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A variety of studies have shown that activation of the cell cyclemachinery requires the participation of multiple signallingpathways. These pathways include Ras-dependent effectorssuch as the extracellular-signal related kinases, otherwiseknown as mitogen-activated protein kinases (ERKs, MAPKs),phosphatidylinositol 3 (PI3)-kinase and p21Ral pathways, aswell as other signalling pathways regulated by the smallGTPases p21Rho, p21Rac and p21Cdc42.

AddressesCancer Research Campaign Centre for Cell and Molecular Biology,Chester Beatty Laboratories, Institute of Cancer Research, LondonSW3 6JB, UK; e-mail: chrism@icr.ac.uk

Current Opinion in Cell Biology 1999, 11:732–736

0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.

AbbreviationsCAK Cdk-activating kinaseCdk cyclin-dependent kinaseCKI Cdk inhibitorERK extracellular-signal-regulated kinaseGSK glycogen synthase kinaseMAPK mitogen-activated protein kinasePI3 phosphatidylinositol 3pRb retinoblastoma protein

IntroductionNumerous studies with cultured cells have demonstratedthat entry of quiescent Go cells into S-phase is dependenton the activities of the G1 cyclin-dependent kinases(Cdks). These kinases consist of Cdk4 and Cdk6 com-plexed with the D-type cyclins D1, D2, D3, and Cdk2complexed with cyclin E. A key regulatory event mediat-ed by the G1 Cdks is the phosphorylation of the ‘pocket’proteins and in particular the retinoblastoma proteinpRb105, which is the product of the Rb tumour suppressorlocus. In its hypophosphorylated form pRb105 associateswith the E2F family of transcription factors and activelypromotes transcriptional repression or sequesters E2Ffrom genes required for entry into S-phase. Followingphosphorylation of pRb105 by the G1 Cdks, E2F is releasedfrom pRb105 and transcription results.

The regulation of the activity of these kinases requires aninterplay between the synthesis of the cyclins, formationof cyclin/Cdk complexes, association with the cyclin-dependent-kinase inhibitors (CKIs), phosphorylation ofCdks by activating kinases (CAKs) and transport of cyclinCdk complexes to the nucleus (Figure 1; for a review, see[1••]). Some of these events may be regulated directly bysignal transduction pathways, whereas others are a conse-quence of the cell cycle machinery becoming activated.Thus, in trying to understand how signal transduction

pathways interface with the cell cycle machinery it isimportant to identify which cell cycle events are directlyregulated by intracellular signals. Considerable evidencesuggests that signal transduction pathways from the smallGTPases of the Ras and Rho family play an important rolein cell cycle control. This review provides an outline ofhow growth-factor-activated small GTPase signalling path-ways control entry into the cell cycle in mammaliansomatic cells. The reader is referred to other reviews for acomprehensive description of signalling pathways regulat-ed by small GTPases [2,3].

Signal transduction and cell cycle progressionA characteristic of mitogenic signalling is that it requiresprolonged exposure to growth factors until the ‘restrictionpoint’ is reached and growth factors are no longer neces-sary. This requirement for prolonged exposure to growthfactors implies that signal transduction also must be sus-tained. The seminal experiments of Stacey andcollaborators [4], using microinjection of a Ras neutralising

How do small GTPase signal transduction pathways regulate cellcycle entry?Chris Marshall

Figure 1

Relationship between cyclins and CKIs in the regulation of Cdkactivity, pRb105 phosphorylation and S-phase entry. ThecyclinD/Cdk4/6 and the cyclinE/Cdk2 complexes phosphorylate pRb,which releases the transcription factor E2F, resulting in entry into S-phase. The activity of Cdk4/6 is inhibited by the p16 family of CKIs,whereas the p21 family (p21Waf1, p27Kip1) inhibits Cdk2 and Cdk4/6.The cyclinE/Cdk2 complex phosphorylates p27Kip1, which is thendegraded by p45Skp2 (SKP2). Degradation of p27Kip1 is an importantcontrol point for entry into the cell cycle. The double-headed arrowbetween Cdk4/6 and the p21 family of CKIs reflects the potential dualrole of these CKIs in assembly of active cyclinD/Cdk4/6 complexesand in their inhibition.

cyclin DCdk4/6 cyclin E

pRb

PO4PO4PO4

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p16 CKIs p21Waf1 p27Kip1

SKP2

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Cdk2

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antibody, showed that cellular Ras function is requiredthroughout most of G1 in order for growth factors to stim-ulate quiescent cells to enter DNA synthesis. Theseobservations are nicely paralleled by the demonstrationthat the activation of Ras occurs in two waves followinggrowth factor stimulation: the first immediately followingaddition of growth factors and the second in mid G1 [5].Ras function is also required for cell cycle progression inasynchronously growing cycling cells, and recent observa-tions indicate that Ras function is actually required in theprevious cell cycle presumably to provide cell cycle com-ponents, such as cyclin D1, for the following cell cycle [6•].

