Inactivation of the Tuberous Sclerosis Complex-1 and -2 Gene Products Occurs by Phosphoinositide 3-Kinase/Akt-dependent and -independent Phosphorylation of Tuberin

Abstract

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. The tuberous sclerosis complex (TSC) 1The abbreviations used are: TSC, tuberous sclerosis complex; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PA, phosphatidic acid; EGF, epidermal growth factor; mTOR, mammalian target of rapamycin; GST, glutathione S-transferase; HA, hemagglutinin; GAP, GTPase-activating protein.1The abbreviations used are: TSC, tuberous sclerosis complex; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PA, phosphatidic acid; EGF, epidermal growth factor; mTOR, mammalian target of rapamycin; GST, glutathione S-transferase; HA, hemagglutinin; GAP, GTPase-activating protein. is a human genetic disorder that leads to the development of benign hamartomatous tumors in the brain, kidneys, heart, eyes, lungs, and skin. The disorder affects about 1 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns. Common symptoms include seizures, mental retardation, behavior problems, and skin abnormalities (for review see Ref. 1Cheadle J.P. Reeve M.P. Kwiakowski D.J. Hum. Genet. 2000; 107: 97-114Crossref PubMed Scopus (277) Google Scholar). Genetic studies have mapped this disorder to the TSC1 and TSC2 genes that encode the protein products hamartin (∼130 kDa) and tuberin (∼200 kDa), respectively. Hamartin and tuberin negatively regulate cell growth and proliferation as a tuberin-hamartin heterodimer (2Green A.J. Smith M. Yates J.R. Nat. Genet. 1994; 6: 193-196Crossref PubMed Scopus (327) Google Scholar, 3Green A.J. Johnson P.H. Yates J.R. Hum. Mol. Genet. 1994; 3: 1833-1834Crossref PubMed Scopus (163) Google Scholar). Drosophila and mouse genetics show that mutations in TSC1 or TSC2/gigas result in increased cell size (an increase in cell mass/cell growth) and cellular proliferation (an increase in cell number) (4Ito N. Rubin G.M. Cell. 1999; 96: 529-539Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 5Onda H. Lueck A. Marks P.W. Warren H.B. Kwiatkowski D.J. J. Clin. Invest. 1999; 104: 687-695Crossref PubMed Scopus (315) Google Scholar, 6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Genetic epistasis analysis in Drosophila places dTSC1/dTSC2 downstream of dPI3K and dAkt (also referred to as protein kinase B) but upstream of Drosophila ribosomal protein S6 kinase (dS6K) (6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 7Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (383) Google Scholar). The loss of function or overexpression of several Drosophila genes recently linked the dPI3K and dTOR signaling pathways to the regulation of cell proliferation and cell growth (8Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (498) Google Scholar), important cellular processes that are inappropriately regulated during tumor formation. These include dPI3K, dAkt, dS6K, and d4E-BP (effectors of dPI3K), and dTOR. Collectively, this work suggested that dTOR and dTSC1/dTSC2 were connected via the PI3K signaling pathway. Recently, a surge of research from many labs revealed that TSC1/TSC2 functioned to inhibit nutrient- and PI3K-dependent signaling through mTOR (also called FRAP or RAFT), resulting in the inhibition of its downstream targets S6K1 and 4E-BP1 (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 10Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, 11Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (562) Google Scholar, 12Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). the of work this the TSC1/TSC2 also function in to inhibit PI3K signaling to S6K1 of mTOR Ref. A. J. M. T. A. J. J. Cell Biol. 2002; PubMed Scopus Google and review see Ref. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). is through the of that PI3K-dependent signaling to inhibit the tuberin-hamartin heterodimer through phosphorylation of tuberin at Ser-939 and Thr-1462 Akt phosphorylation (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, C.J. Xu T. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). that the tuberin-hamartin heterodimer inhibits mTOR and that Akt tuberin, mTOR is regulated by and and is a upstream of S6K1 and 4E-BP1 (for see Ref. Blenis J. 2002; PubMed Scopus Google Scholar). The mTOR S6K1 and 4E-BP1 of S6K1 and 4E-BP1 phosphorylation by of two distinct signaling PI3K and MAPK, an mTOR as rapamycin inhibit the phosphorylation of downstream by both and pathways Blenis J. 2002; PubMed Scopus Google Scholar, C.J. 1994; PubMed Scopus Google Scholar, J. C.J. Blenis J. Cell. Full Text PDF PubMed Scopus Google Scholar). 