Expression of Cyclin E Renders Cyclin D-CDK4 Dispensable forInactivation of the Retinoblastoma Tumor Suppressor Protein,Activation of E2F, and G1-S Phase Progression*

Received for publication, September 21, 2003, and in revised form, November 21, 2003Published, JBC Papers in Press, November 25, 2003, DOI 10.1074/jbc.M310383200

Susan M. Keenan‡§, Nathan H. Lents‡, and Joseph J. Baldassare¶

From the Department of Pharmacological and Physiological Sciences, Saint Louis University School of Medicine,Saint Louis, Missouri 63104

The activation of CDK2-cyclin E in late G1 phase hasbeen shown to play a critical role in retinoblastomaprotein (pRb) inactivation and G1-S phase progressionof the cell cycle. The phosphatidylinositol 3-OH-kinaseinhibitor LY294002 has been shown to block cyclin D1accumulation, CDK4 activity and, thus, G1 progressionin �-thrombin-stimulated IIC9 cells (Chinese hamsterembryonic fibroblasts). Our previous results show thatexpression of cyclin E rescues S phase progression in�-thrombin-stimulated IIC9 cells treated with LY294002,arguing that cyclin E renders CDK4 activity dispensablefor G1 progression. In this work we investigate the abil-ity of �-thrombin-induced CDK2-cyclin E activity to in-activate pRb in the absence of prior CDK4-cyclin D1activity. We report that in the absence of CDK4-cyclinD1 activity, CDK2-cyclin E phosphorylates pRb in vivoon at least one residue and abolishes pRb binding to E2Fresponse elements. We also find that expression of cyclinE rescues E2F activation and cyclin A expression incyclin D kinase-inhibited, �-thrombin-stimulated cells.Furthermore, the rescue of E2F activity, cyclin A expres-sion, and DNA synthesis by expression of E can beblocked by the expression of either CDK2(D145N) orRb�CDK, a constitutively active mutant of pRb. However,restoring four known cyclin E-CDK2 phosphorylationsites to Rb�CDK renders it susceptible to inactivation inlate G1, as assayed by E2F activation, cyclin A expres-sion, and S phase progression. These data indicate thatCDK2-cyclin E, without prior CDK4-cyclin D activity,can phosphorylate and inactivate pRb, activate E2F,and induce DNA synthesis.

The mammalian cell cycle is controlled by two importantfamilies of proteins, the cyclins and the cyclin-dependent ki-nases (CDKs)1 (for reviews, see Refs. 1 and 2). Progressionthrough the cell cycle is governed by the kinase activities of

specific CDKs, which are regulated by association with theregulatory cyclin subunits. The sequential activation of earlyG1 CDK activity, CDK4 or CDK6 together with cyclin D1, D2,or D3, and the late G1 CDK activity, CDK2 together with cyclinE1 or E2, is believed to be required for progression through G1

and into S phase. Because IIC9 cells contain CDK4, but notCDK6, and cyclin D1, but not D2 or D3, CDK4-cyclin D1 acti-vation in early G1 is required for the expression of cyclin E,CDK2 activity, and G1-S phase progression (3–5).

In most cell types inactivation of the retinoblastoma (pRb)protein is essential for passage through G1 and transition ofcells into S phase (2, 6–9). pRb regulates this progression by itsassociation with the E2F family of transcription factors (10–13). In quiescent cells (G0 phase) pRb is unphosphorylated; inearly- to mid-G1 pRb is hypophosphorylated by the D-typeCDKs (14, 15). This hypophosphorylated form of pRb, whichbinds to and inhibits E2F transcription factors, has been shownin vivo to be phosphorylated on 13 of 16 potential CDK phos-phorylation sites, suggesting that hypophosphorylated pRbmay consist of multiple phospho- isoforms (16–18). The hy-pophosphorylation of pRb in early G1 stimulates the release ofHDAC1 and the recruitment of SWI/SNF family members tothe pRb-containing chromatin remodeling complexes, thus al-lowing the expression of cyclin E (19–21). In late G1 and Sphase, pRb is hyperphosphorylated by CDK2-cyclin E and,later, by CDK2-cyclin A (22–26). The hyperphosphorylatedform of pRb is inactivated because it loses affinity for and,therefore, fails to inhibit the E2F transcription factors (7, 23,27). Numerous proteins that are essential for growth, such asthe cyclins E and A, and proteins essential for DNA replication,such as DNA polymerase �, thymidine kinase, dihydrofolatereductase, and histone H2A are controlled at least in part byE2F-responsive promoters (28–35). pRb inhibits these promot-ers by either directly blocking the activation domain of E2F orby acting as a member of a repression complex (21, 36–41).Therefore, the major role of the G1 CDKs, CDK4/6-cyclin D andCDK2-cyclin E in controlling G1-S phase progression, is theinactivation of pRb.

Although the role of CDK4-cyclin D in the inactivation of pRbis well established, the role of CDK2-cyclin E in the inactiva-tion of pRb is less clear. CDK2-cyclin E, in the absence of priorphosphorylation by CDK4-cyclin D, is able to phosphorylatepRb in vitro (16), and overexpression of cyclin E or A canovercome pRb-mediated suppression of proliferation (42). Fur-thermore, Ezhevsky et al. (18) show that CDK2-cyclin E activ-ity phosphorylates pRb in vivo. In agreement with these data,Lundberg and Weinberg (43) also find that CDK2-cyclin Eactivity was necessary for phosphorylation-induced inactiva-tion of pRb. However, these authors and others suggest thatphosphorylation of pRb by CDK2-cyclin E requires pRb to be

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ Contributed equally to this work.§ Present address: Dept. of Pharmacology, University of Medicine

and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675Hoes Lane, Piscataway, NJ 08854.

¶ To whom correspondence should be addressed: Dept. of Pharmaco-logical and Physiological Sciences, Saint Louis University School ofMedicine, Saint Louis, MO 63104. Tel.: 314-577-8543; E-mail:baldasjj@slu.edu.

1 The abbreviations used are: CDK, cyclin-dependent kinase; pRb,retinoblastoma protein; EMSA, electrophoretic mobility shift assay;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, murineembryo fibroblast; dn, dominant negative.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 7, Issue of February 13, pp. 5387–5396, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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hypophosphorylated, and thus, the inactivation of pRb involvessequential phosphorylation by cyclin D-CDK4/6 and cyclin E-CDK2 (24, 44). Conversely, the report of a cyclin E 3 D1“knockin” mouse offers an argument against the strict require-ment for sequential phosphorylation of pRb by the G1 CDK-cyclin complexes (45). These mice, in which the coding sequenceof the cyclin D1 gene is replaced with the coding sequence forcyclin E, reveal that cyclin E expression, which results in a 20%change in the phosphorylation state of pRb, rescues the pheno-typic deficiencies found in the cyclin D1-ablated mouse (46).Although many tissues are unaffected by the loss of cyclin D1because of compensatory functions of cyclin D2 and D3, severaltissues, including retinal and breast tissue are severely defi-cient in growth, presumably because D2 and D3 are not ex-pressed in these tissues and could not compensate. Implicit inthe discovery that cyclin E expression under the cyclin D1promoter reverses the cyclin D1�/� phenotypes is the under-standing that, in the rescued tissues, pRb is inactivated bycyclin E kinase activity without prior hypophosphorylation bycyclin D kinase, with the caveat that the genes for cyclin D2and D3 are still present in these animals.

Previously, we reported that inhibition of phosphatidylinosi-tol 3-OH kinase by LY294002 inhibits cyclin D1 accumulation,CDK4-cyclin D1 activity, and passage through G1 into S phasein IIC9 cells (3). Surprisingly, expression of cyclin E in thepresence of LY294002 rescues cyclin E-CDK2 activity and G1

progression (47). Under these conditions, cyclin D-kinase acti-vation is inhibited, whereas cyclin E-kinase activity is rescued.Therefore, this offers a unique opportunity to question thenecessity of prior phosphorylation of Rb by cyclin D-dependentkinases by examining whether CDK2-cyclin E activity alonecan phosphorylate and inactivate pRb. Here we show thatexpression of cyclin E recovers CDK2, but not CDK4 activationin LY294002-treated cells. Under these conditions, in whichCDK2 can be activated without prior CDK4 activation, wereport that the phosphorylation of pRb on Ser-795, the releaseof pRb from E2F response elements, the activation of E2F, andG1-S phase progression all occur normally. Furthermore, wereport that the cyclin E-mediated rescue of E2F activity can beprevented by co-expression of CDK2(D145N) or Rb�CDK. Fi-nally, we restore four CDK2 phosphorylation sites on Rb�CDK

and show that expression of this construct, Rb��K2, does notblock E2F activation or DNA synthesis. Taken together, thesedata indicate that, in IIC9 cells, prior phosphorylation of pRbby cyclin D-CDK4/6 is not necessary for the phosphorylation ofpRb by cyclin E-CDK2 and the subsequent activation of E2Fand entry into S phase of the cell cycle.

MATERIALS AND METHODS

Cell Culture and Transient Transfection—IIC9 cells, a subclone ofChinese hamster embryo fibroblasts (48, 49), were maintained as pre-viously described (50). Quiescent cells were established by washingsubconfluent (80%) cells once with phosphate-buffered saline followedby a 48-h incubation with �-MEM (minimun Eagle’s medium) contain-ing 2 mM L-glutamine (BioWhittaker) supplemented with 100 units/mlpenicillin and 100 mg/ml of streptomycin (basal media). For transienttransfections IIC9 cells were grown to subconfluency (80%). The cellswere transfected as previously described (51). After 12 h the cells wereserum-arrested for 48 h before stimulation. �80% transfection effi-ciency was determined by co-transfection of green fluorescent protein.LY294002 (Calbiochem) was added 30 min before stimulation to a finalconcentration of 10 �M. Growth-arrested IIC9 cells were stimulatedwith 1 unit/ml human �-thrombin and incubated for the indicatedtimes.

Constructs—Human cyclin E, HU4 fragment, was generously providedby James Roberts; pGL3-TATA-6xE2F-Luc was generously provided byKristian Helin; human CDK2(D145N) was generously provided by Jim Koh;murine p16INK4a cDNA was generously provided by Martine Roussel andCharles Sherr; human wild-type Rb and Rb�CDK was generously provided byJ. Wade Harper. All mutations were confirmed by sequencing analysis.

Rb��K2 was generated from Rb�CDK by site-directed mutagenesis, restoringThr-373, Thr-612, Ser-795, and Thr-821 individually following the manu-facturer’s protocol (QuikChange™ kit, Stratagene). Restoration of the thre-onine or serine codons was confirmed by automated capillary sequencingfollowing the manufacturer’s protocol (Beckman-Coulter).

Cyclin-dependent Kinase Activity Assays—CDK4-cyclin D1 andCDK2-cyclin E assays were performed as previously described (3, 47,50, 52). Briefly, after transfection and serum starvation cells werestimulated with 1 unit/ml �-thrombin for 8 h (for CDK4) or 17 h (forCDK2). Lysates (100 �g) were immunoprecipitated with monoclonalantibodies to cyclin D1 or polyclonal antibodies to CDK4 or CDK2, asindicated. Immunoprecipitates were analyzed for the ability to phos-phorylate GST-Rb (for CDK4 or cyclin D1) or histone H1 (for CDK2) invitro, and [32P]phosphate incorporation was quantified using a Phos-phorImagerTM (Molecular Dynamics). Data are presented as -fold acti-vation over basal level.

Western Blot Analysis—Asynchronously growing IIC9 cells or humanHL60 cells were washed twice with cold phosphate-buffered saline andlysed in cold lysis buffer (52). Lysates were sonicated briefly, andinsoluble material was pelleted by microcentrifugation at 14,000 rpm at4 °C for 2 min. 40 �g of protein lysate was resolved by SDS-polyacryl-amide gel electrophoresis and transferred to a polyvinylidene difluoridemembrane (Millipore Corp., Boston, MA) as recommended by the man-ufacturer. Membranes were probed individually with polyclonal anti-bodies to CDK6, cyclin D1, cyclin D2, or cyclin D3 (all from Santa CruzBiotechnology, Santa Cruz, CA). Immunoreactive bands were visual-ized by enhancer chemiluminescence (ECL) detection (Amersham Bio-sciences) as recommended by the manufacturer.

Growth-arrested IIC9 cells were incubated in the presence or ab-sence of 1 unit/ml �-thrombin for 19 h after pretreatment in the pres-ence or absence of 10 �M LY294002 for 30 min. Cells were then washedtwice with cold phosphate-buffered saline and lysed in cold lysis buffer(52). Lysates were prepared for Western analysis as described andprobed with polyclonal antibodies to phosphorylated Ser-780- or Ser795-pRb (Cell Signaling Technology, Beverly, MA) or cyclin A (SantaCruz Biotechnology, Santa Cruz, CA).

Luciferase Reporter Assay—IIC9 cells were transiently transfectedwith 50 ng/ml pGL3-TATA-6xE2F-Luc, 200 ng/ml �-galactosidase, and1.5–2.0 �g/ml cyclin E, CDK2(D145N), Rb�CDK, or Rb��K2 as indicatedin figure legends. Basal cells were stimulated with 1 unit/ml �-throm-bin where indicated either in the presence or absence of 30 min prein-cubation with 10 �M LY294002. Lysate protein was prepared for lucif-erase activity assay as recommended by manufacturer (Promega,Madison, WI), and 10 �l of room temperature lysate was mixed with 90�l of room temperature luciferase assay buffer reagent (Promega) andplaced in an OpticompII luminometer (MCM Instruments, Baltimore,MD). Light produced was measured and normalized to transfectionefficiency by dividing relative light units by optical density units ob-tained from �-galactosidase activity. �-Galactosidase activity wasmeasure as previously described (5).

Northern Blot Analysis—Quiescent IIC9 cells were incubated in thepresence or absence of 1 unit/ml �-thrombin for indicated lengths oftime after preincubation in the presence or absence of 10 �M LY294002.At the indicated times total RNA was isolated using TRIZOL reagent(Invitrogen) according to the manufacturer’s protocol. RNA (20 �g) wasresolved in a 2%(w/v) agarose-formaldehyde gel. After electrophoresisformaldehyde was removed from the gel by washing in 0.5% ammoniumacetate. RNA was then transferred to a Hybond N� nylon membrane.(Amersham Biosciences) using a TurboblotterTM system (Schleicher &Schuell) and cross-linked onto the membrane using an ultraviolet cross-linker (Amersham Biosciences) as recommended by the manufacturer.Randomly labeled [�-32P]dCTP cDNA probes were made using theRandom Primed DNA labeling kit (Roche Applied Science). Membraneswere preincubated with rapid hybridization buffer (Amersham Bio-sciences) for 1 h at 65 °C and then probed simultaneously with cyclin Aand glyceraldehyde-3-phosphate dehydrogenase probes for 2 h at 65 °C.After hybridization, the membrane was washed twice with 5� SSPE (20mM EDTA, 1 M NaCl, 50 mM NaH2PO4/H2O) with 0.1%(w/v) SDS atroom temperature and once with 1� SSPE, 0.1% SDS at 65 °C. Themembrane was developed using a PhosphorImagerTM.

Electrophoretic Mobility Shift Assays (EMSA)—EMSA were slightlyadapted from established method (12, 39). For nuclear fractionationIIC9 cells were quickly scraped in 3 ml/100-mm2 plate of ice-cold 10 mM

Tris/HCl, pH 7.5, containing 10 mM NaCl, 1 mM EDTA (Buffer A). Thecells were homogenized 20 times in a Potter-Elvehjem homogenizer andspun at 500 � g for 7 min. The nuclear pellet was suspended in BufferA and homogenized 15 times in a Dounce homogenizer with a pestle,layered over 45% sucrose in Buffer A, and centrifuged at 1660 � g for 15

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min. The nuclei were washed once and resuspended in 10 mM Tris/HCl,pH 7.5, containing 10 mM NaCl, 1 mM MgCl2, and 10% sucrose andsonicated to lyse the nuclei. Nuclear proteins (4 �g) were immunopre-cipitated by incubating with 1 �g of anti-RB monoclonal antibody (Cal-biochem). Immunoprecipitated proteins were recovered on protein G-Sepharose beads and dissociated by treatment with deoxycholate (12,39). EMSAs were performed as described previously using pRb immu-noprecipitates and an end-radiolabeled double-stranded DNA fragment(1.5–2 � 105 cpm/assay) containing a single E2F consensus binding sitederived from the dihydrofolate reductase promoter (sc-2507; SantaCruz Biotechnology), termed E2FRE (12, 39). For competition studiesthe DNA binding assays also included 10 ng of unlabeled E2FRE,mutant E2FRE double-stranded oligonucleotide with a CG to AT sub-stitution at the E2F binding motif (E2FREmut, sc-2508) as a nonspe-cific competitor. To identify proteins in complex with the E2F consensussite extracts were preincubated with rabbit polyclonal antibodies (0.5�g each) to E2F1 (sc-193 X) and E2F4 (sc-1083 X) from Santa CruzBiotechnology before EMSA.

Thymidine Incorporation Assay—Performed as previously described(53).

RESULTS

Expression of Cyclin E Rescues CDK2, but Not CDK4 Activityin LY294002-treated cells—Phosphatidylinositol 3-OH kinaseactivity is essential for the accumulation of cyclin D protein andCDK4-cyclin D activity (54). In most cell types, including IIC9cells (3), activation of CDK4-cyclin D in early G1 results inexpression of cyclin E and the activation of CDK2-cyclin E inlate G1. To determine whether ectopic expression of cyclin Erescues CDK2 activity in the absence of CDK4 activity weperformed in vitro kinase assays with histone H or recombi-nant pRb, respectively, as the substrate (Fig. 1). As expected,the activity of CDK4 and CDK2 was negligible in quiescentcells, and �-thrombin stimulated a marked activation of bothCDK4 (Fig. 1A) and CDK2 (Fig. 1B). However, pretreatmentwith LY294002 prevents the activation of both CDK4 andCDK2. These data are consistent with the prevailing notionthat CDK4-cyclin D activity is essential for pRb phosphoryla-tion and the subsequent expression of cyclin E and CDK2-cyclin E activation. Interestingly, ectopic expression of cyclin Erescues the �-thrombin-induced activation of CDK2 (Fig. 1B)but not CDK4 (Fig. 1A). Because IIC9 cells do not expressCDK6 or cyclins D2 or D3 (Fig. 1C), these conditions representa G1 cell in which CDK2 is activated without the prior activa-tion of D-type CDK activity.

In addition to providing an ideal system for studying theability of CDK2-cyclin E alone to phosphorylate and inactivatepRb, these data argue that cyclin D-CDK4/6 activity is dispen-sable for CDK2 activity and G1 progression in the event ofunscheduled cyclin E expression (Ref. 47 and see Fig. 7). Ac-cording to the current model, prior activation of cyclin D-CDKcomplexes has two roles in the activation of cyclin E-CDK2activation. First, the hypophosphorylation of pRb is requiredfor the expression of cyclin E. This role is easily bypassed in oursystem by transfection of cyclin E. An additional function ofcyclin D in the activation of cyclin E is that the formation ofcyclin D-CDK complexes is thought to titrate and sequesterCDK inhibitors, such as p21CIP1/WAF1 and p27KIP1, away fromCDK2 complexes. Transfection of cyclin E also bypasses thissecond function of cyclin D complexes because cyclin E proteinlevels are ectopically maintained at levels above the thresholdrequired for formation of active cyclin E-CDK2 complexes andsubsequent destruction of p27KIP1 by ubiquitin-mediated pro-teolysis, as indicated by the observed activation of CDK2 in Fig.1B. In any event we generated conditions in which CDK2complexes can be activated without prior activation of CDK4complexes, and it is these conditions that we utilized to addressthe inactivation of pRb by cyclin E-CDK2 alone.

Expression of Cyclin E Rescues LY294002- and p16INK4a-inhibited E2F Activation—Ectopic cyclin E expression restores

DNA synthesis in LY294002-treated cells (47) presumably be-cause E2F activation occurs normally in these cells and usherscells into S phase. To examine E2F activity under these condi-

FIG. 1. Expression of cyclin E recovers CDK2, but not CDK4activity in LY294002-treated cells. A–B, IIC9 cells transiently trans-fected with cyclin E followed by serum deprivation for 48–60 h. Cellswere incubated in the presence or absence of 10 �M LY294002 30 minbefore stimulation. A, cells were stimulated with �-thrombin at 1unit/ml for 8 h. Lysates were assayed and quantified for CDK4-cyclin Dactivity as described under “Materials and Methods.” B, cells werestimulated with �-thrombin at 1 unit/ml for 17 h. Lysates were assayedand quantified for CDK2-cyclin E activity as described under “Materialsand Methods.” Error bars represent S.D. (n � 3), and data are repre-sentative of three independent experiments. C, lysates from asynchro-nously growing IIC9 or HL60 cells were analyzed for CDK6, cyclin D1,cyclin D2, or cyclin D3 in separate Western blots.

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tions we made use of an E2F reporter plasmid termed pGL3-TATA-6xE2F-Luc, which is a construct containing six E2Fresponse elements in tandem driving the expression of thefirefly luciferase open reading frame (38, 55). An assay ofrelative luciferase activity from pGL3-TATA-6xE2F-trans-fected cells revealed that in 17 h �-thrombin stimulated a�6-fold increase in E2F activity over that of quiescent cells(Fig. 2A). Not surprisingly, LY294002 abolished this stimula-tion presumably by inhibiting CDK activation and, thus, pRbphosphorylation and subsequent E2F activation. Expression ofcyclin E, however, completely reversed this inhibition, as thelevel of E2F activity is similar to that of uninhibited cells (Fig.2A). This argues that E2F activation is normal in these cellsdespite CDK4-cyclin D inhibition.

In addition to the luciferase reporter of E2F activity we alsoexamined the steady-state mRNA levels of cyclin A, a gene thatis known to contain an E2F response element and is transcrip-tionally regulated by E2F1 (56). Cyclin A mRNA levels reacheda maximum after cells entered S phase, and by Northern blot-ting we observe a 9-fold induction of cyclin mRNA (normalizedfor GAPDH) over basal levels after 17 h of �-thrombin stimu-lation (Fig. 2B). Interestingly, both cyclin A mRNA accumula-tion (Fig. 2B) and E2F reporter activation (Fig. 2A) display G1

time-course kinetics suggestively similar to CDK2-cyclin E ac-tivation (47, 50, 57, 58). Also in concurrence with the data fromthe E2F reporter, we find that the �-thrombin-stimulated in-duction of cyclin A message levels can be largely blocked bypreincubation with LY294002 (Fig. 2C). However, as also seenwith the E2F reporter, this attenuation can be reversed byectopic expression of cyclin E.

To ensure that these observations regarding mRNA levelstranslate to steady-state protein expression, we also assesscyclin A protein levels by Western blotting. In accordance withour observations of mRNA levels, we find that cyclin E expres-sion completely restores the LY294002-attenuated, �-throm-bin-stimulated induction of cyclin A protein expression (Fig.2D). Although treatment of IIC9 cells with LY294002 attenu-ates the activity of CDK4-cyclin D1 to the basal level of detec-tion (Fig. 1A), we utilized a potent inhibitor of D-type CDKs,p16INK4a (59), to ensure complete inhibition of cyclin D1-CDK4.As expected, p16 expression prevents �-thrombin-stimulatedcyclin A protein induction (Fig. 2D). However, cyclin E expres-sion restored cyclin A accumulation in p16-expressing,LY294002-treated cells. These data taken together demon-strate that cyclin E expression renders cyclin D-CDK4 activitydispensable in the initiation of E2F activity.

GW8510 or Co-expression of CDK2(D145N) Eliminates theAbility of Cyclin E to Rescue E2F Activity—Because cyclin E isnot known to bind to or activate other CDKs, the ability of

FIG. 2. CDK2-cyclin E activity drives E2F activation in theabsence of CDK4-cyclin D activity. A, IIC9 cells were transientlytransfected with pGL3-TATA-6xE2F, �-galactosidase, and where indi-cated cyclin E followed by serum deprivation for 48–60 h. Quiescent

cells were incubated in the absence or presence of 10 �M LY294002 or7.5 �M GW8510 followed by stimulation with �-thrombin for 17 h.Lysates were assayed for luciferase and �-galactosidase activity (B-gal),and relative light units (RLU) were divided by units of optical density,respectively, for quantification and normalization. B, quiescent IIC9cells were stimulated with �-thrombin for indicated lengths of time.RNA was isolated and analyzed for cyclin A and GAPDH mRNA andquantified as described under “Materials and Methods”; cyclin A arbi-trary units were divided by GAPDH arbitrary units for normalization.Error bars represent S.D. (n � 3). C–D, IIC9 cells were transientlytransfected with cyclin E and/or p16INK4a where indicated followed by48–60 h of serum deprivation. Cells were incubated in the presence orabsence of 10 �M LY29004 or 7.5 �M GW8510 for 30 min, then stimu-lated with 1 unit/ml �-thrombin for 17 h (C) or 19 h (D). C, RNA wasisolated and analyzed for cyclin A mRNA and quantified, normalizingfor GAPDH mRNA for normalization. D, protein lysates were analyzedfor cyclin A protein levels by Western blotting, as described under“Materials and Methods.” All data are representative of at least threeindependent experiments.

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ectopic cyclin E expression to rescue E2F activity in LY294002-treated cells is presumably through the activation of CDK2. Totest this we made use of a dominant-negative (dnCDK2) con-struct, CDK2(D145N) (60). We find that co-expression ofdnCDK2 prevented the rescue of E2F activity by cyclin E inLY294002-treated, �-thrombin-stimulated cells, as measuredby the luciferase reporter (Fig. 3A) and cyclin A steady-statemRNA levels (Fig. 3B). Similarly, preincubation with 7.5 �M

GW8510 reduces E2F activity in stimulated cells to levelsbelow that of basal cells regardless of ectopic cyclin E expres-sion (Fig. 2A). GW8510 (61) is a non-selective CDK inhibitorthat when preincubated at 7.5 �M inhibited CDK2 activation by95–100% and CDK4 activation by 40–60% in IIC9 cells (data

not shown). These data together with a time-course of E2Factivation that closely mirrors CDK2 activation (Fig. 2A, firstthrough fourth lanes; Fig. 3A) (50, 57, 58, 62) strongly supportthe notion of a function for CDK2 in the activation of E2Fduring G1 progression. We cannot rule out the possibility that,as may be suggested by recent reports, an additional pRbkinase activity exists (see “Discussion”). However, the kinaseactivity in our system requires phosphatidylinositol 3-OH ki-nase for activation, is activated by cyclin E expression, and issensitive to inhibition by GW8510 and dnCDK2, compellingevidence that CDK2 is the pRb kinase responsible for E2Factivation in this study.

CDK2-cyclin E Alone Can Phosphorylate and Inactivate pRbin Vivo—The implication of our observation that cells in which�-thrombin stimulates E2F activation and G1 progression de-spite CDK4 inhibition is that CDK2-cyclin E is able to phos-phorylate and inactivate pRb without prior phosphorylation byCDK4-cyclin D1. However, the possibility remains that theactivation of E2F observed under these conditions is somehowindependent of pRb phosphorylation. To investigate the phos-phorylation of pRb in cells lacking CDK4 activation, we madeuse of two phospho-specific pRb antibodies. One recognizes onlypRb that is phosphorylated on position Ser-795, which can bephosphorylated by either CDK4 or CDK2, and the other recog-nizes pRb that is phosphorylated on position Ser-780, which isonly phosphorylated by CDK4 and not CDK2 (7, 16, 18, 43). Notsurprisingly, very little phosphorylation of these residues isdetected in lysates from serum-starved cells (Fig. 4A) as pRb isthought to be unphosphorylated in quiescence (17, 18, 24, 43,44). The minimal phosphorylation seen in our blots is mostlikely because of a small population of cells that escape serumstarvation. In contrast, robust phosphorylation of pRb is ob-served with both phospho-specific antibodies after 17 h of�-thrombin stimulation. As expected, preincubation withLY294002 diminishes phosphorylation at both sites. Althoughhaving no effect on the LY294002-inhibited phosphorylation ofSer-780, dramatically, ectopic cyclin E expression completelyrestores the level of phosphorylation of Ser-795 to uninhibitedlevels. Thus, a CDK4-specific phosphorylation site remainsunphosphorylated, whereas a site that can be phosphorylatedby CDK2-cyclin E is indeed phosphorylated. Although far froma complete analysis of pRb phosphorylation, this observationdemonstrates that at least a subset of potential CDK2 phos-phorylation sites on pRb is phosphorylated without prior hy-pophosphorylation of pRb by CDK4.

Our evidence of E2F activation under conditions of CDK4inhibition substantiates the assertion that phosphorylation ofpRb by CDK2-cyclin E alone can trigger pRb inactivation (42,45). To more directly test this supposition we next conducted anEMSA according to an established protocol utilizing the E2Fresponse element as the probe (12, 39). Fig. 4B, lane 2, showselectromobility shift of the probe when incubated with pRbimmunoprecipitates from quiescent nuclei. Specificity was en-sured by failure of pRb to shift a mutated probe (not shown)and by competition with unlabeled probe (lane 3). Interest-ingly, when pRb immunoprecipitates from �-thrombin-stimu-lated nuclei are incubated with the probe, electromobility shiftis strikingly reduced (lane 6), an effect that is both blocked byLY294002 (lane 7) and rescued by ectopic expression of cyclin E(lane 8). Because pRb is not thought to directly bind to DNA, weinterpret these data as an indirect observation of the ability ofpRb to bind to E2F-family transcription complexes.

