Tuberous sclerosis complex tumor suppressor–mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent

Abstract

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

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

“…Whereas overexpression of Tsc1-Tsc2 reduces H-motif phosphorylation of coexpressed S6K1 (29), Tsc2-null cells display augmented H-motif phosphorylation of S6K1 relative to wild type cells (36). It remains to be resolved, however, whether T-loop phosphorylation (Thr-229) or phosphorylation of other regulatory sites in the AID is influenced by the loss of Tsc1 or Tsc2.…”

“…Overexpression of the Tsc1-Tsc2 complex in cells has the converse effect, i.e. S6K1 inhibition (28,29,36) (see below). We therefore sought to address whether or not this inhibition is associated with dephosphorylation of the same subset of Tsc1-Tsc2-regulated sites defined by the aforementioned studies.…”

Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway

“…p-p70S6K is generally used in preference to p-mTOR to assess the tumour response to mTOR antagonists and to predict which tumours may be responsive to such agents (for reviews see Boulay et al (2004); MacKenzie and von Mehren (2007)). Indeed, phosphorylated mTOR has been generally found to be expressed in relatively low numbers of tumours, and several studies make no reference to tumour mTOR expression when assessing the effect of rapamycin and or its analogues (Jaeschke et al, 2002;Gao et al, 2003;Krishnan et al, 2006;El-Salem et al, 2007;Lu et al, 2008; Although the TMA was built with 50 chordomas, the total number described for each antibody represents the number that was possible to analyse. The scoring system employed was as follows: negative in the absence of immunoreactivity, 'low' when the immunoreactivity was unequivocal but less strong than the positive control and 'high' when the immunoreactivity was at least as strong as the positive control.…”

Distinct effects of single amino-acid changes to tuberin on the function of the tuberin–hamartin complex

“…5 -7 We expressed tuberin and hamartin in Tsc2 À/À and Tsc1 À/À MEFs and assayed the phosphorylation of S6 in transfected, serum-starved cells by double-label immunofluorescent microscopy, as described previously by others. 6,17 Transfected cells expressing either tuberin or hamartin, with a clear reduction in S6 phosphorylation, were counted 'blind' by two independent observers. Approximately 80% of the Tsc2 À/À MEFs expressing exogenous tuberin and approximately 60% of the Tsc1 À/ À MEFs expressing exogenous hamartin had no detectable phosphorylated S6 (pS6), compared to less than 10% of control cells.…”