Activation Of Insulin Receptor Substrate Research Articles

Angiotensin II Stimulates Tyrosine Phosphorylation and Activation of Insulin Receptor Substrate 1 and Protein-tyrosine Phosphatase 1D in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) and insulin-like growth factor I (IGF I) stimulate intracellular signaling events through binding to their respective G-protein-coupled and growth factor receptors. In rat aortic vascular smooth muscle cells, IGF I (20 ng/ml) induced a sustained (>30 min) increase in the tyrosine phosphorylation of both Src-homology 2 domain-docking insulin receptor substrate 1 (IRS-1) and Src-homology 2-binding tyrosine phosphatase 1D (PTP-1D). In addition, IGF I stimulated PTP-1D phosphatase activity. Ang II (10(-7) M) also increased the tyrosine phosphorylation of IRS-1 (4-fold), PTP-1D (5-fold), and PTP-1D activity (3-4-fold), but with a more transient time course. Ang II also induced PTP-1D.IRS-1 complex formation. These Ang II-induced events were not affected by preincubation with an anti-IGF I antibody, suggesting that Ang II's actions were not mediated via the autocrine secretion of IGF I. Anti-PTP-1D antibody electroporation attenuated Ang II-induced PTP-1D.IRS-1 complex formation and PTP-1D tyrosine phosphorylation and activation. Our findings show that the tyrosine phosphorylation of IRS-1 and PTP-1D represents a convergent intracellular signaling cascade stimulated by both growth factor (i.e. IGF I) and G-protein-coupled (i.e. AT1) receptors.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Neoplastic Transformation Induced by Insulin Receptor Substrate-1 Overexpression Requires an Interaction with Both Grb2 and Syp Signaling Molecules

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.

Phosphorylation of Insulin Receptor Substrate-1 on Multiple Serine Residues, 612, 632, 662, and 731, Modulates Insulin Action

Okadaic acid has been described previously as being a negative regulator of insulin signaling, as it inhibits insulin stimulation of glucose transport. In addition, this drug induces on insulin receptor substrate-1 (IRS-1) a decrease in tyrosine phosphorylation, concomitantly with an increase in serine/threonine phosphorylation. The present work was aimed at the identification of the serine/threonine residues that, upon phosphorylation, might be involved in modulating insulin signaling. To this end, we studied double-point mutants of IRS-1, in which serines 612/632 and 662/731 were replaced with alanine. These are four plausible sites of phosphorylation by mitogen-activated protein kinases and are in the immediate proximity of tyrosine residues, which are potential sites of interaction with phosphatidylinositol 3-kinase Src homology 2 domains. Using transient expression in 293 EBNA cells, we demonstrate that serines 612, 632, 662, and 731 and mitogen-activated protein kinases are not involved in the okadaic acid effect on IRS-1. Rather, these serines appear to play a role in modulating basal and insulin-stimulated IRS-1 tyrosine phosphorylation, association of IRS-1, with p85, and phosphatidylinositol 3-kinase activity in the IRS-1.p85 immune complex, since mutation of these sites enhances these events. Our findings suggest the existence of an IRS-1 desensitization mechanism resulting from serine/threonine phosphorylation, occurring at least on serines 612, 632, 662, and 731.

Effects of contractile activity on tyrosine phosphoproteins and PI 3-kinase activity in rat skeletal muscle

Insulin stimulates signaling reactions that include insulin receptor autophosphorylation and tyrosine kinase activation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and phosphatidylinositol 3-kinase (PI 3-kinase) activation. Muscle contraction has metabolic effects similar to insulin, and contraction can increase insulin sensitivity, but little is known about the molecular signals that mediate the effects of contraction. To investigate the effects of muscle contraction on insulin signaling, rats were studied after contraction of hindlimb muscles by electrical stimulation, maximal insulin injection in the absence of contraction, or contraction followed by insulin injection. Insulin increased tyrosine phosphorylation of the insulin receptor and IRS-1, whereas contraction alone had no effect. Contraction before insulin injection decreased the insulin effect on receptor and IRS-1 phosphorylation by 20-25%. Increased tyrosine phosphorylation of other proteins by insulin and/or contraction was not observed. Contraction alone had little effect on PI 3-kinase activity, but contraction markedly blunted the insulin-stimulated activation of IRS-1 and insulin receptor-immunoprecipitable PI 3-kinase. In conclusion, skeletal muscle contractile activity does not result in tyrosine phosphorylation of molecules involved in the initial steps of insulin signaling. Although contractile activity increases insulin sensitivity and responsiveness in skeletal muscle, contraction causes a paradoxical decrease in insulin-stimulated tyrosine phosphorylation and PI 3-kinase activity.