Multiple Ras Effector Pathways Contribute to G1Cell Cycle Progression

The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G1 progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D1 up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G1, when cyclin D1 expression becomes maximal, implying that other effector pathways may also be important in cyclin D1 induction. We have addressed the involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G1 progression and compared it to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D1 and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D1 transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D1transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle. The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G1 progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D1 up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G1, when cyclin D1 expression becomes maximal, implying that other effector pathways may also be important in cyclin D1 induction. We have addressed the involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G1 progression and compared it to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D1 and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D1 transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D1transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle. Upon reentry of cells into the cell cycle and throughout the G1 phase, mitogenic signals are integrated through the GTPase Ras. Inhibition of Ras function by microinjection of Ras-neutralizing antibodies or by inducible expression of dominant negative Ras arrests cycling cells in G1 and prevents growth factor stimulated cells from leaving G0 to reenter the cell cycle (1Cai H. Szeberenyi J. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5314-5323Crossref PubMed Scopus (180) Google Scholar, 2Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (672) Google Scholar, 3Mulcahy L.S. Smith M.R. Stacey D.W. Nature. 1985; 313: 241-243Crossref PubMed Scopus (587) Google Scholar, 4Downward J. Curr. Biol. 1997; 7: 258-260Abstract Full Text Full Text PDF PubMed Google Scholar). The requirement of Ras function during the G0/G1 transition seems to be conserved from yeast, in which it is necessary for spore germination (5Herman P.K. Rine J. EMBO J. 1997; 16: 6171-6181Crossref PubMed Scopus (93) Google Scholar). Microinjection of anti-Ras antibodies has demonstrated a requirement for Ras up to 2 h prior to S phase entry (6Dobrowolski S. Harter M. Stacey D.W. Mol. Cell. Biol. 1994; 14: 5441-5449Crossref PubMed Scopus (99) Google Scholar, 7Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (367) Google Scholar). Overexpression of mutationally activated Ras leads to cellular transformation and a shortening of the G1 phase of the cell cycle (8Liu J.J. Chao J.R. Jiang M.C. Ng S.Y. Yen J.J. Yang-Yen H.F. Mol. Cell. Biol. 1995; 15: 3654-3663Crossref PubMed Scopus (263) Google Scholar). However, transformation of cells by activated Ras requires other genetic changes, as only immortal cells that have lost cell cycle checkpoints, such as those imposed by cyclin-dependent kinase (cdk) 1The abbreviations used are: cdk, cyclin-dependent kinase; MAP, mitogen-activated protein; ERK, extracellular-regulated kinase; MEK, MAP/ERK kinase; PI, phosphatidylinositol; PIP3, PI 3,4,5-trisphosphate; Rb, retinoblastoma; Rlf, Ral-GDS-like factor; Ral-GDS, Ral guanine nucleotide dissociation stimulator; PKB, protein kinase B; TOR, target of rapamycin.1The abbreviations used are: cdk, cyclin-dependent kinase; MAP, mitogen-activated protein; ERK, extracellular-regulated kinase; MEK, MAP/ERK kinase; PI, phosphatidylinositol; PIP3, PI 3,4,5-trisphosphate; Rb, retinoblastoma; Rlf, Ral-GDS-like factor; Ral-GDS, Ral guanine nucleotide dissociation stimulator; PKB, protein kinase B; TOR, target of rapamycin. inhibitors, retinoblastoma protein, and p53, can be transformed by Ras alone. The expression of activated Ras in primary cells leads to cell cycle arrest via up-regulation of cdk inhibitors and p53. The resulting phenotype resembles that of cellular senescence (9Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3861) Google Scholar). In mouse embryo fibroblasts (10Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (418) Google Scholar, 11Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar) and rat Schwann cells (12Lloyd A.C. Obermuller F. Staddon S. Barth C. McMahon M. Land H. Genes Dev. 1997; 11: 663-677Crossref PubMed Scopus (204) Google Scholar), the Ras effector Raf appears to be sufficient to mediate this effect via induction of p21Waf1/Cip1. Thus, Ras exerts both positive and negative effects on cell growth, depending on cellular context. Recent work from this and other laboratories has shown that Ras is able to interact with multiple effector enzymes, including the Raf protein kinases, the Ral-GDS family of guanine nucleotide exchange factors for Ral, and type I phosphoinositide 3-kinases (13Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (470) Google Scholar). The functions of multiple Ras