Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

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Article

Reference

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

JACOT, Damien, SOLDATI-FAVRE, Dominique

Abstract

Members of the phylum Apicomplexa are responsible for a wide range of diseases in humans and animals. The absence of an effective vaccine or safe curing drugs and the continuous emergence of resistant parasites to available treatments impose a high demand on the identification of novel targets for intervention against the apicomplexans. Protein kinases are considered attractive potential therapeutic targets not only against cancers but also to combat infectious diseases. The scope and aim of this review is to report on the recent progress in dissecting the impact of protein phosphorylation in regulating motility and invasion.

JACOT, Damien, SOLDATI-FAVRE, Dominique. Does protein phosphorylation govern host cell entry and egress by the Apicomplexa? International Journal of Medical Microbiology , 2012, vol. 302, no. 4-5, p. 195-202

DOI : 10.1016/j.ijmm.2012.07.012 PMID : 22951234

Available at:

http://archive-ouverte.unige.ch/unige:23056

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ContentslistsavailableatSciVerseScienceDirect

International Journal of Medical Microbiology

j ourna l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / i j m m

Mini Review

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

Damien Jacot

^∗

, Dominique Soldati-Favre

DepartmentofMicrobiologyandMolecularMedicine,FacultyofMedicine,CMU,1RueMichel-Servet,1211Geneva,Switzerland

a r t i c l e i n f o

Keywords:

Apicomplexa Kinase Phosphorylation Invasion Glidingmotility Signaling

a b s t r a c t

MembersofthephylumApicomplexaareresponsibleforawiderangeofdiseasesinhumansandanimals.

Theabsenceofaneffectivevaccineorsafecuringdrugsandthecontinuousemergenceofresistantpara- sitestoavailabletreatmentsimposeahighdemandontheidentiﬁcationofnoveltargetsforintervention againsttheapicomplexans.Proteinkinasesareconsideredattractivepotentialtherapeutictargetsnot onlyagainstcancersbutalsotocombatinfectiousdiseases.Thescopeandaimofthisreviewistoreport ontherecentprogressindissectingtheimpactofproteinphosphorylationinregulatingmotilityand invasion.

Introduction

MembersofthephylumApicomplexaareresponsibleforawide rangeofdiseasesinhumansandanimals.Thislargegroupofobli- gate intracellular parasites includes Plasmodiumfalciparum,the causativeagent of the most deadly form of malaria that leads tomorethanonemilliondeathsperyear(Murrayetal.,2012);

Toxoplasmagondii,responsibleforseverecongenitaldefectsand life-threatening in immunocompromised individuals (Montoya andLiesenfeld,2004);Babesia,tick-transmitted,hemoprotozoan parasitesthat causediseasein cattle,horses,dogs,andhumans (Homer et al., 2000); Cryptosporidium, causing diarrhea (Xiao, 2010);and ﬁnallyEimeria and Theileria thataccount for a con- siderable economic burden in the poultry and cattle industry, respectively (Morrison,2009; Shirley etal., 2007).Theabsence ofan effectivevaccineorsafecuringdrugs andthecontinuous emergenceofresistantparasitestoavailabletreatmentsimposea highdemandontheidentiﬁcationofnoveltargetsforintervention againsttheapicomplexans.Proteinkinasesareconsideredattrac- tivepotentialtherapeutictargetsnotonlyagainstcancersbutalso tocombatinfectiousdiseases(Petersenetal.,2011).

Proteinphosphorylationis a commonand widespreadpost- translationalmodificationthatimpactsallcellularprocesseswitha prominentroleinsignaltransduction.Similartootherorganisms, Apicomplexaareinaconstant“stateofflux”,whereactivationand regulationofcellularpathwayshavetobeconstantlymodifiedin responsetoexternalstimuli(PawsonandScott,2005).Phospho- rylationisafast,reversiblemodificationnotsimplytoswitchthe

∗Correspondingauthor.Tel.:+41223795771;fax:+41223795702.

E-mailaddress:[email protected](D.Jacot).

activityofagivenproteinonoroffbutalsotoaffectitsstability, localization,orabilitytointeractwithotherproteins(Cohen,2000).

Kinasesandphosphatasescriticallygovernandﬁne-tunethefunc- tionoflargesubsetsofproteinsamongthehugebackgroundof potentiallyphosphorylatedproteins(UbersaxandFerrell,2007).

Twoindependentgenomedatabaseminingandphylogenyanal- ysesledtotheidentiﬁcationoftheP.falciparumkinome,which comprised86(Anamikaetal.,2005)and99(Doerigetal.,2008) kinases,respectively.InT.gondii,thekinomeisslightlylargerand consistsof108predictedactivekinasesand51predictedpseudoki- nases,whichincludeanimportantfamilyofsecretedenzymesthat actashosteffectorsandplayacriticalroleinvirulence(Limetal., 2012;Peixotoetal.,2010).AllmembersoftheApicomplexaalsopos- sessafamilyofcalcium-dependentproteinkinases(CDPKs),which arenormallyfoundinplants.Calciumsignalingisknowntocontrol severalcriticaleventsintheparasite’slifecycleincludingmotility, invasionandegress,andbecauseoftheirabsenceinmammalian hosts,theCDPKsrepresentconceivabledrugtargets(Billkeretal., 2009).

