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Crk is a member of a family of adapter proteins predominantly composed of Src homology 2 and 3 domains, whose role in signaling pathways is presently unclear. Using an in situ electroporation system which permits the introduction of glutathione S-transferase (GST) fusion proteins into cells, we found that c-CrkII bound to p130(cas), but not to paxillin in serum-starved rat-1 fibroblasts overexpressing the human insulin receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130(cas), whereas 13 nM insulin-like growth factor-I, 16 nM epidermal growth factor (EGF), and 10% serum each showed little or no effect. We found that stress fiber formation is consistent with a change in the p130(cas).c-CrkII interactions before and after growth factor stimulation. Microinjection of either GST-Crk-SH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited stress fiber formation before and after insulin-like growth factor-I, EGF, and serum stimulation. Insulin stimulation by itself caused stress fiber breakdown and there was no additive effect of microinjection. Microinjection of anti-p130(cas) antibody also blocked stress fiber formation in quiescent cells. Microinjection of the Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like growth factor-I, EGF, and serum stimulation, but not after insulin. These data suggest that the complex containing p130(cas).c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.

The Grb10 protein appears to be an adapter protein of unknown function that has been implicated in insulin receptor (IR) signaling. The interaction of this protein with the IR has been shown to be mediated in part by the Src homology 2 (SH2) domain of Grb10. Here we demonstrate the existence of a second novel domain within Grb10 that interacts with the IR and insulin-like growth factor receptor in a kinase-dependent manner. This domain was localized to a region of approximately 50 amino acids, and we term it the BPS domain to denote its location between the PH and SH2 domains. The BPS domain does not bear any obvious resemblance to other known protein interaction domains but is highly conserved among the Grb10-related proteins Grb7 and Grb14. We show that the BPS domain interaction is dependent upon receptor tyrosine kinase activity. Furthermore, interaction of the BPS domain requires the kinase domain of the IR, since mutation of the paired tyrosine residues (Y1150F/Y1151F) within the IR activation loop dramatically reduced the interaction. Last, our data suggest that the presence of two distinct protein interaction domains may help to determine the specificity by which Grb10 interacts with different receptors. Specifically, the IR, which appears to interact most strongly with Grb10, interacts well with both the SH2 and BPS domains. Conversely, the insulin-like growth factor receptor and EGFR, which interact less avidly with Grb10, interact well only with the BPS domain or the SH2 domain, respectively. In summary, our findings demonstrate the existence of a previously unidentified tyrosine kinase activity-dependent binding domain located between the Pleckstrin homology and SH2 domains of Grb10.

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

To investigate the mechanisms underlying the enhanced mitogenic activity of the truncated epidermal growth factor receptor (EGFR) lacking the C-terminal autophosphorylation sites (Delta973-EGFR), we studied the intracellular signaling pathways in NR6 cells expressing human wild type EGFR and Delta973-EGFR. Microinjection of dominant/negative p21ras(N17) completely inhibited EGF-induced DNA synthesis in both cell types. EGF stimulated Shc phosphorylation as well as the formation of wild type EGFR.Shc complexes. In contrast, EGF stimulated Shc phosphorylation without formation of Delta973-EGFR.Shc complexes. Tyrosine-phosphorylated Shc formed complexes with Grb2.Sos, and microinjection of anti-Shc antibody and Shc-SH2 GST fusion protein inhibited EGF stimulation of DNA synthesis in both cell lines. EGF markedly increased ErbB2 tyrosine phosphorylation in wild type EGFR cells. In Delta973-EGFR cells, ErbB2 was tyrosine phosphorylated in the basal state and EGFR stimulated further phosphorylation of ErbB2. In addition to ErbB2, additional proteins were tyrosine phosphorylated in Delta973-EGFR cells, mostly in the molecular mass range of 120 170 kDa. Taken together with our findings indicating coupling of ErbB2 to Shc, these data suggest the importance of an alternative signaling pathway in Delta973-EGFR cells mediated by the formation of heterodimeric structures between the truncated EGFR and ErbB2, followed by coupling through Shc to Grb2.Sos and the p21ras pathway, ultimately leading to mitogenesis.

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.

We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.

Syp (SH-PTP2) was recently identified as a phosphotyrosine phosphatase containing two SH2 domains within its primary structure. In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. To assess the importance of Syp in mitogenic signaling, we microinjected mammalian fibroblasts with several reagents designed to interfere with Syp SH2/phosphotyrosine interaction in vivo. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, indicated by bromodeoxyuridine (BrdUrd) incorporation, was dramatically decreased following microinjection of a Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein (approximately 90%) in comparison with cells microinjected with control IgG or glutathione S-transferase (GST), respectively. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1 with an ED50 value of approximately 23 microM, also decreased BrdUrd incorporation in vivo by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide had no effect on serum-stimulated BrdUrd incorporation. In conclusion, disruption of Syp function in living cells inhibited cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction.

Shc proteins contain a single SH2 domain, lack catalytic activity, and are substrates for activated receptors for insulin, insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF). Treatment with these growth factors induced rapid tyrosine phosphorylation of Shc. We investigated the potential role of Shc in mitogenic signaling. Affinity-purified antibodies were microinjected into living Rat1 fibroblasts overexpressing human insulin receptors. Bromodeoxyuridine incorporation into newly synthesized DNA was subsequently studied to assess the importance of Shc. Cellular microinjection of anti-Shc antibody inhibited BrdU incorporation induced by insulin, IGF-1, and EGF, but did not affect cells stimulated by fetal calf serum. Microinjection of an oncogenic p21ras protein (T24) into quiescent cells produced constitutively active mitogenic signaling, and comicroinjection of T24 with the anti-Shc antibody restored insulin and EGF stimulation of DNA synthesis. Immunoprecipitates of Shc from lysates of insulin-stimulated cells removed 70-80% of guanine nucleotide-releasing factor activity. These results indicate that Shc is an important component in a mitogenic signal transduction pathway that is shared by insulin, IGF-1, and EGF. The functional locus of Shc is either upstream of p21ras or lies on a distinct branch of the pathway leading to cell cycle progression.

The heme-sensitive eukaryotic initiation factor (eIF)-2 alpha kinase regulates translational activity in reticulocytes by phosphorylation of the smallest subunit of eukaryotic peptide initiation factor 2, eIF-2. Highly purified preparations of the kinase contain an abundant 90-kDa polypeptide which appears to modulate the activity of the enzyme. The physical properties and structural characteristics of the reticulocyte 90-kDa peptide are similar to those of the 90-kDa heat shock protein (hsp 90) from HeLa and other mammalian cells. The reticulocyte and HeLa cell proteins are shown to be immunologically cross-reactive. A direct comparison of the two proteins by one-dimensional peptide mapping of large peptides generated by limited proteolysis and by reversed-phase high performance liquid chromatography analysis of tryptic peptides indicates that they represent the same protein species. Like the 90-kDa reticulocyte protein, HeLa cell hsp 90 causes increased eIF-2 alpha phosphorylation by the heme-sensitive kinase and is a potent inhibitor of protein synthesis in the reticulocyte lysate system. A potential mechanism for the latter inhibition is inferred. These results implicate hsp 90 in the regulation of protein synthesis via its interaction with and perhaps regulation of the heme-sensitive kinase and phosphorylation of eIF-2 alpha.