Enhancement or inhibition of insulin signaling by insulin receptor substrate 1 is cell context dependent.

Abstract

Insulin treatment of Chinese hamster ovary (CHO) cells expressing high levels of the insulin receptor (CHO/IR cells) activates both c-fos serum response element and activator protein 1 (AP-1) reporter genes approximately 10-fold. In contrast, parental CHO cells display only two-to threefold insulin stimulation of reporter gene activity. Transient transfection of parental CHO cells with an insulin receptor substrate 1 (IRS1) expression plasmid enhanced insulin downstream signaling in a biphasic manner, whereas IRS1 transfection of CHO/IR cells inhibited insulin signaling in a dose-dependent fashion. Further, expression of Grb2 in parental CHO cells had no effect on insulin signaling, whereas Grb2 increased insulin activation of reporter gene expression in CHO/IR cells. These data suggest that the expression levels of various effector molecules can either enhance or inhibit insulin downstream signaling events. To assess the relative effects of various insulin receptor, IRS1, and Grb2 levels on insulin signaling, parental CHO cells were transiently transfected with various combinations of expression plasmids encoding these proteins. Although expression of IRS1 resulted in a biphasic increase of insulin signaling in parental CHO cells, coexpression of IRS1 with the insulin receptor resulted in inhibition of signaling. This inhibition of insulin signaling directly correlated with an increased association of Grb2 with IRS1 and a concomitant sequestration of Grb2 away from Shc. Consistent with the Shc-Grb2 pathway as the major route for insulin-stimulated c-Fos and AP-1 transcriptional activation, the IRS1-mediated inhibition was reversed by transfection with an expression plasmid for Grb2. These data demonstrate that the extent of insulin-stimulated downstream signaling was dependent not only on the levels of individual signaling molecules but also on the formation of multiprotein complexes with specific stoichiometries.The two major proximal intracellular targets for the kinase activity of the insulin and insulin-like growth factor 1 (IGF-1) receptors have been identified as a 185 kDa-protein, termed IRS1 (for insulin receptor substrate 1) (13,36,48), and proteins of 46 and 52 kDa, termed Shc (for Src homology 2/a-collagen related) (26, 32). These molecules contain insulin receptor-specific tyrosine phosphorylation sites responsible for their association with various downstream effector molecules (23, 47). For example, the tyrosine phosphorylation motifs in IRS1 provide recognition sites for the specific binding of SH2 (Src homology 2) domain-containing proteins, including the p85 subunit of the phosphatidylinositol 3-kinase, the small adaptor protein Grb2, and the SH2 domain-containing protein tyrosine-specific phosphatase SHPTP2 (34).The

Growth and Differentiation Signals by the Insulin-like Growth Factor 1 Receptor in Hemopoietic Cells Are Mediated through Different Pathways

“…We can speculate upon the relative proportions of IRS-1 and Shc proteins in determining mitogenesis or differentiation, a hypothesis that was indirectly suggested by the work of Yamauchi and Pessin (39). In agreement with them (39), as already mentioned, the amount of Grb2 that co-precipitates with either IRS-1 or Shc depends on the levels and phosphorylation status of these substrates.…”

“…One important question is whether the effect of IRS-1 on differentiation may be due to its ability to compete with Shc for a common receptor binding site and to sequester substrates common to both IRS-1 and Shc, for instance Grb2 as suggested some years ago by Yamauchi and Pessin (39). For this purpose, we tested the two cell lines described above, both of them overexpressing the IGF-IR and IRS-1 but where one of the cell lines expresses IRS-1 in much larger amounts than the other.…”

Different Effects on Mitogenesis and Transformation of a Mutation at Tyrosine 1251 of the Insulin-like Growth Factor I Receptor

“…Not surprisingly, our experiments do not show gross differences in tyrosyl phosphorylation of the IGF-IR or IRS-1, which are involved in insulin and IGF-I-mediated mitogenesis (Waters et al (1993), Rose et al (1994), Yamauchi and Pessin (1994), and see review by White and Kahn (1994)). We say not surprisingly, because both mutant receptors are fully mitogenic, and, therefore, there is no a priori reason why the known mitogenic pathways of the IGF-IR should be affected.…”

“…But again, Shc phosphorylation was determined in serum-free medium supplemented with IGF-I, whereas the transformation assay is done in 10% serum, which contains other growth factors capable of activating Shc (White and Kahn, 1994). Also one cannot ignore some recent reports that there is some kind of balance between IRS-1 and Shc that is important for the stimulation of DNA synthesis by the IR (Yamauchi and Pessin, 1994), and that a transmembrane mutant of the v-ros oncogene that had lost its transforming activity still retained its ability to phosphorylate Shc (Zong and Wang, 1994).…”

Insulin-Stimulated Disassociation of the SOS-Grb2 Complex

“…We have recently demonstrated that electroporation can be used to express various cDNAs in CHO-IR cells with 85 to 100% transfection efficiency (52). Briefly, CHO-IR cells were electroporated with a total of 40 g of plasmid DNA at 340 V and 960 F in 500 l of phosphate-buffered saline.…”