The study investigates the role of AS160, a 160-kDa protein in adipocytes, in insulin-stimulated glucose transporter (GLUT4) translocation to the plasma membrane. AS160 contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins essential for membrane trafficking. The authors identified six sites of in vivo phosphorylation on AS160, all within the Akt phosphorylation consensus sequence. Insulin treatment increased phosphorylation at five of these sites. Expression of AS160 with mutations at two or more of these sites inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Additionally, inactivating the GAP function of AS160 through a point mutation in the GAP domain largely reversed the inhibitory effect of the phosphorylation site mutants. These findings suggest that insulin-stimulated phosphorylation of AS160 is crucial for GLUT4 translocation and that this phosphorylation likely signals translocation by inactivating the Rab GAP function. The study identifies AS160 as a key component linking the phosphatidylinositol 3-kinase insulin signaling pathway to the vesicle trafficking machinery in GLUT4 translocation.The study investigates the role of AS160, a 160-kDa protein in adipocytes, in insulin-stimulated glucose transporter (GLUT4) translocation to the plasma membrane. AS160 contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins essential for membrane trafficking. The authors identified six sites of in vivo phosphorylation on AS160, all within the Akt phosphorylation consensus sequence. Insulin treatment increased phosphorylation at five of these sites. Expression of AS160 with mutations at two or more of these sites inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Additionally, inactivating the GAP function of AS160 through a point mutation in the GAP domain largely reversed the inhibitory effect of the phosphorylation site mutants. These findings suggest that insulin-stimulated phosphorylation of AS160 is crucial for GLUT4 translocation and that this phosphorylation likely signals translocation by inactivating the Rab GAP function. The study identifies AS160 as a key component linking the phosphatidylinositol 3-kinase insulin signaling pathway to the vesicle trafficking machinery in GLUT4 translocation.