AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain
AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain
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DOI：
10.1042/bj20050887
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发表时间：
2005-10-01
影响因子：
4.1
通讯作者：
Lienhard, GE
中科院分区：
文献类型：
--
作者：
Mîinea, CP;Sano, H;Lienhard, GE
最近，我们描述了一个160 kDa的蛋白质（指定为AS 160，Akt底物的160 kDa）与预测的Rab GAP（GTP酶激活蛋白）结构域，是磷酸化的多个网站上的蛋白激酶Akt。脂肪细胞中AS160的磷酸化是胰岛素刺激葡萄糖转运蛋白GLUT 4向质膜转运所必需的。本研究的目的是确定AS 160是否实际上是Rabs的GAP，如果是，其特异性是什么。我们首先确定了一组16 Rabs在制备的细胞内囊泡含有GLUT4的MS。然后，我们准备了重组GAP结构域的AS 160和检查其对许多这些Rabs的活性，以及其他几个。差距结构域对Rabs 2A、8A、10和14有活性。对其他14只Rabs没有显著的活性。通过发现具有预测的催化精氨酸残基被赖氨酸取代的重组GAP结构域是无活性的，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GLUT 4囊泡中。这些结果表明AS 160是一个Rab GAP，并提示可能参与GLUT 4易位的新Rab。
Recently, we described a 160 kDa protein (designated AS 160, 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 AS 160 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 AS 160 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 AS 160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.