Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.
Previous studies have suggested that 1) atypical protein kinase C (PKC) isoforms are required for insulin stimulation of glucose transport, and 2) 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is required for activation of atypical PKCs. Presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose transporter 4 (GLUT4) to the plasma membrane, during insulin action in transiently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoked increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC-zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression of both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta that 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 activity and HA-GLUT4 translocation to the same extent as kinase-inactive PKC-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were fully 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 to PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 translocation and did not inhibit HA-GLUT4 translocation induced by overexpression of wild-type PDK-1. Our findings suggest that both PDK-1 and its downstream target, Thr-410 in the activation loop of PKC-zeta, are required for insulin-stimulated glucose transport.
Bandyopadhyay
G
G
J.A. Haley Veterans' Hospital Research Service and Department of Internal Medicine, University of South Florida College of Medicine, Tampa 33612, USA.
Standaert
M L
ML
Sajan
M P
MP
Karnitz
L M
LM
Cong
L
L
Quon
M J
MJ
Farese
R V
RV
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D004789:Enzyme Activation; D000939:Epitopes / Q000378:metabolism; D005786:Gene Expression Regulation; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection
D000939:Epitopes; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; C495694:Slc2a4 protein, rat; D013912:Threonine; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; C489591:Prkcq protein, rat; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta