Selective insulin-induced activation of class I(A) phosphoinositide 3-kinase in PIKfyve immune complexes from 3T3-L1 adipocytes.
A diverse range of insulin-regulated cellular processes are dependent on class I(A) phosphatidylinositol 3-kinases (PI 3-Ks) and their association with and activation by up-stream signaling molecules. Here we report on the identification of the phosphoinositide 5'-kinase PIKfyve as a partner of class I(A) PI 3-K. Thus, both p85 and p110 subunits (class I(A)) of PI 3-Ks co-precipitated with anti-PIKfyve antibodies from lysates of resting 3T3-L1 adipocytes and, vice versa, PIKfyve co-precipitated with anti-p85 PI 3-K antibodies. Assignment to class I(A) PI 3-K enzymatic activity was further substantiated by the inhibition of PtdIns 3-P production in PIKfyve immune complexes by low concentrations of wortmannin and Triton X-100, and its preferences for Mg(2+) versus Mn(2+). Insulin but not PDGF or EGF stimulation of 3T3-L1 adipocytes markedly increased the PtdIns 3-P production (4.2-fold) in PIKfyve immune complexes, primarily as a result of increased PI 3-K intrinsic enzymatic activity. Intriguingly, while both insulin and PDGF caused an increase of class I(A) PI 3-K activity co-immunoprecipitated with tyrosine phosphorylated proteins, only insulin treatment yielded an activation of class I(A) PI 3-K in PIKfyve immune complexes. Studies aiming at identifying the underlying mechanism revealed that PIKfyve-class I(A) PI 3-K association and the insulin-induced activation likely operate independently of tyrosine phosphorylated insulin receptor substrate proteins. Together, these results establish PIKfyve as a novel source of activated class I(A) PI 3-K molecules that may be relevant in the insulin-signal transduction pathway.
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Department of Physiology, Wayne State University School of Medicine, 4237 Scott Mall, 540 E. Canfield, Detroit, MI 48201, USA.
Ikonomov
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Shisheva
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D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology*; D000818:Animals; D000936:Antigen-Antibody Complex / Q000187:drug effects* / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects; D007074:Immunoglobulin G / Q000276:immunology; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008970:Molecular Weight; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000145:classification / Q000276:immunology* / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D021122:Protein Subunits; D011972:Receptor, Insulin / Q000378:metabolism; D013379:Substrate Specificity
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D021122:Protein Subunits; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; C116930:Pikfyve protein, mouse; D011972:Receptor, Insulin