title: Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin.
abstract: Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.
authors.affiliation: Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.forename: Isao
authors.initials: I
authors.lastname: Usui
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Satoh
authors.forename: Jennie
authors.initials: J
authors.lastname: Bever
authors.forename: Jerrold M
authors.initials: JM
authors.lastname: Olefsky
mesh_terms: D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction; D022161:Transport Vesicles / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D000099293:Protein Kinase C-lambda
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D007527:Isoenzymes; D016547:Kinesins; D009004:Monosaccharide Transport Proteins; D009025:Morpholines; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099293:Protein Kinase C-lambda; C490202:Kif3a protein, mouse; C495693:Slc2a4 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one