Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation.
Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation.
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DOI：
10.1210/me.2005-0536
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发表时间：
2006-08
期刊：
影响因子：
--
通讯作者：
Sethi JK
中科院分区：
文献类型：
--
作者：
Nawaratne R;Gray A;Jørgensen CH;Downes CP;Siddle K;Sethi JK
胰岛素受体底物（IRS）蛋白丝氨酸残基的磷酸化是一种重要的翻译后修饰，与胰岛素抵抗有关。IRS 1上的几个磷酸丝氨酸位点已被确定;大多数位于磷酸酪氨酸结合结构域附近或关键受体酪氨酸激酶底物和/或Src同源2结构域结合位点附近。在这里，我们报告的N-末端pleckstrin同源（PH）域的IRS 1的丝氨酸磷酸化位点的表征。生物信息学工具将丝氨酸24（Ser 24）鉴定为丝氨酸激酶的蛋白激酶C（PKC）家族的推定底物位点。我们证明，这个网站确实是一个真正的传统PKC基板。在体内，IRS-1也在佛波醇12-肉豆蔻酸酯13-乙酸酯处理细胞后在Ser 24上磷酸化，亚型选择性抑制剂研究表明PKCα参与其中。通过比较佛波醇12-肉豆蔻酸酯13-乙酸酯刺激的Ser 24磷酸化与先前与PKC活性相关的另外两个位点（Ser 307和Ser 612）磷酸化的药理学特征，我们表明PKCα可能直接参与Ser 24磷酸化，但间接参与Ser 307和Ser 612磷酸化。使用Ser 24 Asp IRS-1突变体来模拟磷酸化残基，我们证明Ser 24的磷酸化状态确实在调节磷酸肌醇结合和IRS 1-PH结构域的细胞内定位中发挥重要作用，这最终可以影响胰岛素刺激的葡萄糖摄取。因此，我们提供的证据表明，IRS 1-PH结构域的功能是重要的正常胰岛素信号转导，并调节丝氨酸磷酸化的方式，可能有助于胰岛素抵抗。
Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important post-translational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCα. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCα is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.
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