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REVIEWARTICLE published:17January2013 doi:10.3389/fimmu.2012.00423 Protein kinase C signaling and cell cycle regulation AdrianR.Black*andJenniferD.Black EppleyInstituteforResearchinCancerandAlliedDiseases,UniversityofNebraskaMedicalCenter,Omaha,NE,USA Editedby: AlinkbetweenTcellproliferationandtheproteinkinaseC(PKC)familyofserine/threonine NoahIsakov,Ben-GurionUniversityof kinaseshasbeenrecognizedforabout30years.However,despitethewealthofinformation theNegev,Israel onPKC-mediatedcontrolof Tcellactivation,understandingoftheeffectsofPKCsonthe Reviewedby: cellcyclemachineryinthiscelltyperemainslimited.Studiesinothersystemshaverevealed ChristopherE.Rudd,Universityof importantcellcycle-specificeffectsofPKCsignalingthatcaneitherpositivelyornegatively Cambridge,UK CosimaT.Baldari,UniversityofSiena, impactproliferation.TheoutcomeofPKCactivationishighlycontext-dependent,withthe Italy precisecellcycletarget(s)andoveralleffectsdeterminedbythespecificisozymeinvolved, *Correspondence: the timing of PKC activation, the cell type, and the signaling environment. Although AdrianR.Black,EppleyInstitutefor PKCscanregulateallstagesofthecellcycle,theyappeartopredominantlyaffectG0/G1 ResearchinCancerandAllied and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, Diseases,UniversityofNebraska MedicalCenter,985950Nebraska cyclin-dependent kinases (cdks), cdk inhibitors and cdc25 phosphatases; however, MedicalCenter,Omaha,NE evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of 68198-5950,USA. PKC-regulatedcellcycle-specificeffects.SeveralPKCisozymescantargetCip/Kipproteins e-mail:[email protected] tocontrolG0/G1→Sand/orG2→Mtransit,whileeffectsonD-typecyclinsregulateentry into and progression through G1. Analysis of PKC signaling inT cells has largely focused on its roles inT cell activation; thus, observed cell cycle effects are mainly positive. A prominentroleisemergingforPKCθ,withnon-redundantfunctionsofotherisozymesalso described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in thesecells.Asinothercelltypes,context-dependenteffectsofindividualisozymeshave beennotedinTcells,andCip/KipcdkinhibitorsandD-typecyclinsappeartobemajorPKC targets.Futurestudiesareanticipatedtotakeadvantageofthesimilaritiesbetweenthese varioussystemstoenhanceunderstandingofPKC-mediatedcellcycleregulationinTcells. Keywords:proteinkinaseC,signaltransduction,Tcellactivation,cellcycle,cyclin,cyclin-dependentkinase,cyclin- dependentkinaseinhibitor AnassociationbetweenproteinkinaseC(PKC)signalingandTcell protein kinase C) superfamily of protein kinases (Nieto, 2007; proliferationhasbeenrecognizedforalmostthreedecades.Over Matsuoka etal., 2009; Black, 2010; Rosse etal., 2010; Ryu etal., 30 years ago, it was determined that a combination of phorbol 2010).PKCisozymesarelipid-dependentkinases(requiringphos- estersandelevatedintracellularcalciumpotentlyinducesprolif- phatidylserine binding for activity) and are grouped into three eration of cells of the T cell lineage (e.g., Whitfield etal., 1973; subfamilies based on their structure and requirement for addi- Lyalletal.,1980;Sutherlandetal.,1981).Shortlythereafter,itwas tionalco-factorsandcalcium.Physiologicalactivationofclassical determined that PKC, then recognized as a calcium-dependent PKCs(PKCα,PKCβIandPKCβII,whicharesplicevariantsofthe enzyme,representedthemajorcellularreceptorforphorbolesters prkcbgene,andPKCγ)isinducedbythelipidsecondmessenger (Castagnaetal.,1982)anditwasnotlongbeforetheconnection diacylglycerol (DAG) and calcium, while activation of the novel between these phenomena was made (Isakov etal.,1986; Isakov PKCs (PKCδ, PKCε, PKCθ, and PKCη) requires only DAG. In and Altman,1987). Since then, a central role for PKC in T cell contrast, the atypical PKCs (PKCζ and PKCι/λ) are not depen- receptor(TCR)signalinghasbeenfirmlyestablished.Despitethe dentonlipidsecondmessengersorcalciumforactivity. Instead, longassociationofPKCwithTcellproliferation,detailsonhow theirfunctionisregulatedbyprotein–proteininteractionsmedi- PKCsignalinginteractswiththecellcyclemachineryinthiscell atedbyaPB1domainaswellasacarboxyl-terminalPDZligand typeareonlybeginningtoemerge.Inthisregard,ourknowledge motif. Engagement of growth factor or cytokine receptors leads in T cells lags behind that in other cell types, including other toactivationof phospholipaseC(PLC)βorPLCγ,whichcleave hematopoietic lineages. In this review, we outline our current phosphatidylinositol 4,5-bisphosphate to generate DAG and the understanding of the proliferative effects of PKC signaling in T soluble second messenger inositol trisphosphate (which induces cells within the context of the broader knowledge that has been release of calcium from intracellular stores). The production of gainedinothersystems. DAGrecruitsclassicalandnovelPKCstotheplasmamembrane, wheretheyundergoaconformationalchangeresultinginfullacti- THEPROTEINKINASECFAMILY vation.UnlikeotherAGCkinases,suchasAkt,activationofPKCs Protein kinase C represents a family of serine/threonine kinases doesnotrequireacutephosphorylationoftheenzyme:phospho- thatbelongtotheAGC(cAMP-dependent,cGMP-dependent,and rylations necessary for catalytic competence occur shortly after www.frontiersin.org January2013|Volume3|Article423|1 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 1 — #1 BlackandBlack PKCandthecellcycle synthesisandtheenzymeisconstitutivelyphosphorylatedatthese apotentinhibitorofotherkinasessuchasPRAKandMAPKAP- sites(Matsuokaetal.,2009;Rosseetal.,2010).Asaresult,changes K2 (Soltoff, 2007); thus, any effects of this inhibitor cannot be in phosphorylation do not provide an indication of PKC activ- ascribedtodirectinhibitionofPKCθ. ity; rather signaling-induced translocation of the enzyme to the Despitetheselimitations,ourknowledgeoftherolesofindivid- membrane/particulatefractionrepresentsthemostreliablemeans ualPKCsisemerging.Ofnote,inadditiontotheproliferative/cell ofmonitoringkinaseactivation.Reversalofsignalingcanoccurby cycle effects which are the subject of this review, PKC isozymes metabolismofDAGbyDAGkinaseandreleaseofPKCsfromthe havebeenfoundtoregulatemultiplecellularprocessesof direct membrane,aswellasbyagonist-inducedenzymedegradationor relevance to T cell development and function, including dif- removalofprimingphosphorylationwithsubsequentrapiddegra- ferentiation, migration, survival, apoptosis, endocytosis, and dation(LeontievaandBlack,2004;Newton,2010).Inadditionto secretion/exocytosis(Reyland,2009;Rosseetal.,2010). activation by growth factor signaling, classical and novel PKCs can be stimulated by a number of pharmacological agents that THEMAMMALIANCELLCYCLE mimictheeffectsofDAG,suchasphorbolestersandmacrocyclic Several excellent reviews have been written on the regulation of lactonebryostatins.However,incontrasttoDAG,theseagonists, thecellcycle(SherrandRoberts,2004; Cobrinik,2005; Malum- whichincludephorbol12-myristate13-acetate[PMA;alsoknown bresandBarbacid,2005;DuandPogoriler,2006;Satyanarayana as12-O-tetradecanoylphorbol-13-acetate(TPA)],phorbol12,13- andKaldis,2009)andonlyabriefdescriptionwillbegivenhere. dibutyrate(PDBu),andbryostatin1,arenotrapidlymetabolized Thecellcyclehasbeenclassicallydividedintofourphases,G1(or andthusgiveamoresustainedPKCactivation. Gap1inwhichcellsprepareforDNAsynthesis),Sphase(inwhich Despite limitations related to their lack of specificity for DNAissynthesized),G2(inwhichcellspreparefordivision)and individual PKC isozymes, their ability to promote PKC down- mitosis(orMphase,inwhichsisterchromatidsareseparatedand regulation,andtheexistenceofadditionaltargetsfortheseagents thecelldivides; Figure1). Transitthroughthecellcycleisregu- (Griner and Kazanietz, 2007), use of pharmacological agonists latedbyfourmajorclassesofcyclinswhoseexpressionisstrictly and membrane permeant DAG analogs has provided significant controlledandlimitedtoparticularcellcyclephases.Cyclinsare insightintothedownstreameffectsof PKCactivation. However, theregulatorysubunitsforcyclin-dependentkinases(cdks),whose acompleteunderstandingofPKCsignalingwillrequiredefining activityisabsolutelydependentonassociationwithspecificcyclin thespecificfunction(s)ofindividualPKCisozymes,andprogress partners. Entry of quiescent cells into the cell cycle and transit towardthisgoalhasprovedtechnicallydifficult.Understandingof throughearlyG1isregulatedbyD-typecyclins, whichcomplex thefunctionsofatypicalPKCs,PKCζ,andPKCι,lagsbehindthat withcdk4andcdk6(Musgroveetal.,2011).TherearethreeD-type ofothermembersofthePKCfamily,perhapslargelyduetotheir cyclins,D1,D2,andD3,whichareexpressedtovaryingdegreesin insensitivity to pharmacological activators (e.g., phorbol esters differenttissues;cyclinsD2andD3appeartobethemajorplayers andbryostatins)andsyntheticDAGs.Intheabsenceofisozyme- inTcells.TransitthroughlateG1andprogressionintoSphaseis specificpharmacologicalPKCagonistsandinhibitors,earlystudies regulatedbycyclinEcomplexedwithcdk2(HwangandClurman, reliedonoverexpressionstrategiestodeciphertherolesof indi- 2005; MalumbresandBarbacid,2005). Sphasetransitandearly vidual isozymes, which can result in non-physiological levels of G2areregulatedbycyclinA/cdk2andcyclinA/cdk1complexes, expression, activity, and regulation. RNA interference technol- whereascyclinB,complexedwithcdk1,regulatesprogressioninto ogyandgeneticallyalteredmicearehelpingtocircumventthese Mphase(MalumbresandBarbacid,2005;SanchezandDynlacht, problems,butarenotwithoutdrawbacksoftheirown.Potential 2005).Inadditiontobeingregulatedbycyclinbinding,theactiv- limitationsincludetheneedforahighlevelofsilencingtosuffi- ityofcdksisunderthecontrolofCip/KipandInk4cdkinhibitor cientlydepleteenzymeactivity(e.g.,>80%,Cameronetal.,2008; proteins(ckis;SherrandRoberts,1995).MembersoftheCip/Kip M.A.Pysz,A.R.Black,andJ.D.Black,unpublishedresults),and family,includingp21Cip1,p27Kip1,andp57Kip2,haveadualactivity thefactthatknockdownofonePKCisozymecanaffectaccumu- incellcycleregulation.Theynegativelyregulatecellcycleprogres- lation of other members of the family (M.A. Pysz, A. R. Black, sion by binding to cyclin/cdk2 and cyclin/cdk1 complexes and andJ.D.Black,unpublisheddata).Overlappingrolesofdifferent inhibitingtheirenzymaticactivity.Conversely,theseproteinscan isozymesmeansthatmultiplecrossesof transgenicmicemaybe promote progression by enhancing the association of cyclin D neededtoobservephenotypes. withcdk4andcdk6withoutinhibitingtheactivityofthesecom- Anadditionalsourceof confusionregardingthefunctionsof plexes (Sherr and Roberts,1999). The Ink4 ckis, which include individual PKC isozymes is the fact that many so-called PKC p15Ink4b, p16Ink4a, p18Ink4c, and p19Ink4d, block the activity of inhibitors are of questionable specificity (Griner and Kazanietz, cdk4andcdk6bypreventingtheirassociationwithcyclinD.Cdk 2007; Soltoff,2007). As an example of particular relevance to T activityisalsoregulatedbyphosphorylation:positivephosphory- cellactivation,specialcautionisneededwhenconsideringstud- lationismediatedbycdkactivatingkinase(CAKorcdk7/cyclin iesthathaveusedrottlerintoinfereffectsofsignalingfromPKCθ. H; Fisher and Morgan, 1994), while negative phosphorylation Whilethisagentwasoriginallyconsideredtobeaspecificinhibitor involvesthekinasesWee1andMyt1.Removalofinhibitoryphos- ofnovelPKCs,recentstudieshavedemonstratedthatitdoesnot phorylation,bye.g.,Cdc25phosphatases,isnecessaryforfullcdk inhibitPKCδ(Soltoff,2007).Inkeepingwiththisfinding,theIC activity. 