One approach to understanding how signal transductionpathways are coupled to the cell cycle is to investigatewhether genetic ablation of key regulatory componentsalters the requirements for cell signalling. Two researchgroups have investigated whether pRb105 null cells have areduced requirement for Ras-dependent signalling.Although blocking Ras almost completely inhibited prolif-eration of asynchronous cultures of wild-type cells, it had avery much reduced effect on the null cells [7,8]. Theseexperiments highlight the importance of Ras signalling inactivating the G1 Cdks that phosphorylate and inactivatepRb105. Loss of pRb105 does not completely overcome therequirement for Ras-dependent signalling becauseMittnacht et al. [8] found that pRb105 null cells still showedsome inhibition of proliferation when injected with a Rasneutralising antibody, and Peeper et al. [7] found thatpRb105 null cells still require Ras function for exit from Gointo G1. Possibly this requirement for Ras signalling mayreflect the need for cyclinE/Cdk2 activity which is stillrequired in pRb105 null cells. Experiments with oncogenicRas show that in combination with overexpressed c-Myc itcan lead to the activation of cyclinE/Cdk2 in the absenceof growth factor stimulation [9].

Regulation of cyclin D1 expressionA key event in stimulating cell cycle entry in quiescentcells is the activation of the kinases Cdk4 and Cdk6. In qui-escent cells, expression of the D-type cyclins is low and isstimulated by mitogenic growth factor treatment. Studiesfrom Pouyssegur and collaborators [10] established thatgrowth factor stimulation of cyclin D1 levels requires acti-vation of the ERK MAP kinase pathway. This observationhas been extended to a variety of systems and demonstratesthat one component of Ras-dependent mitogenic signallingis activation of the ERK MAP kinases. Sustained activationof ERK MAP kinases is required for entry into DNA syn-thesis and has been shown to be required for cyclin D1expression [11,12]. The ERK MAP kinase pathway appearsto regulate transcription of the cyclin D1 gene as growthfactor stimulation of cyclin D1 mRNA levels is blocked byinhibiting activation of ERK with the MAP kinasekinase (Mek1) inhibitor PD98059 [11]. Furthermore, acti-vated Raf, a component of the Ras pathway, can stimulateexpression from cyclin D1 reporter constructs [10]. Theprecise mechanism by which ERK MAP kinase signalling

leads to activation of cyclin D1 transcription is not clear. Aclear transcriptional effector of ERK MAP kinase signallingis the AP1 complex of transcription factors, which consistsof Fos related proteins c-Fos, Fos B, Fra-1, Fra-2, and Junrelated proteins c-Jun, JunB and JunD. Thus the linkbetween ERK MAP kinase signalling and cyclin D1 tran-scription might be AP1. Evidence to support such amechanism comes from the observation that mouse embryofibroblasts null for c-Jun grow poorly in culture and fail toinduce cyclin D1 mRNA expression following growth fac-tor stimulation [13•]. However, others have found thatcyclin D1 expression is not reduced in c-Jun null cells andhave argued that the proliferative defect of the null cellsresults from elevated levels of p53 in these cells inducingexpression of the CKI p21Waf1 [14•]. A second transcrip-tional effector of ERK MAP kinase signalling is c-Ets-2 andthis may provide the route to cyclin D1 transcription [15].