Theaimofthis reviewistoreportontherecentprogressin dissecting the impact of protein phosphorylation in regulating motilityandinvasion.Hostcellentryisakeyeventforsurvival andreplicationofanyobligateintracellularpathogen.Inapicom- plexans,thisvitalmulti-stepprocessisinitiatedbytheattachment andreorientationoftheparasitesuchthattheapicalorganelles (micronemesandrhoptries)sequentiallydischargetheircontent at thepoint ofcontact with thehost cell.It is followed byan activepenetrationeventthatcausesinvaginationofthehostcell plasmamembraneandleadstotheformationofaparasitophorous vacuolemembrane(PVM).Duringentry,theparasiteformsaso- calledmovingjunction(alsodescribedastightjunction(Bargieri etal.,2012))thatmaintainsclosecontactbetweenthehostcell 1438-4221/$–seefrontmatter© 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.ijmm.2012.07.012

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196 D.Jacot,D.Soldati-Favre/InternationalJournalofMedicalMicrobiology302 (2012) 195–202

membrane and theparasite and is mediated bysecreted para- site proteins.The rhoptryneckproteins(RONs) are targetedto thehostplasmamembraneandassociatedtothemicronemepro- teinAMA1(apicalmembraneantigen1),whichremainsanchored viaitsmembrane-spanningdomaintotheparasiteplasmamem- brane.Motility,hostcellinvasion,andegressfrominfectedcells arepoweredbytheglideosome,alargemolecularmachineﬁrmly anchoredintheparasitepellicle.Thepellicleconsistsoftheplasma membrane(PM)andtheinnermembranecomplex(IMC)which iscomposedofﬂattenedvesiclesandcloselyopposedtothesub- pellicularmicrotubules(Morrissetteetal.,1997).Theglideosome ishighlyconserved acrossthephylum butbestcharacterizedin T.gondii,where ithasbeenshown tobecomposedofamyosin heavychain(TgMyoA),itstwo associatedlight chains(TgMLC1, TgELC1), two integral inner membrane proteins (TgGAP50 and TgGAP40),and anacylated protein(TgGAP45).The motorcom- plexcomposedofTgMyoA/TgMLC1/TgELC1(Bergmanetal.,2003;

Herm-Gotzetal.,2002;Nebletal.,2011)powersmotilityandpro- pelstheparasiteintothehostcell(Meissneretal.,2002;Wetzel etal.,2003).TgGAP45recruitsandanchorsthemotorcomplexat theIMC,whereitestablishescontactwithTgGAP40andTgGAP50 (Frenaletal.,2010;Gaskinsetal.,2004).Thecurrentmodelsuggests thatthemyosinmotorisanchoredtothesubpellicularcytoskele- tonoftheparasite,whileactin(ACT)interactsviaaldolase(ALD) withthecytoplasmictailsofmicroneme proteins(MICs),them- selvesinteractingoutsidetheparasitewithhostcellreceptorsor theextracellularmatrix.Thisuniquemodeofsubstrate-dependent glidingcriticallyassiststheparasite’smigrationacrossbiological barriersandspreadingthroughtissues(Baumetal.,2006;Daher andSoldati-Favre,2009).Glidingmotility,moredirectlymyosin ATPaseactivity,andactinpolymerizationneedtobetightlyreg- ulatedina timelyandspatialfashion.Severalkeyregulators of actindynamicsincludingproﬁlin(PRF),formins(FRMs),andactin depolymerizationfactor(ADF)werefoundtogovernparasitemotil- ity(Baumetal.,2008;Daheretal.,2010;MehtaandSibley,2011;

Plattneretal.,2008).

Theimportanceof proteinphosphorylationin controlling(i) motorassembly,(ii)actinpolymerization,(iii)organellesecretion, and(iv)movingjunctionformationisstartingtoemergethrough genome-widephosphoproteomicanalysesandfunctionalinvesti- gationsofkeykinasesandtheirtargets.

Phosphoproteome

Toevaluatetheimpactofproteinphosphorylationinregulat- ingagivenbiologicalprocess,itisimportanttofirstidentifythe repertoireoftargetproteinsandthedynamicnatureofthepost- translationalmodifications.Aglobalphosphorylationprofileallows formulationof newhypothesesonwhich proteinsorpathways mightbeselectivelyactivatedataparticularstageoftheparasite.

