The DNA-dependent Protein Kinase Catalytic Subunit Phosphorylation Sites in Human Artemis* S

13 Pages·2005·0.59 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.280,NO.40,pp.33839–33846,October7,2005
©2005byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
The DNA-dependent Protein Kinase Catalytic Subunit
Phosphorylation Sites in Human Artemis*□S
Receivedforpublication,January13,2005,andinrevisedform,June30,2005 Published,JBCPapersinPress,August10,2005,DOI10.1074/jbc.M507113200
YunmeiMa‡,UlrichPannicke§,HaihuiLu‡,DorisNiewolik§,KlausSchwarz§1,andMichaelR.Lieber‡2
Fromthe‡DepartmentsofPathology,BiochemistryandMolecularBiology,BiologicalSciences,andMolecularMicrobiologyand
Immunology,NorrisComprehensiveCancerCenter,UniversityofSouthernCalifornia,LosAngeles,California90089-9176and
§InstituteforClinicalTransfusionMedicineandImmunogenetics,DepartmentofTransfusionMedicine,UniversityofUlm,
D-89081Ulm,Germany
ArtemisproteinhasirreplaceablefunctionsinV(D)Jrecombina- absenceofanyofthesecomponentsinvivotypicallyresultsinanioniz-
tionandnonhomologousendjoining(NHEJ)asahairpinand5(cid:2)and ing radiation sensitivity and severe combined immune deficiency
3(cid:2)overhangendonuclease.ThekinaseactivityoftheDNA-depend- (SCID)phenotype(8).ArtemisisthemostrecentNHEJfactordiscov-
entproteinkinasecatalyticsubunit(DNA-PK )isnecessaryinacti- ered.Itwasclonedfromhumanpatientsthathaveradiosensitivityanda
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vatingArtemisasanendonuclease.Herewereportthatthreebasal SCIDphenotype(RS-SCID)(9).Thehairpinopening(criticalforV(D)J
phosphorylation sites and 11 DNA-PK phosphorylation sites recombination)and5(cid:1)and3(cid:1)overhangprocessing(necessaryforNHEJ)
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within the mammalian Artemis are all located in the C-terminal activitiesofArtemisexplainthedualphenotypeofthehumanpatients
domain.Allbutoneofthesephosphorylationsitesdeviatefromthe (10).AnimalsmissingArtemisalsohavetheRS-SCIDphenotype.The
SQorTQmotifofDNA-PK thatwaspredictedpreviouslyfromin presenceofunresolvedcodingendhairpinsintheseanimalsconfirmed
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vitro phosphorylation studies. Phosphatase-treated mammalian thepivotalroleofArtemisinthehairpinopeningstepofV(D)Jrecom-
ArtemisandArtemisthatismutatedatthethreebasalphosphoryl- bination(11,12).Thefactthathumanpatientsandanimalsdeficientfor
ationsitesstillretainDNA-PK -dependentendonucleolyticactiv- theArtemisproteinbothhavetheRS-SCIDphenotypesuggeststhat
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ities,indicatingthatbasalphosphorylationisnotrequiredforthe othernucleasesdonotfunctionallyfulfilltherolesofArtemis.
activation.InvivostudiesofArtemislackingtheC-terminaldomain Artemisseemstorecognizestructuraldiscontinuityofsingle-todou-
havebeenreportedtobesufficienttocomplementV(D)Jrecombi- ble-strandDNAtransitions.TheactivitiesofArtemisareregulatedby
nationinArtemisnullcells.Therefore,theC-terminaldomainmay associationwithandphosphorylationbyDNA-PK .Artemisinteracts
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have a negative regulatory effect on the Artemis endonucleolytic withDNA-PK invitroandinvivointheabsenceofDNAandATP,and
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activities, and phosphorylation by DNA-PK in the C-terminal itcanonlybeactivatedasahairpinandoverhangendonucleaseonceit
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domainmayrelievethisinhibition. isphosphorylatedbyDNA-PK (10).Itisstillanopenquestionhow
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DNA-PK activatesArtemisbyphosphorylation.
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TheN-terminaldomain(aminoacid1–155,exons1–6)ofArtemis
V(D)J3recombinationisthesomaticDNArecombinationthatoccurs hassignificanthomologytothe(cid:1)-lactamasefoldofseveralmembersof
in precursors of lymphocytes. During this process, the variable (V), themetallo-(cid:1)-lactamasesuperfamily(9)andcontainsmultiplecatalyt-
diversity (D), and joining (J) subexons are recombined to generate a icallyimportantresidues(13,14).Aminoacids156–385(exons7–13)of
functionalvariabledomainexoncodingforimmunoglobulinsandTcell Artemiscomprisethe(cid:1)-CASPregion,foundonlyinmembersofthe
receptors(1–3).V(D)Jrecombinationbeginswiththebindingofthe metallo-(cid:1)-lactamasesuperfamilythatprocessnucleicacid(15).Most
RAGcomplex(RAG1,RAG2,andHMG1)totherecombinationsignal interestingly,ArtemistruncatedfortheC-terminaldomaincanfunc-
sequences.TheRAGcomplexnicksbetweenthecodingendandthe tionallycomplementcellsderivedfromanRS-SCIDpatientforV(D)J
recombination signal sequences and converts the nicks to double- recombination(14).Therefore,itseemsthatthemetallo-(cid:1)-lactamase
strandDNAbreaks,therebycreatinghairpinsealedcodingendsand domainandthe(cid:1)-CASPregiontogetherconstitutethe“catalyticcore”
bluntsignalends(2,4,5).Subsequently,thejoiningofthetwocoding ofArtemis.However,thefunctionoftheremainingC-terminaldomain
endsiscarriedoutbythegeneralNHEJmachinery(6). (aminoacids386–692),composedofasingleexon(exon14),hasyetto
NHEJfactorsincludeKu(composedoftheKu70andKu86subunits), bedetermined.
DNA-PK ,Artemis,andDNAligaseIV-XRCC4complex(6,7).The Inthisstudy,wehavediscovered11DNA-PK phosphorylationsites
cs cs
and3basalphosphorylationsitesofArtemis,allofwhicharelocated
withintheC-terminaldomain.Ourdatasuggestthattheuncharacter-
*Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage ized C-terminal domain of Artemis has important regulatory roles.
charges.Thisarticlemustthereforebeherebymarked“advertisement”inaccordance
□S Twhietho1n8-liUn.eSv.Ce.rSsieocntioofnth17is3a4rtsioclleel(yavtoaiilnadbilceaattehtthtips:/f/awctw.w.jbc.org)containsadditional pThhoesspehroersyullatstiolena.dtoamodelforhowDNA-PKcsactivatesArtemisby
ExperimentalProcedures,Results,Ref.1,andFigs.S1–S2.
1Towhomcorrespondencemaybeaddressed.Tel.:[phone];Fax:[phone]-
575;E-mail:[email protected]. EXPERIMENTALPROCEDURES
2Towhomcorrespondencemaybeaddressed.Tel.:[phone];Fax:[phone];
E-mail:[email protected]. Oligonucleotides—The 46-nt hairpin substrate (YM-164) has been
3Theabbreviationsusedare:V(D)J,variable,diversity,andjoining;DNA-PKcs,DNA-de- describedpreviously(Fig.4AofRef.13).Thesequencesofthe35-bp
pendentproteinkinasecatalyticsubunit;NHEJ,nonhomologousendjoining;wt,wild
type;SCID,severecombinedimmunedeficiency;RS,radiosensitivity;FACS,fluores- DNAusedasaDNA-PKcscofactorarethesameasdescribed(10).
cence-activatedcellsorter;DTT,dithiothreitol;(cid:2)PPase,(cid:2)proteinphosphatase;CIP, ProteinExpressionConstructs—Wild-typeArtemisexpressionplas-
calf intestine phosphatase; IR, ionizing radiation; MS, mass spectrometry; TEV,
mids were constructed as described (13). The triple point mutant
tobaccoetchvirus;ATM,ataxiatelangiectasiamutated;ATR,ATM-andRad3-related
kinase;nt,nucleotide. ARM49(S385A,S516A,andS518A)aswellastheothersubstitution
OCTOBER7,2005•VOLUME280•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33839
This is an Open Access article under the CC BY license.
