Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis.
Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis.
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DOI：
10.1016/j.biocel.2015.02.013
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发表时间：
2015-05
期刊：
影响因子：
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通讯作者：
Koki Nagano;H. Takeuchi;Jing Gao;Y. Mori;Takahito Otani;Daguang Wang;M. Hirata
中科院分区：
文献类型：
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作者：
Koki Nagano;H. Takeuchi;Jing Gao;Y. Mori;Takahito Otani;Daguang Wang;M. Hirata
胰岛素通过在细胞表面获得可用数量的葡萄糖转运蛋白4（GLUT 4）来触发葡萄糖摄取到骨骼肌和脂肪组织中。GLUT 4负载囊泡通过主要由Akt调控的多个运输和融合过程从细胞内储库靶向质膜。然而，胰岛素如何促进细胞表面的GLUT 4表达仍然是未知的。在本研究中，我们确定tomosyn在Ser-783作为一个可能的Akt底物基序，并检查是否在Ser-783磷酸化参与GLUT 4表达的调节。Akt 1和Akt 2磷酸化野生型tomosyn，但不是突变体tomosyn中的Ser-783被替换为Ala。在胰岛素刺激下，完整细胞中tomosyn的Ser-783位也发生磷酸化，这种磷酸化可被PI 3 K抑制剂LY 294002阻断。体外pull-down实验表明，Akt对tomosyn的Ser-783位磷酸化可抑制与syntaxin 4的相互作用。胰岛素刺激增加了CHO-K1细胞表面的GLUT 4，以促进葡萄糖摄取，然而，外源性表达的突变体tomosyn减弱了胰岛素的增加。这些结果表明，tomosyn的Ser-783是Akt的靶点，并参与与syntaxin 4的相互作用。
Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002.In vitropull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.