Although the ERK MAP kinase pathway appears to berequired for growth factor signalling to activate cyclin D1expression in a variety of systems, it is clear that it is not theonly signalling pathway that is required. Activation of thePI3-kinase pathway is also required since activation of ERKMAP kinases resulting from an inducible Mek constructonly results in cyclin D1 expression when a PI3-kinase sig-nal is present [16]. Furthermore, inhibition of thePI3-kinase pathway blocks growth factor stimulated cyclinD1 expression through a rapamycin-insensitive pathway[17]. Transcription from cyclin D1 reporter constructs canbe stimulated by signals from PI3-kinase and the Ral andRac GTPases [18,19•]. In addition, signalling from Cdc42elevates cyclin D1 expression, although it is not clearwhether this is due to an effect on cyclin D1 transcription[20]. These observations demonstrate that multiple smallGTPase signalling pathways are required for cyclin D1expression. The signalling pathways activated by Ral, Racand Cdc42 that contribute to cyclin D1 expression have notbeen elucidated, however. As the activation of PI3-kinasecan lead to the activation of Rac, at least part of the effectsof PI3-kinase on the activation of cyclin D1 reporters maybe mediated by Rac. However, other PI3-kinase signallingpathways may be involved such as protein kinase B (PKB)[19]. Although these observations suggest that a variety ofGTPase signalling pathways impinge on cyclin D1 tran-scription some caution must be exercised. Many of thesestudies have been carried out with reporter constructs intransient assays and using overexpression of constitutivelyactivated signalling components, so there is still much tolearn about the mechanism by which the endogenousD-type cyclin genes are regulated by signalling pathways.

As well as the effects of the PI3-kinase pathway on cyclinD1 transcription, PI3-kinase post-transcriptionally regu-lates the cyclin D1 protein. Phosphorylation of cyclin D1 atThr286 leads to its degradation with the phosphorylatedprotein having a half life of around 10 minutes. Sherr andcollaborators [21••] have identified glycogen synthasekinase-3β (GSK-3β) as the Thr286 kinase and argued that

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inactivation of GSK-3β through phosphorylation by PKBprovides a PI3-kinase- and Ras-dependent route to the sta-bilisation of cyclin D1. PI3-kinase, together with mTor, themammalian target of rapamycin, may also regulate thetranslation of the cyclin D1 mRNA [22]. In cells wherecyclin D1 mRNA levels are high, even in the absence ofgrowth factors, the PI3-kinase-dependent regulation ofcyclin D1 protein stability and translation may be particu-larly important for cell cycle progression and such cells mayshow no requirement for the ERK MAP kinase pathway.Such situations may arise where cyclin D1 transcription iselevated due to gene amplification or translocation.

Signal transduction and CKIsAs well as the regulation of Cdk4 and Cdk6 activity throughthe synthesis of D-type cyclins, the cell cycle machinery is reg-ulated by the CKIs. In quiescent-serum-starved fibroblasts thelevels of the CKI p27Kip1 are high and drop following growthfactor stimulation. The degradation of p27Kip1 appears to be animportant control point for entry into the cell cycle and may bea key regulator of cyclinE/Cdk2 activity [1••]. Studies withinducible dominant negative Ras constructs demonstrate thatthe downregulation of p27Kip1 that occurs late in G1 phaserequires Ras [23]. At this time point ERK MAP kinase sig-nalling is low and the degradation of p27Kip1 appears to bemediated by the PI3-kinase pathway. Phosphorylation ofp27Kip1 at Thr187 by cyclinE/Cdk2 leads to its degradation.Since PI3-kinase activity stabilises cyclin D1 expression, thismay permit cyclinD1/Cdk4 complexes to titrate out p27Kip1

resulting in cyclinE/Cdk2 activity and phosphorylation ofp27Kip1. Recent results show that p27Kip1 phosphorylated atThr187 is recognised by the F-box protein p45Skp2 and thisleads to ubiquitin-dependent proteolysis [24•,25•]. The levelof p45Skp2 is low in quiescent cells and is induced followinggrowth factor stimulation, perhaps this is through a PI3-kinase-dependent step. Another point at which PI3-kinase activitymay be required for p27Kip1 degradation is in the induction of

cyclin E. Cyclin E is an E2F-responsive gene, and PI3-kinasemay elevate cyclin E expression through PKB mediated acti-vation of E2F-dependent transcription [26]. In interleukin-2stimulated T cells, PI3-kinase-mediated activation of E2Ftranscription also involves a rapamycin-sensitive event [27],which may explain why p27Kip1 degradation in T cells isblocked by rapamycin treatment.

