A Quantitative Comparison of Wild-Type and Gatekeeper Mutant Cdk2 for Chemical Genetic Studies with...

ORIGINAL ARTICLE 638J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Cyclin D1 and Cyclin D3 Show
Divergent Responses to DistinctMitogenic Stimulation
ALEXANDRA A. ANDERSON, EMMA S. CHILD, AARATHI PRASAD,
LUCY M. ELPHICK, AND DAVID J. MANN*

Division of Cell and Molecular Biology, Imperial College London, London, UK

D-type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin-dependent kinases (cdks). Here,we show that stimulatingmitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expressionlevels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester-mediated protein kinase C stimulation induces S phaseentry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin-mediated cAMP-dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specificallyincreases the level and activation of cyclinD3. These findings uncover additional levels of complexity in the regulation of the cell cycle at thelevel of the D-type cyclins and thus may have important therapeutic implications in cancers where specific D-cyclins are overexpressed.J. Cell. Physiol. 225: 638–645, 2010. � 2010 Wiley-Liss, Inc.

Alexandra A. Anderson, Emma S. Child and Aarathi Prasadcontributed equally to this work.

Contract grant sponsor: Cancer Research UK.Contract grant sponsor: BBSRC.

*Correspondence to: David J. Mann, Division of Cell and MolecularBiology, Imperial College London, London SW7 2AZ, UK.E-mail: [email protected]

Received 28 February 2010; Accepted 12 April 2010

Published online in Wiley Online Library(wileyonlinelibrary.com.), 8 July 2010.DOI: 10.1002/jcp.22207

Over the past two decades, a detailed understanding has beengained of the processes driving the cell cycle, identifying theindividual cyclins and cyclin-dependent kinases that areactivated at specific stages. Eukaryotic cells commit to divisionduring the G1 phase of the cell cycle in a process regulated inpart by mitogenic stimulation promoting expression of theD-type cyclins (Matsushime et al., 1994; Sherr et al., 1994;Sherr, 1995; Funk and Galloway, 1998). The D-cyclins (D1, D2,and D3) accumulate and associate with cdk4/cdk6 in order topromote phosphorylation-dependent inactivation of membersof the retinoblastoma protein family thereby activating E2Ftranscription factors to enable expression of genes responsiblefor cell-cycle progression. In order for the G1-specific cdks tobecome active they must not only associate with cyclins, butalso translocate to the nucleus and be phosphorylated on athreonine residue in the kinase activation loop by cdk-activatingkinase (Kaldis et al., 1998; Kaldis, 1999; Child and Mann, 2001).Themechanisms underlying the induction of theD-cyclins, theirassociation with cdks and translocation to the nucleus remainunder investigation.

Murine Swiss 3T3 fibroblasts have been widely used as amodel system in which to characterize pro-proliferativesignaling pathways. In general, cell cycle re-entry in quiescentSwiss 3T3 cells requires synergistic interactions betweendefined mitogenic agents: for example, insulin will synergizewith agents that activate either protein kinase C or cAMP-dependent protein kinase whereas any of these treatmentsapplied alone will not elicit S phase entry (Withers et al., 1995;Mann et al., 1997). In Swiss 3T3 cells phorbol ester-mediatedprotein kinase C stimulation results in S phase entry dependenton MAPK activation (Schonwasser et al., 1998). MAPKactivation has been implicated as essential at several levels forthe full activation of G1-specific cyclin/cdks. Indeed cyclin D1expression is known to be dependent on MAPK activation(Albanese et al., 1995; Lavoie et al., 1996; Weber et al., 1997;Cheng et al., 1998; Balmanno and Cook, 1999) as is theassociation of cyclin D1with cdk4 and the nuclear translocationof this complex (Lavoie et al., 1996; Cheng et al., 1999). Incontrast, protein kinase A-mediated mitogenesis isindependent of MAPK (Withers et al., 1995). Indeed, proteinkinase A negatively regulates Raf1, an activating kinase in theMAPK cascade (Dhillon et al., 2002; Dumaz et al., 2002; Dumazand Marais, 2003).

� 2 0 1 0 W I L E Y - L I S S , I N C .

Here we have investigated the signaling pathways leading toD-type cyclin/cdk activation and nuclear translocation usingmitogens that activate distinct signaling pathways. Using thesedistinct mitogens, we demonstrate that stimulation ofproliferation via protein kinase A relies predominantly on cyclinD3/cdk complexes which are produced, assembled andlocalized in an mTOR-dependent manner. Our datademonstrate that different D-type cyclins can be regulatedindependently of each other, with distinct signaling pathwaysleading to activation of cyclin D1/cdk and cyclin D3/cdkcomplexes.

Materials and MethodsAntibodies

Antibodies to cyclin D3 (DCS 28) and a-tubulin (TAT-1) werefrom Cancer Research UK. Antibodies against cyclin D1 (sc-450),cyclin D3 (sc-6283), cdk4 (sc-260), E2F1 (KH95), cdk2 (sc-163),MAPK (sc-93), and p70S6Kinase (sc-230) were from Santa CruzBiotechnology (Santa Cruz, CA). Anti-retinoblastoma protein(G3-245) was from BD Biosciences (San Jose, CA). Anti-Flag (M2)antibody was from Sigma-Aldrich Company Ltd (Dorset, UK).Phospho-MAPK antibodies (E10) were from Cell SignalingTechnology (Danvers, MA). For immunofluorescent detection ofD-cyclins an Alexa Fluor 555-labeled goat anti-mouse IgG fromMolecular Probes (Invitrogen, Ltd, Paisley, UK) was used.Anti-mouse/rabbit HRP-conjugated secondary antibodies werefrom Jackson ImmunoResearch laboratories Inc (WestGrove, PA).

D I V E R G E N T R E S P O N S E S O F D - C Y C L I N S 639

Cell culture and flow cytometry

Swiss 3T3 cells weremaintained inDMEM/10%NCS in a humidifiedatmosphere containing 10% carbon dioxide in air at 378C. Forexperimental purposes, 6–12� 105 cells were plated in 90mmNunc Petri dishes in DMEM/10% NCS and allowed to reachquiescence over the following 7–10 days. Confluent and quiescentcultures of Swiss 3T3 cells were washed in DMEM and incubatedwith DMEM containing supplements at the followingconcentrations: insulin 1mg/ml; PDB 100 nM; forskolin 10mM;IBMX 50mM; U0126 10mM; rapamycin 20 nM; MG132 20mM. Toisolate 3T3 pools ectopically expressing cyclin D3, human FLAG-tagged cyclin D3 lacking the 50 and 30 untranslated regions (ChildandMann, 2001) was subcloned into pLXSN3 and the recombinantplasmid was transfected into GPþ E packaging cells and retroviralsupernatant solutions were used to infect Swiss 3T3 cells prior toselection with G418. For flow cytometry analysis of S phaseentry, cells treated with and without mitogens were incubatedfor the final 30min with 10mM BrdU prior to harvesting.Cells were fixed in 70% ethanol/phosphate-buffered saline(PBS) and resuspended in denaturing solution (2NHCL, 0.5% (v/v)Triton X-100) for 30min at room temperature. They werewashed first in 0.1M Tris, pH 8, and then in blocking solution(0.5% (v/v) Tween-20, 1% (w/v) BSA, in PBS). 1� 106 cells wereincubated with 20ml fluorescein isothiocyanate (FITC)-conjugatedanti-BrdU antibodies (BD Biosciences) for 30min at roomtemperature. Following multiple washes in blocking solution andPBS, cells were stained with 5mg/ml PI for 30min. The BrdU/PIstained samples were analyzed by BD FACSCalibur analyzer (BDBiosciences, San Jose, CA) and a minimum of 10,000 events persample were collected. The cell-cycle profiles were analyzedusing the FlowJo software version 7.2.2. (Tree Star Inc.,Ashland, OR).

Cell extractions and immunological methods

For immunoblotting, cells were washed twice with PBS, lysedin situ in SDS–PAGE sample buffer, and collected by scraping.Samples were boiled and subjected to SDS–PAGE using 37.5:15acrylamide/bis (National Diagnostics, Atlanta, GA) using thefollowing % acrylamide to resolve the indicated proteins:retinoblastoma protein, 7%; all cyclins and cdks, 10%; MAPK, 12.5%(30:0.2 acrylamide/bis). Blots were analyzed by data capture via aFujifilm LAS 3000. All experiments were performed at least threetimes.

Immunoprecipitations and kinase assays

For immunoprecipitations cells were stimulated as described,harvested in PBS, 0.1% Tween, 25mM NaCl at 48C andincubated for 2 h with protein A-Sepharose beads, and cdk4antibody (0.4mg). The beads were washed three times inPBS–Tween, resuspended in SDS–PAGE loading buffer andimmunoblotted for the epitope-tagged cyclin. For kinase assays,Sf9 cells were infected with the appropriate recombinantbaculoviruses and lysed 72 h later in hypotonic kinase buffer(25mM Hepes, pH 7.9, 5mM MgCl, 0.1% 2-mercaptoethanol,0.1mM EDTA) for 10min on ice. Cell debris was removed bycentrifugation. In immunoprecipitation-kinase assays,immunoprecipitations were performed at 48C for 2 h using proteinA-Sepharose beads, cdk4 antibody (0.4mg), and 10ml Sf9 cell lysatein PBS, 0.1% Tween, 25mM NaCl. The beads were washed threetimes in PBS–Tween, then two further times in kinase buffer.Kinase assays were performed in kinase buffer containing 0.5–1mgGST-pRb, 100mM ATP, and 2.5mCi [g-32P]-ATP and incubatedfor 30min at 308C. Reactions were terminated by the additionof an equal volume of 2� SDS–PAGE loading buffer and resolvedby SDS–PAGE. All experiments were performed at least threetimes.

JOURNAL OF CELLULAR PHYSIOLOGY

Immunolocalization of cyclin D1 and cyclin D3

Swiss 3T3 cells were plated onto sterile 13mm glasscoverslips, grown to quiescence and stimulated. Fifteen hourspost-stimulation, cells were fixed in 3.7% formaldehyde for 10min,permeabilized with 0.2% Triton X-100, and treated with blockingbuffer (5% bovine serum albumin) for 45min at room temperatureto block nonspecific interactions prior to addition of the primaryantibody. Cells were incubated with the primary antibody,anti-cyclin D1 or cyclin D3 (1:100; Santa Cruz Biotechnology), for1 h at room temperature; washed; and incubated with thesecondary antibody, Alexa Fluor 555-labeled goat anti-mouseIgG (1:3,000), for 45min at room temperature. Following allfixation, permeabilization, and antibody incubations, cells werewashed (3� 5min) with PBS. Coverslips were mounted withMowiol containing DAPI. Images were captured using an AxioCamcamera on a Zeiss Axiovert 200M inverted microscope. Forquantitation of the immunofluorescence, 150–200 cells inrandom fields of view across the entire coverslip of eachcondition were scored for the presence of either cyclin D1 orcyclin D3 in the nucleus. All experiments were performed at leastthree times.

ResultsD-cyclin expression, assembly with cdk4 andassociated kinase activity after mitogen stimulation

To assess the expression level, complex assembly andassociated kinase activity of D-cyclins, quiescent Swiss 3T3 cellswere stimulated with various agents for 15 h followed byimmunoblotting, immunoprecipitation, and kinase assays. Asreported previously each of the stimuli caused S phase entry asjudged by flow cytometry (Fig. 1B). PDB/insulin preferentiallyelevated cyclin D1 levels, while forskolin/insulin treatment ledto the preferential induction of cyclin D3 (Fig. 1A) (Mann et al.,1997). This preferential increase in expression levels wasmirrored by an increase in the interaction of the D-cyclins withcdk4, as shown by immunoprecipitation of the cyclin/cdkcomplexes through cdk4 (Fig. 1C). Cyclin D/cdk4 complexesrequire cdk4 threonine 172 phosphorylation by cdk-activatingkinase to become catalytically active (Kato et al., 1994). Wemeasured the activity of the D-type cyclin/cdk4 holoenzymesresulting from cdk-activating kinase phosphorylation byimmunoprecipitating cdk4 and assaying for kinase activity. Thisconfirmed that NCS, PBD/insulin, and forskolin/insulinstimulation resulted in active D-cyclin/cdk4 complexes(Fig. 1D).

Localization of D-cyclins following mitogenic stimulation

Activation of D-cyclin/cdk4 complexes depends upon theirtranslocation to the nucleus (Diehl and Sherr, 1997; Benzenoet al., 2006). The effect of PDB/insulin and forskolin/insulinstimulation on the localization of D-cyclins was investigatedusing immunofluorescent quantitation of D-cyclin localization.Quiescent Swiss 3T3 cells were stimulated for 15 h prior toD-cyclin localization. Immunofluorescent detection of theD-cyclins showed that in quiescent cultures only a lowpercentage of cells exhibited nuclear cyclin D1 and cyclin D3(Fig. 2A,B). Stimulation with PDB/insulin substantially increasedthe percentage of cells with nuclear cyclin D1 from21.8� 2.4% in quiescent culture to 56.0� 10.8% (P< 0.025),while forskolin/insulin was less effective in increasing thepercentage of cells exhibiting nuclear cyclin D1 increasing thepercentage of cells to only 49.5� 11.9% (Fig. 2B). In contrast,stimulationwith PDB/insulin had little impact on the localizationof cyclin D3 increasing the percentage of cells with nuclearcyclin D1 from 22.6� 3.3% to 29.3%� 5.7, while stimulationwith forskolin/insulin increased the nuclear cyclin D3 markedly

Fig. 1. D-cyclinexpression,cdk4complexassembly,andactivityaftermitogenstimulation.A:ConfluentandquiescentculturesofSwiss3T3cellswere incubatedat37-Cwith the indicated supplements (0, unstimulated;NCS, 10%normal calf serum;FI, forskolin/insulin; PI, PDB/insulin).After15 h treatment, cells were lysed in 2T SDS–PAGE sample buffer and immunoblotted using antibodies against cyclin D1, cyclin D3, and tubulin.B:Cells, treatedasin(A)wereanalyzedforSphaseentrybymeasuringbromodeoxyuridineincorporationintoDNAbyflowcytometry.C:Cultureswere treated as described in (A). Cells were lysed and immunoprecipitated (IP) through cdk4 and immunoblotted for the indicated cyclin. Totaldenotes the sample prior to immunoprecipitation; FP indicates cells treated with forskolin/PDB;R indicates 10% NCS stimulation. D: Cultureswere stimulated as in (A). Cells were lysed and immunoprecipitated through cdk4 and immune complex kinase assays performed usingretinoblastomaproteinassubstratewiththeproductsof thekinaseassaybeingresolvedbySDS–PAGEfollowedbydetectionviaautoradiography.

640 A N D E R S O N E T A L .

over the level in quiescent cultures to 66.6� 6.9% of cell withnuclear cyclin D3 (P< 0.002).

Cyclin D1 expression and complex formation in responseto PDB/insulin stimulation is MAPK dependent

Stimulation of quiescent Swiss cells with the PDB/insulin orforskolin/insulin revealed significant differences in theresponsiveness of cyclin D1 and cyclin D3 to these mitogens.While cyclin D1 expression has previously been shown to belargely dependent on MAPK activation (Albanese et al., 1995;Lavoie et al., 1996; Weber et al., 1997; Cheng et al., 1998;Balmanno and Cook, 1999), the dependency of cyclin D3 onMAPK has not been thoroughly explored. Studies usingforskolin/insulin as a mitogen in Swiss 3T3 cells havedemonstrated that S phase entry was not dependent on MAPK


activation (Withers et al., 1995). We therefore sought totest the dependency of cyclin D1 and cyclin D3 on MAPKactivation.

Initially we verified the activation status ofMAPK in responseto the various stimuli using antibodies against the activatedphosphorylated species (Fig. 3A). Serum stimulation ortreatment with PDB/insulin resulted in the detection ofsubstantial levels of phospho-MAPK. In contrast, forskolin/insulin treatment did not result in elevation in the levels ofphospho-MAPK over the unstimulated cells after 1 h or longerperiods of time. In order to provide an evenmore rigorous testof the MAPK requirements for mitogenesis, we treatedquiescent Swiss 3T3 cellswith theMEK inhibitorU0126prior tostimulation and verified inhibition immunologically; under theseconditions, forskolin/insulin treatment displayed undetectablelevels of phospho-MAPK (Fig. 3B).

Fig. 2. LocalizationofD-cyclins followingmitogenic stimulation.A,B:Cultureswere treatedasdescribed inFigure1.A:ConfluentandquiescentculturesofSwiss3T3cellswereincubatedat37-Cwiththeindicatedsupplementsbeforefixation,permeabilization,andsequential incubationwithantibodies to cyclinD1/D3andanti-mouseAlexafluorA555. Images capturedusing aZeissAxiovert 200Mmicroscopewith63TPlanNeofluaroilobjective,AxioCam,andAxiovisionsoftware.ScalebarU 20mm.B:QuantitationofthepercentageofcellswithcyclinD1/D3immunofluorescencein the nucleus. Values are meanWSEM for four independent experiments (each quantifying D-cyclin localization of 200 cells for each culturecondition). MP<0.025, MMP<0.002. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


The impact of MAPK activity on the status of G1-specificcyclin/cdkswas then investigated in the absence and presence ofU0126 (Fig. 3C). As shown in Figure 3C, forskolin/insulintreatment preferentially increased the expression of cyclin D3whilst PDB/insulin preferentially elevated cyclin D1 levels.Inhibition of MAPK activity by addition of U0126 largelyattenuated cyclinD1expression in PDB/insulin-stimulated cells.In contrast, induction of cyclin D3 in forskolin/insulin-treatedcells was largely insensitive to inhibition of the MAPK pathway;in PDB/insulin and NCS-treated cells a partial reduction ofcyclin D3 expression was observed (Fig. 3C). The impact of theMAPK pathway in D-type cyclin assembly with cdk4 was thentested by stimulating quiescent Swiss 3T3 with NCS, forskolin/insulin or PDB/insulin and, 15 h later, immunoprecipitating D-type cyclin/cdk4 complexes through the cdk subunit. Theimmunoprecipitates were examined for the presence ofD-typecyclins (Fig. 3C). Whereas cyclin D1/cdk4 complexes wereenriched in NCS- or PDB/insulin-stimulated Swiss 3T3 cells,cyclin D3/cdk4 complexes were evident in cells treated with allmitogenic combinations of agents with most marked complexformation induced in forskolin/insulin-stimulated cells that lackactive MAPK. As a more rigorous test of assembly in theabsence of MAPK activity, these experiments were repeated inthe absence and presence of U0126: cyclin D3/cdk4 assembly


was largely unaffected by the drug whilst cyclin D1/cdk4assembly was strongly repressed (Fig. 3C). These data indicatethat while PDB/insulin induces activation of MAPK and thencepromotes assembly of cyclin D1/cdk4 complexes, elevation ofintracellular levels of cAMP by forskolin/insulin stimulationfacilitates translocation of cyclin D3 into the nucleus in theabsence of MAPK activation. In addition,forskolin/insulin-treated cells (but not PDB/insulin-treatedcells) entered S phase even in the presence of U0126 as judgedby BrdU incorporation (data not shown).

Increased cyclin D3 expression and nuclear localizationin response to forskolin/insulin stimulation ismTOR dependent

Previous work has showed that forskolin-generated cAMP canresult in activation of the protein serine/threonine kinasemTOR which controls translation via phosphorylation of the70-kDa ribosomal protein S6 kinase (p70S6Kinase) andeukaryotic initiation factor 4E-binding protein-1 (Harris andLawrence, 2003; Kwon et al., 2004). To begin to dissect thesignaling underlying forskolin/insulin-stimulated mitogenesis,the impact of mTOR on D-cyclin levels and localization wasassessed. Quiescent Swiss 3T3 cells were stimulated withNCS,

Fig. 3. Cyclin D1 expression and complex formation in response toPDB/insulin stimulation is MAPK dependent. A: Confluent andquiescent cultures of Swiss 3T3 cells were incubated at 37-Cwith theindicated supplements (0, unstimulated; NCS, 10% normal calfserum; FI, forskolin/insulin; PI, PDB/insulin) for the designated times.After treatment, cells were lysed in 2T SDS–PAGE sample buffer andimmunoblotted using antibodies against total MAPK and phosphoMAPK. B: Confluent and quiescent cultures of Swiss 3T3 cells wereincubated at 37-C with the indicated supplements in the absence (�)andpresence (R) ofU0126.After 2 h treatment, cellswere lysed in 2TSDS–PAGE sample buffer and immunoblotted using antibodiesagainst total MAPK and phospho MAPK. C: Confluent and quiescentcultures of Swiss 3T3 cells were treated as above but incubated for15 h prior to lysis or immunoprecipitation (IP) through cdk4 andimmunoblot for the indicated cyclin.


PDB/insulin, or forskolin/insulin in the presence or absence ofrapamycin, an immunosuppressive drug known to inhibitmTOR (Miller et al., 2007). In quiescent cells, a low level ofp70S6Kinase phosphorylation was observed (Fig. 4A). Afterstimulation with PDB/insulin, forskolin/insulin, or NCS, therewas a significant increase in phosphorylated p70S6Kinase (thehyperphosphorylated, slower migrating forms), which wasabolished by incubation with rapamycin (Fig. 4A). Incomparison, rapamycin only partially reduced the response ofcyclin D1 to PDB/insulin and had no impact on the response ofthe D-cyclins to NCS. Assessment of the impact of rapamycinby immunofluorescent quantitation showed no significant effecton the induction of cyclin D1 upon treatment with PBD/insulin


(Fig. 4B). Inmarked contrast, rapamycin abolished the inductionof cyclin D3 expression by forskolin/insulin (Fig. 4A), as well aspreventing the translocation of cyclin D3 to the nucleus,reducing the percentage of cells with nuclear cyclin D3 from64.6� 4.9% to 30.5� 5.7% (P< 0.002) (Fig. 4B,C). Theseresults suggest that the forskolin/insulin-mediated inductionand translocation of cyclin D3, but not that of cyclin D1, ismTOR dependent.

Rapamycin does not increase proteasomal degradationof cyclin D3

To define a possible mechanism underlying the rapamycin-mediated repression of cyclin D3, we investigated the effect ofthe drug on cyclin D3 protein degradation since previousreports have shown that rapamycin can affect D-cyclin levelseither by reducing the mRNA transcript stability or byincreasing proteasome activity (Hashemolhosseini et al., 1998;Pallet et al., 2005). To distinguish between these effects ofrapamycin, quiescent Swiss 3T3 fibroblasts were stimulatedwith forskolin/insulin either in the presence or the absence ofthe proteasome inhibitor MG132, which is known to mediateD-type cyclin degradation (Lahne et al., 2006) (Fig. 5A–C).Immunoblotting of cyclin D3 in the presence of rapamycin andMG132 showed that blockade of proteasomal degradation hadno impact on the suppressive activity of rapamycin (Fig. 5A). Inaddition, application of MG132 did not circumvent thesuppressive activity of rapamycin on the nuclear translocationof cyclin D3 (Fig. 5B,C). These results suggest that down-regulation of cyclin D3 protein levels in rapamycin-treated cellsis not due to increased degradation of cyclin D3, though it maybe acting by reducing cyclinD3mRNA stability or translation. Inorder to support this conclusion, the effect of rapamycin on a3T3 cell line expressing human cyclin D3 lacking 50 and 30

untranslated regions was characterized. As shown inFigure 5D, rapamycin had no effect on cyclin D3 levels in thesecells suggesting that it acts at the mRNA level via noncodingsequences.

Discussion

The D-type cyclins are critical in promoting cell-cycleprogression, making it imperative to understand themechanisms involved in regulating their expression andsignaling requirements for cdk4 activation. The requirement forMAPK activity in the expression of cyclin D1, its assembly withcdk4 and the subsequent nuclear localization and cdk-activatingkinase phosphorylation of the holoenzyme has been previouslyestablished (Matsushime et al., 1994; Albanese et al., 1995;Lavoie et al., 1996;Weber et al., 1997; Cheng et al., 1998, 1999;Balmanno and Cook, 1999; Keenan et al., 2001; Lents et al.,2002). Our data concur with these requirements for cyclin D1,but show the novel requirement for mTOR rather than MAPKactivity for these processes when cyclin D3 is involved. ThoughMAPK-independent D-cyclin induction is somewhatcontroversial, we have shown an increase in cyclin D3 levels,complex formation, and nuclear translocation in response toforskolin/insulin stimulation, which is known to be MAPKindependent (Withers et al., 1995). In this system we havedemonstrated the successful assembly of cyclin D3/cdk4complexes and localization of cyclin D3 to the nucleus and thatthese events can be abrogated by the mTOR inhibitorrapamycin. Thus, the requirement for MAPK activity for thegeneration of active D-type cyclin/cdk4 complexes is not agenerally applicable property of this class of cyclins.

The mechanism by which forskolin/insulin stimulation leadsto cdk4 activation via cyclin D3 awaits further experimentation.Previous experiments have demonstrated thatcAMP-dependent mitogenesis in Swiss 3T3 cells ispredominantly due to activation of protein kinase A (Withers

