Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 inthe activation loop of protein kinase C-zeta

Previous studies have suggested that 1) atypical protein kinase C (PKC) iso forms are required for insulin stimulation of glucose transport; and 2) 3-p hosphoinositide-dependent protein kinase-1 (PDK-1) is required for activati on of atypical PKCa. presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose tr ansporter 4 (GLUT4) to the plasma membrane, during insulin action in transi ently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoke d increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC- zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression o f both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta t hat is mutated at Thr-410, the immediate target of PDK-1 in the activation loop of PKC-zeta, inhibited insulin-induced increases in both HA-PKC-zeta a ctivity and HA-GLUT4 translocation to the same extent as kinase-inactive PK C-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were full y reversed by cotransfection of wild-type PDK-1 and partly reversed by wild -type PKC-zeta but not by wild-type PKB. In contrast to the T410A PKC-zeta mutant, an analogous double mutant of PKB (T308A/S473A) that is resistant t o PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 t ranslocation and did not inhibit HA-GLUT4 translocation induced by overexpr ession of wild-type PDK-1. Our findings suggest that both PDK-1 and its dow nstream target, Thr-410 in the activation loop of PKC-zeta, are required fo r insulin-stimulated glucose transport.