In addition to the PI3-kinase pathway, other signalling path-ways may contribute to p27Kip1 degradation. In vitro p27Kip1

can be phosphorylated by ERK MAP kinases, although thesite(s) of phosphorylation has not been identified. It hasbeen argued that the ERK MAP kinases are involved in thedegradation of p27Kip1 [28]. This requirement for ERK MAPkinase signalling in p27Kip1 degradation could be ascribedmerely to a requirement of the cyclinD/Cdk4/6 pathway forsubsequent cyclinE/Cdk2 activity. However, support for adirect role for ERK MAP kinase activity is provided byexperiments in which activation of ERK in a MEK1-inducible cell line leads to p27Kip1 degradation many hoursbefore any cyclinE/Cdk2 activity is measurable [16]. Thusthe ERK MAP kinase pathway may play a direct role inp27Kip1 degradation. Such a role could be especially impor-tant in cells where the ERK MAP kinase pathway isconstitutively active as a consequence of oncogenic Rasexpression. Interestingly the small GTPase p21RhoA hasbeen shown to be involved in growth factor stimulateddegradation of p27Kip1 as blocking Rho inhibits growth factormediated degradation of p27Kip1 [29]. Rho signalling may beinvolved in the activation of cyclinE/Cdk2 as expression ofconstitutively active RhoA results in stimulation ofcyclinE/Cdk2 activity in the absence of growth factors [30•].These results indicate a RhoA signalling pathway leading top27Kip1 degradation, but an alternative interpretation (seebelow) is that the blockade of Rho signalling results in elevated levels of p21Waf1, which inhibits cyclinE/Cdk2activity and blocks p27Kip1 degradation.

734 Cell multiplication

Figure 2

Small GTPase signalling and the regulation ofG1 Cdk activity. Inhibitory pathways areindicated with a hammerhead, and stimulatorypathways are indicated with an arrow. Rasactivates the PI3-kinase (PI3-K) signallingpathway, resulting in cyclin D1 transcriptionand stability, and degradation of p27Kip1.Cyclin D1 transcription is also activated bythe Ras-activated Ral and MEK/ERKsignalling pathways. It is not clear whetherCdc42 acts transcriptionally or post-transcriptionally to regulate cyclin D1expression. Activation of the ERK pathwayleads to expression of p21Waf1, which inhibitscyclinE/Cdk2 complex activity. Rho signallingis thought to control the cell’s response toactivation of the ERK pathway.

cyclin ECdk2Ras

MEK

ERK

PI3-K

Rac/Cdc42

RalPKB

GSK3β

RafRal-GEFs

TranscriptionCyclin D1Stability

p21Waf1

p27Kip1

Rho

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Although the levels of p27Kip1 are high in quiescent cells,p21Waf1 levels are low, but show modest increases followinggrowth factor stimulation. Why the expression of a CKIshould increase following growth factor stimulation might beexplained by an important role for p21Waf1 and p27Kip1 asassembly factors for Cdk4 and Cdk6 with D-type cyclins andas providers of signals for nuclear import. As these Cdk com-plexes assemble they titrate out p21Waf1 and p27Kip1, andpermit activation of cyclinE/Cdk2. In some systems growthfactor stimulation of p21Waf1 levels is mediated by the ERKMAP kinase pathway [31]. Thus activation of the ERK MAPkinase pathway may be required to stimulate p21Waf1 expres-sion to mediate assembly of the cyclinD1/Cdk4 complex.However, there may be another ERK MAP kinase depen-dent step in assembly of cyclinD1/Cdk4 complexes [32].Signalling events that lead to high levels of p21Waf1 cause cellcycle arrest. Elevated levels of p21Waf1 can result from sus-tained high level activation of the ERK MAP kinase pathway[33] or as a consequence of ERK MAP kinase activity underconditions of low p21Rho signalling [34•]. The requirementfor Rho signalling may ensure that anchorage-dependent cellsonly respond to activation of the ERK MAP kinase pathwaywhen p21Rho is in the active GTP-bound state, for example,when cells are attached to the extracellular matrix [35].

ConclusionsIt is now clear that multiple signal transduction pathwaysfrom small GTPases impinge on the activation of the cellcycle machinery (see Figure 2). Thus, cell cycle progressioncan be viewed as an integrator of signalling pathways. Thisintegration arises because activation of the cell cyclerequires multiple events (e.g. D-type cyclin expression andp27Kip1 degradation) each of which may themselves requiremultiple signalling inputs. For example, cyclin D1 expres-sion can be affected by the ERK MAP kinase, PI3-kinase,Rac and Ral signalling pathways. The regulation of cyclinD1 expression provides an important model, see Figure 2,for how signal pathway integration can occur, since somesignals may regulate transcription, whereas others regulateprotein stability or translation [21••]. How much redundan-cy there is between different signalling pathways remainsto be determined. But it remains a reasonable hypothesisthat a requirement for the activation of multiple signallingpathways may act as a checkpoint to ensure that cell prolif-eration only occurs when it is appropriate.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Sherr CJ, Roberts JM: Cdk inhibitors: positive and negative•• regulators of G1-phase progression. Genes Dev 1999,

13:1501-1512.An excellent review of the role that CKIs play in regulating cyclin-dependentkinase activity.