50 forPKCθinhibitionbyrottlerininthepresenceof 100μMATP Whiletherearemultiplecheckpointsthatallowcellstoundergo is>300μM(Villalbaetal.,1999),aconcentrationfarinexcessof cellcyclearrestinresponsetovariousstresses,themostrelevant thatusedinstudiesonitscellulareffects.Incontrast,rottlerinis to normal tissue homeostasis and differentiation is that which FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|2 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 2 — #2 BlackandBlack PKCandthecellcycle directs entry and exit from the cell cycle in G1 (Prasad etal., repression, allowing for transcription of E2F-dependent genes, 1994;Liuetal.,2012).TheexpressionofD-typecyclinsisacutely oneofwhichiscyclinE.CyclinE/cdk2thencompletesphospho- regulated by mitogenic signals. As such, these proteins are the rylationofpocketproteins,leadingtotheirreleasefromE2Fand mainsensorsforthegrowthenvironmentofthecell,andareinti- robust transcription of growth-related genes. At early stages of matelyinvolvedinregulationof theentryof quiescentcellsinto G1, cells require mitogenic signals to support cyclin expression the cell cycle. Major targets for cyclin D/cdk complexes include andcdkactivity; however, oncesufficientlevelsof cyclinEhave the retinoblastoma protein (pRb) and related pocket proteins, accumulatedtomaintainitsownexpression,cellshavepassedthe p107andp130(Cobrinik,2005).Inthehypophosphorylatedstate, so-called“restrictionpoint”andareabletoproceedthroughtothe pocket proteins bind to E2F transcription factors on the pro- nextcellcyclewithoutfurthermitogenicinput.Inthefaceofloss motersofgrowth-relatedgenes,wheretheyactastranscriptional of mitogenicsignalspriortotherestrictionpointorof negative repressors and actively block expression of genes necessary for growth signals, cell cycle progression is halted and cells eventu- DNA replication (Trimarchi and Lees, 2002; Du and Pogoriler, ally withdraw into G0 phase and quiescence (Grana etal.,1998; 2006;Figure1).Phosphorylationofpocketproteinsrelievesthis ClassonandDyson,2001). FIGURE1|Continued www.frontiersin.org January2013|Volume3|Article423|3 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 3 — #3 BlackandBlack PKCandthecellcycle FIGURE1|Thecellcycle.Thecellcycleconsistsoffourphases,G1,S,G2, (largeredarrow),whichcommitscellstotheproliferativecycle.Ifconditions andM.InearlyG1,hypophosphorylatedpRbbindstheE2Ftranscription arenotoptimaltosignalthistransition,cellsexitthecycleandenterG0or factor,andrecruitshistonedeacetylase(HDAC)andotherfactorstoactively quiescence,areversiblenon-replicativestate.OncecellsenterSphase, represstranscriptionofE2F-regulatedgenesimportantfortransitionintoS cyclinE/cdk2activityisinhibitedbyproteasomaldegradationofcyclinEinthe phaseandDNAreplication(e.g.,PCNA,topoisomeraseI,c-Myc,cyclinE, cytoplasm.Continuedinactivation/hyperphosphorylationofpRballowsthe Cdc25c).ProgressionthroughearlyG1isdependentongrowthfactors,which transcriptionofcyclinAandcyclinB,requiredforsubsequentphasesofthe promoteexpressionofD-typecyclins.FormationofcyclinD/cdk4and cellcycle.CyclinA/cdk2complexesphosphorylateanumberofproteinsto cyclinD/cdk6complexes,whichisfacilitatedbyCip/Kipckis,leadsto facilitateSphasecompletionandtransitintoG2/M.CyclinBisactively phosphorylationofpRbatasubsetofavailablesitesandreleaseofHDAC synthesizedduringG2andassociateswithcdk1totriggermitosis.Cdk1is andotherinhibitoryfactors,relievingrepressionofE2Fandpromoting maintainedinaninactivestatebythekinasesWee1andMyt1.Ascells upregulationofcyclinE.CyclinE/cdk2complexes,relievedfromrepressionby approachMphase,thephosphatasecdc25isactivatedtoremoveinhibitory Cip/KipckisbysequestrationoftheseinhibitorymoleculesincyclinD/cdk phosphatesonTyr14andThr15,drivingthecellsintomitosis.Acheckpointin complexes,completepocketproteinphosphorylationinmidtolateG1, lateG2(largegreenarrow)preventscellsfromenteringMphaseifthe enablingawaveofE2F-dependenttranscriptionalactivityessentialforS genomeisdamaged.ThisDNAdamagecheckpointensuresthatcellsdonot progression.Together,theseeventsdrivecellsthroughtherestrictionpoint initiatemitosisuntiltheyhaverepaireddamagedDNAafterreplication. FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|4 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 4 — #4 BlackandBlack PKCandthecellcycle PKCSIGNALINGANDTCELLPROLIFERATION cellsthatarisefromactivationareeventuallyclearedfromthecir- TCELLDEVELOPMENTANDTCRSIGNALING culation,asmallnumberdevelopintomemoryTcellswhichare + T lymphocytes arise from bone marrow-derived CD34 stem primedforactivationuponsubsequentantigenexposure. cells, which seed the thymus and undergo multistage differenti- Antigen-induced proliferation is a key aspect of both T cell ationtobecomematurecirculatingcells(forreferences,seeKoch differentiation and clonal expansion (Koch and Radtke, 2011). and Radtke, 2011). An early event in this process involves VDJ Thus, mechanisms underlying regulation of the T cell cycle recombination of the TCR-β chain which then complexes with machinery are of critical importance to immune function. As pre-Tα to form the pre-TCR. Signaling from the pre-TCR leads the signaling pathways involved in T cell activation are being to proliferation of pre-T cells and rearrangement of the TCR-α deciphered, increasing evidence is pointing to the importance chain, which combines with the β chain and CD3 to form the of the PKC family in mediating proliferative responses in these TCR.Furtherdifferentiation,accompaniedbynegativeandposi- cells. The following section outlines our current understanding tiveselection,eventuallyleadstothedevelopmentofmaturenaïve of the role of individual PKC isozymes in regulating prolifera- + + Tcells, includingCD4 helperT(Th), CD8 cytotoxicT(Tc), tioninTcellswithinthecontextofknowledgegainedfromother andregulatoryT(Treg)cells.Thesenaïvecellsexitthethymusand systems. remain dormant as they circulate through secondary lymphoid organs until activated by antigen. These organs, which include PKCsANDTHECELLCYCLE the spleen, lymph nodes, and Peyer’s patches, transiently house AsourknowledgeoftheproliferativerolesofthePKCfamilyhas naïve T cells and are the first line of defense against pathogens developed,ithasbecomeincreasinglyapparentthattheeffectsof that traverse the skin or the epithelial lining of the respiratory, thesemoleculesarehighlycontext-dependent.ThefactthatPKCs gastrointestinal,andurogenitaltracts. areactivatedbytumorpromotingphorbolestersandaredown- ActivationofTcellsrequiresinteractionoftheTCRwithmajor stream of growth factor receptors initially led to the idea that histocompatibility complex (MHC) bound antigen on antigen they transduce positive mitogenic signals (Castagna etal., 1982; presentingcells(APCs),suchasdendriticcells,macrophages,and Kikkawa etal., 1983; Leach etal., 1983). Although a number of Bcells(forreferences,seeMarslandandKopf,2008;Smith-Garvin early studies supported this idea (Dicker and Rozengurt, 1978; etal.,2009;Fooksmanetal.,2010;DustinandDepoil,2011).The Rozengurt,1986; Takuwaetal.,1988), itsoonbecameclearthat interfacebetweentheTcellandAPCismarkedbytheformationof PKCscannegativelyandpositivelyregulatecellcycleprogression. astructure,termedtheimmunesynapseorsupramolecularacti- Indeed, regulation of proliferation by the PKC enzyme system vationcluster(SMAC),whichservestoregulateTcellsignaling. exhibitsahighdegreeof complexity,witheffectsinvolvingmul- ProductiveactivationofTcellsrequirestwosignals.Thefirstsig- tiplecellcycleregulatorymolecules,includingcyclins,cdks,and nalisprovidedbytheMHC-boundTCR,whilethesecondsignalis ckis,andimpactingvariousstagesofthecellcycle(Black,2010). providedbyco-stimulatorymoleculessuchasCD28(whichbinds Furthermore, individual isozymes can have opposing effects on toB7proteinsontheAPC).Additionally,cytokinessuchasIL-12 cell cycle progression in different cell types and even within the andtumornecrosisfactoralpha(TNF-α)canprovideathirdsig- samecelltype,dependingonthesignalingenvironment.Asingle nalthatregulatestheresponsetoTcellactivation.Anumberof isozymecantargetdifferentcellcyclemoleculesindifferentcell experimentalmanipulationscanactivateTcellsintheabsenceof types, can have opposite effects on a specific cell cycle target in APCinteraction;theseincludecrosslinkingoftheTCRandCD28 different systems, and can modulate the same target to produce with insoluble antibodies and combined treatment of cells with divergentcellcycleresponses(forreview,seeBlack,2010).Thus, phorbolesterandcalciumionophore. to gain a true understanding of the role of PKCs in regulation Tcellreceptorco-activationleadstotheengagementof mul- of proliferationinanygivensystem,itisimportanttostudythe tiple downstream signaling pathways including those involving mechanismsbywhichindividualisozymesaffectspecificcellcycle phosphatidylinositol 3-kinase (PI-3K), tyrosine kinases such as moleculesinthatsystem. Lck, and PLCγ (for references, see Altman etal., 2000; Mars- TlymphocytesexpressallmembersofthePKCfamilywiththe landandKopf,2008; Smith-Garvinetal.,2009; Fooksmanetal., exceptionofPKCγ(Koretzkyetal.,1989;Chenetal.,1994;Thuille 2010; Dustin and Depoil, 2011). Activation of PLCγ results etal.,2006). A role for PKC isozymes in cell cycle regulation in in production of DAG, which recruits PKCθ to the immune CD3+Tlymphocyteswassuggestedbytheearlyrecognitionthat synapse where it interacts indirectly with CD28 through bind- phorbolesters,inconjunctionwithcalciumionophore,arepotent ing to Lck (Kong etal., 2011; Isakov and Altman, 2012). PKCθ mitogensforthesecells(Altmanetal.,1990).Whilestudieshave then phosphorylates CARMA1, leading to the assembly of the concentratedlargelyontheroleof PKCθinmediatingsignaling CARMA1–BCL10–MALT1(CBM)signalosome.PLCγ-generated fromtheimmunesynapse,aroleforotherPKCisozymesisemerg- inositol trisphosphate releases calcium from intracellular stores. ing.Notably,differentisozymescanhavepro-proliferativeand/or The combined action of downstream TCR signaling eventually anti-proliferative functions, arguing that, as in other cell types, leads to activation of NF-κB, AP1, and nuclear factor of acti- PKCsignalingcanregulateentryintothecellcycle,transitthrough vated T cells (NFAT) transcription factors (Marsland and Kopf, thevariouscellcyclephases,aswellascellcyclewithdrawalinT 2008; Smith-Garvin etal., 2009; Fooksman etal., 2010; Dustin cells.Thefollowingsectionsdiscusscurrentunderstandingofthe and Depoil, 2011). Together, these events promote functional growthregulatoryfunctionsof individualPKCfamilymembers, activationof Tcellswhichismarkedbycellproliferation/clonal followed by a summary of the limited information available on expansion and cytokine secretion. While the majority of the T cellcycle-specificeffectsoftheseisozymesinTcells. www.frontiersin.org January2013|Volume3|Article423|5 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 5 — #5 BlackandBlack PKCandthecellcycle PROLIFERATIVEEFFECTSOFINDIVIDUALPKCFAMILYMEMBERS G1 → S progression in SKRB-3 breast cancer cells (Nakagawa PKCα etal.,2003),whereasPKCαisrequiredforATRA-inducedgrowth Useofselectivepharmacologicalinhibitors,antisensetechnology, arrestinT-47Dbreastcancercells(Choetal.,1997). orsiRNAhasidentifiedananti-proliferativeanddifferentiation- AroleforPKCαinpositiveregulationofproliferationinTcells inducing role of PKCα in multiple cell types, e.g., intestinal wassuggestedbythefindingthat,unlikewild-typecells,Tlym- epithelial cells, keratinocytes, mammary epithelial cells, and phocytes from transgenic mice overexpressing PKCα were able melanoma cells (Black,2000,2010). Anti-proliferative effects of toproliferateinresponsetosolubleanti-CD3antibody(Iwamoto PKCαaffectingG1→Stransitincludedownregulationofcyclin etal.,1992).ThisrolewasconfirmedbystudiesofPKCαknockout D1(Detjenetal.,2000;Hizlietal.,2006;Guanetal.,2007),aswell mice: whilePKCαwasnotrequiredfordifferentiationof CD4+ as induction of p21Cip1 (Frey etal., 1997, 2000; Abraham etal., andCD8+cellsoractivation-inducedIL-2production,PKCα−/− 1998;Slosbergetal.,1999;Black,2000;Detjenetal.,2000;Tibu- T cells showed severe defects in TCR-induced proliferation and dan etal., 2002; Clark etal., 2004; Matsumoto etal., 2006) and IFN-γproduction(Pfeifhoferetal.,2006).Theseeffectswerespe- p27Kip1(Freyetal.,1997,2000;Detjenetal.,2000;Tibudanetal., cifictoTcellssinceBcellproliferationwasunaffected(Pfeifhofer 2002). Inductionof p21Cip1 isalsoinvolvedintheabilityof this etal.,2006;Gruberetal.,2009). isozyme to delay S phase transit and induce G2/M arrest (Frey Interestingly, PKCα and PKCθ cooperate in regulation of T etal.,1997;Olivaetal.,2008).Ouranalysisinintestinalepithelial cell proliferation: while PKCα−/− and PKCθ−/− showed only cells indicated that downregulation of cyclin D1 represents one a mild activation defect in a graft-versus-host model, double of the earliest effects of PKCα signaling (Frey etal., 2004; Hizli PKCα/PKCθknockoutmicehadaseveredefectinalloreactiveT etal.,2006):PKCα-inducedlossofcyclinD1resultsfromtransla- cellproliferation(Gruberetal.,2009).Thiseffectisofdirectphysi- tionalandtranscriptionalinhibition,mediatedbyactivationofthe ologicalrelevancesincethedoubleknockoutmicehadsignificantly translationalrepressor4E-BP1anddownregulationoftheIdfam- improvedtransplantsurvivalcomparedwithsingleknockoutand ily of transcription factors, respectively (Clark etal.,2004; Hizli controlanimals(Gruberetal.,2009).Thesestudiesfurtherindi- etal., 2006; Guan etal., 2007; Hao etal., 2011). Suppression of cated that the cooperative effects of PKCα and PKCθ are due cyclinD1expressionbyPKCαcaninvolvedifferentintermediate to a combinatorial effect on NFAT activation. A role for this signalingevents,includingactivationoftheERK/MAPKpathway pathwayineffectsofPKCαisalsosupportedbythefactthatcon- (Clarketal.,2004;Hizlietal.,2006;Guanetal.,2007;Haoetal., stitutively active PKCα can activate NFAT (and AP1) in T cells 2011)andRORα-mediatedsuppressionofWnt/β-cateninsignal- (Genotetal.,1995). WhilethesestudiesindicatethatPKCαand ing(Birdetal.,1998). Consistentwitharoleof PKCαingrowth PKCθhaveoverlappingfunctionsinregulationofthealloimmune inhibition, activation/membrane association of this isozyme is responseandNFATactivation, theseisozymesclearlyhavenon- detectedinpost-mitoticcellsintheintestinalepithelium(Saxon redundantfunctionsinTcells. PKCα−/− miceshowadefectin etal.,1994;Freyetal.,2000)andepidermis(Tibudanetal.,2002) Th1-dependentIgG2a/bswitching,indicatingthatPKCαispartic- invivo.Furthermore,PKCαknockoutmiceshowincreasedprolif- ularlyimportantinTh1cells(Pfeifhoferetal.,2006),arolewhich erativeactivitywithinintestinalcrypts,andthetumorsuppressive contrastswiththemoreprominentfunctionofPKCθinTh2func- activity of this isozyme in the intestine has been linked directly tion (Salek-Ardakani etal.,2004). These non-redundant actions toitseffectsonthecellcyclemachinery(OsterandLeitges,2006; ofPKCαmayreflectitsrecentlyidentifiedroleinphosphorylation Pyszetal.,2009). ofAktonserine473inTcells(Yangetal.,2010). Therelevance Growth-stimulatory effects of PKCα have been reported in ofthisphosphorylationissupportedbythefindingthatAktlinks glioma cells, osteoblasts, chick embryo hepatocytes, hepatocel- mTORC2toTh1cellswhereasPKCθregulatesmTORC2-mediated lularcarcinomacells,andmyoblasts,amongothers(Black,2000, Th2differentiation(Leeetal.,2010). 2010). Proliferative effects of PKCα on the cell cycle machinery include increased levels of cyclin D1 and cdk4, and enhanced PKCβ cyclin/cdk2complexactivity(Zhouetal.,2002;Alisietal.,2004; Thetwomajorsplicevariantsof thePKCβgene(prkcb), PKCβI Wuetal.,2008;LovattandBijlmakers,2010).PKCαcanalsoelicita andPKCβII,havedifferentfunctions;however,thefactthatearly p21Cip1-dependentenhancementofproliferationasseeninglioma studies did not always differentiate between these forms, and cells(BessonandYong,2000).TheabilityofPKCαtopromotepro- knockdownandknockoutstrategiescanaffectbothisoforms,has liferation has been linked to signaling through the ERK/MAPK complicatedinterpretationoftheirindividualroles. pathway(Schonwasseretal.,1998;Shatosetal.,2008). Thecellcycle-specificeffectsofPKCβII,whichhavebeennoted ConsistentwiththecellcycleeffectsofPKCαdescribedabove, in both G1 and G2/M phases, appear to be largely stimulatory thisisozymeistargetedbyvariousphysiologicalstimulithatelicit (Black, 2010). Effects in G1 have been ascribed to the ability changesinproliferation(Birdetal.,1998;Black,2000,2010).Inter- of PKCβII to enhance transcription of cyclin D1 (Li and Wein- estingly,PKCαcanmediateopposingcellcycle-specificeffectsof stein,2006),promotepRbphosphorylation(Suzumaetal.,2002), these agents depending on context. For example, PKCα appears or to stimulate CAK activity through direct phosphorylation tomediatebothproliferative(Buitragoetal.,2003)andgrowth- (Acevedo-Duncan etal.,2002). Studies by Fields and colleagues inhibitory(Chenetal.,1999;Bikleetal.,2001)effectsofvitamin have established that phosphorylation of lamins contributes to D in different systems. This dichotomy has even been observed the effects of PKCβII on G2 → M transition (Goss etal.,1994; in cells of the same tissue origin: decreased PKCα expression Walkeretal.,1995;ThompsonandFields,1996;MurrayandFields, mediates all-trans retinoic acid (ATRA)-induced inhibition of 1998),whilestudiesbyNewtonandcolleagues(Chenetal.,2004) FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|6 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 6 — #6 BlackandBlack PKCandthecellcycle havealsodeterminedthatPKCβIIcanaffectMphasebyregula- levels of cyclin D1 in colon cancer cells (Cerda etal.,2006) and tionofcytokinesisthroughinteractionwithpericentrin.However, bovineairwaysmoothmusclecells(Pageetal.,2002). PKCδhas PKCβII can also inhibit proliferation and induce differentiation alsobeenshowntoinhibitmitosisinCHOcellsand3Y1murine insomecelltypes, withinductionof p21Cip1 andlossof Cdc25 fibroblasts(Watanabeetal.,1992;Kitamuraetal.,2003). potentiallymediatingthisactivity(Yoshidaetal.,2003;Cejasetal., Although the majority of studies have detected a growth- 2005). The PKCβI splice variant has been implicated in posi- inhibitory role for PKCδ, it can also act as a positive regulator tive and negative regulation of proliferation in fibroblasts and of the cell cycle (Kitamura etal.,2003; Cho etal.,2004; Jackson colon cancer cells, respectively (Housey etal., 1988; Choi etal., andFoster,2004;Czifraetal.,2006).PKCδcanenhanceG1→S 1990; Sauma etal., 1996); however, these findings relied exclu- transitthroughincreasedexpressionofcyclinD1,cyclinE,cyclin sively on overexpression and further work will be required to A,and/orcdk2(Kitamuraetal.,2003;Santiago-Walkeretal.,2005; determinethespecificinvolvementofthePKCβIisozymeinthese Grossonietal.,2007),destabilizationofp21Cip1(Santiago-Walker effects. etal., 2005; Walker etal., 2006), reduced nuclear localization of A number of studies indicate that PKCβI and/or PKCβII p21Cip1 (Sipeki etal., 2002; Ranta etal., 2011), and increased are involved in regulation of T cell proliferation. For example, E2F promoter activity (Nakaigawa etal., 1996). In many cases, antisense-mediatedknockdownhasimplicatedPKCβisozyme(s) these effects are mediated by the ERK/MAPK pathway (Jackson inIL-2signaling(Gomezetal.,1995).Furthermore,PKCβforms and Foster,2004; Grossoni etal.,2007). The opposing effects of are likely involved in cytoskeletal changes following T cell acti- PKCδ on cell cycle progression may be regulated by differential vation.PKCβIIlocalizestoacytoskeletalaggregatethatformsin phosphorylationonTyr155(Acsetal.,2000;Steinberg,2004). close proximity to the microtubule organizing center following T cells from PKCδ knockout mice are hyperproliferative and T cell activation (Black etal., 1988; Gregorio etal., 1992, 1994) produce more IL-2 cytokine upon stimulation in response to and PKCβI has been shown to associate with microtubules in allogeneic MHC. Thus, consistent with a predominant growth- T cells and to play a role in T cell polarization (Volkov etal., inhibitory role of PKCδ in other systems, this isozyme appears 2001).Sincecytoskeletalchangesappeartobeanimportantaspect tonegativelyregulateTcellproliferation,aneffectthathasbeen of T cell activation (Repasky and Black, 1996; Martín-Cófreces ascribedtoattenuationofTCR/CD3-mediatedsignaling(Gruber etal., 2008; Alarcón etal., 2011), these observations are likely etal.,2005a).AsimilarnegativeeffectofPKCδonproliferationis to be relevant to T cell signaling. This idea is supported by the alsoseeninBcells(Miyamotoetal.,2002). finding that antisense-mediated knockdown of PKCβI reduced nuclear translocation of NFAT in TCR/CD28-stimulated Jurkat PKCε T lymphoma cells (Dreikhausen etal., 2003). However, PKCβ PKCεgenerallymediatespro-proliferativeresponses,anditseffects isozymes do not have an essential role in T cell function since appeartobepredominantlyinG1/SratherthanG2/M(Graham PKCβ−/− mice have no appreciable T cell-related defects. This etal.,2000; Balciunaite and Kazlauskas,2001). The enzyme has contrasts with a critical role for PKCβ in B cell receptor sig- been implicated in mediating PDGF-induced G0/G1 → S pro- naling (Thuille etal., 2006) and in dendritic cell differentiation gression(BalciunaiteandKazlauskas,2001).LossofPKCεactivity (Farrenetal.,2010).Thus,anyroleofPKCβI/IIassociationwith inNSCLCcellsisassociatedwithinductionofp21Cip1,prolonged cytoskeletalelementsislikelytoberedundant.Inthisregard,itis G1 → S transition in response to serum, and reduced activa- noteworthythatTcellactivationleadstotranslocationof PKCα tion of cdk2 complexes (Bae etal., 2007), indicating that this and PKCθ to the same PKCβII-associated cytoskeletal aggregate isozyme suppresses p21Cip1 accumulation to facilitate cell cycle describedabove(J.D.BlackandE.A.Repasky,unpublisheddata; progression. PKCε can also induce cyclin D1 transcription and Wangetal.,1999). upregulate cyclin D1 and cyclin E protein (Soh and Weinstein, 2003; F. Hao, M. A Pysz, A. R. Black, and J. D. Black, unpub- PKCδ lished data). Although PKCε is generally downregulated during PKCδ broadly inhibits cell cycle progression in G1 in response differentiation(e.g.,Yangetal.,2003),theenzymepromotesadi- topharmacologicalagonistsandphysiologicalactivatorssuchas pogeniccommitmentandisessentialforterminaldifferentiation ATRA,inositolhexaphosphate(IP6),interferons,andtestosterone of 3T3-F442A preadipocytes (Webb etal., 2003). Its expression (Watanabeetal.,1992;Fukumotoetal.,1997;Ashtonetal.,1999; is also enhanced during myogenic differentiation, resulting in Uddin etal., 2002; Kambhampati etal., 2003; Nakagawa etal., upregulationofcyclinD3(Gaboardietal.,2010). 2005;Vuceniketal.,2005;Cerdaetal.,2006;Bowlesetal.,2007). TheabilityofconstitutivelyactivePKCεtoactivateNFATand Effects on G1 → S phase progression are mediated by direct or AP1inJurkatTlymphomacellspointstoaroleforthisisozymein indirecttargetingofcyclinD1,cyclinE,cyclinA,p21Cip1,and/or Tcellactivation(Genotetal.,1995).Antisense-mediatedknock- p27Kip1 (Fukumoto etal.,1997; Vrana etal.,1998; Ashton etal., downhasalsoimplicatedthisisozymeinIL-2signalinginTcells 1999; Nakagawa etal., 2005; Cerda etal., 2006; Afrasiabi etal., (Gomezetal.,1995).Furthermore,siRNA-mediatedknockdown 2008). CyclinD1expressionisdownregulatedbyPKCδincolon ofPKCεinCD4+Tcellsseverelyreducedproliferationinvitroand cancer cells (Cerda etal., 2006; Pysz etal., 2009), as well as in enhanced the growth-inhibitory effects of transforming growth PKCδ overexpressing vascular smooth muscle cells (Fukumoto factorbeta(TGF-β; Mirandolaetal.,2011). Thesefindingssup- etal., 1997), primary bovine airway smooth muscle cells (Page portapredominantlygrowth-stimulatoryroleofPKCεinTcells, etal.,2002),andNIH3T3cells(SohandWeinstein,2003).Consis- as seen in other systems (see above). However, PKCε−/− mice tentwiththesefindings,lossofPKCδactivityresultedinincreased shownodefectsinTcelldifferentiation,proliferationoractivation, www.frontiersin.org January2013|Volume3|Article423|7 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 7 — #7 BlackandBlack PKCandthecellcycle indicatingthatthefunctionsof thisisotypemaybeinlargepart inTcellactivation,whereitisrequiredforformationoftheCBM redundant,atleastinthemouse(Gruberetal.,2005b).Incontrast complex,whichplaysacentralroleinmediatingdownstreamsig- tothisfinding,analysisof Hashimotothyroiditispatientspoints nalingduringTcellactivation(Rawlingsetal.,2006).Inkeeping toapotentialclinicalrelevanceforproliferativeeffectsofPKCεin withthisrole,PKCθsignalingactivatesanumberoftranscription Tcells.ThesepatientshadsignificantlyhigherexpressionofPKCε factors that regulate T cell activation and proliferation, includ- intheirTcellscomparedwithhealthycontrols(Mirandolaetal., ingAp1,NF-κB,andNFAT(Pfeifhoferetal.,2003).Studiesusing 2011).Furthermore,whileHashimotothyroiditis-derivedTcells prkcq knockoutmicehavedeterminedthatPKCθplaysacentral haddiminishedTGF-βresponsescomparedwithhealthycontrols, roleinmediatingproliferativeresponsesduringTcellactivation. knockdownofPKCεinthesecellsrestorednormalresponsiveness PKCθ-deficientTcellslosetheabilitytoproliferateinresponseto toTGF-β(Mirandolaetal.,2011). TCR/CD28 activation in vitro (Sun etal., 2000; Pfeifhofer etal., 2003). A role for PKCθ in T cell expansion in vivo was also PKCη apparentfromthedefectiveproliferationseeninPKCθ−/− mice PKCηhasbeenassociatedwithpost-mitoticcellsinanumberof during allergic asthmatic reactions and in response to bacter- tissuesincludingsquamousepithelia(Kashiwagietal.,2002;Bre- ial infection (Salek-Ardakani etal., 2004; Sakowicz-Burkiewicz itkreutzetal.,2007),theepidermis(Breitkreutzetal.,2007),and etal.,2008). theintestinalepithelium(Osadaetal.,1993).Consistentwiththis As seen with PKC isozymes in other cell types, the action of localization, PKCη upregulated