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Phosphoinositide 3-Kinase Activation Regulates Cell Division Time by Coordinated Control of Cell Mass and Cell Cycle Progression Rate

2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês

10.1074/jbc.m300663200

ISSN

1083-351X

Autores

Beatriz Álvarez, Elia Garrido, José A. García‐Sanz, Ana C. Carrera,

Tópico(s)

Cancer-related Molecular Pathways

Resumo

Cells must increase their mass in coordination with cell cycle progression to ensure that their size and macromolecular composition remain constant for any given proliferation rate. To this end, growth factors activate early signaling cascades that simultaneously promote cell mass increase and induce cell cycle entry. Nonetheless, the mechanism that controls the concerted regulation of cell growth and cell cycle entry in mammals remains unknown. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway regulates cell cycle entry by inactivating forkhead transcription factors and promoting cyclin D synthesis. PI3K/protein kinase B-derived signals also affect activation of p70 S6 kinase and the mammalian target of rapamycin, enzymes involved in cell growth control. We previously showed that enhancement of PI3K activation accelerates cell cycle entry, whereas reduction of PI3K activation retarded this process. Here we examined whether expression of different PI3K mutants affects cell growth during cell division. We show that diminishing or enhancing the magnitude of PI3K activation in a transient manner reduces or increases, respectively, the protein synthesis rate. Alteration of cell growth and cell cycle entry by PI3K forms appears to be concerted, because it results in lengthening or shortening of cell division time without altering cell size. In support of a central role for PI3K in growth control, expression of a deregulated, constitutive active PI3K mutant affects p70 S6 kinase and mammalian target of rapamycin activities and increases cell size. Together, the results show that transient PI3K activation regulates cell growth and cell cycle in a coordinated manner, which in turn controls cell division time. Cells must increase their mass in coordination with cell cycle progression to ensure that their size and macromolecular composition remain constant for any given proliferation rate. To this end, growth factors activate early signaling cascades that simultaneously promote cell mass increase and induce cell cycle entry. Nonetheless, the mechanism that controls the concerted regulation of cell growth and cell cycle entry in mammals remains unknown. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway regulates cell cycle entry by inactivating forkhead transcription factors and promoting cyclin D synthesis. PI3K/protein kinase B-derived signals also affect activation of p70 S6 kinase and the mammalian target of rapamycin, enzymes involved in cell growth control. We previously showed that enhancement of PI3K activation accelerates cell cycle entry, whereas reduction of PI3K activation retarded this process. Here we examined whether expression of different PI3K mutants affects cell growth during cell division. We show that diminishing or enhancing the magnitude of PI3K activation in a transient manner reduces or increases, respectively, the protein synthesis rate. Alteration of cell growth and cell cycle entry by PI3K forms appears to be concerted, because it results in lengthening or shortening of cell division time without altering cell size. In support of a central role for PI3K in growth control, expression of a deregulated, constitutive active PI3K mutant affects p70 S6 kinase and mammalian target of rapamycin activities and increases cell size. Together, the results show that transient PI3K activation regulates cell growth and cell cycle in a coordinated manner, which in turn controls cell division time. Cell division is the process by which a cell duplicates its DNA content and cell mass to produce two daughter cells. In mammals, cell division is essential during development and in the adult for tissue regeneration. Most cell division is a symmetrical process that gives rise to two virtually identical daughter cells (1Polymenis M. Schmidt E.V. Curr. Opin. Genet. Dev. 1999; 9: 76-80Crossref PubMed Scopus (111) Google Scholar, 2Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar, 3Zetterberg A. Larsson O. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5365-5369Crossref PubMed Scopus (288) Google Scholar, 4Tapon N. Moberg K.H. Hariharan I.K. Curr. Opin. Cell Biol. 2001; 13: 731-737Crossref PubMed Scopus (68) Google Scholar, 5Saucedo L.J. Edgar B.A. Curr. Opin. Genet. Dev. 2002; 12: 565-571Crossref PubMed Scopus (103) Google Scholar). To initiate symmetrical cell division, mitogens trigger a number of early signals that culminate in the activation of G1 cyclin/CDKs (required for the G1-S transition) and induce an increase in cell mass. This increase is required to ensure that macromolecular composition and cell size are conserved in daughter cells (1Polymenis M. Schmidt E.V. Curr. Opin. Genet. Dev. 1999; 9: 76-80Crossref PubMed Scopus (111) Google Scholar, 2Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar, 3Zetterberg A. Larsson O. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5365-5369Crossref PubMed Scopus (288) Google Scholar, 4Tapon N. Moberg K.H. Hariharan I.K. Curr. Opin. Cell Biol. 2001; 13: 731-737Crossref PubMed Scopus (68) Google Scholar, 5Saucedo L.J. Edgar B.A. Curr. Opin. Genet. Dev. 2002; 12: 565-571Crossref PubMed Scopus (103) Google Scholar). Signaling pathways that control the cell mass increase, referred to as cell growth, have recently been elucidated and include proteins such as phosphoinositide 3-kinase (PI3K), 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; mTOR, mammalian target of rapamycin; p70 S6K, p70 S6 kinase; TSC, tuberous sclerosis complex; 4EBP1, initiation factor 4E-binding protein 1; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescence protein; CS, calf serum; TOP, terminal oligopyrimidine tract. p70 S6 kinase (p70 S6K), the mammalian target of rapamycin (mTOR), and the tuberous sclerosis complex (TSC1/2) (4Tapon N. Moberg K.H. Hariharan I.K. Curr. Opin. Cell Biol. 2001; 13: 731-737Crossref PubMed Scopus (68) Google Scholar, 5Saucedo L.J. Edgar B.A. Curr. Opin. Genet. Dev. 2002; 12: 565-571Crossref PubMed Scopus (103) Google Scholar). These proteins regulate synthesis of ribosomal components and activation of the translational machinery (4Tapon N. Moberg K.H. Hariharan I.K. Curr. Opin. Cell Biol. 2001; 13: 731-737Crossref PubMed Scopus (68) Google Scholar, 5Saucedo L.J. Edgar B.A. Curr. Opin. Genet. Dev. 2002; 12: 565-571Crossref PubMed Scopus (103) Google Scholar). It is nonetheless unknown how the two independent processes of cell cycle entry and cell growth are coordinated during cell division, particularly in mammals. Studies in Saccharomyces cerevisiae show that whereas blockage of cell division by inactivation of most cell division cycle (cdc) genes allows cell growth to continue, inhibition of cell growth impairs cell cycle progression (6Johnston G.C. Pringle J.R. Hartwell L.H. Exp. Cell Res. 1977; 105: 79-98Crossref PubMed Scopus (562) Google Scholar). The cell cycle thus appears to be linked to cell growth. Similar results were obtained in Drosophila (7Stocker H. Hafen E. Curr. Opin. Genet. Dev. 2000; 10: 529-535Crossref PubMed Scopus (207) Google Scholar, 8Weinkove D. Neufeld T.P. Twardzik T. Waterfield M.D. Leevers S.J. Curr. Biol. 1999; 9: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 9Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (510) Google Scholar, 10Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (624) Google Scholar). PI3K is an enzyme that transfers phosphate to the 3-position of the inositol ring of membrane phosphoinositides. The PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor stimulation, regulating a variety of cell responses including survival and division (11Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar, 12Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar, 13Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar, 14Zvelebil M.J. MacDougall L. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages F. Koga H. Salim K. Linacre J. Das P. Panaretou C. Wetzler R. Waterfield M.D. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1996; 351: 217-223Crossref PubMed Scopus (89) Google Scholar). The 3-polyphosphoinositides recruit pleckstrin homology domain-containing proteins such as phosphoinositide dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (15Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 16Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1880) Google Scholar, 17Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1828) Google Scholar, 18Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (447) Google Scholar). Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit (11Vanhaesebroeck B. Waterfield M.D. Exp. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar, 12Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar, 13Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar, 14Zvelebil M.J. MacDougall L. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages F. Koga H. Salim K. Linacre J. Das P. Panaretou C. Wetzler R. Waterfield M.D. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1996; 351: 217-223Crossref PubMed Scopus (89) Google Scholar, 19Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (235) Google Scholar, 20Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 21Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 22Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 23Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar). Activation of this enzyme following growth factor receptor stimulation controls cell cycle entry by regulating cyclin D synthesis and inactivation of FOX0 forkhead transcription factors, events required for G1-to-S transition (24Klippel A. Escobedo M.A. Wachowicz M.S. Apell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 18: 5699-5711Crossref PubMed Scopus (241) Google Scholar, 25Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar, 26Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1226) Google Scholar, 27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). Nonetheless, subsequent inactivation of PI3K is also important for completion of the cell cycle, because expression of constitutive active PI3K mutants inhibits forkhead activity in G2, which is required for mitotic progression (27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). In addition to controlling these events, PI3K activation governs cell growth by regulating activation of p70 S6K and mTOR (28Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (657) Google Scholar, 29Reif K. Burgering B.M. Cantrell D.A. J. Biol. Chem. 1997; 272: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 31Manning 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 (1278) Google Scholar, 32Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). mTOR is a large (289 kDa), evolutionarily conserved Ser/Thr kinase that is inhibited by the drug rapamycin (33Zaragoza D. Ghavidel A. Heitman J. Schultz M.C. Mol. Cell. Biol. 1998; 18: 4463-4470Crossref PubMed Scopus (184) Google Scholar, 34Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1026) Google Scholar, 35Fingar D.C. Salama S. Tsou C. Harlow E. Blenis J. Genes Dev. 2002; 16: 1472-1487Crossref PubMed Scopus (862) Google Scholar). mTOR, also termed FRAP (FKBP-12 rapamycin-associated protein), phosphorylates and inactivates the eukaryotic initiation factor 4E-binding protein 1 (4EBP1), an inhibitor of the translation initiation complex (36Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1184) Google Scholar, 37Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (941) Google Scholar). This complex regulates 5′ cap translation, which accounts for the majority of cellular translation (36Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1184) Google Scholar). In addition, mTOR regulates p70 S6K activation (37Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (941) Google Scholar, 38Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar, 39Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (812) Google Scholar). mTOR activity is sensitive to nutrient (amino acid) and energy (ATP) levels and is also regulated by mitogens (40Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 41Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (804) Google Scholar, 42Fang Y. Vilella-Bach M. Bachmann R. Flanigan A. Chen J. Science. 2001; 294: 1942-1945Crossref PubMed Scopus (870) Google Scholar). A number of recent studies show that mTOR action on p70 S6K and 4EBP1 is negatively controlled by the TSC1/2 tumor suppressor complex (30Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 31Manning 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 (1278) Google Scholar, 32Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar, 43Gao 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 (575) Google Scholar). PI3K/PKB controls mTOR function by regulating TSC2 phosphorylation (30Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 31Manning 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 (1278) Google Scholar, 32Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). In fact, the PI3K effector PKB was shown to phosphorylate TSC2; this phosphorylation destabilizes TSC2 and disrupts its association with TSC1, restoring mTOR-regulated phosphorylation of 4EBP1 and p70 S6K (30Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 31Manning 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 (1278) Google Scholar, 32Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). p70 S6K is a Ser/Thr kinase that phosphorylates the 40 S ribosomal protein S6 (44Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (486) Google Scholar). S6 phosphorylation facilitates recruitment of a specific mRNA subset containing a polypyrimidine tract at the 5′ transcriptional start site (5′ TOP) to translating polysomes. 5′ TOP transcripts include those encoding ribosomal proteins (S3, S6, S14, and S24) and translation elongation factors (eEF1A and eEF2) (44Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (486) Google Scholar, 45Thomas G. Nat. Cell Biol. 2000; 2: E71-E72Crossref PubMed Scopus (172) Google Scholar). In addition, p70 S6K regulates eEF2 activity (46Wang X. Li W. Williams M. Terada N. Alessi D.R. Proud C.G. EMBO J. 2001; 20: 4370-4379Crossref PubMed Scopus (630) Google Scholar). p70 S6K is activated by the ordered phosphorylation of residues in the C-terminal pseudosubstrate region, followed by phosphorylation of Thr389 and Thr229 (44Pullen N. Thomas G. FEBS Lett. 1997; 410: 78-82Crossref PubMed Scopus (486) Google Scholar, 45Thomas G. Nat. Cell Biol. 2000; 2: E71-E72Crossref PubMed Scopus (172) Google Scholar). p70 S6K triggering requires activation of both mTOR and PI3K (28Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (657) Google Scholar, 29Reif K. Burgering B.M. Cantrell D.A. J. Biol. Chem. 1997; 272: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 37Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (941) Google Scholar, 38Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar, 39Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (812) Google Scholar). mTOR activity is required for the phosphorylation of p70 S6K in several residues, including Thr389 (37Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (941) Google Scholar, 38Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar, 39Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (812) Google Scholar). PI3K/PKB regulates TSC2 phosphorylation and, in turn, mTOR activation (30Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 31Manning 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 (1278) Google Scholar, 32Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (780) Google Scholar). In addition, PI3K controls p70 S6K activation via mTOR-independent mechanisms, as supported by the observation that PI3K activity is still required for activation of rapamycin-resistant p70 S6K mutants (47Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (223) Google Scholar). The PI3K effectors phosphoinositide-dependent kinase-1 and protein kinase Cζ were shown to stabilize Thr389 phosphorylation, and phosphoinositide-dependent kinase-1 phosphorylates Thr229 (48Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 49Romanelli A. Dreisbach V.C. Blenis J. J. Biol. Chem. 2002; 277: 40281-40289Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Finally, in addition to controlling mTOR and p70 S6K, PI3K also regulates cell growth by controlling translation of 5′ TOP mRNAs via a p70 S6K-independent mechanism (50Stolovich M. Tang H. Hornstein E. Levy G. Cohen R. Bae S.S. Birnbaum M.J. Meyuhas O. Mol. Cell. Biol. 2002; 22: 8101-8113Crossref PubMed Scopus (198) Google Scholar). In addition to the biochemical studies mentioned, genetic experiments in Drosophila support PI3K/PKB, p70 S6K, and mTOR involvement in cell growth control (7Stocker H. Hafen E. Curr. Opin. Genet. Dev. 2000; 10: 529-535Crossref PubMed Scopus (207) Google Scholar, 8Weinkove D. Neufeld T.P. Twardzik T. Waterfield M.D. Leevers S.J. Curr. Biol. 1999; 9: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 9Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (510) Google Scholar, 10Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (624) Google Scholar). In mammals, deregulation of p70S6K, mTOR, or the PI3K/PKB pathway was shown to affect cell size (35Fingar D.C. Salama S. Tsou C. Harlow E. Blenis J. Genes Dev. 2002; 16: 1472-1487Crossref PubMed Scopus (862) Google Scholar, 51Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar, 52Crackower M.A. Oudit G.Y. Kozieradzki I. Sarao R. Sun H. Sasaki T. Hirsch E. Suzuki A. Shioi T. Irie-Sasaki J. Sah R. Cheng H.Y. Rybin V.O. Lembo G. Fratta L. Oliveira-dos-Santos A.J. Benovic J.L. Kahn C.R. Izumo S. Steinberg S.F. Wymann M.P. Backx P.H. Penninger J.M. Cell. 2002; 110: 737-749Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 53Shioi T. McMullen J.R. Kang P.M. Douglas P.S. Obata T. Franke T.F. Cantley L.C. Izumo S. Mol. Cell. Biol. 2002; 22: 2799-2809Crossref PubMed Scopus (447) Google Scholar). Nonetheless, despite the well demonstrated function of PI3K/PKB, p70 S6K, and mTOR in cell growth control, little is known of how cell growth induction is linked to cell cycle progression. Based on the capacity of PI3K to regulate pathways that control cell growth and cell cycle entry, we hypothesized that PI3K activation may contribute to the concerted regulation of these processes during cell division in mammals. We previously described the consequences on cell cycle progression of interfering with physiological PI3K regulation in NIH 3T3 cells by expressing different p85/p110 PI3K forms (27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). These studies indicated that enhancement of PI3K activation in a transient manner accelerates cell cycle progression, whereas reduction of PI3K activation decreases this process (27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). Here we show that expression of p65PI3K, a mutant that enhances transient PI3K activation, augmented the protein synthesis rate of cycling cells. This increase was concerted with the cell cycle progression rate, because p65PI3K expression shortened division time without altering cell size. Accordingly, expression of the recombinant p85α regulatory subunit, which reduces the magnitude of transient PI3K activation, increased cell division time without altering cell size. These observations illustrate the concerted regulation of cell growth and cell cycle progression rates by PI3K, thereby controlling cell division time. The key role of PI3K in growth control is supported by the observation that expression of a deregulated, constitutive active PI3K form altered p70 S6K and mTOR activation kinetics, giving rise to larger cells. cDNA Constructs, Antibodies, and Materials—pcDNA3-TSC1 and pcDNA3-TSC2 cDNA and anti-TSC2 antibodies were kindly provided by Mark Nellist (54van Slegtenhorst M. Nellist M. Nagelkerken B. Cheadle J. Snell R. van den Ouweland A. Reuser A. Sampson J. Halley D. van der Sluijs P. Hum. Mol. Genet. 