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.

We thank David Kwiatkowski for providing wild-type and TSC1- and TSC2-null MEFs; Peter van der Geer (University of California, San Diego), John Blenis (Harvard Medical School), Paul Worley (Johns Hopkins University), and David Sabatini (Whitehead Institute) for cDNAs; Jongsoon Lee (Harvard Medical School) for the pIRS1-[S³⁰⁷] antibody used in this study; and Jill Meisenhelder for help with peptide mapping.

O.J.S. was supported by National Institutes of Health Grant T32-CA09523, by an NRS Award (GM67407), and a Pioneer Fellowship. This study was supported by USPHS grants CA14195 and CA82683 to T.H. from the National Cancer Institute. T.H. is a Frank and Else Schilling American Cancer Research Professor.