Mîinea Cristinel P, Sano Hiroyuki, Kane Susan, Sano Eiko, Fukuda Mitsunori, Peränen Johan, Lane William S, Lienhard Gustav E
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Biochem J. 2005 Oct 1;391(Pt 1):87-93. doi: 10.1042/BJ20050887.
Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.
最近，我们描述了一种160 kDa的蛋白质（命名为AS160，即160 kDa的Akt底物），它具有一个预测的Rab GAP（GTP酶激活蛋白）结构域，可被蛋白激酶Akt在多个位点磷酸化。脂肪细胞中AS160的磷酸化是胰岛素刺激葡萄糖转运蛋白GLUT4转位到质膜所必需的。本研究的目的是确定AS160是否实际上是Rabs的GAP，如果是，其特异性是什么。我们首先通过质谱在含有GLUT4的细胞内囊泡制剂中鉴定出一组16种Rabs。然后我们制备了AS160的重组GAP结构域，并检测了其对许多这些Rabs以及其他几种Rabs的活性。该GAP结构域对Rab 2A、8A、10和14具有活性。对其他14种Rabs没有明显活性。通过将预测的催化精氨酸残基替换为赖氨酸的重组GAP结构域无活性这一发现，进一步验证了GAP活性。最后，通过免疫印迹发现Rab 2A、8A和14存在于GLUT4囊泡中。这些结果表明AS160是一种Rab GAP，并提示可能参与GLUT4转位的新型Rabs。
