Insulin receptor substrate-1 enhances growth hormone induced proliferations

GH exerts a variety of metabolic and growth-promoting effects. GH induces a ctivation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, JAK2, resulting in tyrosine phosphorylation of the GHR and activation of ST AT (signal transducer and activator of transcription), Ras-mitogen-activate d protein kinase, and phosphoinositol 3-kinase signaling pathways, among ot hers. GH-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins has been demonstrated in vitro and in vivo. IRS-1 is a multi ply phosphorylated cytoplasmic docking protein involved in metabolic and pr oliferative signaling by insulin, IL4, and other cytokines, but the physiol ogical role of IRS-1 in GH signaling is unknown. In this study, as noted by others, we detected in murine 3T3-F442A preadipocytes GH-dependent tyrosin e phosphorylation of IRS-1 and specific GH-induced coimmunoprecipitation wi th JAK2 of a tyrosine phosphoprotein consistent with IRS-1. We further exam ined this interaction by in vitro affinity precipitation experiments with g lutathione-S-transferase fusion proteins incorporating regions of rat IRS-l and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins con taining amino-terminal regions of IRS-I that include the pleckstrin homolog y, phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains, but not those containing other IRS-1 regions or glutathione-S-transferase alone, b ound JAK2 from cell extracts. Tyrosine-phosphorylated JAK2 resulting from G H stimulation was included in the amino-terminal IRS-1 fusion precipitates; however, neither tyrosine phosphorylation of JAK2 nor treatment of cells w ith GH before extraction was necessary for the specific JAK2-IRS-1 interact ion to be detected. In contrast, in this assay, specific insulin receptor a ssociation with the IRS-1 phosphotyrosine-binding, and Shc and IRS-I NPXY-b inding domains was insulin and phosphotyrosine dependent, as previously sho wn. To test for significance of IRS-1 with regard to GH signaling, IRS- and GHR-deficient 32D cells were stably reconstituted with the rabbit (r) GHR, either alone (32D-rGHR) or with IRS-1 (32D-rGHR-IRS-1). As assayed by thre e independent methods. GH induced proliferation in 32D-rGHR cells, even in the absence of transfected IRS-I. Notably, however, GH-induced proliferatio n was markedly enhanced in cells expressing IRS-I. Similarly, GH-induced mi togen-activated protein kinase activation was significantly augmented in IR S-l-expressing cells relative to that in cells harboring no IRS-1. These re sults indicate that IRS-1 enhances GH-induced proliferative signaling.