4PS/Insulin Receptor Substrate (IRS)-2 Is the Alternative Substrate of the Insulin Receptor in IRS-1-deficient Mice

Abstract

(1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.Stimulation of the insulin and IGF-1 1 receptor tyrosine kinases results in rapid autophosphorylation and subsequent phosphorylation of cytoplasmic substrates. A major substrate of the insulin receptor is IRS-1, a cytoplasmic protein of 160 -185 kDa on SDS-PAGE (1-3). Following insulin/IGF-1 stimulation, IRS-1 is rapidly phosphorylated on multiple tyrosines (4). This results in docking of several SH2 domain proteins, including: the p85 subunit of PI 3-kinase (5-8), an upstream element in insulin-stimulated glucose transport and activation of p70 S6 kinase (9, 10); Grb 2, an adapter molecule linking IRS-1 to activation of Ras and mitogen-activated protein kinase (11-13); and the tyrosine phosphatase SHPTP2 (14, 15). Insulin and IGF-1 receptors can also phosphorylate other cytoplasmic proteins. These include Shc, a cytoplasmic protein which binds to Grb 2 (16), a p62 protein which associates with Ras-GAP (17), and a 55-60-kDa protein which associates with PI 3-kinase (18,19).Abundant evidence from Xenopus oocytes (20), cell culture systems (21-23), and animal models (24,25) has demonstrated the central role of IRS-1 in mediating downstream effects of insulin and IGF-1. Recently, we (26) and others (27) have shown that mice made IRS-1-deficient by targeted gene knockout exhibit hyperinsulinemia, glucose intolerance, and marked growth retardation. However, IRS-1-deficient mice continue to exhibit some insulin-stimulated glucose disposal and phosphotyrosine-associated PI 3-kinase activation, suggesting the presence of an IRS-1-independent pathway of signaling. Immunoblots from both liver and muscle tissue of IRS-1 (Ϫ/Ϫ) animals reveal a ϳ180-kDa protein (tentatively designated IRS-2) which is tyrosine-phosphorylated within 1 min of insulin stimulation and binds to PI 3-kinase...

Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor

“…After activation of receptor tyrosine kinase, a number of substrates and signalling pathways are activated. IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine residues [14][15][16], as are the cytosolic proteins p46Shc and p52Shc [17,18]. IRS-1, IRS-2 and Shc link the activated receptor to downstream signalling pathways.…”

Crk protein binds to PDGF receptor and insulin receptor substrate-1 with different modulating effects on PDGF- and insulin-dependent signaling pathways

“…Many of the e ects of insulin are thought to be mediated by the interaction of IRS-1 and IRS-2 (Sun et al, 1991) with SH2-containing enzymes and adaptor molecules . IRS-2 is the alternative substrate of IR in IRS-1 de®cient mice and has signi®cant structural similarity to IRS-1 (Patti et al, 1995). Recently another insulin receptor substrate (termed IRS3) was described in rat adipocytes (Smith-Hall et al, 1997).…”

The Insulin‐Like Growth Factor System

“…All conserved tyrosine residues that are phosphorylated in the IR in response to insulin are also phosphorylated in the IGF-IR in response to IGF-I. Thus, the IGF-IR and IR share many substrates, such as the members of the IR substrate (IRS) family (IRS-1 to IRS-4), Gab-1, and Shc (Fantin et al, 1998;Lavan and Lienhard, 1993;Patti et al, 1995;Pelicci et al, 1992;Winnay et al, 2000). IRS-1 is the best characterized of the IRS family members.…”