Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking.
PKCzeta (protein kinase Czeta) 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 in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 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 PKCzeta 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 PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 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, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.
Hodgkinson
Conrad P
CP
School of Biological Sciences, University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK.
Mander
Ann
A
Sale
Graham J
GJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D019556:COS Cells; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D005959:Glucosidases; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D051938:Munc18 Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques; D000099221:Protein Kinase C zeta
D002135:Calcium-Binding Proteins; D005947:Glucose; D051275:Glucose Transporter Type 4; D005959:Glucosidases; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D010750:Phosphoproteins; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta; C512949:Prkcsh protein, mouse; C070368:PRKCSH protein, human