Insulin receptor substrate-1 enhances growth hormone induced proliferations

被引:42
作者
Liang, L
Zhou, T
Jiang, J
Pierce, JH
Gustafson, TA
Frank, SJ
机构
[1] Univ Alabama Birmingham, Dev Res & Endocrinol Branch, Univ Alabama Stn, Dept Med,Div Endocrinol & Metab, Birmingham, AL 35294 USA
[2] Univ Alabama Birmingham, Dept Med, Div Rheumatol, Birmingham, AL 35294 USA
[3] Univ Alabama Birmingham, Dept Cell Biol, Birmingham, AL 35294 USA
[4] Vet Affairs Med Ctr, Birmingham, AL 35294 USA
[5] NCI, Cellular & Mol Biol Lab, NIH, Bethesda, MD 20892 USA
[6] Metabolex Inc, Hayward, CA 94545 USA
关键词
D O I
10.1210/en.140.5.1972
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
GH exerts a variety of metabolic and growth-promoting effects. GH induces activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, JAK2, resulting in tyrosine phosphorylation of the GHR and activation of STAT (signal transducer and activator of transcription), Ras-mitogen-activated protein kinase, and phosphoinositol 3-kinase signaling pathways, among others. GH-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins has been demonstrated in vitro and in vivo. IRS-1 is a multiply phosphorylated cytoplasmic docking protein involved in metabolic and proliferative signaling by insulin, IL4, and other cytokines, but the physiological 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 tyrosine phosphorylation of IRS-1 and specific GH-induced coimmunoprecipitation with JAK2 of a tyrosine phosphoprotein consistent with IRS-1. We further examined this interaction by in vitro affinity precipitation experiments with glutathione-S-transferase fusion proteins incorporating regions of rat IRS-l and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins containing amino-terminal regions of IRS-I that include the pleckstrin homology, phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains, but not those containing other IRS-1 regions or glutathione-S-transferase alone, bound JAK2 from cell extracts. Tyrosine-phosphorylated JAK2 resulting from GH stimulation was included in the amino-terminal IRS-1 fusion precipitates; however, neither tyrosine phosphorylation of JAK2 nor treatment of cells with GH before extraction was necessary for the specific JAK2-IRS-1 interaction to be detected. In contrast, in this assay, specific insulin receptor association with the IRS-1 phosphotyrosine-binding, and Shc and IRS-I NPXY-binding domains was insulin and phosphotyrosine dependent, as previously shown. 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 three independent methods. GH induced proliferation in 32D-rGHR cells, even in the absence of transfected IRS-I. Notably, however, GH-induced proliferation was markedly enhanced in cells expressing IRS-I. Similarly, GH-induced mitogen-activated protein kinase activation was significantly augmented in IRS-l-expressing cells relative to that in cells harboring no IRS-1. These results indicate that IRS-1 enhances GH-induced proliferative signaling.
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页码:1972 / 1983
页数:12
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