Sano Hiroyuki, Kane Susan, Sano Eiko, Mîinea Cristinel P, Asara John M, Lane William S, Garner Charles W, Lienhard Gustav E
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 2003 Apr 25;278(17):14599-602. doi: 10.1074/jbc.C300063200. Epub 2003 Mar 11.
Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.
胰岛素可刺激脂肪和肌肉细胞中GLUT4亚型的细胞内葡萄糖转运体快速转运至质膜。已知的胰岛素信号通路与这一膜转运过程的蛋白质机制之间的联系尚未完全明确。最近，我们在脂肪细胞中鉴定出一种160 kDa的蛋白质，命名为AS160，它可被胰岛素激活的激酶Akt磷酸化。该蛋白质含有一个针对Rabs的GTPase激活结构域（GAP），Rabs是膜转运所需的小G蛋白。在本研究中，我们鉴定出AS160在体内的六个磷酸化位点。这些位点位于Akt磷酸化的特征基序中，胰岛素处理可增加其中五个位点的磷酸化。将AS160的两个或更多这些位点突变为丙氨酸后进行表达，可显著抑制3T3-L1脂肪细胞中胰岛素刺激的GLUT4转运。此外，当磷酸化位点突变体中的GAP功能通过点突变失活时，这种抑制作用并未发生。这些发现有力地表明，AS160的胰岛素刺激磷酸化是GLUT4转运所必需的，并且这种磷酸化通过失活Rab GAP功能来信号传导转运。
