, and we have found it to be present in a variety of other cellular proteins. In this study we used a dephosphorylation protection assay, competition analysis with phosphotyrosine-containing synthetic peptides, and epidermal growth factor receptor (EGFR) mutants to determine the binding sites of the PI domain of Shc on the EGFR. We demonstrate that the PI domain of Shc binds the LXNPXpY motif that encompasses Y-1148 of the activated EGFR. We conclude that the PI domain imparts to Shc its ability to bind the NPXpY motif.

Shc binding to nerve growth factor receptor is mediated by the phosphotyrosine interaction domain.

Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2.Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXN-PXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine-phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways.

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.

Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.

Nck associates with the SH2 domain-docking protein IRS-1 in insulin-stimulated cells.

Nck, an oncogenic protein composed of one SH2 and three SH3 domains, is a common target for various cell surface receptors. Nck is thought to function as an adaptor protein to couple cell surface receptors to downstream effector molecules that regulate cellular responses induced by receptor activation. In this report, we show that Nck forms a stable complex in vivo with IRS-1 in insulin-stimulated cells. The interaction between IRS-1 and Nck is mediated by the binding of the SH2 domain of Nck to tyrosine-phosphorylated IRS-1. Although Nck associates with IRS-1, Nck phosphorylation is not affected by insulin stimulation. Furthermore, in vitro and in vivo studies show that the SH2 domains of Nck, GRB2, and p85 bind distinct phosphotyrosine residues in IRS-1. After insulin stimulation all three signaling molecules can be found complexed to a single IRS-1 molecule. These findings provide further evidence that, in response to insulin stimulation, IRS-1 acts as an SH2 docking protein that coordinates the regulation of various different signaling pathways activated by the insulin receptor.

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.

Characterization of two tyrosine-specific protein kinases from pig spleen. Substrate-specific effect of autophosphorylation.

Spontaneously active tyrosine-specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51,000 for TyrK I and two protein bands of Mr 55,000 and 54,000 for TyrK II. After incubation in the presence of [gamma-32P]ATP, the bands corresponding to both protein kinases contained phosphotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).