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
mutantsweregeneratedusingtheQuickChangesite-directedmutagen- InVitroNucleaseAssay—25nMofthe5(cid:1)-labeledsubstratewasincu-
esiskit(Stratagene,LaJolla,CA). batedwith100nMArtemisin25mMTris,pH8.0,10mMKCl,10mM
FortheArtemisFLAGbaculovirus,full-lengthArtemiscontaininga MgCl2,1mMDTT,0.25mMATP,and50(cid:3)g/mlbovineserumalbumin
SapI site in front of its stop codon was generated and cloned into inatotalvolumeof10(cid:3)l.Whereindicated,50nMDNA-PKcsand0.25
pENTR1a-TEV(amodifiedversionpENTR1a(Invitrogen)).Theresult- (cid:3)Mof35-bpDNA(nonspecificDNA-PKcscofactor)wereincludedin
ing plasmid (pENTR1a-TEV-Artemis-SapI) was recombined with thereactions.Reactionswereincubatedfor1hat37°Candthendena-
pDEST8-FLAG (a modified version of pDEST8 (Invitrogen), which turedfor5minat100°Cinanequalvolumeofdenaturinggelloading
containsaSapIsiteinfrontofaTEVproteaserecognitionsitefollowed dye(98%formamide,10mMEDTA,0.025%ofbromphenolblue,and
byaFLAGepitope)vectorusingtheGatewaysystem(Invitrogen).The 0.025%ofxylenecyanolFF).Reactionmixtureswereresolvedby12%
resultingplasmidwasdigestedwithSapIandligatedbacktogetherto denaturingPAGE,andthegelimagewasobtainedwithPhosphorIm-
generateanin-framefusiontotheTEVcleavagesiteandtheFLAGtag agerSI445(AmershamBiosciences).
at the C terminus. This construct was then used in the Bac-to-Bac DNA-PK Phosphorylation and Phosphatase Dephosphorylation
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system(Invitrogen)togeneratethebaculovirusfollowingthemanufac- Assays—For the DNA-PK phosphorylation reactions, Artemis was
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turer’srecommendation. incubatedwithDNA-PKcsin10mMTris,pH7.5,10mMMgCl2,1mM
InordertogeneratethemammalianexpressionplasmidforArtem- EDTA,1mMDTT,0.25mMATP,and1(cid:3)M35-bpDNAat37°Cfor30
isFLAG,full-lengthArtemiswasamplifiedbyPCRusingPfuTurboHot min. If the kinase reactions are followed by dephosphorylation, the
Startpolymerase(Stratagene,LaJolla,CA)andprimersArtemis-Bam- Artemisimmunobeadswerewashedin25mMHEPES,pH7.9,650mM
HI.R (5(cid:1)-CGCGGATCCCTACTTGTCATCGTCATCCTTGTAAT- KCl,10mMMgCl2,and0.1%NonidetP-40threetimes,andin25mM
CTGCGGTATCTAAGAGTGAGCATTTTCTTTT-3(cid:1))andArtemis- HEPES,pH7.9,10mMMgCl2,and2mMDTTtwice,andtheimmuno-
SbfI.R (5(cid:1)-CATGCCTGCAGGATGAGTTCTTTCGAGGGGCAGA- beadswerethenaliquotedandtreatedwitheither(cid:2)proteinphospha-
TGG-3(cid:1)).ThePCRproductwasthenclonedintotheBamHIandSbfI tase ((cid:2)PPase) (New England Biolabs, Beverly, MA) or calf intestine
sitesofpEXAL1.1.TheArtemisFLAGinsertwassequencedtoconfirm phosphatase(CIP)(RocheAppliedScience)underconditionssuggested
that no mutations were introduced during the cloning process. The bythevendor.DNA-PKcsorproteinphosphatase-treatedsampleswere
DNA-PK expressionplasmidpPK1wasconstructedasdescribed(16). resolvedby7or8%SDS-PAGEandstainedwithCoomassieBlueR-250.
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InVivoV(D)JRecombinationAssay—ThecellularV(D)Jrecombina- Immunoprecipitation Assay—The immunoprecipitation assay was
tionassayandFACSanalysisoftransfectedcellswerecarriedoutas doneasdescribed(10).DNA-PKcswasdetectedwithanti-DNA-PKcs
described(13). antibody25-4(Neomarkers,Fremont,CA).Myc-taggedArtemiswas
ProteinPurification—DNA-PK andArtemis-myc-Hiswerepurified detectedwithanti-Mycantibody(Invitrogen).Equaltransfectioneffi-
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asdescribed(10).pEXAL1.1-ArtemisFLAGwastransfectedinto293T ciencieswereconfirmedbyco-transfectionofanenhancedgreenfluo-
cellsbycalciumphosphateprecipitation,andthecellswereharvested rescentproteinexpressionplasmidandFACSanalysis.SV40T-antigen
48 h after transfection. Artemis-FLAG was immunoprecipitated as wasusedasasampleloadingcontrolandwasdetectedwithananti-
described(10)withananti-FLAGM2monoclonalantibody(Sigma). SV40T-Agantibody(SantaCruzBiotechnology,SantaCruz,CA).
TheFLAG-taggedArtemiswasleftontheresinor,inthecasethatthe
proteinwaseluted,theimmunobeadswerewashedthreetimesin25 RESULTS
mMTris,pH8.0,50mMNaCl,10mMMgCl2,10%glycerol,and1mM ArtemisCanBePhosphorylatedatMultiplePositionsintheC-termi-
DTT,andthentheproteinwaselutedbyincubatingthebeadsin0.1 nalDomainbyDNA-PK —DNA-PK ishomologoustophosphatidy-
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mg/mlofFLAGpeptide(Sigma)inthesamebufferfor1hat4°Cwith linositol3-kinases,yetinvitroitphosphorylatesmanyproteintargetson
constantmixing.ThesupernatantwascollectedasthesolubleArtemis- serineandthreonineresidues(17,18).IthasbeenshownthattheDNA-
FLAGprotein. PK kinaseactivityisnecessarytoactivateArtemisasahairpinand5(cid:1)
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MassSpectrometry—ProteinstobeanalyzedwereresolvedbySDS- and3(cid:1)overhangendonuclease(10),andtheseactivitiesareverylikelyto
PAGEandstainedwithCoomassieBlueR-250.Thebandscorrespond- becriticaltoV(D)JrecombinationandNHEJinvivo.Wewereinter-
ingtothesampleswereexcised,andthegelpiecesweredestainedin50% estedinmappingthephosphorylationpositionsofArtemisbyDNA-
methanol.Next,thegelpiecesweredehydratedinacetonitrileandrehy- PK invitrobymassspectrometry(MS).Artemis-myc-Hisimmuno-
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dratedin10mMdithiothreitol(DTT)and0.1Mammoniumbicarbon- beads were incubated with DNA-PK under kinase permissive
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ate.Subsequently,thesampleswerealkylatedin50mMiodoacetamide conditionsandthenanalyzedbySDS-PAGEandCoomassiestaining
and0.1Mammoniumbicarbonate.Thegelpieceswerethendehydrated (see“ExperimentalProcedures”).Inordertoconfirmthatthisinvitro
inacetonitrile,rehydratedin0.1Mammoniumbicarbonate,dehydrated phosphorylationsystemmimicsotherDNA-PK kinasereactions,we
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againinacetonitrile,andcompletelydriedbyvacuumcentrifugation. firstanalyzedtheproteinbandcorrespondingtoautophosphorylated
Trypsinizationofthedriedpieceswascarriedoutin20ng/(cid:3)ltrypsinin DNA-PK . Nine phosphoserines and phosphothreonines were
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50 mM ammonium bicarbonate. The sample was digested overnight, detectedbyMS(Fig.1A).Sixofthesesites(Thr-2609,Ser-2612,Thr-
andthepeptideswereextractedfromthepolyacrylamidein2aliquotsof 2624,Thr-2638,Thr-2647,andSer-3205)havebeenreportedbyDou-
50%acetonitrile,5%formicacid. glasetal.(19),andthree(Ser-3821,Ser-4026,andThr-4102)arenovel.
Trypsinized samples were analyzed with the liquid chromatogra- Next, the untreated and DNA-PK -phosphorylated mammalian
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phy-MSsystem,whichconsistedofaFinniganLCQiontrapmassspec- ArtemiswasanalyzedbyMS.WhereastheuntreatedArtemis(purified
trometersystemwithaProtanananosprayionsourceinterfacedtoa frommammaliancells)hasonly3phosphorylatedserines,thephospho-
PhenomenexJupiter10-(cid:3)mC18reversed-phasecapillarycolumn.The rylatedArtemishas14phosphorylatedserinesandthreonines(includ-
data were analyzed by data base searching using the Sequest search ingthethreebasalphosphorylatedresidues)(Fig.1B).Becauseofthe
algorithm.Peptidesthatwerenotmatchedbythisalgorithmwereinter- limitationsofMS,phosphorylationpositionsonsomeoftheadjacent
preted manually and searched versus the EST data bases using the serinesorthreoninescannotbedefinitivelydetermined(seeFig.1B).It
Sequestalgorithm. wassuggestedpreviouslythattheDNA-PK consensusphosphoryla-
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33840 JOURNALOFBIOLOGICALCHEMISTRY VOLUME280•NUMBER40•OCTOBER7,2005
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
FIGURE1.PositionsofphosphorylatedserinesandthreoninesinDNA-PK andArtemisidentifiedbymassspectroscopy.A,DNA-PK autophosphorylationsitesidentifiedbyMS.The
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positionsofphosphorylatedserinesandthreoninesareshownonthetopoftheschematicDNA-PK protein.TheautophosphorylationsitesidentifiedbyDouglasetal.(19)areshownonthe
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bottomforcomparison.Theboldfaceanditalictext(T2620)indicatestheonlyunconfirmedphosphorylationsite.B,phosphorylationsitesofArtemisidentifiedbyMS.Theschematicstructure
ofArtemisisshowntoemphasizethemetallo-(cid:1)-lactamasedomain(darkgray),the(cid:1)-CASPdomain(lightgray),andtheC-terminaldomain(white).Positionsofphosphorylatedserinesfrom
untreatedArtemisareshowninthetoppanel;andpositionsofphosphorylatedserinesandthreoninesofDNA-PK phosphorylatedArtemisareshowninthebottompanelwiththethreebasal
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phosphorylationsiteshighlightedbyboldfaceanditalicletters.ThepositionsoftheSQandTQsitesarelabeledonthebottom.ThesourceofthecellsfromwhichArtemiswaspurifiedandthe
treatmentofArtemisareindicatedontheright.AdjacentserinesandthreoninesthatcannotbedistinguishedbyMSarenoted.
TABLE ONE
V(D)JrecombinationassayinArtemis-deficienthumanprimaryskinfibroblastscomplementedwithArtemisserineorthreoninepointmutants
Humanprimaryskinfibroblastswereco-transfectedwithV(D)JrecombinationsubstrateplasmidandplasmidsexpressingRAG1,RAG2,andoneoftheArtemis
proteins(wild-typeormutant).Asnegativecontrols,thesameco-transfectionswithouttheplasmidsexpressingRAG2((cid:2)RAG2)orwild-typeArtemis((cid:2)wtArt)
were performed. Wild-type Artemis was used as a positive control ((cid:3)wt Art). A pre-recombined substrate plasmid was transfected in order to evaluate
thetransfectionefficiency(T.E.).Ineachassay5(cid:4)104fibroblastswereanalyzedbyFACSanalysis.Thepercentagesofrecombination-positivecellsoutofthe
subpopulationoftransfectedfibroblastsareshown.
