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Molecular and Cellular Biology, July 2003, p. 4892-4900, Vol. 23, No. 14
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.14.4892-4900.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Insulin-Induced GLUT4 Translocation Involves Protein Kinase C-{lambda}-Mediated Functional Coupling between Rab4 and the Motor Protein Kinesin

Takeshi Imamura,1 Jie Huang,1 Isao Usui,1 Hiroaki Satoh,1 Jennie Bever,1 and Jerrold M. Olefsky1,2*

Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California 92093,1 San Diego Veterans Administration Medical Research Service and the Whittier Diabetes Institute, La Jolla, California 920372

Received 23 September 2002/ Returned for modification 7 April 2003/ Accepted 17 April 2003

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}-32P]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.

* Corresponding author. Mailing address: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Phone: (858) 534-6651. Fax: (858) 534-6653. E-mail: jolefsky{at}ucsd.edu.

Molecular and Cellular Biology, July 2003, p. 4892-4900, Vol. 23, No. 14
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.14.4892-4900.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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