Insulin stimulates the phosphorylation of the exocyst protein Sec8 in adipocytes.
The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.
Lyons
Patrick D
PD
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Peck
Grantley R
GR
Kettenbach
Arminja N
AN
Gerber
Scott A
SA
Roudaia
Liya
L
Lienhard
Gustav E
GE
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D018274:Electroporation; D000939:Epitopes / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006388:Hemagglutinins / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008565:Membrane Proteins; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics; D013997:Time Factors
D002232:Carbocyanines; D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D000939:Epitopes; D005456:Fluorescent Dyes; D051275:Glucose Transporter Type 4; D006388:Hemagglutinins; D007004:Hypoglycemic Agents; D007328:Insulin; D008565:Membrane Proteins; C489608:Sec8l1 protein, mouse; C495693:Slc2a4 protein, mouse; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins