14-3-3-BETA PROTEIN ASSOCIATES WITH INSULIN-RECEPTOR SUBSTRATE-1 AND DECREASES INSULIN-STIMULATED PHOSPHATIDYLINOSITOL 3'-KINASE ACTIVITY IN 3T3L1 ADIPOCYTES

The 14-3-3 protein family has been implicated in growth factor signali ng. We investigated whether 14-3-3 protein is involved in insulin sign aling in 3T3L1 adipocytes. A significant amount of insulin receptor su bstrate 1 (IRS-1) was immunodetected in the immunoprecipitate with ant i-14-3-3 beta antibody at the basal condition. 100 nM insulin increase d the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3 beta fusion protein di rectly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Be cause the recombinant IRS-1 was not phosphorylated on its tyrosine res idues, the results suggest that serine/threonine phosphorylation of IR S-1 is responsible for the association, When the cells are treated wit h insulin, phosphatidylinositol 3'-kinase (PI3K) is supposed to comple x either 14-3-3 beta-IRS-1 or IRS-1. The 14-3-3 beta-IRS-1-PI3K and IR S-1-PI3K complexes were separately prepared by a sequential immunoprec ipitation, first with anti-14-3-3 beta and then with anti-IRS-1 antibo dies. The specific activity of the PI3K in the former was approximatel y half of that in the latter, suggesting that 14-3-3 beta protein boun d to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K i n 3T3L1 adipocytes.