2. Van Aelst L, D’Souza-Schorey C: Rho GTPases and signalingnetworks. Genes Dev 1997, 11:2295-2322.

3. Khosravi-Far R, Campbell S, Rossman KL, Der CJ: Increasingcomplexity of Ras signal transduction: involvement of Rho familyproteins. Adv Cancer Res 1998, 72:57-107.

4. Dobrowoski S, Harter M, Stacey DW: Cellular Ras activity isrequired for passage through multiple points of the GO-G1 phasein BALB-c 3T3 cells. Mol Cell Biol 1994, 14:5441-5449.

5. Taylor SJ, Shalloway D: Cell cycle-dependent activation of Ras.Curr Biol 1996, 6:1621-1627.

6. Hitomi M, Stacey DW: Cellular Ras and cyclin D1 are required• during different cell cycle periods in cycling NIH3T3 cells.

Mol Cell Biol 1999, 19:4623-4632.This paper makes the interesting observation that in cycling NIH3T3 cellsRas function is only required in the preceding cell cycle.

7. Peeper DS, Upton TM, Ladha MH, Neuman E, Zalvide J, Bernards R,DeCaprio JA, Ewen ME: Ras signalling linked to the cell-cyclemachinery by the retinoblastoma protein. Nature 1997,386:177-181.

8. Mittnacht S, Paterson H, Olson MF, Marshall CJ: Ras signalling isrequired for inactivation of the tumour suppressor pRb cell-cyclecontrol protein. Curr Biol 1997, 7:219-221.

9. Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR: Myc and Rascollaborate in inducing accumulation of active cyclin E/Cdk2 andE2F. Nature 1997, 387:422-426.

10. Lavoie JN, L’Allemain G, Brunet A, Muller R, Pouyssegur J: Cyclin D1expression is regulated positively by the p42/p44-MAPK andnegatively by the p38/HOG-MAPK pathway. J Biol Chem 1996,271:20608-20616.

11. Weber JD, Raben DM, Phillips PJ, Baldassare JJ: Sustainedactivation of extracellular-signal-regulated kinase 1 (ERK1) isrequired for the continued expression of cyclin D1 in G1 phase.Biochem J 1997, 326:61-68.

12. Balmanno K, Cook SJ: Sustained MAP kinase activation is requiredfor the expression of cyclin D1, p21Cip1 and a subset of AP-1proteins in CCL39 cells. Oncogene 1999, 18:3085-3097.

13. Wisdom R, Johnson RS, Moore C: c-Jun regulates cell cycle• progression and apoptosis by distinct mechanisms. EMBO J

1999, 18:188-197.See annotation [14•].

14. Schreiber M, Kolbus A, Piu F, Szabowski A, Mohle-Steinlein U, Tian J,• Karin M, Angel P, Wagner EF: Control of cell cycle progression by

c-Jun is p53 dependent. Genes Dev 1999, 13:607-619.Both Schreiber et al. [14•] and Wisdom et al. [13•] use c-Jun null cells butcome to opposite conclusions on whether c-Jun is required for cyclin D1expression. Why?

15. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A,Pestell RG: Transforming p21Ras mutants and c-Ets-2 activate theCyclin D1 promoter through distinguishable regions. J Biol Chem1995, 270:23589-23597.

16. Treinies I, Paterson HF, Hooper S, Wilson R, Marshall CJ: ActivatedMEK stimulates expression of AP1 components independently ofphosphatidylinositol 3-kinase (PI3-kinase) but requires aPI3-kinase signal to stimulate DNA synthesis. Mol Cell Biol 1999,19:321-329.

17. Takuwa N, Fukui Y, Takuwa Y: Cyclin D1 expression mediated byphosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signalling in growth factor-stimulated NIH 3T3fibroblasts. Mol Cell Biol 1999, 19:1346-1358.

18. Westwick JK, Lambert QT, Clark GJ, Symons M, Van AL, Pestell RG,Der CJ: Rac regulation of transformation, gene expression, andactin organization by multiple, PAK-independent pathways.Mo Cell Biol 1997, 17:1324-1335.

