title: Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis.
abstract: The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.
authors.affiliation: Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: O Jameel
authors.initials: OJ
authors.lastname: Shah
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000172:deficiency / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D013347:Subcellular Fractions; D058570:TOR Serine-Threonine Kinases; D014402:Tuberous Sclerosis / Q000473:pathology*; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000172:deficiency
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D009479:Neuropeptides; D010750:Phosphoproteins; D011506:Proteins; C490211:RHEB protein, human; C463659:RPTOR protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; C000624651:Tsc1 protein, mouse; C000624654:Tsc2 protein, mouse; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D010768:Phosphoserine; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins