The principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, we created IRS-1-deficient mice by targeted gene mutation. These mice had no IRS-1 and showed no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also had a 50 per cent reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlated with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. Our results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signalling and for the existence of an alternative substrate of these receptor kinases.
Autoimmunity has long been linked to myocarditis and its sequela, dilated cardiomyopathy, the leading causes of heart failure in young patients. However, the underlying mechanisms are poorly defined, with most clinical investigations focused on humoral autoimmunity as the target for intervention. Here, we show that the α-isoform of myosin heavy chain (α-MyHC, which is encoded by the gene Myh6) is the pathogenic autoantigen for CD4 + T cells in a spontaneous mouse model of myocarditis. Further, we found that Myh6 transcripts were absent in mouse medullary thymic epithelial cells (mTECs) and peripheral lymphoid stromal cells, which have been implicated in mediating central and peripheral T cell tolerance, respectively. Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis, demonstrating that α-MyHC is a primary autoantigen in this disease process. Remarkably, we found that humans also lacked α-MyHC in mTECs and had high frequencies of α-MyHC-specific T cells in peripheral blood, with markedly augmented T cell responses to α-MyHC in patients with myocarditis. Since α-MyHC constitutes a small fraction of MyHC in human heart, these findings challenge the longstanding notion that autoimmune targeting of MyHC is due to its cardiac abundance and instead suggest that it is targeted as a result of impaired T cell tolerance mechanisms. These results thus support a role for T cell-specific therapies for myocarditis.
(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...
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