Ultimately,todissectthemechanisticsignificance ofthismodi- fication,itwouldbeinstrumentaltomappreciselythemodified residuesandidentifythekinasesimplicated.Recentadvancesin phosphopeptideenrichmentcoupledwithhigh-throughputmass spectrometry have considerably increased the power of global phosphoproteomic analysis. Phosphoproteomes for “intracellular”and“extracellular”T.gondiitachyzoites(Treecketal.,2011) as well as for P. falciparum schizont stage have been recently reported(Solyakovetal.,2011;Treecketal.,2011).Additionally, aphosphoproteomewiththousandsofphosphorylationsitesinP.

falciparumerythrocyticstageswillbereleasedshortly (Lasonder etal.,2012).Thehighreproducibilitybut alsothecomplemen- taryresultsobtainedbyTreeck,Solyakov,andLasonderillustrate theimportanceof generatingindependentdatasets inorderto assurehighcoverageandhighconﬁdenceinphosphorylationsite assignment.

Treecketal.(2011)identiﬁed8463phosphorylationsiteslocal- ized in 1673 proteins in P. falciparum, meaning that 26% of predicted proteinsare phosphorylated at least on one residue.

12,793phosphorylationsitesmatching2793proteinswerefound forintracellularT.gondiitachyzoites.Duetotheabsenceofhost cellmaterial,theanalysisofpurified“extracellular”parasitessam- plewasconsiderablymoresensitive,with24,298phosphorylation sitesidentifiedfor3506proteins(42%ofthepredictedproteomeis phosphorylated).Theseresultsareconsistentwithwhatwaspre- viouslyreportedinotherorganisms,whereitwasestimatedthat 30%ofallcellularproteinsaremodifiedonatleastoneresiduein humans(Cohen,2000).Theinvasomeandexportomewereranked atthetopofaclassificationofthemostfrequentlyphosphorylated groupsofproteins,bothinT.gondiiandP.falciparum.Severalkey componentsoftheglideosome,regulatorsofactindynamicsand proteinsimplicatedinthetightjunctionformationwereindeed abundantlyphosphorylated(TablesS1andS2).

In Solyakov et al. (2011), a kinome-wide reverse genetics approachwasconductedinparalleltoidentifytheessentialmalaria kinases (Solyakov et al.,2011).Someof thepredictedessential kinases were previously implicated in motility such as cAMP- dependentproteinkinaseA(PKA)(Kurokawaetal.,2011;Leykauf etal.,2010),calcium-dependantproteinkinase1(CDPK1)(Green etal.,2008;Katoetal.,2008;Winteretal.,2009),proteinkinaseB (PKB)(Vaidetal.,2008),andproteinkinaseG(PKG)(Hoppetal., 2012).Thesedatasetsthereforeconstituteanimportantresource forfuturehypothesesontheregulationofproteinfunctionbyphos- phorylationandtheirimplicationsonhostcellentry.

Glideosome:myosinmotorassemblyandfunction

The phosphoproteome revealed that all components of the glideosomearephosphorylated,andtoalargeextent,thesitesare inconservedregionsacrosstheproteinsofthephylumApicomplexa (Fig.1,TableS1).Moreover,acomparativeanalysisofthephos- phoproteomefollowing apulseof calciumstimulationrevealed a selectiveincrease inphosphorylationof residuesinTgGAP45, TgMLC1,andTgMyoA(TableS1)(Nebletal.,2011).

Inhighereukaryotes,regulationofthemyosinATPaseactivity iscriticallycontrolledbyphosphorylationofmyosinlightchains (ScruggsandSolaro,2011;Stulletal.,2011),butverylittleisknown aboutMyoA.AconservedphosphorylationsitebetweenP.falci- parumandT.gondiiwasidentiﬁedintheN-terminalpartofMyoA outsideoftheheaddomain.Severaladditionalsiteswerefoundin T.gondii,includingonelocatedclosetotheIQmotif,responsiblefor bindingtoTgMLC1.TgMLC1anditshomologinthemalariapara- sitePfMTIP(MyoAtail-interactingprotein)arephosphorylatedon severalresidues,includingoneconservedinbothproteins(Fig.1, TablesS1andS2).Despitetheimportanceofphosphorylationfor theregulationofMLCsinotherorganisms,theallelicreplacement ofTgMLC1withphospho-mimeticorphospho-nullmutantsdidnot affectthelocalization oftheprotein; itsassemblywiththerest oftheglideosomeandthemotilityandinvasionofthetransgenic parasiteswereunaltered(Jacotetal.,unpublisheddata).

