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.

FIG. 1.

The TSC/Rheb/Raptor-mTOR/S6K regulates IRS1 serine phosphorylation. (A) TSC1-null, TSC2-null, or wild-type (+/+) MEFs were cultured in serum-free medium overnight, followed by the addition of 100 nM rapamycin for 1 h. IRS1 was immunoprecipitated and immunoblotted with the indicated antibodies. (B) HEK293 cells were cotransfected with Flag-IRS1 and either empty vector (pRK5) or individual Myc-Rheb constructs as indicated. Lysates were prepared from serum-starved cells, and immunoblot analysis of exogenous and endogenous proteins was carried out. Under these conditions, endogenous IRS1 phosphorylation is insignificant. (C) A dominant-interfering allele of Myc-mTOR (KD) was included in transfections of HEK293 with Flag-IRS1 and Myc-Rheb. (D) Flag-IRS1 was coexpressed with Myc-Rheb in HEK293 cells in the presence or absence of wild-type or dominant-interfering alleles of HA-S6K1 or HA-S6K2. Cells were deprived of serum overnight prior to harvest. (E) Flag-IRS1 was coexpressed with Myc-Rheb and a rapamycin-resistant allele of HA-S6K1 (ED₃E). Serum-starved cells were treated or not treated with 100 nM rapamycin for 1 h prior to lysis.

FIG. 2.

In vitro phosphorylation of IRS1 by S6K1. (A) Wild-type Flag-IRS1 was expressed in HEK293T cells, purified, and dephosphorylated as indicated in Materials and Methods. HA-S6K1 was separately coexpressed with Myc-Rheb in HEK293T cells, which were treated or not for 1 h with 100 nM rapamycin. HA-S6K1 was subsequently purified and combined with dephosphorylated Flag-IRS1. Phosphopeptide mapping of the in vitro phosphorylation product was then carried out. Panels show S6K1 phosphorylation of IRS1 before (i) and after (ii) rapamycin treatment. Panel iii schematically represents the distribution of rapamycin-sensitive phosphopeptides. (B) Phosphoamino acid analysis of in vitro S6K1-phosphorylated Flag-IRS1 before (left panel) and after (middle panel) rapamycin treatment. The right panel shows schematically the distribution of rapamycin-sensitive phosphoamino acids. (C) HA-S6K1 was prepared as described in panel A and used to phosphorylate an RXRXXS/T peptide substrate. The relative kinase activity toward this peptide is presented. (D) Wild-type, S307A, S312A, and 4A (S616A/S636A/S666A/S736A) Flag-IRS1 constructs were expressed in HEK293T cells, purified, and dephosphorylated as in panel A. These substrates were mixed with HA-S6K1 expressed in and purified from HEK293T cells coexpressing Myc-Rheb before and after treatment with 100 nM rapamycin for 1 h. In vitro phosphorylation was then carried out, and ³²P incorporation was monitored by autoradiography. Aliquots from the same reaction were subjected to immunoblot analysis to determine expression level and site-specific phosphorylation.

FIG. 3.

Depletion of IRS1 from the HSP fraction in TSC1/2-null or Rheb-overexpressing cells. (A) TSC1^−/−, TSC2^−/−, and wild-type MEFs were deprived of serum for 4 h and then lysed under hypotonic conditions and fractionated into HSP and cytosolic (CYT) components. Equivalent amounts of cell protein were loaded, and the ratio of HSP to cytosolic IRS1 was calculated (as shown in a histogram). (B) IRS1 was detected in the HSP fraction from TSC2^+/+ MEFs, TSC2^−/− MEFs, TSC2^−/− MEFs treated with 100 nM rapamycin for 24 h, and two clonal lines of TSC2^−/− MEFs reconstituted with retroviral TSC2 (R2 and R4). (C) HEK293 cells were transfected with empty vector or Myc-Rheb and serum deprived overnight in the presence or absence of 100 nM rapamycin. Quantitation of the HSP/cytosolic ratio of endogenous IRS1 was calculated and is presented in the accompanying histogram. (D) HEK293 cells were cotransfected with either empty vector or Myc-Rheb and wild-type or 6A (S307A/S312A/S616A/S636A/S666A/S736A) Flag-IRS1. Equivalent amounts of protein were loaded for each panel. However, the abundance of detectable Flag-IRS1 is lower in the cytosolic fraction, presumably because it is not as tightly regulated when overexpressed. The relative ratios of HSP to cytosolic exogenous Flag-IRS1 were then calculated and are presented in the histogram to the right.

FIG. 4.

IRS1 depletion from the HSP in HEK293T cells. (A) HEK293T cells expressing Flag-IRS1 and/or Myc-Rheb were serum deprived for 4 h and then fractionated and immunoblotted with anti-Flag antibody. (B) HEK293T cells transfected with Flag-IRS1 were cotransfected with empty vector or Myc-Rheb and Myc-mTOR (KD) as indicated. Cells were either left untreated or cultured in the presence 100 nM rapamycin or 5 μM clasto-lactacystin-β-lactone (CLβL) for 48 h. (C) Myc-Rheb was coexpressed with a panel of Flag-IRS1 mutants (defined in the legend of Fig. 2D and 3D) as indicated. The HSP fraction was prepared and immunoblotted with anti-Flag antibodies. The expressions of these mutants are similar at steady state (data not shown). (D) Proposed model for IRS1 regulation in the context of TSC deficiency. When TSC1 or TSC2 is inactivated, signaling through the Rheb/Raptor-mTOR/S6K axis is constitutive. S6K directly phosphorylates IRS1 on the RXRXXS/T site, S307 and potentially S527 (solid arrows) but not S1101. Raptor-mTOR or other kinases phosphorylate IRS1 on the S/T,P sites, S312, S616, and S636/9, which is somehow indirectly regulated by S6K1 (hatched arrows. The phosphorylation of these sites collectively contributes to the depletion of IRS1 from the HSP fraction. This either involves phosphorylation-mediated redistribution of IRS1 from the HSP to the cytosol, where it is subsequently degraded, or degradation directly in the HSP. The mode of IRS1 turnover is likely to be cell type dependent. IRS1 turnover produces cell-autonomous insensitivity to insulin or IGF-I.