Fig. 4. Increased cyclinD3 expression and nuclear localization in response to forskolin/insulin stimulation ismTor dependent. A:Confluent andquiescent cultures of Swiss 3T3 cells were incubated at 37-Cwith the indicated supplements in the absence (�) and presence (R) of rapamycin.After 15 h treatment, cellswere lysed in 2TSDS–PAGE sample buffer and immunoblotted as indicated. B:Quantitationof the percentage of cellswith cyclinD1/D3 immunofluorescence in the nucleus under the indicated conditions. Values aremeanWSEM for four independent experiments(each quantifying 200 cells).MP<0.002. C: Representative images of cyclin D3 immunofluorescence. Images captured using Zeiss Axiovert 200Mfluorescence microscope and Axiocam. Scale barU 20mm. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]


et al., 1996). To begin to further dissect this signaling, we havetested downstream effectors of protein kinase A usingpharmacological inhibitors. The small GTPase Rap1B isactivated by protein kinase A (Tsygankova et al., 2001);blockade of this enzyme with GGTI-298 showed nodifferentiation in the response of cyclin D1 and cyclin D3 (datanot shown). Protein kinase Cd is also activated by proteinkinase A but again inhibition of this novel protein kinase usingrottlerin failed to specifically perturb the cyclin D3 responses(data not shown). However, use of the mTOR inhibitorrapamycin specifically blocked cyclin D3 responses without


affecting those of cyclin D1. Previous studies in otherspecialized cell settings have revealed conflicting data about theaffect of rapamycin on cyclin D3: at a transcriptional level inmegakaryocytes (Raslova et al., 2006), at a translational levelin lymphocytes (Hleb et al., 2004) and at a protein stability levelin HER2-positive breast cancer cells (Garcia-Morales et al.,2006). Our data indicate that in Swiss 3T3 cells, a post-transcriptional mechanism is important that is independent ofcyclin D3 proteasomal degradation. One key mechanism bywhich mTOR can effect protein levels is via the regulation oftranslation of mRNAs containing 50-terminal oligopyrimidine

Fig. 5. Rapamycindoesnot increaseproteosomaldegradationofcyclinD3.A–D:Culturesweretreatedasdescribed inFigure4.A:Confluentandquiescent cultures of Swiss 3T3 cellswere incubated at 37-Cwith the indicated supplements for 15 hprior to lysis in 2TSDS–PAGEsample buffer.B: Quantitation of the percentage of cells with cyclin D3 immunofluorescence in the nucleus. Values are meanWSEM for three independentexperiments (eachmeasuring 200 cells for each condition). C: Representative images of cyclin D3 immunofluorescence. Images captured usingZeiss Axiovert 200M fluorescence microscope and Axiocam. Scale barU 20mm. D: Confluent and quiescent cultures of Swiss 3T3 ectopicallyexpressing cyclin D3were incubated at 37-Cwith the indicated supplements for 15h prior to immunoprecipitation (IP) through the FLAG tag ofcyclin D3 and immunoblot for cyclin D3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


tracts (50-TOPs) (Price et al., 1992; Dumont et al., 1994;Brown et al., 1995). mTOR inhibition via rapamycin leads torapid dephosphorylation and inactivation of p70S6Kinasewhich substantially reduces translation of mRNAscontaining 50-TOPs. Given the rapamycin insensitivity of acyclin D3 transgene lacking its 50-UTR, this mechanismappears plausible although inspection of the sequence failed toreveal a TOP-like sequence. Additionally, mTOR hasrecently been implicated in the regulation of cyclin D2 inbeta cells through protein synthesis and stability (Balcazaret al., 2009) although again 50-TOP sequences are not readilyevident.

In summary, our study demonstrates that in Swiss 3T3 cellsdifferent mitogenic stimuli can induce disparate responses in


cyclin D1 and cyclin D3, both of which are competent in causingcell-cycle progression. Phorbol ester-mediated protein kinaseC stimulation results in MAPK activation and thesubsequent generation of active cyclin D1/cdk4 complexeswhile forskolin-mediated protein kinase A stimulation results inmTOR activation leading to the generation of active cyclin D3/cdk4 species. The differences in the responsiveness of the cyclinD1 and cyclin D3 may have important therapeutic implications.A number ofmodels have suggested thatmutations affecting theexpression levels and nuclear translocation of cyclin D1/cdk4can trigger neoplastic transformations (Kim and Diehl, 2009).Similarly, cyclin D3 overexpression is implicated in oncogenesisin diseases such as multiple myeloma (Liebisch and Dohner,2006). Thus, gaining a more in-depth understanding of the


mechanisms underlying the specific induction of individualD-cyclins may allow the development of more preciselytargeted therapies.

Acknowledgments

We thank Gordon Peters, London Research Institute, CancerResearch UK for the generous gift of antibodies. This work wasfunded by Cancer Research UK and the BBSRC.

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