Our interpretation is supported by the observation that an-tibodies to E2F1 (lane 4) and E2F4 (lane 5) super-shift thepRb-retarded band. Failure of saturating amounts of theseantibodies to induce complete super-shift is consistent with the

FIG. 3. Dominant-negative CDK2 blocks the cyclin E rescue ofE2F activity in LY29004-treated cells. A, IIC9 cells were transientlytransfected with pGL3-TATA-6xE2F, �-galactosidase, and where indi-cated cyclin E and/or CDK2(D145N) followed by serum deprivation for48–60 h. Quiescent cells were incubated in the absence or presence of10 �M LY294002 followed by stimulation with �-thrombin for 17 h.Lysates were assayed for luciferase and �-galactosidase (B-gal) activity,and relative light units (RLU) were divided by units of optical density,respectively, for quantification and normalization. B, IIC9 cells weretransiently transfected with cyclin E and/or CDK2(D145N) where indi-cated followed by 48–60 h of serum deprivation. Cells were incubated inthe presence or absence of 10 �M LY29004 for 30 min, then stimulatedwith 1 unit/ml �-thrombin for 17 h. Lysates were analyzed for cyclin Aand GAPDH mRNA and quantified; cyclin A arbitrary units were di-vided by GAPDH arbitrary units for normalization. All data are repre-sentative of three independent experiments.

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notion that in quiescent cells pRb exists in multiple complexes(7, 63). Antibodies to E2F1 and E2F4 were chosen for studybecause they are thought to be binding partners of pRb inquiescent cells (7, 64–66). When coalesced with our observa-tions regarding E2F activation, these data regarding pRb phos-phorylation and E2F-responsive promoter binding make astrong case that hypophosphorylation by cyclin D-dependentkinase, once thought to be prerequisite, is actually dispensablefor the phosphorylation and inactivation of pRb by cyclin E-de-pendent kinase under the condition of constitutive cyclin Eexpression.

Expression of Rb�CDK Blocks the Activation of E2F by CDK2-Cyclin E—Given our data regarding activation of E2F andrelease of pRb from E2F transcription complexes in the absenceof cyclin D1-CDK4 activity, we reasoned that expression ofRb�CDK, a hyper-repressive mutant of pRb, would block cyclinE-dependent rescue of E2F activation in our system. Rb�CDK

has 14 of the 16 known CDK phosphorylation sites mutated toalanine and, therefore, functions as a constitutively active cellcycle repressor immune to the inactivating effects of the G1

cyclin-CDKs (67). This construct, when transfected into IIC9cells, prevents the �-thrombin-induced activation of the E2F-luciferase reporter plasmid (not shown). Interestingly, expres-

sion of Rb�CDK further prevents the ectopic cyclin E-mediatedrescue of E2F activation in LY294002-treated cells (Fig. 5A),whereas expression of wild-type pRb is ineffective in blockingE2F activation, presumably because wild-type pRb is subject toinactivation by the CDKs as cells progress through G1 (Fig. 6Aand data not shown). In accordance with this result, Rb�CDK

significantly attenuates the cyclin E rescue of cyclin A induc-

FIG. 4. CDK2-cyclin E phosphorylates and inactivates pRb invivo in the absence of prior CDK4-cyclin D activity. IIC9 cellstransiently transfected with cyclin E where indicated followed by serumdeprivation for 48–60 h. Cells were incubated in the presence or ab-sence of 10 �M LY29004 30 min before stimulation with 1 unit/ml�-thrombin for 17 h. A, lysates were assayed for phospho-pRb(Ser-780)(p-Ser-780) and phospho-pRb(Ser-795) (p-Ser-795) by Western blotting.B, pRb-immunoprecipitates were prepared from isolated nuclei as de-scribed under “Materials and Methods” and assayed for their ability toretard electro-migration of a 32P-labeled E2F response element probe byEMSA. Lane 1, free probe; lanes 2–5, probe incubated with pRb immu-noprecipitates from basal nuclei; lane 3, competition with unlabeledprobe; lanes 4–5, super-shift with antibodies to E2F1 (lane 4) and E2F4(lane 5); lanes 6–8, probe incubated with pRb immunoprecipitates from�-thrombin-stimulated nuclei. Data are representative of at least twoindependent experiments.

FIG. 5. Rb�CDK blocks the cyclin E-mediated rescue of E2Factivation in LY29004-treated cells. A, IIC9 cells were transientlytransfected with pGL3-TATA-6xE2F, �-galactosidase, and where indi-cated cyclin E and/or Rb�CDK followed by serum deprivation for 48–60h. Quiescent cells were incubated in the absence or presence of 10 �M

LY294002 followed by stimulation with �-thrombin for 17 h. Lysateswere assayed for luciferase and �-galactosidase (B-gal) activity, andrelative light units (RLU) were divided by units of optical density,respectively, for quantification and normalization. B, IIC9 cells weretransiently transfected with cyclin E and/or Rb�CDK where indicatedfollowed by 48–60 h of serum deprivation. Cells were incubated in thepresence or absence of 10 �M LY29004 for 30 min, then stimulated with1 unit/ml �-thrombin for 17 h. Lysates were analyzed for cyclin A andGAPDH mRNA and quantified; cyclin A arbitrary units were divided byGAPDH arbitrary units for normalization. Error bars represent S.D.(n � 3), and data are representative of two independent experiments.

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tion in LY294002-treated cells (Fig. 5B). Because this non-phosphorylatable mutant of pRb blocks the function of cyclin Ein these experiments, these data add further evidence that therole of ectopic cyclin E in the rescue of E2F activity is tofacilitate the phosphorylation of pRb. This result, when coupledwith the observations that dnCDK2 (Fig. 3) and GW8510 (Fig.2) also reverse the cyclin E rescue, strongly implicates CDK2-cyclin E in the observed inactivation of pRb.