effectors are required for cellular transformation; in addition to Raf, both the PI 3-kinase and the Ral-GDS pathways cooperate to achieve efficient transformation of immortalized cells (14White M.A. Nicolette C. Minden A. Polverino A. Van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (626) Google Scholar, 15Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). Therefore, several Ras effector pathways may interface with the cell cycle machinery. Ras has been implicated in the positive regulation of the cyclin D1 promoter. The conditional expression of oncogenic Ras induces cyclin D1 protein production in growth factor-deprived cells (16Filmus J. Robles A.I. Shi W. Wong M.J. Colombo C.J. 1994; Google Scholar). However, the resulting cyclin may in the of growth factors 1996; Google Scholar). of a of Raf to the of the and conditional expression of activated of the MAP/ERK MEK, the effect of oncogenic Ras on cyclin and this can be by of a kinase or of Therefore, Ras effects have been to the activation of Raf MEK, and A. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C.J. S. A. PubMed Scopus Google Scholar, W. J.J. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, there are which activity may be for cyclin D1 production as the cyclin in fibroblasts J.J. J. 1997; PubMed Scopus Google Scholar). has been that of Raf kinase activity by cyclin D1 and Raf activity cell cycle progression through production of cdk inhibitors (9Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3861) Google Scholar, A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (418) Google Scholar, 11Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar, A.C. Obermuller F. Staddon S. Barth C. McMahon M. Land H. Genes Dev. 1997; 11: 663-677Crossref PubMed Scopus (204) Google Scholar). However, it is also that the effects of Ras on are mediated by effector which be to regulation by with other signaling Therefore, which of the Ras effectors interface with the G1 cell cycle machinery. We that activation of PI 3-kinase is required for cyclin D1 protein expression and S phase entry in fibroblasts. The PI 3-kinase also to the activation of transcription for progression through activated of Ral-GDS-like factor which has been implicated in the induction of the also activated cyclin D1 Thus, multiple pathways in addition to kinase mediate the effects of Ras on cyclin D1 expression and E2F activity in to G1 progression. The and has been B. S. A. 1985; PubMed Scopus Google Scholar). The of the cyclin including the into the of and by for Ral-GDS-like factor EMBO J. 1997; 16: PubMed Scopus Google Scholar) and L.A. EMBO J. 1996; 15: PubMed Scopus Google Scholar) have been the on The activated Nature. 1995; PubMed Scopus Google Scholar) and J. B.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) by B. for activated PI 3-kinase B.M. P. S. Warne P.H. Downward J. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Google Scholar), and have been P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). Raf is activated of in which Raf is to the by a from and also by of and to it by and NIH 3T3 cells in serum and for h in serum used at The of and used at with inhibitors for prior to addition of serum to used to with of h activity by and activity as The and the of to and The expression of by in the in and at in in the activity is by Ras signaling of E2F activity by of a the E2F with various Ras effectors into NIH 3T3 activity h are as the induction of activity to the in cells that have been only with the In to cell cycle NIH 3T3 cells with and in at and at in and with for at with and by The cyclin D1 a from used for and a serum from Ras Ras C. A. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) in and in and The and at to and The protein on NIH 3T3 cells in the but The with in for at in and Ras by kinase from NIH 3T3 cell and kinase as a as B.M. P. S. Warne P.H. Downward J. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Google Scholar). Ras function has been shown to be required throughout G1 up to the which cells to S In to Ras activity at later stages in G1, of the of the GTPase with the Ras of Raf kinase D. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). NIH 3T3 cells stimulated with and Ras on to to protein the Ras of Ras activation to be with a of activity that at h from the of Ras activity up to stimulation with little activity be at h also D. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). is to be to the induction of kinase as a of the early phase of growth factor and Ras activation S. M.R. S. M.H. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). of the functions in cell cycle progression to Ras in cells from is the induction of cyclin D1 protein as a of However, cyclin D1 expression only at h stimulation and at h and with B.M. 1995; 10: Google Scholar). Thus, cyclin D1 induction with the phase of Ras activity, which is with The serine/threonine kinase is activated by phosphatidylinositol which is activation of the Ras effector PI 3-kinase. In to the of activation is with a in G1 kinase activity at various during the G1 shown in activity during G1, and of activity the of Ras In to which Ras effector pathways are required for G1 of inhibitors for and PI 3-kinase it is that also of PI such as TOR, the also NIH 3T3 cells with and stimulated with serum in the of the cell cycle at and h serum both and in S phase cells with able to by However, in the of cells into S phase at of the cells in compared with well for for cells and those with or rapamycin. In to the of the cell with or of the activity of effectors of PI 3-kinase and Raf less of serum for in the of or for in the of at the effect of the PI 3-kinase on The of for h a of arrest to that in growth factor The of G1 progression at which is to the for of stimulated activity B.M. P. S. Warne P.H. Downward J. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Google Scholar). that PI 3-kinase activity is for the progression of cells through the G1 phase of the cell cycle. of the effects of serum stimulation to cell cycle reentry in fibroblasts is the expression of cyclin D1 In to the effects of the various inhibitors on cyclin D1 NIH 3T3 cells with inhibitors as and the expression of cyclin D1 that the of the PI 3-kinase cyclin D1 expression at The of the to a expression of cyclin D1 to cells at but there effect at later The in cyclin D1 expression at and but by this effect less for cyclin D1 expression that the effects of the PI 3-kinase with of the target of rapamycin. The PI 3-kinase of the in In to the of in G1 during which PI 3-kinase activity is required for cyclin D1 the at of cells from by serum that h serum cyclin D1 expression becomes of PI 3-kinase activity but that of PI 3-kinase at during the h of serum in of cyclin D1 Inhibition of through the of has only a effect on cyclin D1 which is lost is h serum addition, that the of activity in G1 are important for cyclin D1 that the of used in this sufficient to of the target of The of to the that PI 3-kinase to the regulation of there are multiple pathways including protein kinase that are to of PI 3-kinase. Therefore, to be required for S phase entry in in with Cooper G.M. 1996; Google Scholar). The effects of on serum stimulation also In with the shown in a in induction of cyclin only a The kinase is to be activated by PI 3-kinase through a The of the to the to the it is at and that can only when is to has been and is on for activity P. C. P. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Google Scholar, D. F. 1997; PubMed Scopus Google Scholar). The of the kinase is at Thus, is required for at least in the activation the of which can be by the kinase to the as is in in which the is is but to the effects of PI 3-kinase cell and to the regulation of cyclin in the of that expression in a of cyclin D1 expression in the of with h of serum cyclin D1 but the effect less at and by h the of cyclin D1 to that in the cells of cyclin D1 in the of growth Therefore, the of cyclin D1 expression in cell cyclin D1 expression in the of growth at cells Thus, activity may mediate but of the effects of Ras on cyclin D1 work on the regulation of cyclin D1 transcription has shown that the is growth and can be activated by oncogenic of Ras (16Filmus J. Robles A.I. Shi W. Wong M.J. Colombo C.J. 1994; Google 1996; Google Scholar, B. M. M. 1994; Google Scholar). to in cyclin D1 protein in the of or expression of activated the regulation of cyclin D1 transcription in to the expression of various Ras We used a to the of cyclin shown in Ras transcription from the dominant negative of Ras effectors used to to this negative of Ral and both activated Ras induction of the cyclin D1 activated a PI 3-kinase also able to the cyclin D1 as M. Van Aelst L. C.J. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar), a dominant negative of the only a of that function of Ras in cyclin D1 The of Ras expression and to when cells with dominant negative In addition, as a kinase activity in in to of the activity of with Ras. well with the of of kinase activity by by that the dominant negative on other Activated of the various effectors of Ras used to effects on cyclin D1 activated PI 3-kinase as well as both signals of to that expression of activated of Raf kinase the Ras effectors signaling cooperate to cyclin Ras Rlf, also in this expression of activated of Raf, Rlf, and PI 3-kinase on the effect of Raf and expression of PI 3-kinase and Raf as well as PI 3-kinase and in cyclin D1 that pathways to D1 the of the PI 3-kinase cyclin D1 expression and that the PI 3-kinase activity may be required for the of E2F activity from Therefore, used E2F from the to E2F activity B. S. A. 1985; PubMed Scopus Google Scholar). that the of E2F activity can be by of that the function of the Ras effectors Ral-GDS and PI 3-kinase a of also induction of Activated of PI 3-kinase and to a The PI 3-kinase of that is the PI 3-kinase effector required for cell cycle dominant negative the effects of activated Raf or The that the in E2F activity be by of cdk that PI 3-kinase by cyclin D1 to dominant negative Ral able to the E2F activated of Ral as well as also Ral, activated of Ral, only to a induction. of Ral with a for nucleotide that GTPase activity, Ral also The exchange of leads to a of this this has been shown to to activation of S. D. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). Ral to a but in E2F activity, to that expression of activated that part of the activation mediated by Ras is mediated by a Ral but that a from the PI 3-kinase and pathway. of oncogenic of Ras leads to induction of cell cycle of quiescent cells from G0 and through at least G1 and S in cell J. Curr. Biol. 1997; 7: 258-260Abstract Full Text Full Text PDF PubMed Google Scholar). In it has clear that activated Ras are of a of families of effector in the activation of several signaling the kinase the Ral-GDS and the PI 3-kinase (13Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (470) Google Scholar). The of the kinase has been for to the expression of cell cycle but the involvement of the other pathways in cell cycle is less well the of the Ras effector on cell cycle in to the function of Ras pathways can be with the of or the activity of the pathways is integrated at of the that can been in the expression of cell cycle following of fibroblasts from serum is in the expression of cyclin of activated Ras cause this in the of serum (8Liu J.J. Chao J.R. Jiang M.C. Ng S.Y. Yen J.J. Yang-Yen H.F. Mol. Cell. Biol. 1995; 15: 3654-3663Crossref PubMed Scopus (263) Google Scholar, J. Robles A.I. Shi W. Wong M.J. Colombo C.J. 1994; Google Scholar, 1996; Google Scholar). least in this is by activation of the kinase in a of cell activation of Raf or has been to stimulate expression of cyclin D1 D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar, A. J. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C.J. S. A. PubMed Scopus Google Scholar, E. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). The in that in NIH 3T3 activated Raf is able to cyclin D1 less activated Ras. activated of the other Ras effectors and PI 3-kinase both cyclin D1 transcription at least as as of those PI to act is that transcription of the cyclin can be via a of pathways of Ras and that in to may the function of with PI 3-kinase endogenous cyclin D1 expression in NIH 3T3 cells from serum appears to this of quiescent cells with the PI 3-kinase in a in the expression of cyclin D1 following serum effect is the is up to h the addition of of the kinase by the of the in a in cyclin D1 that the function of PI 3-kinase signaling pathways is important in the serum regulation of cyclin D1 protein is the kinase pathway. have that the of growth factor I to cyclin in cells B. J. B. P.H. J. 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Biol. 1997; 17: PubMed Scopus Google Scholar). is that multiple pathways of PI 3-kinase can to cyclin endogenous cyclin D1 are in cells activated Ras or activated Akt/PKB, it is clear that Ras expression of cyclin activated is of only a induction and and cause induction In the of expression of endogenous cyclin multiple pathways of Ras may be the that regulation of cyclin at a of in addition to the regulation of cyclin D1 expression of a activated of Akt/PKB, can only the effect of this may that other pathways of PI such as important or that is of PI 3-kinase the for to to the is by the on the requires by the and which are at least in part on PI 3-kinase activity J. PubMed Scopus Google Scholar). D1 is only the of the cell cycle to be by Ras. other of regulation of the cell cycle by Ras are at later later that is is the regulation of E2F transcription factors from following E2F target from the used as a of E2F activity following activation of Ras a but that for cyclin D1 transcription is is that activated and PI 3-kinase are of E2F activity, Ras and PI 3-kinase induction of cyclin D1 expression is less this that may act through other pathways in addition to cyclin D1 to E2F is the regulation of the cyclin-dependent kinase the expression of which is following serum of quiescent cells in a Ras activity and is also in Ras transformed is of as to the kinase can to in with that it can D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar, E. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar) and that it M. C.J. S. A. PubMed Scopus Google Scholar). has been to the of growth factors to P. B.M. 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In this it is important to the in which transformed cells a activated Ras and that of cells that endogenous type Ras protein to signals from various growth activation of the Raf, Ral-GDS, PI Akt/PKB, or pathways by of the activated or of oncogenic Ras may be sufficient to stimulate that be activated by activation of pathway. shown in serum stimulation of NIH 3T3 cells leads to activation of endogenous Ras protein, as has been D. Curr. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of endogenous kinase and at well with the of Ras this is several other pathways are in the regulation of both enzymes, including protein kinase and pathways in kinase activation Mol. Cell. Biol. PubMed Scopus Google Scholar), induction of in kinase H. Cell. 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