Cell Biology and Metabolism
Growth Hormone, Interferon-γ, and Leukemia Inhibitory Factor Promoted Tyrosyl Phosphorylation of Insulin Receptor Substrate-1

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The identification of JAK2 as a growth hormone (GH) receptor-associated, GH-activated tyrosine kinase has established tyrosyl phosphorylation as a signaling mechanism for GH. In the present study, GH is shown to stimulate tyrosyl phosphorylation of insulin receptor substrate 1 (IRS-1), the principle substrate of the insulin receptor. Tyrosyl phosphorylation of IRS-1 is a critical step in insulin signaling and provides binding sites for proteins with the appropriate Src homology 2 domains, including the 85-kDa regulatory subunit of phosphatidylinositol (PI) 3′-kinase. In 3T3-F442A fibroblasts, GH-dependent tyrosyl phosphorylation of IRS-1 was detected by 1 min and at GH concentrations as low as 5 ng/ml (0.23 nM). Tyrosyl phosphorylation of IRS-1 was transient, with maximal stimulation detected at 30 min and diminished signal detected at 60 min. The ability of GH receptor (GHR) to transduce the signal for IRS-1 tyrosyl phosphorylation is mediated by the intracellular region of GHR between amino acids 295 and 380 by a mechanism not involving the two tyrosines in this region. This region of GHR is required for GH-dependent JAK2 association and activation (VanderKuur, J. A., Wang, X., Zhang, L., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C.(1994) J. Biol. Chem. 269, 21709-21717). When other cytokines that activate JAK2 were tested for the ability to stimulate the tyrosyl phosphorylation of IRS-1, stimulation was detected with interferon-γ and leukemia inhibitory factor. The correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation in response to GH, interferon-γ, and leukemia inhibitory factor and in cells expressing different GHR mutants, provides evidence that IRS-1 may interact with JAK2 or an auxiliary molecule that binds to JAK2. GH is also shown to stimulate binding of IRS-1 to the 85-kDa regulatory subunit of PI 3′-kinase. The ability of GH to stimulate tyrosyl phosphorylation of IRS-1 and its association with PI 3′-kinase provides a biochemical basis for responses shared by insulin and GH including the well characterized insulin-like metabolic effects of GH observed in a variety of cell types.

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This work was supported in part by research Grants RO1-DK34171 (to C. C.-S.) and DK 43808 (to M. F. W.) from the National Institutes of Health and by Joslin's Diabetes and Endocrinology Research Center, Grant DK36836. Computer studies were supported in part by the General Clinical Research Center at The University of Michigan, funded by Grant M01 RR00042 from the National Center for Research Resources, National Institutes of Health, United State Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Recipient of Postdoctoral Fellowship IF32-DK08737 from the National Institutes of Health.

Fellow of the Markey Program in Cell and Developmental Biology, Division of Medical Sciences, Harvard Medical School.