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Address correspondence to Richard Lamb, Cancer Research UK Centre for Cell and Molecular Biology, Institute for Cancer Research, 237 Fulham Rd., London SW36JB, UK. Tel.: 44-207-970-6096. Fax.: 44-207-352-5630. E-mail: rlamb{at}icr.ac.uk; or George Thomas, Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058, Basel, Switzerland. Tel.: 41-61-6973012. Fax: 41-61-6973976. E-mail: gthomas{at}fmi.ch
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Abstract |
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Key Words: TSC; S6K1; 4E-BP1; PI3K; rapamycin
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Introduction |
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The effects of TSC deficiency that could provide clues to the functions of the TSC1-2 complex in mammalian tissues have been hampered by the embryonic lethality caused by homozygous TSC1 or -2 deficiency in knockout mice (Kobayashi et al., 1999, 2001; Onda et al., 1999; Kwiatkowski et al., 2002). However, recent studies in Drosophila have provided compelling evidence that the fly orthologues of TSC1 and -2 regulate cell size both in larval and adult tissues (Gao and Pan, 2001; Potter et al., 2001; Tapon et al., 2001). Additional effects on the cell cycle in Drosophila were also reported in these studies. These include an increase in cell number and shortening of the G1 phase during eye imaginal disc development (Tapon et al., 2001), as well as inappropriate cell proliferation of post-mitotic cells (Potter et al., 2001; Tapon et al., 2001). The effect of TSC deficiency on cell size has been particularly amenable to genetic epistasis analysis in the fly. This approach indicates that TSC1-2 functions in insulin/phosphatidylinositide-3-OH kinase (PI3K)regulated signaling pathways, which control cell size upstream of Drosophila p70 S6 kinase (S6K) (Gao and Pan, 2001; Potter et al., 2001; Tapon et al., 2001). Three models were proposed to explain the observed genetic epistatic interactions between TSC1-2 and S6K (Potter et al., 2001): (1) TSC1-2 is a direct downstream target of protein kinase B (PKB), until recently a presumed upstream effector of mammalian target of rapamycin (mTOR) (Radimerski et al., 2002); (2) TSC1-2 acts on a parallel pathway that integrates the insulin signaling pathway at the level of S6K; or (3) TSC1-2 acts on a common downstream target of S6K. Here we provide evidence of a biochemical link between TSC1-2 and S6K1, and establish that mTOR and PI3K signaling act separately to activate S6K1, the latter by repressing TSC1-2.
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Results and discussion |
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TSC1-2 acts independently of mTOR on GST-C-S6K1
That both S6K1 and 4E-BP1 are constitutively phosphorylated in TSC2-/- MEFs and sensitive to rapamycin, argues that TSC1-2 may act directly to inhibit mTOR function. However, these findings do not exclude the possibility that TSC1-2 functions either in a parallel pathway or downstream of mTOR to regulate the activities of both S6K1 and 4EBP-1. If TSC1-2 acts as a negative regulator of S6K1 through inhibiting mTOR function, S6K1 inactivation by TSC1-2 overexpression should occur in an mTOR-dependent manner. To test this, we first developed an assay in which epitope-tagged variants of human TSC1 and TSC2 were ectopically expressed in Cos cells together with a wild-type S6K1 reporter. Under these conditions, expression of TSC1-2 reduced basal and insulin-induced S6K1 reporter T389 phosphorylation and kinase activation (Fig. 3 a). In contrast, in a separate experiment, cotransfection of either TSC1 or TSC2 alone or the human TSC2 N1643K mutant (Fig. 1 b) with TSC1 had no significant effect on insulin-induced S6K1 activation (Fig. 3 b). To determine whether inhibition of S6K1 by TSC1-2 was mTOR dependent, advantage was taken of a recently described variant of S6K1, GSTC-S6K1, in which the COOH-terminal autoinhibitory domain has been deleted and GST fused to the NH2 terminus (Dennis et al., 2001). Consistent with earlier findings, insulin-induced GST
C-S6K1 T389 phosphorylation and activation are sensitive to the PI3K inhibitor wortmannin, but resistant to rapamycin, and therefore uncoupled from mTOR (Fig. 3 c). Unexpectedly, using the Cos cell overexpression assay, coexpression of TSC1 and wild-type TSC2 also suppressed insulin-induced GST
C-S6K1 reporter T389 phosphorylation and activation (Fig. 3 d), and thus phenocopied wortmannin but not rapamycin treatment. To confirm these results, we used an alternative assay in which TSC1-2 regulates S6K1. Because reintroduction of wild-type TSC2 by microinjection into TSC2-/- MEFs restores serum-regulated S6K1 activity, presumably through forming complexes with endogenous TSC1 (Fig. 1, b and c), we reasoned that it may similarly regulate the activity of either ectopically expressed S6K1 or GST
C-S6K1. However, given the findings above we would expect GST
C-S6K1 to be resistant to the effects of rapamycin. Indeed, introduction of TSC2 together with either S6K1 or GST
C-S6K1 led to kinase inhibition in serum-derived TSC2-/- MEFs in the presence or absence of rapamycin as assessed by phospho-S6 staining (Fig. 3 e). In contrast, only GST
C-S6K1 was active when introduced with control vector in the presence of rapamycin, confirming that this S6K1 variant is indeed mTOR independent, but still regulated by TSC2. Taken together, these findings argue that TSC1-2 inhibition of S6K1 is unlikely to be via direct regulation of mTOR.
