14-3-3beta 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 signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3beta antibody at the basal condition. 100 nM insulin increased 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-14-3-3beta fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3'-kinase (PI3K) is supposed to complex either 14-3-3beta-IRS-1 or IRS-1. The 14-3-3beta-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3beta and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3beta protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.
Kosaki
A
A
Clinical Research Unit, Diabetes Center, Kyoto National Hospital, Fushimi-ku, Kyoto 612, Japan. akosaki@osk2.3web.ne.jp
Yamada
K
K
Suga
J
J
Otaka
A
A
Kuzuya
H
H
D048948:14-3-3 Proteins; D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects* / Q000201:enzymology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014446:Tyrosine 3-Monooxygenase; D000077191:Wortmannin
D048948:14-3-3 Proteins; D000730:Androstadienes; D004791:Enzyme Inhibitors; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D000077191:Wortmannin; C526295:Irs1 protein, mouse