Liang L, Zhou T, Jiang J, Pierce J H, Gustafson T A, Frank S J
Department of Medicine, University of Alabama, Birmingham 35294, USA.
Endocrinology. 1999 May;140(5):1972-83. doi: 10.1210/endo.140.5.6724.
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, IL-4, 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 pre-adipocytes 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-1 and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins containing amino-terminal regions of IRS-1 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-1 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-1. Notably, however, GH-induced proliferation was markedly enhanced in cells expressing IRS-1. Similarly, GH-induced mitogen-activated protein kinase activation was significantly augmented in IRS-1-expressing cells relative to that in cells harboring no IRS-1. These results indicate that IRS-1 enhances GH-induced proliferative signaling.
生长激素（GH）发挥多种代谢和促进生长的作用。GH诱导与生长激素受体（GHR）相关的细胞质酪氨酸激酶JAK2活化，导致GHR的酪氨酸磷酸化以及信号转导子和转录激活子（STAT）、Ras-丝裂原活化蛋白激酶和磷脂酰肌醇3-激酶信号通路等的激活。体外和体内实验均已证实GH刺激胰岛素受体底物（IRS）蛋白的酪氨酸磷酸化。IRS-1是一种多重磷酸化的细胞质对接蛋白，参与胰岛素、白细胞介素-4和其他细胞因子的代谢和增殖信号传导，但IRS-1在GH信号传导中的生理作用尚不清楚。在本研究中，正如其他人所指出的，我们在小鼠3T3-F442A前脂肪细胞中检测到了IRS-1的GH依赖性酪氨酸磷酸化以及与JAK2特异性共免疫沉淀的一种与IRS-1一致的酪氨酸磷酸化蛋白。我们通过体外亲和沉淀实验进一步研究了这种相互作用，该实验使用了包含大鼠IRS-1区域的谷胱甘肽-S-转移酶融合蛋白以及作为JAK2来源的3T3-F442A细胞提取物。含有IRS-1氨基末端区域（包括普列克底物蛋白同源性、磷酸酪氨酸结合、Shc和IRS-1 NPXY结合结构域）的融合蛋白能够结合细胞提取物中的JAK2，而包含其他IRS-1区域或单独的谷胱甘肽-S-转移酶的融合蛋白则不能。GH刺激产生的酪氨酸磷酸化JAK2包含在氨基末端IRS-1融合沉淀物中；然而，检测到特异性JAK2-IRS-1相互作用既不需要JAK2的酪氨酸磷酸化，也不需要在提取前用GH处理细胞。相反，在该实验中，胰岛素受体与IRS-1磷酸酪氨酸结合、Shc和IRS-1 NPXY结合结构域的特异性结合如先前所示依赖于胰岛素和磷酸酪氨酸。为了测试IRS-1在GH信号传导方面的重要性，用兔（r）GHR单独（32D-rGHR）或与IRS-1（32D-rGHR-IRS-1）稳定重建了IRS-和GHR缺陷的32D细胞。通过三种独立方法检测，即使在没有转染IRS-1的情况下，GH也能诱导32D-rGHR细胞增殖。然而，值得注意的是，在表达IRS-1的细胞中，GH诱导的增殖明显增强。同样，相对于不含有IRS-1的细胞，在表达IRS-1的细胞中，GH诱导的丝裂原活化蛋白激酶激活显著增强。这些结果表明IRS-1增强了GH诱导的增殖信号传导。