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TSC1-2 mechanism of action in regulating S6K1 and 4E-BP1
Mitogen-induced S6K1 activation is mediated by the phosphorylation of residues residing in its linker and conserved catalytic domains in a hierarchical manner via mTOR and the phosphoinositide-dependent protein kinase 1, respectively (Isotani et al., 1999). Despite prevailing models that both kinases are downstream effectors of PI3K with respect to S6K1 activation (Blume-Jensen and Hunter, 2001), recent studies indicate that this is not the case for either kinase (Pullen et al., 1998; Dennis et al., 2001). Instead, both kinases appear constitutively active regardless of mitogenic ligand stimulation. Our findings demonstrate that in normal cells the PI3K input to S6K1 is the suppression of TSC1-2, which we hypothesize may normally act as a positive effector of a S6K1 phosphatase. Alternatively, given the potential role of TSC2 in trafficking to the plasma membrane (Kleymenova et al., 2001), TSC1-2 complexes may act to partition S6K1 in a compartment of the cell where access by activating kinases is limited.
However, it also should be noted that in a few cell types and in Drosophila (Radimerski et al., 2002), S6K1 activation can be PI3K independent, suggesting other mechanisms may exist to regulate TSC1-2 function. During the completion of these studies, others reported that S6K1 activity was elevated in TSC1-deficient cells and blocked by rapamycin and LY294002 (Kwiatkowski et al., 2002), a PI3K inhibitor (Vlahos et al., 1994). This led to the conclusion that PI3K induces S6K1 activation through mTOR and that mTOR is the target of TSC1-2 suppression. However, at the concentrations employed, LY294002 has been demonstrated to be a potent inhibitor of both PI3K and mTOR (Brunn et al., 1996). Thus, the fact that LY294002 inhibits phosphorylation of S6K1 in TSC1-deficient cells is most likely explained by inhibitory affects of the drug on mTOR rather than PI3K. Such an explanation would clarify why we observe little to no inhibitory affect of wortmannin or microinjection of either a kinase-inactive PI3K or a deletion mutant of the p85 subunit on S6K1 activity.
Our findings indicate that in normal cells, signaling via PI3K antagonizes the TSC1-2 complex and its ability to repress S6K1. During the review process, three other reports have appeared that suggest a mechanism of the PI3K-regulated TSC1-2 inactivation described here. Activation of the PI3K-regulated serine-threonine kinase Akt/PKB has been shown in these studies to lead to phosphorylation of TSC2 (Inoki et al., 2002; Manning et al., 2002; Potter et al., 2002), although the suggested mechanism of the resulting inhibition of the TSC1-2 complex varies between studies. Interestingly, these new reports place Akt/PKB upstream of S6K1, despite previous data to the contrary (Dufner et al., 1999, Radimerski et al., 2002). Further studies are clearly necessary to establish whether inactivation of TSC1-2 by the activated alleles of Akt/PKB commonly used in these types of studies is equivalent to physiological ligand-induced activation of PI3K signaling for S6K1 regulation. More importantly, it will be critical to establish whether such a mode of inactivation of the TSC1-2 complex by phosphorylation in normal cells is equivalent to the mode of inactivation of TSC1-2 by pathogenic TSC2 mutations. Finally, in one of these reports (Inoki et al., 2002), use of different rapamycin-resistant S6K1 constructs has allowed the authors to reach opposite conclusions from ours regarding whether TSC1-2 regulates S6K1 by regulating mTOR activity, or acts directly on S6K1. With the emerging importance of the mTORS6K4E-BP signaling pathway in cancer (Gingras et al., 2001), it will be critical to resolve this issue and establish the molecular mechanisms by which TSC1-2 acts as a tumor suppressor in this pathway. Importantly, given the selective effects of rapamycin on the proliferation of TSC2-/- MEFs described here and its efficacious use in the treatment of solid tumors in phase 1 clinical trials (Hidalgo and Rowinsky, 2000), the use of this drug could be explored for the treatment of TSC and TSC-associated maladies.
