AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.
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.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Cristinel P
CP
Mîinea
Hiroyuki
H
Sano
Susan
S
Kane
Eiko
E
Sano
Mitsunori
M
Fukuda
Johan
J
Peränen
William S
WS
Lane
Gustav E
GE
Lienhard
D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011994:Recombinant Proteins; D022161:Transport Vesicles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D011994:Recombinant Proteins; C505647:Tbc1d4 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D020691:rab GTP-Binding Proteins