Critical Role of T-Loop and H-Motif Phosphorylation in the Regulation of S6 Kinase 1 by the Tuberous Sclerosis Complex

Abstract

The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (Hmotif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex.

Tuberous Sclerosis Complex Proteins 1 and 2 Control Serum-Dependent Translation in a TOP-Dependent and -Independent Manner

“…In agreement with previous reports (26,30,38,45,64,68), we found that mTORC1 regulation by growth factors requires an intact TSC1/TSC2 complex, since cells deficient for Tsc1 or Tsc2 fail to downregulate mTORC1 function in response to serum deprivation, and rescue experiments restore mTORC1 sensitivity to serum withdrawal. Both growth factor and nutrient inputs are necessary for the full activation of mTORC1 in WT MEFs, whereas in MEFs lacking either Tsc1 or Tsc2 either input maintains partial or full mTORC1 activity.…”

Therapeutic targets: MTOR and related pathways

“…Collectively, these data suggest that tumor cells sensitive to mTOR inhibition are likely those with aberrant signaling through PI3K-Akt pathway such as those with activating mutations and/or amplification of growth factor receptors, PI3K, Akt, or through loss of PTEN [38][39][40] or TSC1/2. 41 Sensitive cells may be triggered to undergo G 1 arrest or apoptosis. The apoptotic effect may be determined, in part, by p53, p21 Cip1 , p27 Kip and bcl-2 function.…”

Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

“…In TSC1/2-null cells and in cells ectopically expressing Rheb, S6K1 is highly active and yet remains sensitive to rapamycin (11,19,21,30,36,47). In fact, by virtue of its TOS motif and C-terminal tail, S6K1 is a direct substrate of Raptor-mTOR, which phosphorylates T389 within the S6K1 hydrophobic motif (1).…”

“…This is remarkably overt in cell-based and animal models of the tumor predisposition syndrome, tuberous sclerosis, wherein loss-of-function of the TSC1-TSC2 complex gives rise to pathological hyperactivation of mTOR and S6K (7,11,19,20,36,45,47) and produces cell-autonomous insulin resistance (13,20,32,39). In mouse embryo fibroblasts (MEFs) derived from TSC1-or TSC2-deficient animals, IRS1 and IRS2 are transcriptionally repressed (13,39) through a mechanism requiring S6Ks (13).…”