Growth Hormone, Interferon-γ, and Leukemia Inhibitory Factor Utilize Insulin Receptor Substrate-2 in Intracellular Signaling

Nils Billestrup,Morris F. White,Gunnar Norstedt,Lawrence S. Argetsinger,Christin Carter-Su

doi:10.1074/jbc.271.46.29415

Abstract

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.

Highlights

IRS-11 is a major cytoplasmic substrate of insulin receptor [1] and several cytokine receptors that are coupled to Janus kinases
To determine if insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated in response to growth hormone (GH), 3T3-F442A fibroblasts were incubated for 15 min with different concentrations of GH
Consistent with this phosphoprotein being IRS-2, it co-migrates with a protein in the immunoprecipitate that is recognized by ␣IRS-2 in immunoblots (Fig. 1, lane A), co-migrates with IRS-2 phosphorylated in response to IGF-1 and insulin (Fig. 1, lanes G and H), and migrates as a slightly larger protein than IRS-1 (Fig. 1, lane I)

Summary

Introduction

IRS-11 is a major cytoplasmic substrate of insulin receptor [1] and several cytokine receptors that are coupled to Janus kinases. IRS-2 has substantial structural similarity to IRS-1 with multiple potential tyrosyl phosphorylation sites, 13 of which are identical or show substantial identity to sites present in IRS-1 Conserved sites include those previously seen to bind Grb, the protein-tyrosine phosphatase SHP2, and the 85-kDa regulatory subunit of PI 3Ј-kinase. GH is shown to promote association of IRS-2 with both PI 3Ј-kinase and SHP2 This and recent reports of IRS-2 tyrosyl phosphorylation in response to IL-2, IL-4, and IL-7 (cytokines that activate JAK1 and JAK3; Ref. 10), and IFN␣ (a cytokine that activates JAK1 and Tyk; Ref. 11) suggest that signaling through IRS-2 may be a common element in signaling for many members of the cytokine family of receptors that activate JAK tyrosine kinases

Results

Discussion

Conclusion