Role of IRS-1-GRB-2 complexes in insulin signaling.

Abstract

GRB-2 is a small S112-and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p2l1 during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-i. Coexpression of IRS-1 or IRS-1F-s95 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p2l1 and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21r' and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-s95, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21r'-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.IRS-1 is a 131-kDa protein that undergoes tyrosine phosphorylation during stimulation of cells with insulin, insulin-like growth factor 1 (IGF-1), and interleukin 4 (IL-4) and immediately associates with signaling molecules which contain Src homology 2 domains (SH2 proteins) (23,25,36,37,40,41). Overexpression of IRS-1 increases the proliferative response of Chinese hamster ovary cells to insulin and IGF-1 (36), whereas reduction of the IRS-1 level reduces the response (42). Moreover, 32-D myeloid progenitor cells are insensitive to insulin, IGF-1, and IL-4 because they lack IRS-1 or a functionally related molecule called 4PS (41). Expression of IRS-1 in 32-D cells rescues the mitogenic response to IGF-1 and IL-4, whereas coexpression of IRS-1 and insulin receptors (IRs) rescues the insulin response (40,41). Thus, IRS-1 appears to play an essential role for some insulin responses by linking the receptor tyrosine kinase to key SH2 proteins.IRS-1 contains more than 20 potential tyrosine phosphorylation sites, several of which bind specifically to the SH2 domains in various signaling proteins, including phosphatidylinositol (Ptdlns) 3'-kinase, GRB-2, Syp, and nck (17,18,33,37). The SH2 domains in Ptdlns 3'-kinase preferentially bind to phosphopeptides containing Y-460, Y-608, Y-939, and Y-987, whereas GRB-2 and SH-PTP2 bind phosphopeptides containing Y-895 and Y-1172, respectively (35). Tyrosine phosphorylation of IRS-1 acts as the switch to control these interactions, whereas the surrounding amino acid sequences select the ...

Retinoic acid-induced growth arrest of MCF-7 cells involves the selective regulation of the IRS-1/PI 3-kinase/AKT pathway

“…We looked at the effect of RA on phosphorylation of the p44 and p42 (erk1 and erk2) members of the MAPK pathway and phosphorylation of AKT, a serine/threonine kinase activated downstream of PI 3-kinase. Phosphorylated IRS-1 can activate erk1 and erk2 by binding Grb2 and activating the ras/MAPK pathway (Myers et al, 1994). In spite of decreased levels of phosphorylated IRS-1, we found that The effects of RA on the serine phosphorylation status of AKT kinase were not due to a change in the level of total AKT (Figure 4c).…”

c-Jun N-terminal Kinase (JNK) Mediates Feedback Inhibition of the Insulin Signaling Cascade

“…MAP kinases are activated by dual-specificity MAP kinase kinases that phosphorylate adjacent tyrosine and threonine residues (29). During insulin stimulation, ERK1 and ERK2 are strongly activated by the dualspecificity kinases MEK1 or MEK2, which are activated when Grb2/Sos bound to Irs1 or Shc activates the Ras 3 Raf cascade (53,54); however, a molecular pathway linking the receptors for proinflammatory cytokines or insulin to JNK is difficult to establish.…”

Janus Kinase-dependent Activation of Insulin Receptor Substrate 1 in Response to Interleukin-4, Oncostatin M, and the Interferons

“…32D cells expressing transfected IRS proteins were as described (51,52) and maintained in medium containing 5-10 mM histidinol (Sigma).…”