Le Maithao N, Kohanski Ronald A, Wang Lu-Hai, Sadowski Henry B
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Mol Endocrinol. 2002 Dec;16(12):2764-79. doi: 10.1210/me.2002-0017.
Insulin stimulates signal transducer and activator of transcription 5 (Stat5) activation in insulin receptor (IR)-overexpressing cell lines and in insulin target tissues of mice. Stat5b and insulin receptor substrate 1 (IRS-1) interact with the same autophosphorylation site in the IR [phosphotyrosine (pY) 972] in yeast two-hybrid assays, and the IR phosphorylates Stat5b in vitro. These data suggest that Stat5 proteins might be recruited to, and phosphorylated by, the activated IR in vivo. Nevertheless, insulin activates Janus kinases (JAKs) in IR-overexpressing cell lines and in insulin target tissues. To determine whether Stat5 proteins must be recruited to the pY972LSA motif in the IR for insulin-stimulated activation in mammalian cells, we generated and tested a series of IR mutants. The L973R/A975D mutation abolishes the ability of the IR to induce Stat5 activation, whereas IRS-1 phosphorylation is unaffected. In contrast, the N969A/P970A mutation in the IR has no effect on Stat5 activation but significantly reduces IRS-1 phosphorylation. In coimmunoprecipitation assays, insulin-stimulated Stat5 activation correlates with Stat5 recruitment to the IR. We also find that insulin stimulates tyrosine phosphorylation of JAKs that are constitutively associated with the IR. Expression of dominant-negative (DN) JAKs, the JAK inhibitor suppressor of cytokine signaling 1, or pretreatment with the JAK inhibitor, AG490, reduces, but does not eliminate, insulin-induced Stat5 activation. Expression of the appropriate pair of DN JAKs in each of the singly JAK-deficient cell lines further establishes a component of insulin-stimulated Stat5 activation that is JAK independent. This likely represents phosphorylation of Stat5 proteins by the IR, as we find that IR kinase domain phosphorylates Stat5b in vitro on Y699 as efficiently as JAK2. Increasing the concentration of Stat5 proteins in cells favors the direct phosphorylation of Stat5 by the IR kinase where the DN-JAK inhibition of insulin-stimulated Stat5 activation becomes insignificant. At physiological levels of Stat5 however, we propose that JAKs and the IR both contribute to the insulin-induced phosphorylation of Stat5.
胰岛素可刺激信号转导及转录激活因子5（Stat5）在胰岛素受体（IR）过表达细胞系及小鼠胰岛素靶组织中的激活。在酵母双杂交试验中，Stat5b与胰岛素受体底物1（IRS-1）可与IR中相同的自磷酸化位点[磷酸酪氨酸（pY）972]相互作用，并且IR可在体外使Stat5b磷酸化。这些数据表明，Stat5蛋白可能在体内被募集至激活的IR并被其磷酸化。然而，胰岛素可在IR过表达细胞系及胰岛素靶组织中激活Janus激酶（JAK）。为了确定在哺乳动物细胞中，胰岛素刺激激活Stat5蛋白是否必须被募集至IR中的pY972LSA基序，我们构建并测试了一系列IR突变体。L973R/A975D突变消除了IR诱导Stat5激活的能力，而IRS-1磷酸化不受影响。相反，IR中的N969A/P970A突变对Stat5激活无影响，但显著降低了IRS-1磷酸化。在免疫共沉淀试验中，胰岛素刺激的Stat5激活与Stat5募集至IR相关。我们还发现，胰岛素可刺激与IR组成型相关的JAK的酪氨酸磷酸化。表达显性负性（DN）JAK、细胞因子信号转导抑制因子1（JAK抑制剂）或用JAK抑制剂AG490预处理，可降低但不能消除胰岛素诱导的Stat5激活。在每个单JAK缺陷细胞系中表达合适的一对DN JAK，进一步证实了胰岛素刺激的Stat5激活存在一个不依赖JAK的成分。这可能代表IR对Stat5蛋白的磷酸化，因为我们发现IR激酶结构域在体外可与JAK2一样高效地使Stat5b在Y699位点磷酸化。增加细胞中Stat5蛋白的浓度有利于IR激酶对Stat5的直接磷酸化，此时DN-JAK对胰岛素刺激的Stat5激活的抑制作用变得微不足道。然而，在Stat5的生理水平下，我们认为JAK和IR均参与胰岛素诱导的Stat5磷酸化。