Assemblyofthemyosinmotoratthepellicleiscriticallydepen- dent upon GAP45, a protein extensively modified by acylation and phosphorylationboth in T. gondiiand P.falciparum (Fig.1, TableS1).TgGAP45wasshowntobephosphorylatedonresidues S163 and S167 in vivo (Gilk et al., 2009). These modifications appeared toplay a role in thefinal step of glideosomeassem- bly,since transient expressionof double phospho-null/mimetic mutantsfollowed by co-immunoprecipitationrevealed thatthe phospho-mimetic mutant failed to associate with TgGAP50. In T.gondiithebulkofphosphorylationsitesisclusteredinadisor- deredregionlocatedbetweenthecoiled coilandtheconserved

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PfGAP45 TgGAP45

TgMLC1

PfMTIP

TgMyoA

1 93 774 831

S7 S20/21* S517/518 S561 S748 S823

PfMyoA

1 91 772 818

S19*

1 24 327 385

TMD

head domain Head Domain

T11 T353

S358 S355

S357 S384

TgGAP40

PfGAP40

1 23 326 456

S372 S382

Y385 S394

S395 S399

S405 S409S415

S420S445 S390 S446

S393 S454

1 213

1 44 70

S55 S57 T98 S113*

S132 S124

T182/3 Y114

DR

1 204

1 15 120 158

S89 S121

S142 S149

S156 S103 T158

S107 S109

S173 S198*

coiled coil DR

S31

1 245

1 15 152 199

S153 Y158

S163 S167

S169 T171

T172 T175

S184 S185

T189 S192

S239*

DR globular

globular

2 degenerated EF hands coiled coil

1 204

1 41 67

S47 S51 S85/6 S108*/109

Nt DR Nt

head domain 2 degenerated EF hands

IQ IQ

Fig.1.Schematicrepresentationofthephosphorylationsitedistributionintheglideosomeproteins.AllproteindomainswerepredictedusingSMARTEMBLifnototherwise speciﬁed.Underlinedphosphorylatedsiteswerefoundonlyinsilico.DR:disorderedregion(Nebletal.,2011),Nt:N-terminalextension,TMD:transmembranedomain,Star:

conservedphosphorylatedresidues.ProteinsarepresentedwiththeN-terminusontheleft.PhosphorylationsitereferencesarelistedinTableS1.

C-terminalglobular domain(Fig.1).Theglobular domainalone wasshowntobesufﬁcientforassemblywiththeTgMLC1/TgMyoA complexand targeting totheIMC (Frenal etal., 2010).In con- sequenceonecanpredictthatphosphorylationinthatregionof TgGAP45isunlikelytohaveadramaticimpactontheassemblyof thecomplex.MoreoverthecomplementationofTgGAP45inducible knockoutwithamutantversionofTgGAP45lackingthisdisordered regionrescuedgliding,invasion,andegressphenotypes(Jacotetal., unpublisheddata).InP.falciparum,tworecentstudiesidentiﬁed multiplephosphorylatedresiduesonPfGAP45,buttheexpression ofthecorrespondingphospho-mutants(nullandmimetic)showed noapparenteffectonPfGAP45assemblyintothemotororitssub- cellularlocation(Ridzuanetal.,2012;Thomasetal.,2012).Taken together,theseresultsfailsofartoidentifyakeycontributionof phosphorylationtomotorrecruitmentandfunction.

GAP40 is a polytopic IMC resident protein containing 10 transmembranedomains(TMDs)andahighlyphosphorylatedC- terminaltailpredictedtobecytosolic.Aphosphorylationsiteatthe N-terminalregionwasalsomappedonTgGAP40(Fig.1,TableS1).

ThesecondresidentIMCproteinknowntobeassociatedtothe glideosomeisGAP50.ThispredictedtypeItransmembranepro- teinismainlylocalizedtothelumenoftheIMC,withonly6amino acidsfacingthecytosol.Auniquephosphorylationsitehasbeen identiﬁedexclusivelyonTgGAP50andnotinthemalariahomolog.

Thissiteislocatedinthephosphatasedomainthatispredictedto beinthelumenoftheIMCandinactivesincekeycatalyticresidues arelacking.

Fractalinformationiscurrentlyavailableontheidentityofthe kinasesimplicatedinglideosomephosphorylation.Invitrophos- phorylationassaysofPfGAP45andPfMTIPidentiﬁedPfCDPK1as

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apotentialkinase(Green etal.,2008; Katoetal.,2008;Winter etal.,2009).PfCDPK1islocalizedattheperipheryoftheP.fal- ciparummerozoiteandisthereforecorrectlypositionedtofulﬁll suchafunction(Greenetal.,2008).ThehomologofPfCDPK1inT.

gondii,TgCDPK3canphosphorylateTgALDinvitroandislocatedat theapicalpoleofextracellularparasites(Sugietal.,2009).Another enzyme,theproteinkinaseB(PfPKB),wasreportedtointeractwith PfGAP45viaimmunoprecipitationassaysandwasalsoshownto phosphorylatePfGAP45invitro(Thomasetal.,2012;Vaidetal., 2008).PfPKBharborsacalmodulinbindingdomainthatcouldmake thelinkwithanupstreamactivationcascadeviacalcium.Further- more,Go6983,aPKCinhibitor(PfPKBcatalyticdomainisclosely relatedtoPKC)isabletoaffectinvitrorecombinantPfPKBactiv- ityandreducetheabilityofP.falciparumtoinvadeerythrocytes (Kumaretal.,2004).Consistentwithavitalroleinparasitedevel- opment,bothPKBandCDPK1arerefractorytogenedisruptionin P.bergheiandP.falciparum(Katoetal.,2008;Solyakovetal.,2011;

Tewarietal.,2010).Usingabroad-rangeserine/threoninekinase inhibitor(staurosporine),amarkedreductionofphosphorylation ofPfGAP45wasobserved(Jonesetal.,2009).Surprisingly,whenthe phosphorylationofPfGAP45isimpaired,immunoprecipitationof themotorcomplexusingGAP45antibodiesshowedstrongreduc- tionoftheimmunoprecipitatedPfMTIPandPfMyoA.Thissuggests apotentiallinkbetweenphosphorylationandtheformingrateof themotorcomplex.

Theextensivephosphorylationoccurringonthecomponents oftheglideosome(Tg-36andPf-30,TableS2)is suggestiveofa potentiallyimportantroleinitsassembly,stability,andfunction.

Despitethis,nokinasehasyet beenshown toselectivelyaffect motility.Itis worthmentioningthatgiven theessential contri- butionofmicronemeproteinsingliding,itisexperimentallynot trivialtodifferentiateandmeasuretheimpactonglidinginsit- uations where microneme secretionis concomitantlyimpaired.

PreliminaryobservationsonTgMLC1and TgGAP45suggest that phosphorylationisnotessentialforglideosomeassemblyandfunc- tion,howevertheimportanceofphosphorylationonMyoAand GAP40remainstobeinvestigated.

Actindynamics

Inapicomplexanparasites,hostcellentryusuallytakes10–30s, andthereforeactin-basedmotilityneedstobetightlycontrolled inspaceandtime.Furthermore,parasitesbecomereadilymotile uponinducedegress andstopmovingequallyfastimmediately aftersealing theparasitophorous vacuole.A rather unique fea- tureofapicomplexanactinisthatitis largelymaintainedinan un-polymerizedstate(Angrisanoetal.,2012;Dobrowolskietal., 1997). Filamentous actin is hence predictably highly dynamic, short,andtherateofpolymerizationanddepolymerizationmust be tightly controlled. Several key players have been identified including profilin (PRF), formins (FRMs), and actin depolymerizationfactor(ADF).Profilinwasfoundtocriticallycontributeto egress,gliding,andhostcellinvasion(Plattneretal.,2008).Inthe absenceofanARP2/3complex,theroleoftheactinnucleatoris carriedoutbytwoforminsinPlasmodiumspp.andthreeinT.gondii (Baumetal.,2008;Daheretal.,2010,2012).Topromotemotility, thetwoforminsTgFRM1andTgFRM2actinconcertattheparasite pellicletogeneratepresumablyshort,transientfilaments(Daher etal.,2010).InP.falciparum,PfFRM1wasreportedtolocalizeat theapicalpoleoffreemerozoitesandtofollowthetightjunction wheretheglideosomeispredictedtopowermovement.TheFH2 domainsofthesetwoforminsenhanceactinpolymerizationinthe presenceofToxoplasmaactininvitro(Skillmanetal.,2012).Inthose sameassays,thebiochemicalpropertiesofTgPRFindicatethatthe proteinsequestratesactinmonomersanddoesnotcontributeto polymerization(Skillman et al.,2012).TgADF is anotherhighly

potentfactorthatleadstoactindepolymerization,anddeletionof thegeneresultsintheaccumulationofactinﬁlamentsinT.gondii (Mehta and Sibley, 2011). This abnormal actin polymerization causesseveredefectsinegress,gliding,andhostcellinvasion.All oftheseregulatorsofactindynamicswerefoundtobephospho- rylatedwiththeexceptionofADF(TableS2).Moreover,actinitself wasfoundtobephosphorylatedinbothorganisms(TableS2).To date,thereisnoreportoftheimportanceofphosphorylationin regulationofactinpolymerizationinapicomplexans.

In mammals, the actin nucleating activity of the formins is tightlycontrolled,andnumerousforminsactasRhoGTPaseeffec- tors.RhobindstotheforminN-terminalGTPase-bindingdomains (GBDs)and causestheadjacentDia-inhibitory domain(DID)to release the C-terminal Dia-autoregulatory domain (DAD) that ﬂankstheFH2domain.ThisreleaseallowstheFH2domaintonucle- ateactin(Schonichenand Geyer,2010; Wallaret al.,2006).All apicomplexanparasiteslacktheseregulatorydomains,butthere isabundantliteraturecoveringstudiesinmammalsandyeastthat highlighthowphosphorylationregulatesforminsactivity(Cheng etal.,2011;Iskratschetal.,2010;Wangetal.,2009).Inthiscon- textitisintriguingandlikelyrelevanttonoticethelargecluster ofphosphorylationsitesontheC-terminalregionof TgFRM2.A recentinvestigationofTgFRM3hasuncoveredtheimportanceof theC-terminalregionincontrollingthenucleatingactivityofthis formin(Daheretal.,2012).

