ABSTRACT
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.
Subject(s)
GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/metabolism , Glucose Transporter Type 4/metabolism , Proto-Oncogene Proteins c-akt/metabolism , rab GTP-Binding Proteins/metabolism , Adipocytes/metabolism , Animals , Cell Line , Dogs , Enzyme Activation , Humans , Mice , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins , Transport Vesicles/metabolismABSTRACT
Insulin causes the rapid translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane in fat and muscle cells. There is considerable evidence that the signaling to this trafficking process is downstream of the insulin-activated protein kinase Akt. One Akt substrate that connects signaling to trafficking is a 160 kDa GTPase activating protein for Rabs. Another potential connecting substrate is the protein Synip, which associates with the SNARE syntaxin4. A recent study presents evidence that Akt phosphorylates Synip on serine 99, at least in vitro, and proposes that this phosphorylation enables GLUT4 translocation by causing the dissociation of Synip from syntaxin4. In the present study we show that marked overexpression of Synip mutant S99A, which lacks this phosphorylation site, has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. This finding is strong evidence that phosphorylation of Synip on serine 99 is not required for GLUT4 translocation.
Subject(s)
Insulin/pharmacology , Monosaccharide Transport Proteins/metabolism , Muscle Proteins/metabolism , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolism , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Amino Acid Substitution , Animals , Binding Sites , Biological Transport, Active/drug effects , Cell Membrane/metabolism , Gene Expression , Glucose Transporter Type 4 , Membrane Proteins/metabolism , Mice , Monosaccharide Transport Proteins/genetics , Muscle Proteins/genetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Qa-SNARE Proteins , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine/chemistry , Signal Transduction , Transfection , Vesicular Transport Proteins/geneticsABSTRACT
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.