Restoration of Four CDK2 Phosphorylation Sites to Rb�CDK

Renders It Susceptible to Inactivation by Cyclin E-CDK2—Byall accounts the phosphorylation of pRb by CDK2 is less exten-sive than the phosphorylation of pRb by CDK4 (16). Therefore,when CDK4 is inhibited but E2F is activated nonetheless byexpression of cyclin E it can be argued that pRb is phosphoryl-ated on a relatively small number of residues. To demonstratethat this minimal phosphorylation of pRb is sufficient to relievepRb-mediated repression of E2F activity, we created a con-struct termed Rb��K2. This construct was generated by restor-ing the following four reported in vitro CDK2-cyclin E phos-phorylation sites to Rb�CDK by site-directed mutagenesis:Thr-373, Ser-612, Ser-795, and Thr-821. It should be noted thatof these, Thr-373 and Ser-795 can also be phosphorylated byCDK4-cyclin D in vitro (16, 68).

To ascertain whether Rb��K2 is inactivated by cyclin E-CDK2 activity we first examine the ability of cyclin E-CDK2 toactivate E2F in the presence of Rb��K2. In contrast to Rb�CDK,expression of Rb��K2 does not significantly block the activationof E2F (Fig. 6A) in the condition of constitutive cyclin E expres-sion. To confirm that CDK2-cyclin E alone can inactivateRb��K2 we again generate conditions in which cyclin D-depend-ent kinases are inhibited by preincubation with LY294002, butcyclin E expression is provided (Fig. 6B). Under these condi-tions expression of Rb��K2 fails to repress �-thrombin-stimu-lated E2F activation. Furthermore, expression of Rb��K2 doesnot prevent the accumulation of cyclin A steady-state proteinlevel as does Rb�CDK (Fig. 6C). These results argue that thephosphorylation of Rb��K2 by CDK2-cyclin E on just four res-idues is sufficient to neutralize pRb-mediated repression ofE2F activity.

Ultimately, we examine the effect of Rb�CDK and Rb��K2 onG1 progression. First, we confirm our earlier observation thatLY294002-inhibited S phase entry can be rescued by ectopicexpression of cyclin E in �-thrombin-stimulated cells (Fig. 7A).In addition to phosphatidylinositol 3-OH kinase inhibition byLY294002, we express p16INK4a to ensure that cyclin D-CDKcomplexes are inhibited. Confirming our previous report (47),we find that unscheduled cyclin E expression rescues S phaseprogression in LY294002-treated p16INK4a-expressing cells.Not surprisingly, expression of Rb�CDK blocks �-thrombin-stimulated DNA synthesis despite cyclin E expression, becauseit is a constitutive repressor of E2F activity, immune to inac-tivation by the CDKs (Fig. 7B). However, Rb��K2 is not domi-nantly repressive, as levels of [3H]thymidine incorporation in�-thrombin-stimulated cells expressing this construct are sim-ilar to those expressing wild-type pRb. Again, our interpreta-tion of this observation is that the phosphorylation of pRb onthe four CDK2-preferred sites is sufficient to mediate release ofpRb from E2F transcription complexes and, thus, functionallyinactivate pRb, allowing for E2F-mediated transcription of S-phase genes and the initiation of DNA synthesis.

DISCUSSION

Unscheduled Cyclin E Expression Renders Cyclin D-CDKActivity Dispensable for G1 Progression—CDK2-cyclin E activ-ity has been long thought to be essential for the progressionfrom G1 to S phase of the cell cycle (2, 69). In cells expressingpRb, hyperphosphorylation of pRb by the G1 CDKs, which

FIG. 6. Expression of Rb��K2 does not block E2F activation in�-thrombin-stimulated cyclin E-expressing cells. A–B, IIC9 cellswere transiently transfected with pGL3-TATA-6xE2F, �-galactosidase,and where indicated pcDNA3.1 (vector), Rb, Rb�CDK, Rb��K2, and/orcyclin E followed by serum deprivation for 48–60 h. Quiescent cellswere incubated in the absence or presence of 10 �M LY294002 or 7.5 �M

GW8510 followed by stimulation with �-thrombin for 17 h. Lysateswere assayed for luciferase and �-galactosidase activity (B-gal), andrelative light units (RLU) were divided by units of optical density,respectively, for quantification and normalization. C, IIC9 cells weretransiently transfected with cyclin E, Rb�CDK, and/or Rb��K2 whereindicated followed by 48–60 h of serum deprivation. Cells were incu-bated in the presence or absence of 10 �M LY29004 for 30 min, thenstimulated with 1 unit/ml �-thrombin for 19 h. Lysates were harvestedand analyzed for cyclin A protein levels by Western blotting, as de-scribed under “Materials and Methods.” All data are representative ofat least three independent experiments.

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occurs in a cell cycle-dependent manner and results in pRbinactivation, is important for progression from G1 to S phase.Although the direct phosphorylation of pRb by CDK4-cyclin Din vivo is not disputed, the role CDK2-cyclin E plays in thephosphorylation of pRb in vivo has remained unclear. In thiswork, we have examined the role of cyclin E-CDK2 in thephosphorylation and inactivation of pRb. We have used anti-bodies to specific forms of phosphorylated pRb and found thatCDK2-cyclin E activity can phosphorylate pRb in the absence ofCDK4-cyclin D1 activity, suggesting that this phosphorylationof pRb by CDK2-cyclin E is not dependent on prior phospho-rylation by CDK4-cyclin D. These data are consistent with invitro experiments showing the ability of CDK2-cyclin E tophosphorylate unphosphorylated pRb (16) and with studies inthe E 3 D1 knockin mouse (45) that found a significant in-crease in the phosphorylation state of pRb in the absence ofcyclin D1 activity. These researchers reported that CDK2-cy-

clin E activity restores all the deficiencies associated with theablation of cyclin D1 in the cyclin D1 knockout mouse (46). Inaddition to demonstrating that cyclin E-CDK2 can directlyphosphorylate pRb in vivo in the absence of prior phosphoryl-ation by cyclin D-CDK4, we also found that this phosphoryla-tion results in the activation of E2F and the expression of cyclinA, which are essential for S-phase progression.