Micronemesecretionandegress

Motilityistriggeredbysignalingeventsinvolvinganincreasein intracellularcalciumthatleadstomicronemesecretion(Carruthers andSibley, 1999;MorenoandDocampo,2003;Wassermanand Chaparro,1996; Wetzelet al.,2004)and alsotoconoidprotru- sion in Coccidia. The dischargeof microneme proteinsleadsto theestablishment ofa bridge betweentheextracellular matrix or host cell receptors and the glideosome,hence allowingthe parasitetomoveontothesubstrate.InT.gondii,micronemesecre- tioncanbeartiﬁciallytriggeredbyethanolorcalciumionophores (Carruthersetal.,1999;CarruthersandSibley,1999;Endoetal., 1982;Wetzeletal.,2004).During glidingmotilitycytosoliccal- ciumlevelsundergofastanddramaticchangesthatreﬂectmultiple sequentialmicronemedischargeevents.However,whenthepar- asite initiateshostcell penetration, theintracellular calcium is rapidlyquenched(LovettandSibley,2003).InP.falciparum,inva- sionofredbloodcellsbymerozoitesisinhibitedbyintracellular calciumchelators(Joneset al.,2009;Wassermanand Chaparro, 1996).Free merozoitesalsoexhibit a highcalciumlevel, which dropsfollowingredbloodcellattachment(Singhetal.,2010).The interactionofthetransmembraneerythrocytebindingantigen-175 (EBA175,amicronemeprotein)withitsreceptorrestoresthebasal calciumlevelandisbelievedtotriggerthesecretionofrhoptries.

Thisinteractionmightalsoserveasanegativefeedbacklooptoshut downthesecretionofmicronemes(Singhetal.,2010).

T.gondiicalcium-dependentproteinkinase1(TgCDPK1)was identiﬁedasacriticaltransducerdownstreamofcalciumsignal- ingthatgovernsmicronemeexocytosis.ATet-inducibleknockout ofTgCDPK1blocksthesecretionofmicronemes,whichresultsin impairedglidingmotility,invasion,andegress(Louridoetal.,2010).

SeveralspeciﬁcinhibitorsweredesignedagainstTgCDPK1,includ- ingpyrazolopyrimidinederivativesforwhichagatekeepermutant wasshowntorestoremicronemesecretioninthepresenceofthe drug(Johnsonetal.,2012;Kieschnicketal.,2001;Ojoetal.,2010;

Sugietal.,2010).

Inhibition of the T. gondii cGMP-dependent protein kinase G(PKG) by compound 1 wasalso foundto inhibit microneme dischargewithaminimaleffectonintracellularparasitegrowth

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(Wiersmaetal.,2004).Moreover,thePKGlocuswasprovedtobe refractorytogenedisruption,suggestingavitalfunction(Donald etal.,2002).TgPKGandTgCDPK1couldactindependentlyandin parallelandmightrepresentredundantsignalingpathwaysforcon- trollingthiscriticalevent(Louridoetal.,2010).Infact,manyof theprocessescontrolledbycGMPappeartobealsoregulatedby calcium,andhence thetwo pathwaysarelikelyinterconnected (Hopp et al., 2012).In T. gondii, PKG is believed toact down- streamofcalcium,ascalciumionophoresarenotabletoinduce microneme secretion in parasites treated with PKG inhibitors (Wiersmaetal.,2004).InPlasmodiumspp.,thepictureismorecom- plicatedduetothemultipleeffectsresultingfromPKGinhibition.

InP.berghei,PKGisakeyregulatorofookinetegliding,andthe geneisrefractorytodisruptioninintra-erythrocyticstages(Moon et al., 2009). A conditional disruptionof PbPKG in sporozoites leadstoasevereintracellular blockintheliverstage,although themutated sporozoites werestill ableto invade (Falae et al., 2010).PfPKG is also implicated in mediating differentiation of male and female gametocytes into mature gametes withinthe mosquito(McRobertetal.,2008).TreatmentofP.falciparumring stagewithcompound1allowednormaldevelopmentupto30h postinfection(pi), andsegmentedschizontswereabletoform.

Parasitestreatedonlyintheschizontstagebecamedysmorphic, wereunable torupture, and themerozoites releasedwere not invasive(Tayloretal.,2010).Othershavefoundthatschizonttreat- mentwithcompound1inhibitedprocessingofthemajorsurface protein1(PfMSP1)bytheproteasesubtilisin1(PfSUB1),indicat- ingthatPKGactsupstreamoftheproteasecascade(Dvorinetal., 2010).EgressofPlasmodiumreliesonproteaseactivityinorderto escapefromthePVM(Blackman,2008).PfCDPK5,a membrane- associatedkinaseexpressedinlateschizontsandmerozoiteswas recentlydemonstratedtoactdownstreamofthisproteasecascade (Dvorin etal., 2010).PfCDPK5-deficient parasites werearrested inmatureschizontswithnoapparentultrastructuredefects,and theprocessing of PfMSP1 by PfSUB1 wasunaffected. Mechani- callyegressedPfCDPK5-deficientmerozoitesretainedtheirability toinvade,excludingablockinmicronemesecretion.Theauthors thereforeproposedamodelofparasiteegresswheretheprotease cascadeisnecessarybutnotsufficientandwherethefinalstages oftheruptureofthePVMandtheerythrocyteplasmamembrane aredownstreamofPfCDPK5(Dvorinetal.,2010).

