Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation
Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation
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DOI：
10.1074/jbc.m114.565945
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发表时间：
2014-09-19
影响因子：
4.8
通讯作者：
Mizuno, Kensaku
中科院分区：
文献类型：
--
作者：
Homma, Yuta;Kanno, Shin-ichiro;Mizuno, Kensaku
Cofilin通过其切断肌动蛋白细丝的活性来增强肌动蛋白细丝的动力学，从而在细胞迁移和形态发生中发挥重要作用。Singshot-1(SSH1)是一种蛋白磷酸酶，通过去磷酸化和重新激活cofilin来调节肌动蛋白的动态变化。在本研究中，我们发现胰岛素受体底物(IRS)-4是一种新的SSH1结合蛋白。共沉淀分析表明，Irs4与SSH1直接内源性结合。IRS4，而不是IRS1或IRS2与SSH1结合。IRS4主要通过靠近IRS4磷酸酪氨酸结合区C末端的独特区域(氨基酸335-400)与SSH1结合。SSH1的N-末端A、B和磷酸酶结构域独立地与Irs4结合。而体外磷酸酶检测表明，Irs4对SSH1的cofilin磷酸酶活性没有直接影响，敲除irs4可以增加培养细胞中cofilin的磷酸化。Irs4基因的敲除降低了磷脂酰肌醇3-激酶(PI3K)的活性，而PI3K的抑制剂处理增加了Cofilin的磷酸化。AKT优先使SSH1在Thr-826处磷酸化，但SSH1的非磷酸化突变体T826A的表达不影响胰岛素诱导的Cofilin去磷酸化，Akt的抑制剂也不增加Cofilin的磷酸化。这些结果表明，Irs4通过顺序激活PI3K和SSH1而不是通过Akt来促进Cofilin去磷酸化。此外，Irs4与SSH1共定位于胰岛素刺激细胞富含F-肌动蛋白的膜突起中，提示Irs4与SSH1的结合有助于膜突起中cofilin的局部激活。
Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament- severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.