p21Cip1 and p27Kip1, decreased PKCθinproliferationappearstobehighlycontext-dependent.For cdk2kinaseactivity,andinducedgrowtharrestinNIH3T3cells example,whileaclearroleforthisisozymeinregulationofTh2cell andkeratinocytes(Livnehetal.,1996;Ishinoetal.,1998;Cabodi proliferationinvivoisseenintheallergicasthmaticresponse,this etal.,2000).However,thisisozymecanalsoenhanceproliferation wasnotthecaseforTh1cells(Salek-Ardakanietal.,2004). Fur- asseeninMCF-7breastcancercells,whereitupregulatedcyclinD thermore,PKCθ-deficiencydoesnotaffectTcellproliferationin andcyclinElevelsandpromotedaredistributionofp21Cip1 and responsetoviralinfection(Giannonietal.,2005)andcanmediate p27Kip1fromcdk2tocdk4complexes(Fimaetal.,2001). growth-inhibitoryeffectsofcytokinewithdrawal(Lietal.,2006b). PKCηisrecruitedtotheimmunesynapse,pointingtoinvolve- Notably,whilePKCθgenerallyplaysapositiveroleinproliferation mentofthisisozymeinTcellactivation(FuandGascoigne,2012). of effector T cells, it has the opposite effect in Treg cells, where ThisrolewasconfirmedbythefindingthatPKCη−/−Tcellshave itissequesteredfromtheimmunesynapseandpromotesgrowth adefectiveproliferativeresponsetoanti-CD3stimulationinvitro inhibition(Zanin-Zhorovetal.,2010). (Fuetal.,2011).Asomewhatmoresevereproliferativedefectwas A recent study has given insight into possible explanations alsoobservedinresponsetoantigenpresentationbothinvitroand for divergent functions of PKCθ (Kong etal., 2011). PKCδ and invivo(Fuetal.,2011).ConsistentwitharoleforPKCηinmediat- PKCθarehighlyhomologous;yet,asnotedabove,PKCδisgrowth ingTCRsignaling,activatedPKCη−/−Tcellsshowedareduction inhibitoryinTcells.Inkeepingwiththesedifferences,PKCδisnot incalciumfluxandNF-κBtranslocation(Fuetal.,2011).While targetedtotheimmunesynapse, disruptssignalosomeassembly theseeffectsarelargelyredundantwithPKCθ, specificeffectsof andcannotsubstituteforPKCθinTcellfunction. Thesediffer- PKCηwereseeninTcellhomeostaticproliferation,whichinvolves ences are due to a proline-rich motif in theV3 region of PKCθ self-antigenrecognitionandIL-7andIL-15signaling(FuandGas- thatmediatesindirectinteractionwithCD28throughLck.Muta- coigne,2012).Notably,nodefectinhomeostaticproliferationwas tionofthissequenceblockslocalizationofPKCθtotheimmune seeninPKCθ−/−mice,indicatingthatthiseffectislargelyspecific synapse;conversely,aPKCδmutantcontainingthissequencewas toPKCη,althoughdoubleknockoutsdidhaveasomewhatmore targeted to the immune synapse and could substitute for PKCθ severephenotype. in T cell signaling (Kong etal.,2011; Isakov andAltman,2012). Thesefindingspointtotheimportanceofalterationsinprotein– PKCθ protein interactions and localization in dictating the effects of PKCθ has been implicated as a positive regulator of prolifera- PKCsignaling,andofferamechanismforthedivergentrolesof tioninanumberof celltypesincludinggastrointestinalstromal PKC isozymes in different cell types and in different signaling tumorcellsandbreastcancercells,whereitrepressesexpressionof environments. p21Cip1and/orp27Kip1(BelguiseandSonenshein,2007;Ouetal., 2008),andincapillaryendothelialcells,whereitpromotesG2/M AtypicalPKCisozymes progression(Tangetal.,1997). WhileanalysisofthefunctionsofatypicalPKCsislessadvanced Alargebodyofevidencehasemergedtosupportacriticalrole thanthatofotherPKCisotypes,PKCιandPKCζgenerallyappear for PKCθ in T cell activation. The functions of this isozyme are topromotecellcycleprogression.Consistentwithacellcyclestim- the subject of several excellent reviews in this issue (e.g., Free- ulatory role of PKCζ, keratin-induced blockade of HaCaT cell leyandLong,2012; IsakovandAltman,2012;Wangetal.,2012) cycleprogressioninvolvedinhibitionofPKCζactivity,areduction and will only be discussed briefly here. While PKCθ is dispens- in cyclin D1 and cyclin E levels, and pRb hypophosphorylation ablefordifferentiationofCD4+andCD8+Tcells,itisintimately (Paramioetal.,2001).PKCζcanmediatetranscriptionalactivation involvedinTcellactivationandtransducespro-proliferativesig- of cyclinD1downstreamof Ras(Kampferetal.,2001), andcan nalsinmultiplepathways,includingthosetriggeredbytheTCR, inducephosphorylationandproteasome-dependentdegradation CD28, and TNF-α (Altman etal.,2000; So and Croft,2012). As ofp21Cip1downstreamofPI-3K(Scottetal.,2002).Theabilityof mentionedabove,PKCθisrecruitedtotheimmunesynapseearly PKCζtomodulatethesubcellulardistributionofp27Kip1 during FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|8 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 8 — #8 BlackandBlack PKCandthecellcycle cellcyclereentryof quiescentMCF7cellsisalsodownstreamof whichdirectlyimpactimmunefunction,involvebothredundant PI-3K(Castoriaetal.,2004).PKCζmayalsoenhancecdc25activ- and non-redundant functions of individual PKC family mem- itytopromoteG2/MtransitinA549lungepithelialcells,aneffect bers, and a high degree of cooperation between different PKC associated with changes in cdk2 activity (Lee etal., 2011; Kang isozymes is becoming apparent. As in other systems, the effects etal.,2012).ExcitingstudiesbyMurray,Fieldsandcolleagueshave ofPKCsignalingarehighlycontext-dependent,withthereliance recentlyidentifiedPKCιasanoncogenewhichisrequiredforthe onindividualisozymesdifferingbetweenTcellsubtypes. While transformedgrowthofvarioushumancancercelltypes(Fieldsand the majority of the characterized effects of PKC signaling in T Regala,2007;Murrayetal.,2011).Consistentwiththesefindings, cells have been pro-proliferative, negative effects are also seen: PKCιisupstreamof PKCζinRas-relatedupregulationof cyclin PKCδappearstoplayapredominantlyinhibitoryroleandPKCθ D1(Kampferetal.,2001).PKCιalsophosphorylatesandactivates canhavenegativeproliferativeeffectsdependentonthesignaling CAKinresponsetoPI-3Ksignalingingliomaandneuroblastoma environmentandcelltype. cells(Acevedo-Duncanetal.,2002; Pillaietal.,2011; Desaietal., AlthougheffectsofPKCsignalinghavebeennotedinallstages 2012)andmaytargetcyclinEinovariancancer(Ederetal.,2005). ofthecellcycle,thepredominantactionsofPKCisozymesarein IncontrasttoPKCαandPKCε,constitutivelyactivePKCζhad G1andG2phases.Similarly,whilePKCscanmodulatetheactivity no effect on AP1 and NFAT in Jurkat cells (Genot etal., 1995). ofmultiplecellcycleregulatorymolecules,consistentwitheffects However,workofGruberetal.(2008)pointstoaroleforatypi- inG1andG2,D-typecyclinsandCip/Kipcdkinhibitors(p21Cip1 calPKCsinPKCθ-mediatedpro-proliferativesignalinginTcells. and p27Kip1) are emerging as important targets of PKC control. ThesestudiesfoundthatPKCζphysicallyinteractswithPKCθina Inkeepingwiththeinvolvementoftheseproteinsinregulationof yeast two-hybrid screen and that PKCζ is a substrate for PKCθ. quiescence,accumulatingevidenceindicatesthatcontrollingcell This physical interaction likely occurs in vivo since PKCζ and cycleentryandexitisanimportantroleforPKCsignaling. The PKCι are constitutively localized in lipid rafts to which PKCθ abilityofPKCstopromoteG →G progressionhasbeennoted 0 1 is recruited following activation of primary T cells and Jurkat inseveralcelltypes(Chiuetal.,2002,2003;Santiago-Walkeretal., cells. Use of dominant negative mutant proteins further impli- 2005).PKCsignalinghasalsobeenshowntopromotecellcycleexit cated the atypical isozymes in NF-κB induction by PKCθ. In inanumberof systems,includingintestinalepithelialcells,ker- keepingwiththeircommonlocalizationandstructure,itappears atinocytes,PKC-overexpressingfibroblasts,andleukemiccelllines thatPKCιandPKCζcansubstituteforeachotherinmostTcell (Black,2000,2010).Studiesinleukemiacells(ZhangandChellap- functions. Nonetheless, PKCζ function appears to be particu- pan,1996;Vranaetal.,1998;Wangetal.,1998),non-transformed larly important for activation of Th2 cells (Martin etal., 2005): intestinalepithelialcells(Freyetal.,2000),pancreaticcancercells while PKCζ knockout did not result in proliferative or signal- (Detjenetal.,2000),andkeratinocytes(Tibudanetal.,2002)indi- ing defects in naïve T cells, it dramatically inhibited activation catethatPKCfamilymembersarecapableofactivatingacomplete of Th2 cells. This effect was reflected in disruption of STAT6, programofcellcyclewithdrawal,whichcanincludedownregula- NFAT,andNF-κBactivationfollowingstimulationwithanti-CD3. tionofcyclinD1,upregulationofp21Cip1andp27Kip1,alterations ThedramaticupregulationofPKCζnotedduringTh2celldiffer- in the expression and phosphorylation of the pocket proteins entiation may account for the inability of PKCι to compensate p107,pRb,andp130,andchangesinE2Fexpressionandcomplex forlossof PKCζinthesecells(Martinetal.,2005; Gruberetal., formation (Zhang and Chellappan,1996; Saunders etal.,1998). 2008).ThephysiologicalrelevanceofPKCζsignalinginTh2cellsis WhiletheabilityofPKCsignalingtopromoteexitfromquiescence seenintheimpairedallergicasthmaticresponseinPKCζ−/−mice followingTCR/CD28andpre-TCRengagementisestablished,fur- (Martinetal.,2005). therstudiesarerequiredtodefineitsroleinpromotingcellcycle exitduringTcelldevelopmentandtheestablishmentofquiescent Summaryanddiscussion memoryTcells. Fromtheabovediscussion,itisapparentthatPKCsignalingplays an important role in regulation of cell proliferation in a broad SPECIFICCELLCYCLETARGETSOFPKCSIGNALINGINTCELLS spectrumofcelltypesincludingTcells.PKCactivationcaneither Antigen-inducedproliferationisakeyaspectofbothTcelldiffer- promoteorinhibittransitthroughmultiplestagesofthecellcycle. entiationandclonalexpansion(KochandRadtke,2011).Thus,the The precise effect of PKCs on the cell cycle is highly context- mechanismsunderlyingPKCisozyme-specificeffectsonthecell dependent, and is influenced by the specific isozyme involved, cyclemachineryinTcellsareof criticalimportancetoimmune thetiminganddurationofPKCactivation,thecelltype,andthe function. As noted above, the cell cycle is tightly regulated by signalingenvironmenttowhichthecellisexposed;however,some coordinated actions of cyclins, cdks and ckis, which modulate themesarebeginningtoemerge.WithregardtoindividualPKC the activity of the retinoblastoma family and thus expression of familymembers,accumulatingevidenceindicatesthatPKCαcan E2F-dependent genes (Figure 1). Proliferative T cell signaling exertcontext-dependentinhibitoryorstimulatoryeffects. While affects multiple members of this control network. For example, PKCδcanhavepositiveeffectsoncellcycleprogression,itseffects proliferation induced by TCR/CD28 costimulation is associated are generally inhibitory. On the other hand, effects of PKCβII, with increased pRb phosphorylation by cyclin D2/3 and cyclin PKCε, and atypical PKCs appear to be mainly pro-proliferative, E, and enhanced transcription of E2F-dependent genes such as whilethoseofPKCηaregenerallyinhibitory. cyclinsEandA(Colombettietal.,2006).Analysisofmechanisms InTcells,multiplePKCisozymesmediateproliferativesignals underlying these changes has pointed to a particularly impor- associatedwithTCR/CD28engagement(Figure2).Theseeffects, tant role for cyclin D3, cdk6, and p27Kip1 in regulation of T www.frontiersin.org January2013|Volume3|Article423|9 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 9 — #9 BlackandBlack PKCandthecellcycle FIGURE2|ProliferativeeffectsofPKCisozymesinTcells.Positiveand ThesetargetsreflecttheimportanceofD-typecyclinsandCip/Kipckisas negativeproliferativeeffectsofindividualPKCisozymesareindicatedby targetsforPKCsignaling,asseeninothersystems(notethatinpre-Tcells, greenarrowsandredbarredlines,respectively.Proposedcellcycletargets cyclinD1appearstobethetargetforPKCθ).Thedashedlinesindicatethelack inthegrowth-inhibitoryandgrowth-stimulatoryeffectsofPKCθareshown. ofknowledgeofspecificcellcycletargetsforotherPKCisozymesinTcells. cell proliferation. For example, cyclin D3 and cdk6 knockout WhilePKCactivationmediatesTCRsignalingtoNF-κB,NFAT, miceshowdefectsinTcellproliferation,whereascdk4andcdk2 and Ap1, transcription factors that have been shown to have a knockoutmicedonot(Sicinskaetal.,2003;Huetal.,2009),and directroleinregulationofthecellcyclemachineryinTcells,the p27Kip1 nullTcellsshowreducedmitogenrequirementsandare function of specific PKCs in these effects remains largely unex- resistanttoanergy(Mohapatraetal.,2001;Rowelletal.,2005;Li plored.However,limitedinformationisemergingtoindicatethat, etal.,2006a). as in other cell types, D-type cyclins and Cip/Kip proteins are FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|10 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 10 — #10

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REVIEWARTICLE published:17January2013 doi:10.3389/fimmu.2012.00423 Protein kinase C signaling and cell cycle regulation AdrianR.Black*andJenniferD.Black EppleyInstituteforResearchinCancerandAlliedDiseases,UniversityofNebraskaMedicalCenter,Omaha,NE,USA Editedby: AlinkbetweenTcellproliferationandtheproteinkinaseC(PKC)familyofserine/threonine NoahIsakov,Ben-GurionUniversityof kinaseshasbeenrecognizedforabout30years.However,despitethewealthofinformation theNegev,Israel onPKC-mediatedcontrolof Tcellactivation,understandingoftheeffectsofPKCsonthe Reviewedby: cellcyclemachineryinthiscelltyperemainslimited.Studiesinothersystemshaverevealed ChristopherE.Rudd,Universityof importantcellcycle-specificeffectsofPKCsignalingthatcaneitherpositivelyornegatively Cambridge,UK CosimaT.Baldari,UniversityofSiena, impactproliferation.TheoutcomeofPKCactivationishighlycontext-dependent,withthe Italy precisecellcycletarget(s)andoveralleffectsdeterminedbythespecificisozymeinvolved, *Correspondence: the timing of PKC activation, the cell type, and the signaling environment. Although AdrianR.Black,EppleyInstitutefor PKCscanregulateallstagesofthecellcycle,theyappeartopredominantlyaffectG0/G1 ResearchinCancerandAllied and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, Diseases,UniversityofNebraska MedicalCenter,985950Nebraska cyclin-dependent kinases (cdks), cdk inhibitors and cdc25 phosphatases; however, MedicalCenter,Omaha,NE evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of 68198-5950,USA. PKC-regulatedcellcycle-specificeffects.SeveralPKCisozymescantargetCip/Kipproteins e-mail:[email protected] tocontrolG0/G1→Sand/orG2→Mtransit,whileeffectsonD-typecyclinsregulateentry into and progression through G1. Analysis of PKC signaling inT cells has largely focused on its roles inT cell activation; thus, observed cell cycle effects are mainly positive. A prominentroleisemergingforPKCθ,withnon-redundantfunctionsofotherisozymesalso described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in thesecells.Asinothercelltypes,context-dependenteffectsofindividualisozymeshave beennotedinTcells,andCip/KipcdkinhibitorsandD-typecyclinsappeartobemajorPKC targets.Futurestudiesareanticipatedtotakeadvantageofthesimilaritiesbetweenthese varioussystemstoenhanceunderstandingofPKC-mediatedcellcycleregulationinTcells. Keywords:proteinkinaseC,signaltransduction,Tcellactivation,cellcycle,cyclin,cyclin-dependentkinase,cyclin- dependentkinaseinhibitor AnassociationbetweenproteinkinaseC(PKC)signalingandTcell protein kinase C) superfamily of protein kinases (Nieto, 2007; proliferationhasbeenrecognizedforalmostthreedecades.Over Matsuoka etal., 2009; Black, 2010; Rosse etal., 2010; Ryu etal., 30 years ago, it was determined that a combination of phorbol 2010).PKCisozymesarelipid-dependentkinases(requiringphos- estersandelevatedintracellularcalciumpotentlyinducesprolif- phatidylserine binding for activity) and are grouped into three eration of cells of the T cell lineage (e.g., Whitfield etal., 1973; subfamilies based on their structure and requirement for addi- Lyalletal.,1980;Sutherlandetal.,1981).Shortlythereafter,itwas tionalco-factorsandcalcium.Physiologicalactivationofclassical determined that PKC, then recognized as a calcium-dependent PKCs(PKCα,PKCβIandPKCβII,whicharesplicevariantsofthe enzyme,representedthemajorcellularreceptorforphorbolesters prkcbgene,andPKCγ)isinducedbythelipidsecondmessenger (Castagnaetal.,1982)anditwasnotlongbeforetheconnection diacylglycerol (DAG) and calcium, while activation of the novel between these phenomena was made (Isakov etal.,1986; Isakov PKCs (PKCδ, PKCε, PKCθ, and PKCη) requires only DAG. In and Altman,1987). Since then, a central role for PKC in T cell contrast, the atypical PKCs (PKCζ and PKCι/λ) are not depen- receptor(TCR)signalinghasbeenfirmlyestablished.Despitethe dentonlipidsecondmessengersorcalciumforactivity. Instead, longassociationofPKCwithTcellproliferation,detailsonhow theirfunctionisregulatedbyprotein–proteininteractionsmedi- PKCsignalinginteractswiththecellcyclemachineryinthiscell atedbyaPB1domainaswellasacarboxyl-terminalPDZligand typeareonlybeginningtoemerge.Inthisregard,ourknowledge motif. Engagement of growth factor or cytokine receptors leads in T cells lags behind that in other cell types, including other toactivationof phospholipaseC(PLC)βorPLCγ,whichcleave hematopoietic lineages. In this review, we outline our current phosphatidylinositol 4,5-bisphosphate to generate DAG and the understanding of the proliferative effects of PKC signaling in T soluble second messenger inositol trisphosphate (which induces cells within the context of the broader knowledge that has been release of calcium from intracellular stores). The production of gainedinothersystems. DAGrecruitsclassicalandnovelPKCstotheplasmamembrane, wheretheyundergoaconformationalchangeresultinginfullacti- THEPROTEINKINASECFAMILY vation.UnlikeotherAGCkinases,suchasAkt,activationofPKCs Protein kinase C represents a family of serine/threonine kinases doesnotrequireacutephosphorylationoftheenzyme:phospho- thatbelongtotheAGC(cAMP-dependent,cGMP-dependent,and rylations necessary for catalytic competence occur shortly after www.frontiersin.org January2013|Volume3|Article423|1 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 1 — #1 BlackandBlack PKCandthecellcycle synthesisandtheenzymeisconstitutivelyphosphorylatedatthese apotentinhibitorofotherkinasessuchasPRAKandMAPKAP- sites(Matsuokaetal.,2009;Rosseetal.,2010).Asaresult,changes K2 (Soltoff, 2007); thus, any effects of this inhibitor cannot be in phosphorylation do not provide an indication of PKC activ- ascribedtodirectinhibitionofPKCθ. ity; rather signaling-induced translocation of the enzyme to the Despitetheselimitations,ourknowledgeoftherolesofindivid- membrane/particulatefractionrepresentsthemostreliablemeans ualPKCsisemerging.Ofnote,inadditiontotheproliferative/cell ofmonitoringkinaseactivation.Reversalofsignalingcanoccurby cycle effects which are the subject of this review, PKC isozymes metabolismofDAGbyDAGkinaseandreleaseofPKCsfromthe havebeenfoundtoregulatemultiplecellularprocessesof direct membrane,aswellasbyagonist-inducedenzymedegradationor relevance to T cell development and function, including dif- removalofprimingphosphorylationwithsubsequentrapiddegra- ferentiation, migration, survival, apoptosis, endocytosis, and dation(LeontievaandBlack,2004;Newton,2010).Inadditionto secretion/exocytosis(Reyland,2009;Rosseetal.,2010). activation by growth factor signaling, classical and novel PKCs can be stimulated by a number of pharmacological agents that THEMAMMALIANCELLCYCLE mimictheeffectsofDAG,suchasphorbolestersandmacrocyclic Several excellent reviews have been written on the regulation of lactonebryostatins.However,incontrasttoDAG,theseagonists, thecellcycle(SherrandRoberts,2004; Cobrinik,2005; Malum- whichincludephorbol12-myristate13-acetate[PMA;alsoknown bresandBarbacid,2005;DuandPogoriler,2006;Satyanarayana as12-O-tetradecanoylphorbol-13-acetate(TPA)],phorbol12,13- andKaldis,2009)andonlyabriefdescriptionwillbegivenhere. dibutyrate(PDBu),andbryostatin1,arenotrapidlymetabolized Thecellcyclehasbeenclassicallydividedintofourphases,G1(or andthusgiveamoresustainedPKCactivation. Gap1inwhichcellsprepareforDNAsynthesis),Sphase(inwhich Despite limitations related to their lack of specificity for DNAissynthesized),G2(inwhichcellspreparefordivision)and individual PKC isozymes, their ability to promote PKC down- mitosis(orMphase,inwhichsisterchromatidsareseparatedand regulation,andtheexistenceofadditionaltargetsfortheseagents thecelldivides; Figure1). Transitthroughthecellcycleisregu- (Griner and Kazanietz, 2007), use of pharmacological agonists latedbyfourmajorclassesofcyclinswhoseexpressionisstrictly and membrane permeant DAG analogs has provided significant controlledandlimitedtoparticularcellcyclephases.Cyclinsare insightintothedownstreameffectsof PKCactivation. However, theregulatorysubunitsforcyclin-dependentkinases(cdks),whose acompleteunderstandingofPKCsignalingwillrequiredefining activityisabsolutelydependentonassociationwithspecificcyclin thespecificfunction(s)ofindividualPKCisozymes,andprogress partners. Entry of quiescent cells into the cell cycle and transit towardthisgoalhasprovedtechnicallydifficult.Understandingof throughearlyG1isregulatedbyD-typecyclins, whichcomplex thefunctionsofatypicalPKCs,PKCζ,andPKCι,lagsbehindthat withcdk4andcdk6(Musgroveetal.,2011).TherearethreeD-type ofothermembersofthePKCfamily,perhapslargelyduetotheir cyclins,D1,D2,andD3,whichareexpressedtovaryingdegreesin insensitivity to pharmacological activators (e.g., phorbol esters differenttissues;cyclinsD2andD3appeartobethemajorplayers andbryostatins)andsyntheticDAGs.Intheabsenceofisozyme- inTcells.TransitthroughlateG1andprogressionintoSphaseis specificpharmacologicalPKCagonistsandinhibitors,earlystudies regulatedbycyclinEcomplexedwithcdk2(HwangandClurman, reliedonoverexpressionstrategiestodeciphertherolesof indi- 2005; MalumbresandBarbacid,2005). Sphasetransitandearly vidual isozymes, which can result in non-physiological levels of G2areregulatedbycyclinA/cdk2andcyclinA/cdk1complexes, expression, activity, and regulation. RNA interference technol- whereascyclinB,complexedwithcdk1,regulatesprogressioninto ogyandgeneticallyalteredmicearehelpingtocircumventthese Mphase(MalumbresandBarbacid,2005;SanchezandDynlacht, problems,butarenotwithoutdrawbacksoftheirown.Potential 2005).Inadditiontobeingregulatedbycyclinbinding,theactiv- limitationsincludetheneedforahighlevelofsilencingtosuffi- ityofcdksisunderthecontrolofCip/KipandInk4cdkinhibitor cientlydepleteenzymeactivity(e.g.,>80%,Cameronetal.,2008; proteins(ckis;SherrandRoberts,1995).MembersoftheCip/Kip M.A.Pysz,A.R.Black,andJ.D.Black,unpublishedresults),and family,includingp21Cip1,p27Kip1,andp57Kip2,haveadualactivity thefactthatknockdownofonePKCisozymecanaffectaccumu- incellcycleregulation.Theynegativelyregulatecellcycleprogres- lation of other members of the family (M.A. Pysz, A. R. Black, sion by binding to cyclin/cdk2 and cyclin/cdk1 complexes and andJ.D.Black,unpublisheddata).Overlappingrolesofdifferent inhibitingtheirenzymaticactivity.Conversely,theseproteinscan isozymesmeansthatmultiplecrossesof transgenicmicemaybe promote progression by enhancing the association of cyclin D neededtoobservephenotypes. withcdk4andcdk6withoutinhibitingtheactivityofthesecom- Anadditionalsourceof confusionregardingthefunctionsof plexes (Sherr and Roberts,1999). The Ink4 ckis, which include individual PKC isozymes is the fact that many so-called PKC p15Ink4b, p16Ink4a, p18Ink4c, and p19Ink4d, block the activity of inhibitors are of questionable specificity (Griner and Kazanietz, cdk4andcdk6bypreventingtheirassociationwithcyclinD.Cdk 2007; Soltoff,2007). As an example of particular relevance to T activityisalsoregulatedbyphosphorylation:positivephosphory- cellactivation,specialcautionisneededwhenconsideringstud- lationismediatedbycdkactivatingkinase(CAKorcdk7/cyclin iesthathaveusedrottlerintoinfereffectsofsignalingfromPKCθ. H; Fisher and Morgan, 1994), while negative phosphorylation Whilethisagentwasoriginallyconsideredtobeaspecificinhibitor involvesthekinasesWee1andMyt1.Removalofinhibitoryphos- ofnovelPKCs,recentstudieshavedemonstratedthatitdoesnot phorylation,bye.g.,Cdc25phosphatases,isnecessaryforfullcdk inhibitPKCδ(Soltoff,2007).Inkeepingwiththisfinding,theIC activity. 