InT.gondii,TgCDPK1andTgPKGhaveclearlybeenimplicated inmicronemesecretion,whereasinP.falciparumtheexact con- tributionofPKGinparasiteegressremainstobedetermined.The identiﬁcationofthesubstrateswillbeinstrumentalinelucidating whetherornotthis kinasefulﬁllshomologousfunctionsinboth parasitesandotherapicomplexans.

Movingjunctionformation

TheidentificationofAMA1complexedwithseveralRONpro- teinsprovidedthefirstgraspofthemolecularnatureofthemoving junctionandofferedanelegantmodeltoexplainhowtheparasites hijackthehostplasmamembrane(Besteiroetal.,2011;Lamarque etal.,2011;Tonkinetal.,2011;Tyleretal.,2011).Intriguingly,the rhoptryproteinsTgRON2,TgRON4,TgRON5,andTgRON8arephos- phorylatedpriortosecretioninT.gondii(TableS2),implyingthat aluminalkinaseisresponsibleforthemodification.

TgMIC2is majordeterminantof glidingmotility,connecting itscytoplasmictailviaTgALDtotheactomyosinsystem(Brossier andDavidSibley,2005;HuynhandCarruthers,2006;Jewettand Sibley, 2003). A phosphorylation site has been mapped in the highlyacidicregionoftheTgMIC2tailimplicatedinbindingto TgALD;henceitisappealingtospeculatethatduringinvasionthe phosphorylationofTgMIC2mightcontroltherecruitmentofthe

glycolytic enzymein a timely,restrictedfashion (Starnes etal., 2006)(Fig. 2).TgMIC8 was foundto be dispensablefor egress andglidingmotilitybutessentialformovingjunctionformation.

TgMIC8ispresumablyinvolvedinasignalingcascadethatleadsto thesecretionofrhoptries(Kessleretal.,2008).InP.falciparum,a similarfunctionappearstobecarriedoutbyEBA175,theligandfor theerythrocytesurfaceproteinglycophorinA.Intuitively,sucha signalingfunctionislikelytobecoupledtophosphorylation,and indeedTgMIC8cytosolictailisphosphorylated,whereasEBA175 hasnotbeenreportedtobemodiﬁed(Fig.2).

AMA1isakeyplayerforinvasionandisthemostconserved micronemeproteinacrossthephylumofApicomplexa.(Hehletal., 2000; Triglia et al., 2000). Analysis of a conditional knock-out of TgAMA1 established its important role in invasion and was foundtobedispensableforglidingmotility(Mitaletal.,2005).

InT.gondii,AMA1iscleavedduringinvasionintoitsmembrane- spanning domain by the rhomboid protease ROM4 (Buguliskis et al., 2010), whereas in Plasmodium a juxtamembrane cleavage is performed by the subtilisin protease (PfSUB2) (Harris etal.,2005;Howelletal.,2003;Olivieri etal.,2011).BothinT.

gondiiandP.falciparum,AMA1wasfoundtobephosphorylated (Fig.2andTableS1).

Usingacomplementationassaybasedonstrain-speciﬁcinhi- bition,thecytosolictailofPfAMA1wasfoundtobedispensable forcorrecttrafﬁckingbutessentialforthefunction(Treecketal., 2009).Abioinformaticsscreenperformedonthetailpredicted6 aminoacidstobephosphorylated(2ofthemwerealsofoundin Treecketal.,2011:S590andS610)andaphospho-mutant,gen- erated by replacing all the residues by alanine, was unable to complement. Investigationof PfAMA1phosphorylationwasfur- therconductedbyLeykaufetal.(2010),where2DEgelanalysis ofmatureschizontstagerevealedthepresenceofatleast3phos- phorylatedresiduesdependentonthephosphorylationofresidue S610.Timecourseexperimentsrevealedthatphosphorylationof PfAMA1 occurs mainlyin late schizontsor even in merozoites.

Moreover, ring stage parasites showeda simpler phosphoryla- tionpattern,suggestingadephosphorylationmechanismafteror duringinvasion.Furthermore,aninvitrophosphorylationassay revealedaphosphorylationincreasefollowingcAMPstimulation, stronglyindicatingtheimplicationofthecAMP-dependentprotein kinase(PKA). Indeed,puriﬁed PfPKA wasable tophosphorylate arecombinantPfAMA1tail. Usingthesamestrain-speciﬁccom- plementation assay, a phospho-null mutant S610A wasunable tocomplement,andparasiteswerearrestedafterre-orientation.

