Insulin stimulates the translocation of intracellular glucose transporters (GLUT4) to the plasma membrane in fat and muscle cells. This process, known as GLUT4 translocation, involves the generation of specialized vesicles containing GLUT4, their movement to the plasma membrane, and their fusion with the membrane. The signaling pathways that lead to this process have not been fully defined. However, it is known that insulin activates the Akt kinase, which phosphorylates a 160-kDa protein called AS160. AS160 contains a Rab GTPase-activating protein (GAP) domain, which is essential for membrane trafficking. This study shows that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation. Phosphorylation of AS160 inhibits its GAP activity, which is necessary for the translocation of GLUT4. The study identified six phosphorylation sites on AS160, and insulin treatment increased phosphorylation at five of these sites. Mutations in these sites significantly inhibited insulin-stimulated GLUT4 translocation. Additionally, mutations that inactivated the GAP function of AS160 did not inhibit translocation, indicating that the GAP function is required for the translocation process. The study also shows that the Rab GAP domain of AS160 is critical for the translocation of GLUT4. The arginine residue in the GAP domain is essential for its activity, and mutating this residue to lysine abolishes the GAP function. The study further demonstrates that the inhibitory effect of phosphorylation site mutations on GLUT4 translocation is reversed when the GAP domain is also mutated. This suggests that the GAP function of AS160 is necessary for the translocation of GLUT4. 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 also suggests that AS160 may act on Rab4 and Rab11, which are present in the intracellular vesicles containing GLUT4. Future studies will focus on identifying the specific Rab(s) on which AS160 acts and determining whether phosphorylation of AS160 directly inhibits its GAP activity.Insulin stimulates the translocation of intracellular glucose transporters (GLUT4) to the plasma membrane in fat and muscle cells. This process, known as GLUT4 translocation, involves the generation of specialized vesicles containing GLUT4, their movement to the plasma membrane, and their fusion with the membrane. The signaling pathways that lead to this process have not been fully defined. However, it is known that insulin activates the Akt kinase, which phosphorylates a 160-kDa protein called AS160. AS160 contains a Rab GTPase-activating protein (GAP) domain, which is essential for membrane trafficking. This study shows that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation. Phosphorylation of AS160 inhibits its GAP activity, which is necessary for the translocation of GLUT4. The study identified six phosphorylation sites on AS160, and insulin treatment increased phosphorylation at five of these sites. Mutations in these sites significantly inhibited insulin-stimulated GLUT4 translocation. Additionally, mutations that inactivated the GAP function of AS160 did not inhibit translocation, indicating that the GAP function is required for the translocation process. The study also shows that the Rab GAP domain of AS160 is critical for the translocation of GLUT4. The arginine residue in the GAP domain is essential for its activity, and mutating this residue to lysine abolishes the GAP function. The study further demonstrates that the inhibitory effect of phosphorylation site mutations on GLUT4 translocation is reversed when the GAP domain is also mutated. This suggests that the GAP function of AS160 is necessary for the translocation of GLUT4. 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 also suggests that AS160 may act on Rab4 and Rab11, which are present in the intracellular vesicles containing GLUT4. Future studies will focus on identifying the specific Rab(s) on which AS160 acts and determining whether phosphorylation of AS160 directly inhibits its GAP activity.