Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates

InsulinstimulatesthetranslocationoftheglucosetransporterGLUT4fromintracellularlocationstotheplasmamembraneinadipose and muscle cells. Prior studies have shown that Aktphosphorylation of the Rab GTPase-activating protein, AS160(160-kDa Akt substrate; also known as TBC1D4), triggersGLUT4 translocation, most likely by suppressing its RabGTPase-activatingproteinactivity.However,theregulationofaverysimilarprotein,TBC1D1(TBCdomainfamily,member1),which is mainly found in muscle, in insulin-stimulated GLUT4translocation has been unclear. In the present study, we haveidentifiedlikelyAktsitesofinsulin-stimulatedphosphorylationof TBC1D1 in C2C12 myotubes. We show that a mutant ofTBC1D1,inwhichseveralAktsiteshavebeenconvertedtoala-nine, is considerably more inhibitory to insulin-stimulatedGLUT4 translocation than wild-type TBC1D1. This result thusindicates that similar to AS160, Akt phosphorylation ofTBC1D1 enables GLUT4 translocation. We also show that inaddition to Akt activation, activation of the AMP-dependentprotein kinase partially relieves the inhibition of GLUT4 trans-locationbyTBC1D1.Finally,weshowthattheR125WvariantofTBC1D1, which has been genetically associated with obesity, isequally inhibitory to insulin-stimulated GLUT4 translocation,as is wild-type TBC1D1, and that healthy and type 2 diabeticindividualsexpressapproximatelythesamelevelofTBC1D1inbiopsies of vastus lateralis muscle. In conclusion, phosphoryla-tionofTBC1D1isrequiredforGLUT4translocation.Thus,theregulation of TBC1D1 resembles that of its paralog, AS160.