50 forPKCθinhibitionbyrottlerininthepresenceof 100μMATP Whiletherearemultiplecheckpointsthatallowcellstoundergo is>300μM(Villalbaetal.,1999),aconcentrationfarinexcessof cellcyclearrestinresponsetovariousstresses,themostrelevant thatusedinstudiesonitscellulareffects.Incontrast,rottlerinis to normal tissue homeostasis and differentiation is that which FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|2 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 2 — #2 BlackandBlack PKCandthecellcycle directs entry and exit from the cell cycle in G1 (Prasad etal., repression, allowing for transcription of E2F-dependent genes, 1994;Liuetal.,2012).TheexpressionofD-typecyclinsisacutely oneofwhichiscyclinE.CyclinE/cdk2thencompletesphospho- regulated by mitogenic signals. As such, these proteins are the rylationofpocketproteins,leadingtotheirreleasefromE2Fand mainsensorsforthegrowthenvironmentofthecell,andareinti- robust transcription of growth-related genes. At early stages of matelyinvolvedinregulationof theentryof quiescentcellsinto G1, cells require mitogenic signals to support cyclin expression the cell cycle. Major targets for cyclin D/cdk complexes include andcdkactivity; however, oncesufficientlevelsof cyclinEhave the retinoblastoma protein (pRb) and related pocket proteins, accumulatedtomaintainitsownexpression,cellshavepassedthe p107andp130(Cobrinik,2005).Inthehypophosphorylatedstate, so-called“restrictionpoint”andareabletoproceedthroughtothe pocket proteins bind to E2F transcription factors on the pro- nextcellcyclewithoutfurthermitogenicinput.Inthefaceofloss motersofgrowth-relatedgenes,wheretheyactastranscriptional of mitogenicsignalspriortotherestrictionpointorof negative repressors and actively block expression of genes necessary for growth signals, cell cycle progression is halted and cells eventu- DNA replication (Trimarchi and Lees, 2002; Du and Pogoriler, ally withdraw into G0 phase and quiescence (Grana etal.,1998; 2006;Figure1).Phosphorylationofpocketproteinsrelievesthis ClassonandDyson,2001). FIGURE1|Continued www.frontiersin.org January2013|Volume3|Article423|3 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 3 — #3 BlackandBlack PKCandthecellcycle FIGURE1|Thecellcycle.Thecellcycleconsistsoffourphases,G1,S,G2, (largeredarrow),whichcommitscellstotheproliferativecycle.Ifconditions andM.InearlyG1,hypophosphorylatedpRbbindstheE2Ftranscription arenotoptimaltosignalthistransition,cellsexitthecycleandenterG0or factor,andrecruitshistonedeacetylase(HDAC)andotherfactorstoactively quiescence,areversiblenon-replicativestate.OncecellsenterSphase, represstranscriptionofE2F-regulatedgenesimportantfortransitionintoS cyclinE/cdk2activityisinhibitedbyproteasomaldegradationofcyclinEinthe phaseandDNAreplication(e.g.,PCNA,topoisomeraseI,c-Myc,cyclinE, cytoplasm.Continuedinactivation/hyperphosphorylationofpRballowsthe Cdc25c).ProgressionthroughearlyG1isdependentongrowthfactors,which transcriptionofcyclinAandcyclinB,requiredforsubsequentphasesofthe promoteexpressionofD-typecyclins.FormationofcyclinD/cdk4and cellcycle.CyclinA/cdk2complexesphosphorylateanumberofproteinsto cyclinD/cdk6complexes,whichisfacilitatedbyCip/Kipckis,leadsto facilitateSphasecompletionandtransitintoG2/M.CyclinBisactively phosphorylationofpRbatasubsetofavailablesitesandreleaseofHDAC synthesizedduringG2andassociateswithcdk1totriggermitosis.Cdk1is andotherinhibitoryfactors,relievingrepressionofE2Fandpromoting maintainedinaninactivestatebythekinasesWee1andMyt1.Ascells upregulationofcyclinE.CyclinE/cdk2complexes,relievedfromrepressionby approachMphase,thephosphatasecdc25isactivatedtoremoveinhibitory Cip/KipckisbysequestrationoftheseinhibitorymoleculesincyclinD/cdk phosphatesonTyr14andThr15,drivingthecellsintomitosis.Acheckpointin complexes,completepocketproteinphosphorylationinmidtolateG1, lateG2(largegreenarrow)preventscellsfromenteringMphaseifthe enablingawaveofE2F-dependenttranscriptionalactivityessentialforS genomeisdamaged.ThisDNAdamagecheckpointensuresthatcellsdonot progression.Together,theseeventsdrivecellsthroughtherestrictionpoint initiatemitosisuntiltheyhaverepaireddamagedDNAafterreplication. FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|4 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 4 — #4 BlackandBlack PKCandthecellcycle PKCSIGNALINGANDTCELLPROLIFERATION cellsthatarisefromactivationareeventuallyclearedfromthecir- TCELLDEVELOPMENTANDTCRSIGNALING culation,asmallnumberdevelopintomemoryTcellswhichare + T lymphocytes arise from bone marrow-derived CD34 stem primedforactivationuponsubsequentantigenexposure. cells, which seed the thymus and undergo multistage differenti- Antigen-induced proliferation is a key aspect of both T cell ationtobecomematurecirculatingcells(forreferences,seeKoch differentiation and clonal expansion (Koch and Radtke, 2011). and Radtke, 2011). An early event in this process involves VDJ Thus, mechanisms underlying regulation of the T cell cycle recombination of the TCR-β chain which then complexes with machinery are of critical importance to immune function. As pre-Tα to form the pre-TCR. Signaling from the pre-TCR leads the signaling pathways involved in T cell activation are being to proliferation of pre-T cells and rearrangement of the TCR-α deciphered, increasing evidence is pointing to the importance chain, which combines with the β chain and CD3 to form the of the PKC family in mediating proliferative responses in these TCR.Furtherdifferentiation,accompaniedbynegativeandposi- cells. The following section outlines our current understanding tiveselection,eventuallyleadstothedevelopmentofmaturenaïve of the role of individual PKC isozymes in regulating prolifera- + + Tcells, includingCD4 helperT(Th), CD8 cytotoxicT(Tc), tioninTcellswithinthecontextofknowledgegainedfromother andregulatoryT(Treg)cells.Thesenaïvecellsexitthethymusand systems. remain dormant as they circulate through secondary lymphoid organs until activated by antigen. These organs, which include PKCsANDTHECELLCYCLE the spleen, lymph nodes, and Peyer’s patches, transiently house AsourknowledgeoftheproliferativerolesofthePKCfamilyhas naïve T cells and are the first line of defense against pathogens developed,ithasbecomeincreasinglyapparentthattheeffectsof that traverse the skin or the epithelial lining of the respiratory, thesemoleculesarehighlycontext-dependent.ThefactthatPKCs gastrointestinal,andurogenitaltracts. areactivatedbytumorpromotingphorbolestersandaredown- ActivationofTcellsrequiresinteractionoftheTCRwithmajor stream of growth factor receptors initially led to the idea that histocompatibility complex (MHC) bound antigen on antigen they transduce positive mitogenic signals (Castagna etal., 1982; presentingcells(APCs),suchasdendriticcells,macrophages,and Kikkawa etal., 1983; Leach etal., 1983). Although a number of Bcells(forreferences,seeMarslandandKopf,2008;Smith-Garvin early studies supported this idea (Dicker and Rozengurt, 1978; etal.,2009;Fooksmanetal.,2010;DustinandDepoil,2011).The Rozengurt,1986; Takuwaetal.,1988), itsoonbecameclearthat interfacebetweentheTcellandAPCismarkedbytheformationof PKCscannegativelyandpositivelyregulatecellcycleprogression. astructure,termedtheimmunesynapseorsupramolecularacti- Indeed, regulation of proliferation by the PKC enzyme system vationcluster(SMAC),whichservestoregulateTcellsignaling. exhibitsahighdegreeof complexity,witheffectsinvolvingmul- ProductiveactivationofTcellsrequirestwosignals.Thefirstsig- tiplecellcycleregulatorymolecules,includingcyclins,cdks,and nalisprovidedbytheMHC-boundTCR,whilethesecondsignalis ckis,andimpactingvariousstagesofthecellcycle(Black,2010). providedbyco-stimulatorymoleculessuchasCD28(whichbinds Furthermore, individual isozymes can have opposing effects on toB7proteinsontheAPC).Additionally,cytokinessuchasIL-12 cell cycle progression in different cell types and even within the andtumornecrosisfactoralpha(TNF-α)canprovideathirdsig- samecelltype,dependingonthesignalingenvironment.Asingle nalthatregulatestheresponsetoTcellactivation.Anumberof isozymecantargetdifferentcellcyclemoleculesindifferentcell experimentalmanipulationscanactivateTcellsintheabsenceof types, can have opposite effects on a specific cell cycle target in APCinteraction;theseincludecrosslinkingoftheTCRandCD28 different systems, and can modulate the same target to produce with insoluble antibodies and combined treatment of cells with divergentcellcycleresponses(forreview,seeBlack,2010).Thus, phorbolesterandcalciumionophore. to gain a true understanding of the role of PKCs in regulation Tcellreceptorco-activationleadstotheengagementof mul- of proliferationinanygivensystem,itisimportanttostudythe tiple downstream signaling pathways including those involving mechanismsbywhichindividualisozymesaffectspecificcellcycle phosphatidylinositol 3-kinase (PI-3K), tyrosine kinases such as moleculesinthatsystem. Lck, and PLCγ (for references, see Altman etal., 2000; Mars- TlymphocytesexpressallmembersofthePKCfamilywiththe landandKopf,2008; Smith-Garvinetal.,2009; Fooksmanetal., exceptionofPKCγ(Koretzkyetal.,1989;Chenetal.,1994;Thuille 2010; Dustin and Depoil, 2011). Activation of PLCγ results etal.,2006). A role for PKC isozymes in cell cycle regulation in in production of DAG, which recruits PKCθ to the immune CD3+Tlymphocyteswassuggestedbytheearlyrecognitionthat synapse where it interacts indirectly with CD28 through bind- phorbolesters,inconjunctionwithcalciumionophore,arepotent ing to Lck (Kong etal., 2011; Isakov and Altman, 2012). PKCθ mitogensforthesecells(Altmanetal.,1990).Whilestudieshave then phosphorylates CARMA1, leading to the assembly of the concentratedlargelyontheroleof PKCθinmediatingsignaling CARMA1–BCL10–MALT1(CBM)signalosome.PLCγ-generated fromtheimmunesynapse,aroleforotherPKCisozymesisemerg- inositol trisphosphate releases calcium from intracellular stores. ing.Notably,differentisozymescanhavepro-proliferativeand/or The combined action of downstream TCR signaling eventually anti-proliferative functions, arguing that, as in other cell types, leads to activation of NF-κB, AP1, and nuclear factor of acti- PKCsignalingcanregulateentryintothecellcycle,transitthrough vated T cells (NFAT) transcription factors (Marsland and Kopf, thevariouscellcyclephases,aswellascellcyclewithdrawalinT 2008; Smith-Garvin etal., 2009; Fooksman etal., 2010; Dustin cells.Thefollowingsectionsdiscusscurrentunderstandingofthe and Depoil, 2011). Together, these events promote functional growthregulatoryfunctionsof individualPKCfamilymembers, activationof Tcellswhichismarkedbycellproliferation/clonal followed by a summary of the limited information available on expansion and cytokine secretion. While the majority of the T cellcycle-specificeffectsoftheseisozymesinTcells. www.frontiersin.org January2013|Volume3|Article423|5 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 5 — #5 BlackandBlack PKCandthecellcycle PROLIFERATIVEEFFECTSOFINDIVIDUALPKCFAMILYMEMBERS G1 → S progression in SKRB-3 breast cancer cells (Nakagawa PKCα etal.,2003),whereasPKCαisrequiredforATRA-inducedgrowth Useofselectivepharmacologicalinhibitors,antisensetechnology, arrestinT-47Dbreastcancercells(Choetal.,1997). orsiRNAhasidentifiedananti-proliferativeanddifferentiation- AroleforPKCαinpositiveregulationofproliferationinTcells inducing role of PKCα in multiple cell types, e.g., intestinal wassuggestedbythefindingthat,unlikewild-typecells,Tlym- epithelial cells, keratinocytes, mammary epithelial cells, and phocytes from transgenic mice overexpressing PKCα were able melanoma cells (Black,2000,2010). Anti-proliferative effects of toproliferateinresponsetosolubleanti-CD3antibody(Iwamoto PKCαaffectingG1→Stransitincludedownregulationofcyclin etal.,1992).ThisrolewasconfirmedbystudiesofPKCαknockout D1(Detjenetal.,2000;Hizlietal.,2006;Guanetal.,2007),aswell mice: whilePKCαwasnotrequiredfordifferentiationof CD4+ as induction of p21Cip1 (Frey etal., 1997, 2000; Abraham etal., andCD8+cellsoractivation-inducedIL-2production,PKCα−/− 1998;Slosbergetal.,1999;Black,2000;Detjenetal.,2000;Tibu- T cells showed severe defects in TCR-induced proliferation and dan etal., 2002; Clark etal., 2004; Matsumoto etal., 2006) and IFN-γproduction(Pfeifhoferetal.,2006).Theseeffectswerespe- p27Kip1(Freyetal.,1997,2000;Detjenetal.,2000;Tibudanetal., cifictoTcellssinceBcellproliferationwasunaffected(Pfeifhofer 2002). Inductionof p21Cip1 isalsoinvolvedintheabilityof this etal.,2006;Gruberetal.,2009). isozyme to delay S phase transit and induce G2/M arrest (Frey Interestingly, PKCα and PKCθ cooperate in regulation of T etal.,1997;Olivaetal.,2008).Ouranalysisinintestinalepithelial cell proliferation: while PKCα−/− and PKCθ−/− showed only cells indicated that downregulation of cyclin D1 represents one a mild activation defect in a graft-versus-host model, double of the earliest effects of PKCα signaling (Frey etal., 2004; Hizli PKCα/PKCθknockoutmicehadaseveredefectinalloreactiveT etal.,2006):PKCα-inducedlossofcyclinD1resultsfromtransla- cellproliferation(Gruberetal.,2009).Thiseffectisofdirectphysi- tionalandtranscriptionalinhibition,mediatedbyactivationofthe ologicalrelevancesincethedoubleknockoutmicehadsignificantly translationalrepressor4E-BP1anddownregulationoftheIdfam- improvedtransplantsurvivalcomparedwithsingleknockoutand ily of transcription factors, respectively (Clark etal.,2004; Hizli controlanimals(Gruberetal.,2009).Thesestudiesfurtherindi- etal., 