Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling
Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling
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DOI：
10.1074/jbc.m410610200
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发表时间：
2005-02-11
影响因子：
4.8
通讯作者：
Eldar-Finkelman, H
中科院分区：
文献类型：
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作者：
Liberman, Z;Eldar-Finkelman, H
糖原合成酶激酶-3（GSK-3）磷酸化胰岛素受体底物-1（IRS-1）的能力是2型糖尿病胰岛素抵抗的潜在抑制机制。然而，IRS-1中GSK-3磷酸化的丝氨酸位点尚未确定。使用N-末端缺失的IRS-1突变体和两个IRS-1片段PTB-1（1-320）和PTB-2（1-350），我们将GSK-3磷酸化位点定位在氨基酸序列320-350内。位于PTB-2或IRS-1内GSK-3共有基序（SXXXS）中的丝氨酸332或336突变为丙氨酸消除了GSK-3对它们的磷酸化。这表明Ser（332）是GSK-3磷酸化位点，并且Ser（336）充当GSK-3作用通常所需的“引发”位点。事实上，IRS-1的去磷酸化阻止了GSK-3的磷酸化。此外，与不被GSK-3磷酸化的非磷酸化肽相反，源自IRS-1序列的磷酸化肽容易被GSK-3磷酸化。当IRS-1突变体S332 A（IRS-1）、S336 A（IRS-1）或S332 A/336 A（IRS-1）在过表达胰岛素受体的中国仓鼠卵巢细胞中表达时，与野生型（WT）IRS-1相比，其胰岛素诱导的酪氨酸磷酸化水平增加。这种作用在双突变体S332 A/336 A（IRS-1）中更强，并导致胰岛素介导的蛋白激酶B活化增强。最后，用针对IRS-1在Ser（332）处磷酸化的多克隆抗体的免疫印迹分析证实了培养细胞中IRS-1的磷酸化。此外，治疗GSK-3抑制剂锂减少丝氨酸332磷酸化，而GSK-3过表达增强这种磷酸化。综上所述，我们的研究确定Ser（332）为IRS-1中GSK-3磷酸化的靶点，表明其生理相关性并证明其在胰岛素信号传导中的新抑制作用。
The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1(1-320) and PTB-2(1-350), we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser(332) is a GSK-3 phosphorylation site and that Ser(336) serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser(332) confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser 332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser(332) as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.