Moreover,aphospho-mimeticmutantwasunabletocomplement, suggestinga dynamicturnoverofphosphorylationfor thefunc- tion. Using a completelydifferentapproach in T. gondii, Santos etal.(2011)foundacriticalcontributionofTgAMA1asarhomboid substrate triggeringintracellular parasitereplication.Theyused adominantnegativeversionoftherhomboidprotease,TgROM4, inwhichthecatalyticresiduewasmutatedinordertoblockthe cleavageofthesubstrates.Theobservedgrowtharrestphenotype couldbeovercomebyconditionalexpressionofTgAMA1tailand PfAMA1 tail asa productof ROM4 cleavage.The phospho-null mutantS610A ofthePfAMA1tailwasalsoabletocomplement (Santosetal.,2011).Inthephospho-proteomespublishedbyTreeck et al. (2011) and Solyakov et al. (2011), S610 was conﬁrmed aswellas4 additionalsitesin thetail, andtwo newphospho- rylated serine residues were identiﬁed in TgAMA1 (Fig. 1 and Table S1).Notably, there is noconservation of the phosphorylated residuesbetween the2 organisms. Furtherinvestigations intoP.falciparumareneededinordertounravelhowphospho- rylation ofS610 rendersthemerozoite competentfor hostcell entry. It is not known if the phosphorylation turnover of the PfAMA1 tail could impact intracellular growth via a signaling pathway.

(7)

TgAMA1

1 484506 569

TMD

S527 S537

PfAMA1

1 547 566 622

TMD

622 S588

S590 S601

S610 T612

TgMIC2

S764

1 74 251 769

A domain

273 649

TMD CT

CT CT

TSP1 domains

TgMIC8

TMD

1 67 806 877899 952

S925 S935

CT EGF-like domains

Fig.2.SchematicrepresentationofphosphorylationsitedistributioninAMA1andTgMIC2/8.AllproteindomainswerepredictedusingSMARTEMBL.TMD:transmembrane domain,CT:cytosolictail,EGF:epidermalgrowthfactor-likedomain,Adomain:vonWillebrandfactor(vWF)typeAdomain,TSP1:thrombospondintype1.Proteinsare presentedwiththeN-terminusontheleft.PhosphorylationsitereferencesarelistedinTableS1.

Conclusionandperspectives

InT.gondii,TgCDPK1andTgPKGareimplicatedinmicroneme secretionwhileinP.falciparum,PfPKG,PfPKA,andPfPKBareasso- ciatedwithhostinvasion;however, inboth organisms there is nodirect evidence regarding an impact of phosphorylationon glideosomeassemblyorfunction.Nevertheless,thehighlevelof phosphorylationidentiﬁed onproteins participatingin motility (150forT.gondiiand57forP.falciparum,TableS2)suggeststhatthis posttranslationalmodiﬁcationplaysarolein controllinggliding motility.

Aclassicwaytoinvestigatesite-specificphosphorylationisto generateaseriesofphospho-mutantstoeithersuppressormimic thephosphorylation.However,thistechniquehasseverallimita- tions;eachsiteorcombinationsofthemhavetobeinvestigated onebyoneandrepresentagainaninfinitenumberofcombina- tions,especiallyinthecaseoftheglideosomewhereatleastfive differentiallyphosphorylatedproteinsareassembledinonelarge machine.Furthermore,bychangingtheaminoacidcomposition, thestructure/stabilityof theprotein canbeaffected,and these changesmightnotberelatedtophosphorylation.Lastlyandmore importantly,theexpressionofanadditionalmutatedcopycould notbesufficienttoidentifyphenotypesandassessthefunction;

thereforegenereplacementorgenerationof conditionalknock- outstrainsforcomplementationwouldberequired.Largescale, quantitativeMS-methodssuchasSILAC(stableisotopelabelingin cellculture)willundoubtedlybefurtherdevelopedtogetamore generalpictureofphosphorylationandidentifytheunderlyingnet- worksandtheirinterconnections.Thenextstepwouldconsistof interferingselectivelyandrapidlywithagivenkinaseactivityand recordthechangesinthephosphoproteome.Thedeterminationof phosphorylationsitesiscrucialforacompleteunderstandingof proteinkinasesandkinase-substraterelationships,andwealready havepartoftheanswer.Thecurrentchallengeistofurtherinves- tigatewhichkeysteps(theglideosome,theactinpolymerization, theorganellesecretionand/orthemovingjunction)aregoverned bythis modiﬁcationandrevealhowphosphorylationtailorsthe function(s)oftheproteinsimplicatedinthesefourkeyevents.

Acknowledgments

WethankDr.KarineFrenalandtheothermembersofthelab- oratoryforconstructive discussions.DominiqueSoldati-Favreis

supportedbytheSwissNationalFoundation(FN3100A0-116722) andDamienJacotbytheIndo-SwissJointResearchProgramme.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.ijmm.

2012.07.012.

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