Our results further the understanding of the recognized rolethat deregulation of cyclin E plays in tumorigenesis. In addi-tion to the observation that many cancer cell lines overexpressfull-length cyclin E or an amino-truncated variant (70–73),deregulated cyclin E has been reported in primary tumors ofmany different cell types (74). The clinical impact of cyclin Eexpression appears most dramatic in the pathology of breastcancer. A recent report from Keyomarsi et al. (75) argues con-vincingly that cyclin E expression levels are the single mostpowerful clinical parameter in predicting long term prognosisin breast cancer patients, with high levels of cyclin E having aneven higher hazard ratio than the classic prognostic indicatorscyclins D1 and D3, HER-2/neu, estrogen/progesterone receptorstatus, and lymph node metastases (75). Supplementing thatargument, Span et al. (76) recently reported that the expressionlevel of cyclin E is a strong predictor of endocrine therapyfailure in breast cancer patients, a result that builds uponmounting that evidence that cyclin E is a critical target ofendocrine signaling in promoting steroid hormone-dependenttumor growth (77). Finally, 10 percent of mice that express ahuman cyclin E transgene develop carcinoma (78), and cyclin Eknockout MEFs are strikingly resistant to transformation withoncogenic Ras (Ref. 79 and see next paragraph). The datareported here are consistent with the notion that the oncogenicpotential of atypical cyclin E expression is due to inappropriatecell cycle re-entry through the inactivation of pRb.

Two recent controversial reports seemingly call into questionthe necessity of CDK2-cyclin E activity for cell cycle progres-sion. The two reports that CDK2 knockout mice develop nor-mally and, despite meiotic defects and sterility, are viable andhealthy (80, 81) and that cyclin E1/E2 double knockouts can beborn alive by placental rescue of cyclin E expression (79) sug-gest that CDK2-cyclin E activity is dispensable for cell-auton-omous murine embryogenesis and cell proliferation. Thesefindings, although intriguing and no doubt warrant re-exami-nation of current cell cycle dogma, do not challenge the obser-vations reported here. This work is particularly concerned withwhat unscheduled cyclin E expression can do in terms of pRbphosphorylation, E2F activation, and cell cycle progression.The observation that cyclin E-null MEFs cannot be stimulatedto proliferate following serum starvation suggests a crucial rolefor cyclin E in cell cycle re-entry from quiescence, an event thatmay be critical during in vivo tumorigenesis (79). In any event,whether or not CDK2-cyclin E is the normal mediator of E2Factivation awaits a more comprehensive study and is beyondthe scope addressed in the present study. However, given ourfindings a strong case can be made for the ability of aberrantcyclin E expression to drive the inactivation of pRb and, thus,G1 progression, despite CDK4-cyclin D inhibition, a findingthat augments the known role of cyclin E in tumor progressionand prognosis.

Interestingly, another recent report has challenged previousinterpretations regarding the role of pRb in G1 cell cycle con-trol. By using “acute” conditional loss of pRb to cells in culture,a system more accurately representing the sporadic inactiva-tion of RB alleles seen in the genesis of a tumor, Sage et al. (82)convincingly reaffirm that pRb is the critical mediator of thesenescence program in cell culture-induced cell cycle exit inMEFs. By escaping the complications of developmental com-

FIG. 7. Cyclin E-CDK2 without prior cyclin D-CDK activity issufficient for entry into S phase. IIC9 cells were transiently trans-fected with p16INK4a, cyclin E, pcDNA3.1, pRb, Rb�CDK, and/or Rb��K2

where indicated followed by serum deprivation for 48–60 h. Cells wereincubated in the presence or absence of 10 �M LY29004 or 7.5 �M

GW8510 30 min before stimulation with 1 unit/ml �-thrombin for 17 h.Cells were then pulsed with 0.5 �Ci/ml [3H]thymidine for 4 h andassayed for trichloroacetic acid (TCA)-precipitable counts as described.

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pensation and plasticity, this report further highlights thetumor-promoting potential of pRb inactivation in quiescentsomatic cells; that is, cells that have not recalibrated theirsenescence regulators to compensate for the loss of pRb. Inlight of these results, aberrant cyclin E expression could pro-vide just the window of opportunity needed to initiate tumorformation in vivo.

Going further, it would be interesting to analyze the phos-phorylation status of all of the CDK2-preferred sites on pRb inCDK2 or cyclin E knockout cells. Provocatively, in cyclin E1/E2-null MEFs, pRb does appear to become phosphorylatedsometime in late G1, suggesting the compensatory or perhapsredundant activity of a cyclin E-independent G1 kinase (79).Even more provocatively, pRb appears phosphorylated on Thr-821 in CDK2�/� MEFs, a site thought to be phosphorylated byCDK2, suggesting that the crucial inactivation of pRb is some-how endogenously recovered in these cells (80). Further com-plicating this issue is the fact that a mutant form of cyclin Ethat cannot bind to CDK2 transforms rat embryo fibroblastswhen expressed with oncogenic H-Ras (83), a result not easilyexplained by the current understanding of cell cycle dynamics.Clearly, further studies are required to elucidate the role ofCDK2, cyclin E, and possibly an undiscovered G1 kinase innormal cell proliferation and in tumorigenesis. However, thereality remains that cyclin E expression and pRb inactivationare intimately connected and most likely have a causal rela-tionship to the development and morbidity of human cancer.

Acknowledgments—We thank James Roberts, Kristian Helin,Jim Koh, Martine Roussel, Charles Sherr, and J. Wade Harper for agenerous supply of cDNAs. N. H. L. thanks Jaqueline Lees andJ. Alan Diehl for encouragement and helpful discussions andLeroy Wheeler for critical reading of the manuscript.

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Susan M. Keenan, Nathan H. Lents and Joseph J. BaldassareProgression

-S Phase1Retinoblastoma Tumor Suppressor Protein, Activation of E2F, and GExpression of Cyclin E Renders Cyclin D-CDK4 Dispensable for Inactivation of the

doi: 10.1074/jbc.M310383200 originally published online November 25, 20032004, 279:5387-5396.J. Biol. Chem. 

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