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Materials and methods |
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Transfection and retroviral reintroduction of TSC2 into TSC2-/- MEFs
For transient transfection of Cos cells, cells were seeded in 6-well plates at 2 x 105 per well. Transfection was performed using Fugene 6 from Roche. 24 h after transfection, cells were serum starved for 48 h and extracted directly or after stimulation with 200 nM insulin, with or without the addition of 20 nM rapamycin or 100 nM wortmannin. For retroviral transfection, the complete coding regions of wild-type TSC2 and the TSC N1643K and G294E mutants were excised as BamH1 fragments from pCMVTag2 TSC2 vectors and cloned into a modified retroviral vector based on pRevTRE (CLONTECH Laboratories, Inc.), in which the TRE-minimal CMV promoter was replaced with the minimal tk promoter from pBLCAT2. BOSC cells were used to package retroviruses and retroviral supernatants used to infect TSC2-/-/p53-/- MEFs. Retrovirally transduced populations were selected with hygromycin and 3T3 cell lines generated by limit dilution cloning.
Microinjection
TSC2+/+ or TSC2-/- MEFs on coverslips were nuclear microinjected with 0.1 mg/ml plasmids together with 5 mg/ml biotindextran (Molecular Probes) as an injection marker. For Fig. 1 c, TSC-/- MEFs were microinjected with 0.2 mg/ml empty pCMVTag vector or the same vector expressing wild-type TSC2 or TSC2 mutants. For Fig. 2 e, cells were injected with 0.5 mg/ml pCMVTag empty vector or pCMVTag wild-type TSC2 together with 0.1 mg/ml pRK5-GSTS6K1 or 0.3 mg/ml pRK5-GSTC-S6K1. In both Fig. 1 c and Fig. 3 e, cells were serum starved after microinjection for 12 h and left untreated or treated with 10 nM rapamycin for a further 1 h before fixation. Expression of S6K1 was detected using a monoclonal antibody to GST (Santa Cruz Biotechnology, Inc.) and goat antimouse FITC. For Fig. 4 e, empty pCMVTag vector or expression plasmids encoding PI3K deltap85 (pcDNA3) or kinase-dead p110 (pEFBos; both from Dr. Julian Downward) were injected at 0.1 mg/ml. After serum starvation for 12 h, TSC2-/- were left unstimulated whereas TSC2+/+ cells were stimulated with 200 nM insulin for 1 h before fixation. All cells were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min with PBS/0.4% Triton X-100, blocked for 15 min with PBS/1% BSA, and stained with an antibody to phospho-S6 (Ser235/236; Cell Signaling) followed by goat antirabbit TRITC (Molecular Probes). Injected cells were detected with Alexa®350-conjugated Streptavidin (Molecular Probes). Images were acquired on a ZEISS Axioplan 2 imaging microscope on 20x or 40x objectives with Axiovision software and processed either as greyscale or dual color TIFF images in Adobe Photoshop®.
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Footnotes |
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* Abbreviations used in this paper: MEF, mouse embryo fibroblast; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositide-3-OH kinase; PKB, protein kinase B; S6K, S6 kinase; TSC, tuberous sclerosis complex.
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Acknowledgments |
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These studies were supported by grants from Cancer Research UK, the Tuberous Sclerosis Association (UK), and the LAM Foundation (J. Hardkamp, W. Roworth, and R. Lamb) and a grant from the Swiss Cancer League to A. Jaeschke, T. Nobukuni, and G. Thomas. M. Saitoh was supported by a fellowship from the European Molecular Biology Organization, and G. Thomas was supported by the Novartis Research Foundation.