19. Gille H, Downward J: Multiple Ras effector pathways contribute to• G(1) cell cycle. J Biol Chem 1999, 274:22033-22040.Cyclin D1 reporter constructs are used to show that Ral, Raf and PI-3 kinasepathways may all influence cyclin D1 transcription.

20. Gjoerup O, Lukas J, Bartek J, Willumsen BM: Rac and Cdc42 arepotent stimulators of E2F-dependent transcription capable ofpromoting retinoblastoma susceptibility gene producthyperphosphorylation. J Biol Chem 1998, 273:18812-18818.

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21. Diehl JA, Cheng M, Roussel MF, Sherr CJ: Glycogen synthase•• kinase-3ββ regulates cyclin D1 proteolysis and subcellular

localization. Genes Dev 1998, 12:3499-3511.Diehl et al. show that GSK-3β phosphorylates Cyclin D1 at Thr286 and tar-gets it for degradation. PI3-kinase signalling through PKB inhibits GSK-3βand stabilises cyclin D1.

22. Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE,Tsichlis PN, Rosen N: Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem 1998, 273:29864-29872.

23. Takuwa N, Takuwa Y: Ras activity late in G1 phase required forp27Kip1 downregulation, passage through the restriction point,and entry into S phase in growth factor-stimulated NIH 3T3fibroblasts. Mol Cell Biol 1997, 17:5348-5358.

24. Sutterluty H, Chatelain E, Marti A, Wirbelauer C, Senften M, Muller U• et al.: p45SKP2 promotes p27Kip1 degradation and induces S

phase in quiescent cells. Nat Cell Biol 1999, 1:207-214.See annotation [25•].

25. Carrano A, Eytan E, Hershko A, Pagano M: SKP2 is required for• ubiquitin-mediated degradation of the Cdk inhibitor p27.

Nat Cell Biol 1999, 1:193-199.Sutterluty et al. [24•] and Carrano et al. [25•] demonstrate the importanceof p45Skp2 in mediating the ubiquitin-dependent degradation of p27Kip1.

26. Brennan P, Babbage JW, Burgering BM, Groner B, Reif K, CantrellDA: Phosphatidylinositol 3-kinase couples the interleukin-2receptor to the cell cycle regulator E2F. Immunity 1997, 7:679-689.

27. Brennan P, Babbage JW, Thomas G, Cantrell D: p70(s6k) integratesphosphatidylinositol 3-kinase and rapamycin-regulated signalsfor E2F regulation in T lymphocytes. Mol Cell Biol 1999,19:4729-4738.

28. Kawada M, Yamagoe S, Murakami Y, Suzuki K, Mizuno S, Uehara Y:Induction of p27Kip1 degradation and anchorage independence by

Ras through the MAP kinase signaling pathway. Oncogene 1997,15:629-637.

29. Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I,Oeda T, Tahara K, Terano T, Narumia et al.: Geranylgeranylated Rhosmall GTPase(s) are essential for the degradation of p27Kip1 andfacilitate the progression from G1 to S Phase in growth-stimulated Rat FRTL-5 cells. J Biol Chem 1997, 272:13-16.

30. Hu W, Bellone CJ, Baldassare JJ: RhoA stimulates p27Kip1

• degradation through its regulation of cyclin E/Cdk2 activity.J Biol Chem 1999, 274:3396-3401.

This paper, along with [29], indicates a role for p21RhoA in regulatingp27Kip1 via activation of cyclin E/Cdk2.

31. Zezula J, Sexl V, Hutter C, Karel A, Schutz W, Freissmuth M: Thecyclin-dependent kinase inhibitor p21cip1 mediates the growthinhibitory effect of phorbol esters in human venous endothelialcells. J Biol Chem 1997, 272:29967-29974.

32. Cheng M, Sexl V, Sherr CJ, Roussel MF: Assembly of cyclinD-dependent kinase and titration of p27Kip1 regulated bymitogen-activated protein kinase kinase (MEK1).Proc Natl Acad Sci USA 1998, 95:1091-1096.

33 Lloyd AC: Ras versus cyclin-dependent kinase inhibitors.Curr Opin Genet Dev 1998, 8:43-48.

34. Olson MF, Paterson HF, Marshall CJ: Signals from Ras and Rho• GTPases interact to regulate expression of p21Waf1/Cip1. Nature

1998, 394:295-299.This study shows how Rho and Ras dependent signalling interact by con-trolling expression of the CKI p21Waf1/cip1.

35. Ren XD, Kiosses WB, Schwartz MA: Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J1999, 18:578-585.

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