4E-BP1 to the protein to inhibit (for review see Ref. B. N. Genes Dev. 2001; 15: PubMed Scopus Google Scholar), is 4E-BP1 is The of 4E-BP1 from leads to the of to and the initiation of protein that both S6K1 and of mammalian cell size D.C. S. C. Blenis J. Genes Dev. 2002; PubMed Scopus Google Scholar). The research with the Drosophila genetic studies (8Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (498) Google Scholar), that signaling to both S6K1 and to the and size of TSC that phosphorylation of S6K1 and 4E-BP1 are regulated by PI3K by pathways J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. A. Blenis J. 1994; PubMed Scopus Google Scholar, T.P. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Tee A.R. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We phorbol 12-myristate 13-acetate a phorbol and a tumor to S6K1 and 4E-BP1 phosphorylation via of the and this we show that the TSC1/TSC2 inhibits PKC/MAPK signaling toward 4E-BP1 and not PI3K-dependent signaling in the cells used in this the that TSC1/TSC2 inhibits mTOR We that tuberin is on the sites and and on distinct phosphorylation sites in the of PI3K signaling and is by the protein kinase inhibitors toward and PKC This reveals that the PKC/MAPK signaling to tuberin is distinct from that by work an important as to S6K1 is PI3K-independent that cell signaling through PI3K and PKC/MAPK pathways the tuberin-hamartin heterodimer as a result of tuberin phosphorylation at sites. and and were from and bisindolylmaleimide I was from PMA, and were from and epidermal growth factor was from was from were from and was from were from and mammalian were as A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). The tuberin and Thr-1462 to was by B. D. Manning and C. Cantley was human 4E-BP1 was a from N. S6K1 was as C. A. K.L. Blenis J. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and cells were and in with were by as Blenis J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). was used that or of or respectively. to was as H. J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with and with 1 1 of The cell were at and at to of cell of cell and of cell were Akt or to that the cells were to cell The in this are of at of were to to and with the by a of 4E-BP1 and tuberin, the were on and respectively. were by were from was by M. of and and were from Cell The was from were from were as J. A. A. Blenis J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). was by on as D.C. S. C. Blenis J. Genes Dev. 2002; PubMed Scopus Google Scholar). analysis of hamartin and tuberin the the cell were as A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). and studies of cell were with to protein were as C. A. K.L. Blenis J. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). S6K1 kinase was in by of ribosomal protein as a as C. A. K.L. Blenis J. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of of the was on a with of tuberin was in cells hamartin and The cells were in and the cells were and with of hamartin and tuberin were with an to protein 1 were as the S6K1 kinase and to and of Hamartin and 4E-BP1 Phosphorylation and of we that of both hamartin and tuberin 4E-BP1 phosphorylation of cells A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). this cell signaling through the PI3K pathway but not the pathway A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). that of the pathway by phorbol in the phosphorylation of 4E-BP1 that is on mTOR but of PI3K T.P. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Tee A.R. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), we hamartin tuberin 4E-BP1 phosphorylation. Hamartin and tuberin were with 4E-BP1 in cells that were and with as a to PI3K-dependent we show that of this cell the PKC/MAPK signaling pathway but not the as by the of to phosphorylation of extracellular signal-regulated kinase (also as phosphorylation of Akt on The of hamartin and tuberin the cell were and revealed that the of hamartin and tuberin protein was was with the as A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, M. B. A.J. D.J. Hum. Mol. Genet. 