1998; 7: 1053-1057Crossref PubMed Scopus (493) Google Scholar). Prk5-Myc-D3Ep70S6K was kindly donated by George Thomas (55Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (727) Google Scholar). Anti-p70 S6K antibodies were from Santa Cruz Biotechnology, and anti-Thr(P)389 and anti-Thr(P)421/Ser(P)424 p70 S6K antibodies were from New England Biolabs. Horseradish peroxidase-conjugated antibodies were from Dako, and the enhanced chemiluminiscence developing kit was from Amersham Biosciences. Rapamycin was from Calbiochem. Cell Culture and Transfection—NIH 3T3 cells were cultured (37 °C, 10% CO2) in Dulbecco's modified Eagle's medium (DMEM; BioWhittaker) with 10% calf serum (Invitrogen). Stable NIH 3T3 cell lines expressing p110caax, p65PI3K and p85α have been described (56Jimenez C. Jones D.R. Rodriguez-Viciana P. Gonzalez-Garcia A. Leonardo E. Wennstrom S. von Kobbe C. Toran J.L. Rodriguez-Borlado L. Calvo V. Copin S.G. Albar J.P. Gaspar M.L. Diez E. Marcos M.A. Downward J. Martinez A.C. Merida I. Carrera A.C. EMBO J. 1998; 17: 743-753Crossref PubMed Scopus (237) Google Scholar, 57Jimenez C. Portela R.A. Mellado M. Rodriguez-Frade J.M. Collard J. Serrano A. Martinez A.C. Avila J. Carrera A.C. J. Cell Biol. 2000; 151: 249-262Crossref PubMed Scopus (201) Google Scholar). Stable cell lines expressing D3Ep70S6K were obtained by transfection of cells with Prk5-Myc-D3Ep70S6K cDNA combined with p-Pur cDNA (Clontech); clones were selected in medium containing 2 μg/ml puromycin (Sigma). Transient transfection was performed using LipofectAMINE Plus (Invitrogen) according to manufacturer's instructions. Cell cycle arrest was as described (27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). Briefly, for G0 phase arrest, cells were incubated without serum for 20 h. For G2 phase arrest, cells were incubated (20 h) with 5 μm etoposide (Sigma), which yielded 40–50% cells in G2. For M phase arrest, cells were incubated (20 h) with 0.1 μg/ml colcemid (Invitrogen), yielding ∼70% cells in M phase. For G1 samples, cells were arrested in G0 for 19 h and incubated with serum for 1 h. Extract Preparation and Western Blotting—Cells were lysed in 50 mm HEPES pH 8, 150 mm NaCl and 1% Triton X-100 containing phosphatase and protease inhibitors (27Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar, 58Gonzalez-Garcia A. Garrido E. Hernandez C. Alvarez B. Jimenez C. Cantrell D.A. Pullen N. Carrera A.C. J. Biol. Chem. 2002; 277: 1500-1508Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). For p70S6K immunoblotting, cells were lysed in 10 mm Hepes pH 7.8, 20 mm β glycerol phosphate, 15 mm KCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 0.2% Nonidet P-40 containing phosphatase and protease inhibitors (58Gonzalez-Garcia A. Garrido E. Hernandez C. Alvarez B. Jimenez C. Cantrell D.A. Pullen N. Carrera A.C. J. Biol. Chem. 2002; 277: 1500-1508Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Protein concentration was estimated by the BCA assay (Pierce) and equal protein amounts were resolved in SDS-PAGE. Gels were transferred to nitrocellulose and probed with the indicated antibodies. Cell Labeling—Cells were washed in methionine/cysteine-free RPMI (BioWhittaker) and incubated in this medium supplemented with 10% dialyzed fetal calf serum for 2 h prior addition of 35S Met/Cys (20 μCi; Amersham Biosciences) for the times indicated. For 35S Met/Cys labeling of cells in G0 and G2, cells were incubated 16 h in serum-free medium or in medium containing 10% serum and 5 μm etoposide, respectively, then labeled as above. For G1 labeling, cells were incubated as for G0 conditions, labeled, and then incubated with 10% dialyzed calf serum for 1 h. The cells were collected and lysed in Triton X-100 lysis buffer (50 mm HEPES pH 8, 150 mm NaCl and 1% Triton X-100 containing phosphatase and protease inhibitors, 58Gonzalez-Garcia A. Garrido E. Hernandez C. Alvarez B. Jimenez C. Cantrell D.A. Pullen N. Carrera A.C. J. Biol. Chem. 2002; 277: 1500-1508Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Protein concentration was estimated and 20 μg of total protein were resolved in SDS-PAGE and autoradiographed. Cell Size Determinations—To examine cell size after transient transfection and sorting, cells were seeded in 60 mm dishes (2.5 × 105 cells/plate), transfected the following day at 80% confluence using 0.5 μg pEGFP C1 (Clontech) plus 2 μg of plasmids encoding p110caax or p70S6K (58Gonzalez-Garcia A. Garrido E. Hernandez C. Alvarez B. Jimenez C. Cantrell D.A. Pullen N. Carrera A.C. J. Biol. Chem. 2002; 277: 1500-1508Abstract Full Text Full Text PDF

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