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.Insulin rapidly stimulates glucose transport into fat and muscle cells by causing the insertion of additional glucose transporters of the GLUT4 isotype into the plasma membrane, in a process referred to as GLUT4 translocation. The overall process consists of generation of the specialized vesicles containing GLUT4 from the endosomal system, the movement of these vesicles from the perinuclear region to the plasma membrane, and the fusion of the vesicles with the plasma membrane (1). The steps in this process that insulin accelerates, and the complete signaling pathways from the insulin receptor that lead to their acceleration, have not yet been fully defined. One partial insulin signaling pathway that has been established to be required for GLUT4 translocation is the pathway that proceeds from the receptor through tyrosine phosphorylation of the insulin receptor substrates to activation of phosphatidylinositol 3-kinase and generation of phosphatidylinositol 3,4,5-trisphosphate. The latter leads to the activation of the protein kinase Akt and also protein kinase C /, and there is evidence that GLUT4 translocation requires the activation of one or both of these kinases (reviewed in Refs. 2 and 3). However, although a substrate linking either kinase to GLUT4 translocation has been sought for several years, hitherto none has been clearly identified.Recently, we reported the discovery of a new substrate for insulin-activated Akt in 3T3-L1 adipocytes, which was designated AS160 for Akt subtrate of 160 kDa (4). The most prominent feature of AS160 is the presence of a GTPase activating domain for a Rab. Since Rabs are small G proteins that play critical roles in vesicle formation, movement, and fusion (5), we investigated the role of AS160 in GLUT4 translocatio...

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.

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