YMXM Motifs and Signaling by an Insulin Receptor Substrate 1 Molecule without Tyrosine Phosphorylation Sites

Abstract

Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) by the activated receptors for insulin, IGF-1, and various cytokines creates binding sites for signaling proteins with Src homology 2 domains (SH2 proteins). Determining the role of specific SH2 proteins during insulin signaling has been difficult because IRS-1 possesses as many as 18 potential tyrosine phosphorylation sites, several of which contain redundant motifs. Using 32D cells, which contain no endogenous IRS proteins, we compared the signaling ability of an IRS-1 molecule in which 18 potential tyrosine phosphorylation sites were replaced by phenylalanine (IRS-1^F18) with two derivative molecules which retained three YMXM motifs (IRS-1^3YMXM) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1^YCT). During insulin stimulation, IRS-1^F18 failed to undergo tyrosine phosphorylation or mediate activation of the phosphotidylinositol (PI) 3′-kinase or p70^s6k; IRS-1^YCT was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1^3YMXM nor IRS-1^YCT mediated activation of mitogen-activated protein kinases. IRS-1^F18 and IRS-1^YCT partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-1^YCT do not augment this signal. IRS-1^3YMXM mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1^3YMXM with PI 3′-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3′-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.