2006; Guan etal., 2007; Hao etal., 2011). Suppression of cated that the cooperative effects of PKCα and PKCθ are due cyclinD1expressionbyPKCαcaninvolvedifferentintermediate to a combinatorial effect on NFAT activation. A role for this signalingevents,includingactivationoftheERK/MAPKpathway pathwayineffectsofPKCαisalsosupportedbythefactthatcon- (Clarketal.,2004;Hizlietal.,2006;Guanetal.,2007;Haoetal., stitutively active PKCα can activate NFAT (and AP1) in T cells 2011)andRORα-mediatedsuppressionofWnt/β-cateninsignal- (Genotetal.,1995). WhilethesestudiesindicatethatPKCαand ing(Birdetal.,1998). Consistentwitharoleof PKCαingrowth PKCθhaveoverlappingfunctionsinregulationofthealloimmune inhibition, activation/membrane association of this isozyme is responseandNFATactivation, theseisozymesclearlyhavenon- detectedinpost-mitoticcellsintheintestinalepithelium(Saxon redundantfunctionsinTcells. PKCα−/− miceshowadefectin etal.,1994;Freyetal.,2000)andepidermis(Tibudanetal.,2002) Th1-dependentIgG2a/bswitching,indicatingthatPKCαispartic- invivo.Furthermore,PKCαknockoutmiceshowincreasedprolif- ularlyimportantinTh1cells(Pfeifhoferetal.,2006),arolewhich erativeactivitywithinintestinalcrypts,andthetumorsuppressive contrastswiththemoreprominentfunctionofPKCθinTh2func- activity of this isozyme in the intestine has been linked directly tion (Salek-Ardakani etal.,2004). These non-redundant actions toitseffectsonthecellcyclemachinery(OsterandLeitges,2006; ofPKCαmayreflectitsrecentlyidentifiedroleinphosphorylation Pyszetal.,2009). ofAktonserine473inTcells(Yangetal.,2010). Therelevance Growth-stimulatory effects of PKCα have been reported in ofthisphosphorylationissupportedbythefindingthatAktlinks glioma cells, osteoblasts, chick embryo hepatocytes, hepatocel- mTORC2toTh1cellswhereasPKCθregulatesmTORC2-mediated lularcarcinomacells,andmyoblasts,amongothers(Black,2000, Th2differentiation(Leeetal.,2010). 2010). Proliferative effects of PKCα on the cell cycle machinery include increased levels of cyclin D1 and cdk4, and enhanced PKCβ cyclin/cdk2complexactivity(Zhouetal.,2002;Alisietal.,2004; Thetwomajorsplicevariantsof thePKCβgene(prkcb), PKCβI Wuetal.,2008;LovattandBijlmakers,2010).PKCαcanalsoelicita andPKCβII,havedifferentfunctions;however,thefactthatearly p21Cip1-dependentenhancementofproliferationasseeninglioma studies did not always differentiate between these forms, and cells(BessonandYong,2000).TheabilityofPKCαtopromotepro- knockdownandknockoutstrategiescanaffectbothisoforms,has liferation has been linked to signaling through the ERK/MAPK complicatedinterpretationoftheirindividualroles. pathway(Schonwasseretal.,1998;Shatosetal.,2008). Thecellcycle-specificeffectsofPKCβII,whichhavebeennoted ConsistentwiththecellcycleeffectsofPKCαdescribedabove, in both G1 and G2/M phases, appear to be largely stimulatory thisisozymeistargetedbyvariousphysiologicalstimulithatelicit (Black, 2010). Effects in G1 have been ascribed to the ability changesinproliferation(Birdetal.,1998;Black,2000,2010).Inter- of PKCβII to enhance transcription of cyclin D1 (Li and Wein- estingly,PKCαcanmediateopposingcellcycle-specificeffectsof stein,2006),promotepRbphosphorylation(Suzumaetal.,2002), these agents depending on context. For example, PKCα appears or to stimulate CAK activity through direct phosphorylation tomediatebothproliferative(Buitragoetal.,2003)andgrowth- (Acevedo-Duncan etal.,2002). Studies by Fields and colleagues inhibitory(Chenetal.,1999;Bikleetal.,2001)effectsofvitamin have established that phosphorylation of lamins contributes to D in different systems. This dichotomy has even been observed the effects of PKCβII on G2 → M transition (Goss etal.,1994; in cells of the same tissue origin: decreased PKCα expression Walkeretal.,1995;ThompsonandFields,1996;MurrayandFields, mediates all-trans retinoic acid (ATRA)-induced inhibition of 1998),whilestudiesbyNewtonandcolleagues(Chenetal.,2004) FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|6 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 6 — #6 BlackandBlack PKCandthecellcycle havealsodeterminedthatPKCβIIcanaffectMphasebyregula- levels of cyclin D1 in colon cancer cells (Cerda etal.,2006) and tionofcytokinesisthroughinteractionwithpericentrin.However, bovineairwaysmoothmusclecells(Pageetal.,2002). PKCδhas PKCβII can also inhibit proliferation and induce differentiation alsobeenshowntoinhibitmitosisinCHOcellsand3Y1murine insomecelltypes, withinductionof p21Cip1 andlossof Cdc25 fibroblasts(Watanabeetal.,1992;Kitamuraetal.,2003). potentiallymediatingthisactivity(Yoshidaetal.,2003;Cejasetal., Although the majority of studies have detected a growth- 2005). The PKCβI splice variant has been implicated in posi- inhibitory role for PKCδ, it can also act as a positive regulator tive and negative regulation of proliferation in fibroblasts and of the cell cycle (Kitamura etal.,2003; Cho etal.,2004; Jackson colon cancer cells, respectively (Housey etal., 1988; Choi etal., andFoster,2004;Czifraetal.,2006).PKCδcanenhanceG1→S 1990; Sauma etal., 1996); however, these findings relied exclu- transitthroughincreasedexpressionofcyclinD1,cyclinE,cyclin sively on overexpression and further work will be required to A,and/orcdk2(Kitamuraetal.,2003;Santiago-Walkeretal.,2005; determinethespecificinvolvementofthePKCβIisozymeinthese Grossonietal.,2007),destabilizationofp21Cip1(Santiago-Walker effects. etal., 2005; Walker etal., 2006), reduced nuclear localization of A number of studies indicate that PKCβI and/or PKCβII p21Cip1 (Sipeki etal., 2002; Ranta etal., 2011), and increased are involved in regulation of T cell proliferation. For example, E2F promoter activity (Nakaigawa etal., 1996). In many cases, antisense-mediatedknockdownhasimplicatedPKCβisozyme(s) these effects are mediated by the ERK/MAPK pathway (Jackson inIL-2signaling(Gomezetal.,1995).Furthermore,PKCβforms and Foster,2004; Grossoni etal.,2007). The opposing effects of are likely involved in cytoskeletal changes following T cell acti- PKCδ on cell cycle progression may be regulated by differential vation.PKCβIIlocalizestoacytoskeletalaggregatethatformsin phosphorylationonTyr155(Acsetal.,2000;Steinberg,2004). close proximity to the microtubule organizing center following T cells from PKCδ knockout mice are hyperproliferative and T cell activation (Black etal., 1988; Gregorio etal., 1992, 1994) produce more IL-2 cytokine upon stimulation in response to and PKCβI has been shown to associate with microtubules in allogeneic MHC. Thus, consistent with a predominant growth- T cells and to play a role in T cell polarization (Volkov etal., inhibitory role of PKCδ in other systems, this isozyme appears 2001).Sincecytoskeletalchangesappeartobeanimportantaspect tonegativelyregulateTcellproliferation,aneffectthathasbeen of T cell activation (Repasky and Black, 1996; Martín-Cófreces ascribedtoattenuationofTCR/CD3-mediatedsignaling(Gruber etal., 2008; Alarcón etal., 2011), these observations are likely etal.,2005a).AsimilarnegativeeffectofPKCδonproliferationis to be relevant to T cell signaling. This idea is supported by the alsoseeninBcells(Miyamotoetal.,2002). finding that antisense-mediated knockdown of PKCβI reduced nuclear translocation of NFAT in TCR/CD28-stimulated Jurkat PKCε T lymphoma cells (Dreikhausen etal., 2003). However, PKCβ PKCεgenerallymediatespro-proliferativeresponses,anditseffects isozymes do not have an essential role in T cell function since appeartobepredominantlyinG1/SratherthanG2/M(Graham PKCβ−/− mice have no appreciable T cell-related defects. This etal.,2000; Balciunaite and Kazlauskas,2001). The enzyme has contrasts with a critical role for PKCβ in B cell receptor sig- been implicated in mediating PDGF-induced G0/G1 → S pro- naling (Thuille etal., 2006) and in dendritic cell differentiation gression(BalciunaiteandKazlauskas,2001).LossofPKCεactivity (Farrenetal.,2010).Thus,anyroleofPKCβI/IIassociationwith inNSCLCcellsisassociatedwithinductionofp21Cip1,prolonged cytoskeletalelementsislikelytoberedundant.Inthisregard,itis G1 → S transition in response to serum, and reduced activa- noteworthythatTcellactivationleadstotranslocationof PKCα tion of cdk2 complexes (Bae etal., 2007), indicating that this and PKCθ to the same PKCβII-associated cytoskeletal aggregate isozyme suppresses p21Cip1 accumulation to facilitate cell cycle describedabove(J.D.BlackandE.A.Repasky,unpublisheddata; progression. PKCε can also induce cyclin D1 transcription and Wangetal.,1999). upregulate cyclin D1 and cyclin E protein (Soh and Weinstein, 2003; F. Hao, M. A Pysz, A. R. Black, and J. D. Black, unpub- PKCδ lished data). Although PKCε is generally downregulated during PKCδ broadly inhibits cell cycle progression in G1 in response differentiation(e.g.,Yangetal.,2003),theenzymepromotesadi- topharmacologicalagonistsandphysiologicalactivatorssuchas pogeniccommitmentandisessentialforterminaldifferentiation ATRA,inositolhexaphosphate(IP6),interferons,andtestosterone of 3T3-F442A preadipocytes (Webb etal., 2003). Its expression (Watanabeetal.,1992;Fukumotoetal.,1997;Ashtonetal.,1999; is also enhanced during myogenic differentiation, resulting in Uddin etal., 2002; Kambhampati etal., 2003; Nakagawa etal., upregulationofcyclinD3(Gaboardietal.,2010). 2005;Vuceniketal.,2005;Cerdaetal.,2006;Bowlesetal.,2007). TheabilityofconstitutivelyactivePKCεtoactivateNFATand Effects on G1 → S phase progression are mediated by direct or AP1inJurkatTlymphomacellspointstoaroleforthisisozymein indirecttargetingofcyclinD1,cyclinE,cyclinA,p21Cip1,and/or Tcellactivation(Genotetal.,1995).Antisense-mediatedknock- p27Kip1 (Fukumoto etal.,1997; Vrana etal.,1998; Ashton etal., downhasalsoimplicatedthisisozymeinIL-2signalinginTcells 1999; Nakagawa etal., 2005; Cerda etal., 2006; Afrasiabi etal., (Gomezetal.,1995).Furthermore,siRNA-mediatedknockdown 2008). CyclinD1expressionisdownregulatedbyPKCδincolon ofPKCεinCD4+Tcellsseverelyreducedproliferationinvitroand cancer cells (Cerda etal., 2006; Pysz etal., 2009), as well as in enhanced the growth-inhibitory effects of transforming growth PKCδ overexpressing vascular smooth muscle cells (Fukumoto factorbeta(TGF-β; Mirandolaetal.,2011). Thesefindingssup- etal., 1997), primary bovine airway smooth muscle cells (Page portapredominantlygrowth-stimulatoryroleofPKCεinTcells, etal.,2002),andNIH3T3cells(SohandWeinstein,2003).Consis- as seen in other systems (see above). However, PKCε−/− mice tentwiththesefindings,lossofPKCδactivityresultedinincreased shownodefectsinTcelldifferentiation,proliferationoractivation, www.frontiersin.org January2013|Volume3|Article423|7 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 7 — #7 BlackandBlack PKCandthecellcycle indicatingthatthefunctionsof thisisotypemaybeinlargepart inTcellactivation,whereitisrequiredforformationoftheCBM redundant,atleastinthemouse(Gruberetal.,2005b).Incontrast complex,whichplaysacentralroleinmediatingdownstreamsig- tothisfinding,analysisof Hashimotothyroiditispatientspoints nalingduringTcellactivation(Rawlingsetal.,2006).Inkeeping toapotentialclinicalrelevanceforproliferativeeffectsofPKCεin withthisrole,PKCθsignalingactivatesanumberoftranscription Tcells.ThesepatientshadsignificantlyhigherexpressionofPKCε factors that regulate T cell activation and proliferation, includ- intheirTcellscomparedwithhealthycontrols(Mirandolaetal., ingAp1,NF-κB,andNFAT(Pfeifhoferetal.,2003).Studiesusing 2011).Furthermore,whileHashimotothyroiditis-derivedTcells prkcq knockoutmicehavedeterminedthatPKCθplaysacentral haddiminishedTGF-βresponsescomparedwithhealthycontrols, roleinmediatingproliferativeresponsesduringTcellactivation. knockdownofPKCεinthesecellsrestorednormalresponsiveness PKCθ-deficientTcellslosetheabilitytoproliferateinresponseto toTGF-β(Mirandolaetal.,2011). TCR/CD28 activation in vitro (Sun etal., 2000; Pfeifhofer etal., 2003). A role for PKCθ in T cell expansion in vivo was also PKCη apparentfromthedefectiveproliferationseeninPKCθ−/− mice PKCηhasbeenassociatedwithpost-mitoticcellsinanumberof during allergic asthmatic reactions and in response to bacter- tissuesincludingsquamousepithelia(Kashiwagietal.,2002;Bre- ial infection (Salek-Ardakani etal., 2004; Sakowicz-Burkiewicz itkreutzetal.,2007),theepidermis(Breitkreutzetal.,2007),and etal.,2008). theintestinalepithelium(Osadaetal.,1993).Consistentwiththis As seen with PKC isozymes in other cell types, the action of localization, PKCη upregulated p21Cip1 and p27Kip1, decreased