2001; 10: PubMed Scopus Google Scholar). The of hamartin and tuberin to 4E-BP1 phosphorylation was by a in with that 4E-BP1 on and The and of 4E-BP1 as on The and the the and of respectively. of tuberin its the phosphorylation of was hamartin was with Hamartin and tuberin the phosphorylation of 4E-BP1 at to the of Phosphorylation of is the phosphorylation of the sites and (for review see Ref. B. N. Genes Dev. 2001; 15: PubMed Scopus Google Scholar). and are the of 4E-BP1 from in Ref. B. N. Genes Dev. 2001; 15: PubMed Scopus Google Scholar), the of phosphorylation with its from A.R. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). the of and phosphorylation on 4E-BP1 hamartin and tuberin 4E-BP1 to was on and the of 4E-BP1 to was not treatment of cells hamartin and These that 4E-BP1 phosphorylation and from are by tuberin-hamartin in a to that of rapamycin 1 and to and in overexpression of tuberin and hamartin by mTOR and from TSC to 4E-BP1 tuberin, mutants of tuberin are to PI3K signaling toward downstream of mTOR K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, 12Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). We two tuberin mutants and were of 4E-BP1 phosphorylation was that the tuberin the tuberin to and hamartin from the to the the tuberin this function M. B. A.J. D.J. Hum. Mol. Genet. 2001; 10: PubMed Scopus Google Scholar). with the tuberin increased the of hamartin the the of tuberin not The of the tuberin was as M. B. A.J. D.J. Hum. Mol. Genet. 2001; 10: PubMed Scopus Google Scholar), to in the from The phosphorylation of 4E-BP1 was by the tuberin with hamartin with the as by the of 4E-BP1 on and of and phosphorylation of the tuberin with hamartin not or phosphorylation and phosphorylation of 4E-BP1 with the tuberin the tuberin of the tuberin to inhibit 4E-BP1 phosphorylation. and Hamartin S6K1 the of hamartin and tuberin to downstream mTOR signaling the of S6K1 was from cells with in the of protein kinase inhibitors of PI3K mTOR and PKC was by bisindolylmaleimide a PKC inhibitor that the PKC of by bisindolylmaleimide I to wortmannin Akt and S6K1 wortmannin S6K1 by of hamartin and tuberin also S6K1 that was cells were with wortmannin that the inhibition of S6K1 by tuberin-hamartin and wortmannin are that tuberin-hamartin inhibits S6K1 through a PI3K-independent as A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). and Hamartin S6K1 of hamartin and tuberin inhibits S6K1 via a PI3K-independent was to S6K1 in the of PI3K we cells with either or in the of wortmannin signaling through that wortmannin phosphorylation of the or of S6K1 in the of wortmannin of of S6K1 was by both rapamycin and and and a both mTOR and to S6K1 in the of that bisindolylmaleimide I not phosphorylation or S6K1 the and that the PI3K-independent increased of S6K1 by on of signaling PKC this that the of bisindolylmaleimide I used not inhibit signaling to or S6K1 Hamartin and tuberin the PI3K-independent of S6K1 by was with the treatment that the PKC/MAPK pathway S6K1 was by rapamycin and by either or bisindolylmaleimide I and that were to block phosphorylation Hamartin and tuberin the PI3K-independent of S6K1 by was reduced to that of the with either or bisindolylmaleimide I and We show that hamartin and tuberin in with treatment and of S6K1 either or treatment that tuberin-hamartin inhibits signaling through wortmannin was used in with either or that hamartin and tuberin S6K1 of mTOR by Hamartin and tuberin-hamartin inhibits we cells with wortmannin to inhibit signaling mTOR signaling with to mTOR signaling through the of to the of mTOR Y. M. A. J. 2001; PubMed Scopus Google Scholar). of hamartin and tuberin the of S6K1 treatment Y. M. A. J. 2001; PubMed Scopus Google Scholar), not either Akt or that S6K1 treatment was a result of increased mTOR signaling toward S6K1 the that the tuberin-hamartin inhibits in Phosphorylation of of to S6K1 of PI3K that a PI3K-independent inhibit the tuberin-hamartin heterodimer that S6K1 by PKC/MAPK the function of tuberin-hamartin is through phosphorylation of tuberin (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 10Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, C.J. Xu T. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, M. M. Yeung R.S. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), we to tuberin was also of the PKC/MAPK signaling pathway Akt was Phosphorylation of tuberin its on as cells were with Phosphorylation of tuberin by was either or by the cells with and bisindolylmaleimide respectively. The phosphorylation of tuberin was by wortmannin and reveals that both PKC and signaling leads to the phosphorylation of tuberin in the of PI3K in increase of tuberin with both and and from not the of tuberin on The that are on the or the is This was used to show that Akt tuberin on Ser-939 and Thr-1462 and that both to the (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar). the of tuberin treatment through the phosphorylation of as sites that are distinct from by Akt treatment caused an increase in tuberin phosphorylation at the Akt phosphorylation sites by the increased of tuberin with the Akt from that to phosphorylation of tuberin at Thr-1462 from of phosphorylation with the was to wortmannin but by both and bisindolylmaleimide that Akt is not the kinase in the phosphorylation of tuberin at Thr-1462 or the sites This reveals that tuberin is in by a protein kinase regulated by the PKC/MAPK signaling pathway that a phosphorylation to that of the of tuberin phosphorylation treatment in we tuberin in vivo. phosphorylation of tuberin by in the of we the of tuberin treatment in the or of or bisindolylmaleimide in with wortmannin of tuberin, hamartin was with tuberin to increase the of the tuberin-hamartin heterodimer the the phosphorylation of tuberin, was by the cells with either or bisindolylmaleimide was increase of hamartin that the PKC/MAPK signaling pathway the function of the tuberin-hamartin heterodimer by tuberin The in that the phosphorylation of tuberin treatment at sites that overlap with and are to that are by this we a of tuberin, the two Akt phosphorylation sites and to to as and Akt but not the of tuberin but not the that the Akt phosphorylation sites. the that tuberin at treatment the of the and the tuberin protein. phosphorylation of the was by with either or bisindolylmaleimide cell that were to phosphorylation of These that two signaling and to the phosphorylation of and distinct sites on S6K1 tuberin phosphorylation at Ser-939 and Thr-1462 the function of the protein we the of tuberin and to block S6K1 We that the tuberin was at S6K1 with the tuberin The tuberin S6K1 by the S6K1 by This that tuberin phosphorylation at Ser-939 and Thr-1462 through the PKC/MAPK signaling pathway to inhibit Phosphorylation of kinase (also referred to as to tuberin on Y. K. P. Guan K.L. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that the phosphorylation tuberin is the is not to the is that the tuberin on of the signaling pathway tuberin phosphorylation on the Akt we cells with to as a Cell with phosphorylation but not result in tuberin phosphorylation on the and tuberin phosphorylation on the sites that were by with wortmannin block or bisindolylmaleimide I block respectively. of the signaling pathway not tuberin phosphorylation on the sites that are of either the or PKC/MAPK signaling work (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 10Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, 11Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (562) Google Scholar, 12Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, A. J. M. T. A. J. J. Cell Biol. 2002; PubMed Scopus Google that the inhibits signaling toward S6K1 and as to tuberin-hamartin inhibits PI3K signaling through an or (for review see Ref. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). The that signaling is the target of We that tuberin-hamartin inhibits 4E-BP1 phosphorylation and and S6K1 and and cell that the mTOR signaling pathway of also to inhibit S6K1 the of to cells K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, 11Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (562) Google Scholar, 12Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google and to with mTOR K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar). Collectively, the the that mTOR signaling is the target of This work of TSC as a tumor suppressor mammalian We that tuberin-hamartin signaling to the downstream of mTOR, and is that both S6K1 and function to cell growth and proliferation D.C. S. C. Blenis J. Genes Dev. 2002; PubMed Scopus Google Scholar, D. A. N. Google Scholar). that tuberin is and by a PKC/MAPK signaling is S6K1 and function to is that the of tuberin through PKC/MAPK signaling to signaling inhibits the tumor suppressor function of tuberin-hamartin through the phosphorylation of tuberin that at Ser-939 and Thr-1462 (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 10Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, C.J. Xu T. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, M. M. Yeung R.S. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of sites also of the PKC/MAPK signaling pathway to the tumor suppressor function of the tuberin-hamartin We a both and PKC/MAPK signaling pathways on the tuberin-hamartin and phosphorylation of sites tuberin the tumor suppressor heterodimer to mTOR of we show that the by Akt (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar), inhibits of S6K1 tuberin phosphorylation of sites tuberin by and signaling are important the regulation of the tuberin and is that phosphorylation of tuberin to the inhibition of the tuberin-hamartin kinase that tuberin at was ribosomal S6 kinase 1 overexpression of cells not the phosphorylation of tuberin not that is that is the studies have to to the tuberin phosphorylation We are to kinase phosphorylates the sites tuberin of PKC/MAPK kinase include and and During the of this a of studies in as a target of and H. T. B. P. P. S. Nat. Cell Biol. PubMed Scopus Google Scholar, X. Li Pan D. Nat. Cell Biol. PubMed Scopus Google Scholar, Y. X. Ru B. Pan D. Nat. Cell Biol. PubMed Scopus Google Scholar, A. T. M. M. H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. Manning B.D. Cantley L.C. Blenis J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Drosophila genetics epistasis analysis downstream of and upstream of dTOR H. T. B. P. P. S. Nat. Cell Biol. PubMed Scopus Google Scholar, X. Li Pan D. Nat. Cell Biol. PubMed Scopus Google Scholar). by Zhang Y. X. Ru B. Pan D. Nat. Cell Biol. PubMed Scopus Google as a GTPase-activating protein from that signaling toward S6K1 and 4E-BP1 that was by tuberin and hamartin overexpression A.R. Manning B.D. Cantley L.C. Blenis J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). mammalian studies show that tuberin is a A. T. M. M. H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. Manning B.D. Cantley L.C. Blenis J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the of tuberin to function as a is with its hamartin A.R. Manning B.D. Cantley L.C. Blenis J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These studies that is an upstream of mTOR and a downstream target of that is and the phosphorylation of tuberin regulate the tuberin-hamartin is phosphorylation of tuberin the of tuberin and hamartin to tuberin phosphorylation to of the tuberin-hamartin or Akt overexpression reduced the of hamartin that with tuberin in cell (6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). a tuberin protein the phosphorylation sites were to with hamartin K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar). two research (9Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, M. M. Yeung R.S. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google that the phosphorylation of tuberin mammalian cells was not to the TSC tumor suppressor complex. we not of the tuberin-hamartin heterodimer tuberin phosphorylation not also that phosphorylation of tuberin the of both tuberin and hamartin K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2331) Google Scholar, M. M. Yeung R.S. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We in cells tuberin to or signaling the of tuberin, and we that of cells in the loss of tuberin an not This a of overexpression that the of research M. C. B. A. A.J. P. D.J. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. K. Yeung Guan K.L. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. 2002; Google Scholar, Li Y. Y. J. Biol. Full Text Full Text PDF PubMed Scopus Google have also that to the of tuberin, and kinase was to tuberin on and to tuberin Y. K. P. Guan K.L. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These studies an with tuberin inhibit the tumor suppressor complex and target tuberin is that is the kinase that phosphorylates tuberin PKC/MAPK signaling as the signaling pathway is not cell with of the signaling pathway not result in the phosphorylation of tuberin at the phosphorylation sites that the and and kinase are downstream of are to tuberin phosphorylation at sites. the that tuberin and hamartin function as a heterodimer to inhibit mTOR signaling and that tuberin is through both PI3K-dependent and is to that the tuberin-hamartin heterodimer as a of and PKC/MAPK signaling pathways on the of either the of mTOR is the by tuberin-hamartin to inhibit mTOR, research that is a target of and an upstream of to see this complex with We The and The We also C. Fingar of the and of