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Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking

By Conrad P. Hodgkinson, Ann Mander and Graham J. Sale

Abstract

PKC? (protein kinase C? ) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane instimulated cells. How PKC? modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length humanPKC? identified80K-Hprotein as an interactor withPKC? . GST (glutathione S-transferase) pull-down assays with GSTtagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKC? from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3–5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKC? , with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKC? , 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKC?, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest amodelwhereby insulin triggers the formation of a PKC?–80K-H–munc18c complex that enhances GLUT4 translocation to the plasma membrane

Topics: QH301
Year: 2005
OAI identifier: oai:eprints.soton.ac.uk:24607
Provided by: e-Prints Soton
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