PKCθinproliferationappearstobehighlycontext-dependent.For cdk2kinaseactivity,andinducedgrowtharrestinNIH3T3cells example,whileaclearroleforthisisozymeinregulationofTh2cell andkeratinocytes(Livnehetal.,1996;Ishinoetal.,1998;Cabodi proliferationinvivoisseenintheallergicasthmaticresponse,this etal.,2000).However,thisisozymecanalsoenhanceproliferation wasnotthecaseforTh1cells(Salek-Ardakanietal.,2004). Fur- asseeninMCF-7breastcancercells,whereitupregulatedcyclinD thermore,PKCθ-deficiencydoesnotaffectTcellproliferationin andcyclinElevelsandpromotedaredistributionofp21Cip1 and responsetoviralinfection(Giannonietal.,2005)andcanmediate p27Kip1fromcdk2tocdk4complexes(Fimaetal.,2001). growth-inhibitoryeffectsofcytokinewithdrawal(Lietal.,2006b). PKCηisrecruitedtotheimmunesynapse,pointingtoinvolve- Notably,whilePKCθgenerallyplaysapositiveroleinproliferation mentofthisisozymeinTcellactivation(FuandGascoigne,2012). of effector T cells, it has the opposite effect in Treg cells, where ThisrolewasconfirmedbythefindingthatPKCη−/−Tcellshave itissequesteredfromtheimmunesynapseandpromotesgrowth adefectiveproliferativeresponsetoanti-CD3stimulationinvitro inhibition(Zanin-Zhorovetal.,2010). (Fuetal.,2011).Asomewhatmoresevereproliferativedefectwas A recent study has given insight into possible explanations alsoobservedinresponsetoantigenpresentationbothinvitroand for divergent functions of PKCθ (Kong etal., 2011). PKCδ and invivo(Fuetal.,2011).ConsistentwitharoleforPKCηinmediat- PKCθarehighlyhomologous;yet,asnotedabove,PKCδisgrowth ingTCRsignaling,activatedPKCη−/−Tcellsshowedareduction inhibitoryinTcells.Inkeepingwiththesedifferences,PKCδisnot incalciumfluxandNF-κBtranslocation(Fuetal.,2011).While targetedtotheimmunesynapse, disruptssignalosomeassembly theseeffectsarelargelyredundantwithPKCθ, specificeffectsof andcannotsubstituteforPKCθinTcellfunction. Thesediffer- PKCηwereseeninTcellhomeostaticproliferation,whichinvolves ences are due to a proline-rich motif in theV3 region of PKCθ self-antigenrecognitionandIL-7andIL-15signaling(FuandGas- thatmediatesindirectinteractionwithCD28throughLck.Muta- coigne,2012).Notably,nodefectinhomeostaticproliferationwas tionofthissequenceblockslocalizationofPKCθtotheimmune seeninPKCθ−/−mice,indicatingthatthiseffectislargelyspecific synapse;conversely,aPKCδmutantcontainingthissequencewas toPKCη,althoughdoubleknockoutsdidhaveasomewhatmore targeted to the immune synapse and could substitute for PKCθ severephenotype. in T cell signaling (Kong etal.,2011; Isakov andAltman,2012). Thesefindingspointtotheimportanceofalterationsinprotein– PKCθ protein interactions and localization in dictating the effects of PKCθ has been implicated as a positive regulator of prolifera- PKCsignaling,andofferamechanismforthedivergentrolesof tioninanumberof celltypesincludinggastrointestinalstromal PKC isozymes in different cell types and in different signaling tumorcellsandbreastcancercells,whereitrepressesexpressionof environments. p21Cip1and/orp27Kip1(BelguiseandSonenshein,2007;Ouetal., 2008),andincapillaryendothelialcells,whereitpromotesG2/M AtypicalPKCisozymes progression(Tangetal.,1997). WhileanalysisofthefunctionsofatypicalPKCsislessadvanced Alargebodyofevidencehasemergedtosupportacriticalrole thanthatofotherPKCisotypes,PKCιandPKCζgenerallyappear for PKCθ in T cell activation. The functions of this isozyme are topromotecellcycleprogression.Consistentwithacellcyclestim- the subject of several excellent reviews in this issue (e.g., Free- ulatory role of PKCζ, keratin-induced blockade of HaCaT cell leyandLong,2012; IsakovandAltman,2012;Wangetal.,2012) cycleprogressioninvolvedinhibitionofPKCζactivity,areduction and will only be discussed briefly here. While PKCθ is dispens- in cyclin D1 and cyclin E levels, and pRb hypophosphorylation ablefordifferentiationofCD4+andCD8+Tcells,itisintimately (Paramioetal.,2001).PKCζcanmediatetranscriptionalactivation involvedinTcellactivationandtransducespro-proliferativesig- of cyclinD1downstreamof Ras(Kampferetal.,2001), andcan nalsinmultiplepathways,includingthosetriggeredbytheTCR, inducephosphorylationandproteasome-dependentdegradation CD28, and TNF-α (Altman etal.,2000; So and Croft,2012). As ofp21Cip1downstreamofPI-3K(Scottetal.,2002).Theabilityof mentionedabove,PKCθisrecruitedtotheimmunesynapseearly PKCζtomodulatethesubcellulardistributionofp27Kip1 during FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|8 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 8 — #8 BlackandBlack PKCandthecellcycle cellcyclereentryof quiescentMCF7cellsisalsodownstreamof whichdirectlyimpactimmunefunction,involvebothredundant PI-3K(Castoriaetal.,2004).PKCζmayalsoenhancecdc25activ- and non-redundant functions of individual PKC family mem- itytopromoteG2/MtransitinA549lungepithelialcells,aneffect bers, and a high degree of cooperation between different PKC associated with changes in cdk2 activity (Lee etal., 2011; Kang isozymes is becoming apparent. As in other systems, the effects etal.,2012).ExcitingstudiesbyMurray,Fieldsandcolleagueshave ofPKCsignalingarehighlycontext-dependent,withthereliance recentlyidentifiedPKCιasanoncogenewhichisrequiredforthe onindividualisozymesdifferingbetweenTcellsubtypes. While transformedgrowthofvarioushumancancercelltypes(Fieldsand the majority of the characterized effects of PKC signaling in T Regala,2007;Murrayetal.,2011).Consistentwiththesefindings, cells have been pro-proliferative, negative effects are also seen: PKCιisupstreamof PKCζinRas-relatedupregulationof cyclin PKCδappearstoplayapredominantlyinhibitoryroleandPKCθ D1(Kampferetal.,2001).PKCιalsophosphorylatesandactivates canhavenegativeproliferativeeffectsdependentonthesignaling CAKinresponsetoPI-3Ksignalingingliomaandneuroblastoma environmentandcelltype. cells(Acevedo-Duncanetal.,2002; Pillaietal.,2011; Desaietal., AlthougheffectsofPKCsignalinghavebeennotedinallstages 2012)andmaytargetcyclinEinovariancancer(Ederetal.,2005). ofthecellcycle,thepredominantactionsofPKCisozymesarein IncontrasttoPKCαandPKCε,constitutivelyactivePKCζhad G1andG2phases.Similarly,whilePKCscanmodulatetheactivity no effect on AP1 and NFAT in Jurkat cells (Genot etal., 1995). ofmultiplecellcycleregulatorymolecules,consistentwitheffects However,workofGruberetal.(2008)pointstoaroleforatypi- inG1andG2,D-typecyclinsandCip/Kipcdkinhibitors(p21Cip1 calPKCsinPKCθ-mediatedpro-proliferativesignalinginTcells. and p27Kip1) are emerging as important targets of PKC control. ThesestudiesfoundthatPKCζphysicallyinteractswithPKCθina Inkeepingwiththeinvolvementoftheseproteinsinregulationof yeast two-hybrid screen and that PKCζ is a substrate for PKCθ. quiescence,accumulatingevidenceindicatesthatcontrollingcell This physical interaction likely occurs in vivo since PKCζ and cycleentryandexitisanimportantroleforPKCsignaling. The PKCι are constitutively localized in lipid rafts to which PKCθ abilityofPKCstopromoteG →G progressionhasbeennoted 0 1 is recruited following activation of primary T cells and Jurkat inseveralcelltypes(Chiuetal.,2002,2003;Santiago-Walkeretal., cells. Use of dominant negative mutant proteins further impli- 2005).PKCsignalinghasalsobeenshowntopromotecellcycleexit cated the atypical isozymes in NF-κB induction by PKCθ. In inanumberof systems,includingintestinalepithelialcells,ker- keepingwiththeircommonlocalizationandstructure,itappears atinocytes,PKC-overexpressingfibroblasts,andleukemiccelllines thatPKCιandPKCζcansubstituteforeachotherinmostTcell (Black,2000,2010).Studiesinleukemiacells(ZhangandChellap- functions. Nonetheless, PKCζ function appears to be particu- pan,1996;Vranaetal.,1998;Wangetal.,1998),non-transformed larly important for activation of Th2 cells (Martin etal., 2005): intestinalepithelialcells(Freyetal.,2000),pancreaticcancercells while PKCζ knockout did not result in proliferative or signal- (Detjenetal.,2000),andkeratinocytes(Tibudanetal.,2002)indi- ing defects in naïve T cells, it dramatically inhibited activation catethatPKCfamilymembersarecapableofactivatingacomplete of Th2 cells. This effect was reflected in disruption of STAT6, programofcellcyclewithdrawal,whichcanincludedownregula- NFAT,andNF-κBactivationfollowingstimulationwithanti-CD3. tionofcyclinD1,upregulationofp21Cip1andp27Kip1,alterations ThedramaticupregulationofPKCζnotedduringTh2celldiffer- in the expression and phosphorylation of the pocket proteins entiation may account for the inability of PKCι to compensate p107,pRb,andp130,andchangesinE2Fexpressionandcomplex forlossof PKCζinthesecells(Martinetal.,2005; Gruberetal., formation (Zhang and Chellappan,1996; Saunders etal.,1998). 2008).ThephysiologicalrelevanceofPKCζsignalinginTh2cellsis WhiletheabilityofPKCsignalingtopromoteexitfromquiescence seenintheimpairedallergicasthmaticresponseinPKCζ−/−mice followingTCR/CD28andpre-TCRengagementisestablished,fur- (Martinetal.,2005). therstudiesarerequiredtodefineitsroleinpromotingcellcycle exitduringTcelldevelopmentandtheestablishmentofquiescent Summaryanddiscussion memoryTcells. Fromtheabovediscussion,itisapparentthatPKCsignalingplays an important role in regulation of cell proliferation in a broad SPECIFICCELLCYCLETARGETSOFPKCSIGNALINGINTCELLS spectrumofcelltypesincludingTcells.PKCactivationcaneither Antigen-inducedproliferationisakeyaspectofbothTcelldiffer- promoteorinhibittransitthroughmultiplestagesofthecellcycle. entiationandclonalexpansion(KochandRadtke,2011).Thus,the The precise effect of PKCs on the cell cycle is highly context- mechanismsunderlyingPKCisozyme-specificeffectsonthecell dependent, and is influenced by the specific isozyme involved, cyclemachineryinTcellsareof criticalimportancetoimmune thetiminganddurationofPKCactivation,thecelltype,andthe function. As noted above, the cell cycle is tightly regulated by signalingenvironmenttowhichthecellisexposed;however,some coordinated actions of cyclins, cdks and ckis, which modulate themesarebeginningtoemerge.WithregardtoindividualPKC the activity of the retinoblastoma family and thus expression of familymembers,accumulatingevidenceindicatesthatPKCαcan E2F-dependent genes (Figure 1). Proliferative T cell signaling exertcontext-dependentinhibitoryorstimulatoryeffects. While affects multiple members of this control network. For example, PKCδcanhavepositiveeffectsoncellcycleprogression,itseffects proliferation induced by TCR/CD28 costimulation is associated are generally inhibitory. On the other hand, effects of PKCβII, with increased pRb phosphorylation by cyclin D2/3 and cyclin PKCε, and atypical PKCs appear to be mainly pro-proliferative, E, and enhanced transcription of E2F-dependent genes such as whilethoseofPKCηaregenerallyinhibitory. cyclinsEandA(Colombettietal.,2006).Analysisofmechanisms InTcells,multiplePKCisozymesmediateproliferativesignals underlying these changes has pointed to a particularly impor- associatedwithTCR/CD28engagement(Figure2).Theseeffects, tant role for cyclin D3, cdk6, and p27Kip1 in regulation of T www.frontiersin.org January2013|Volume3|Article423|9 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 9 — #9 BlackandBlack PKCandthecellcycle FIGURE2|ProliferativeeffectsofPKCisozymesinTcells.Positiveand ThesetargetsreflecttheimportanceofD-typecyclinsandCip/Kipckisas negativeproliferativeeffectsofindividualPKCisozymesareindicatedby targetsforPKCsignaling,asseeninothersystems(notethatinpre-Tcells, greenarrowsandredbarredlines,respectively.Proposedcellcycletargets cyclinD1appearstobethetargetforPKCθ).Thedashedlinesindicatethelack inthegrowth-inhibitoryandgrowth-stimulatoryeffectsofPKCθareshown. ofknowledgeofspecificcellcycletargetsforotherPKCisozymesinTcells. cell proliferation. For example, cyclin D3 and cdk6 knockout WhilePKCactivationmediatesTCRsignalingtoNF-κB,NFAT, miceshowdefectsinTcellproliferation,whereascdk4andcdk2 and Ap1, transcription factors that have been shown to have a knockoutmicedonot(Sicinskaetal.,2003;Huetal.,2009),and directroleinregulationofthecellcyclemachineryinTcells,the p27Kip1 nullTcellsshowreducedmitogenrequirementsandare function of specific PKCs in these effects remains largely unex- resistanttoanergy(Mohapatraetal.,2001;Rowelletal.,2005;Li plored.However,limitedinformationisemergingtoindicatethat, etal.,2006a). as in other cell types, D-type cyclins and Cip/Kip proteins are FrontiersinImmunology|TCellBiology January2013|Volume3|Article423|10 “fimmu-03-00423” — 2013/1/17 — 10:57 — page 10 — #10

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