YMXM Motifs and Signaling by an Insulin Receptor Substrate

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-1F18) with two derivative molecules which retained three YMXM motifs (IRS-13YMXM) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1YCT). During insulin stimulation, IRS-1F18 failed to undergo tyrosine phos-phorylation or mediate activation of the phosphotidylinositol (PI) 3*-kinase or p70s6k; IRS-1YCT was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-13YMXM nor IRS-1YCT mediated activation of mitogen-activated protein kinases. IRS-1F18 and IRS-1YCT partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phos-photyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-1YCT do not augment this signal. IRS-13YMXM 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-13YMXM with P