Submitted: 25 June 2002
Revised: 16 September 2002
Accepted: 24 September 2002
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References |
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Brunn, G.J., J. Williams, C. Sabers, G. Wiederrecht, J.C. Lawrence, Jr., and R.T. Abraham. 1996. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 15:52565267.[Abstract]
Brunn, G.J., C.C. Hudson, A. Sekulic, J.M. Williams, H. Hosoi, P.J. Houghton, J.C. Lawrence, Jr., and R.T. Abraham. 1997. Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science. 277:99101.
Burnett, P.E., R.K. Barrow, N.A. Cohen, S.H. Snyder, and D.M. Sabatini. 1998. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA. 95:14321437.
Dennis, P.B., A. Jaeschke, M. Saitoh, B. Fowler, S.C. Kozma, and G. Thomas. 2001. Mammalian TOR: a homeostatic ATP sensor. Science. 294:11021105.
Dufner, A., M. Andjelkovic, B.M. Burgering, B.A. Hemmings, and G. Thomas. 1999. Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation. Mol. Cell. Biol. 19:45254534.
The European Chromosome 16 Tuberous Sclerosis Consortium. 1993. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell. 75:13051315.
Gao, X., and D. Pan. 2001. TSC1 and TSC2 tumor suppressors antagonize insulin signalling in cell growth. Genes Dev. 15:13831392.
Gingras, A.C., B. Raught, and N. Sonenberg. 2001. Regulation of translation initiation by FRAP/mTOR. Genes Dev. 15:807826.
Gomez, M.R., J.R. Sampson, and V.H. Whittemore. 1999. Tuberous Sclerosis. Third edition. Oxford University Press, New York. 340 pp.
Haruta, T., T. Uno, J. Kawahara, A. Takano, K. Egawa, P.M. Sharma, J.M. Olefsky, and M. Kobayashi. 2000. A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. Mol. Endocrinol. 14:783794.
Hodges, A.K., S. Li, J. Maynard, L. Parry, R. Braverman, J.P. Cheadle, J.E. DeClue, and J.R. Sampson. 2001. Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin. Hum. Mol. Genet. 10:28992905.
Isotani, S., K. Hara, C. Tokunaga, H. Inoue, J. Avruch, and K. Yonezawa. 1999. Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase in vitro. J. Biol. Chem. 274:3449334498.
Kleymenova, E., O. Ibraghimov-Beskrovnaya, H. Kugoh, J. Everitt, H. Xu, K. Kiguchi, G. Landes, P. Harris, and C. Walker. 2001. Tuberin-dependent membrane localization of polycystin-1: a functional link between polycystic kidney disease and the TSC2 tumor suppressor gene. Mol. Cell. 7:823832.[CrossRef][Medline]
Kobayashi, T., O. Minowa, J. Kuno, H. Mitani, O. Hino, and T. Noda. 1999. Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line TSC2 mutation in mice. Cancer Res. 59:12061211.
Kobayashi, T., O. Minowa, Y. Sugitani, S. Takai, H. Mitani, E. Kobayashi, T. Noda, and O. Hino. 2001. A germ-line TSC1 mutation causes tumor development and embryonic lethality that are similar. but not identical, to those caused by TSC2 mutation in mice. Proc. Natl. Acad. Sci. USA. 98:87628767.
Lamb, R.F., C. Roy, T.J. Diefenbach, H.V. Vinters, M.W. Johnson, D.G. Jay, and A. Hall. 2000. The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat. Cell Biol. 2:281287.[CrossRef][Medline]
Onda, H., A. Lueck, P.W. Marks, H.B. Warren, and D.J. Kwiatkowski. 1999. TSC2(+/-) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J. Clin. Invest. 104:687695.
Potter, C.J., L.G. Pedraza, and T. Xu. 2002. Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4:658665.[CrossRef][Medline]
Pullen, N., P.B. Dennis, M. Andjelkovic, A. Dufner, S. Kozma, B.A. Hemmings, and G. Thomas. 1998. Phosphorylation and activation of p70s6k by PDK1. Science. 279:707710.
Reif, K., B.M. Burgering, and D.A. Cantrell. 1997. Phosphatidylinositol 3-kinase links the interleukin-2 receptor to protein kinase B and p70 S6 kinase. J. Biol. Chem. 272:1442614433.
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. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet. 7:10531057.
Vlahos, C.J., W.F. Matter, K.Y. Hui, and R.F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:52415248.
von Manteuffel, S.R., P.B. Dennis, N. Pullen, A.C. Gingras, N. Sonenberg, and G. Thomas. 1997. The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k. Mol. Cell. Biol. 17:54265436.[Abstract]