14-3-3 facilitates insulin-stimulated intracellular trafficking of insulin receptor substrate 1.
The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.
Xiang
Xiaoqin
X
Diabetes and Metabolism Research Unit, Section of Endocrinology, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Yuan
Mingsheng
M
Song
Ying
Y
Ruderman
Neil
N
Wen
Rong
R
Luo
Zhijun
Z
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D019319:Okadaic Acid / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*
D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019319:Okadaic Acid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D010768:Phosphoserine; D010769:Phosphothreonine; D019000:Phosphotyrosine; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate; D014446:Tyrosine 3-Monooxygenase; C526295:Irs1 protein, mouse