Controls
T.E. T91A T251A S362A S516A S534A S538A S548A S553A S560A S645A
(cid:1)RAG2 (cid:1)wtArt (cid:2)wtArt
Artemis-positivefibroblasts
Exp.1 57.25 0.02 3.54 3.19
Exp.2 57.63 0.05 4.06 4.24
Artemis-deficientfibroblasts
Exp.1 56.47 0.04 0.10 5.30 3.22 3.87 3.70 3.05 3.50 1.73 2.87 3.26 3.42 3.96
Exp.2 62.18 0.11 0.11 4.67 3.58 3.39 4.67 4.26 3.73 4.17/3.18a 3.64 4.19 3.76 4.02
aTwoindependentvalueswereascertained.
tionsitesareserinesorthreoninesfollowedbyaglutamine(SQorTQ) atSQorTQintoanalanineresidue(SertoAlaorThrtoAlamutations).
(20).However,noneoftheArtemisphosphorylationsitesbyDNA-PKcs Next, the 10 plasmids expressing different point mutants of Artemis
isatanSQorTQ,despitethefactthatthereisaclusterofSQandTQ weretestedfortheirabilitytofunctionallycomplementhumanprimary
sitesintheC-terminaldomainofArtemis(Fig.1B).Onlyoneofthe
skinfibroblastsdeficientforArtemisinaV(D)Jrecombinationassay
basalphosphorylationsiteswithinArtemisisatanSQsite(Ser-516).
(TABLE ONE) as described (13). All the mutants showed wild-type
It is interesting that even untreated Artemis purified from human
levelsofcomplementation,indicatingthatmutationsattheseSQand
293Tcellshasthreebasalphosphorylationsites.Inordertodetermine
TQsitesindividuallydonothaveaneffectonthefunctionsofArtemis.
whether this modification is conserved, human Artemis protein
ThisisinagreementwithourfindingthatDNA-PK phosphorylation
expressedfromabaculovirusininsectcellswassubjectedtoMSanaly- cs
sis.FLAG-taggedArtemispurifiedfrominsectcellsisalsophosphoryl- sitesofArtemisarenotatSQorTQsites(Fig.1B).
atedatthethreebasalphosphorylationsitesinadditiontoafourthone Full-length Artemis Purified from Mammalian and Insect Cells Has
(Fig.1B).Therefore,thekinaseactivitythatisresponsibleforthebasal DNA-PKcs-dependent Hairpin Opening and 5(cid:1) Overhang Processing
phosphorylationofArtemisexistsininvertebrates. Activities—It is interesting that insect cell-purified Artemis has similar
AsaninitialefforttoscreenforcriticalDNA-PK phosphorylation basalphosphorylationasthemammaliancell-purifiedArtemis(Fig.1B).
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site(s)ofArtemis,wemutatedeachserineorthreonineresiduelocated Wewereinterestedintestingiftheinsect-cellderivedArtemisisfunctional.
OCTOBER7,2005•VOLUME280•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33841
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
HumanArtemisexpressionconstructswereintroducedintodiffer- tionsfromthesesourcesweresubjectedtoaninvitronucleaseassay.
entproteinexpressionsystems.FLAG-taggedArtemisfrominsectand Artemis-FLAGpurifiedfrombaculovirus-infectedinsectcellsdiddem-
mammalian cells were generated. Subsequently, the protein prepara- onstrateDNA-PK -dependenthairpinopeningand5(cid:1)overhangproc-
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essingactivitiessimilartothewild-typeprotein(Fig.2,AandB,com-
parelanes4and6).
IntheabsenceofDNA-PK ,Artemishasexonucleolyticactivity(Fig.
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2,lanes3and5).Inordertoruleoutthepossibilitythattheexonucleo-
lyticactivitywasfromaproteinco-purifyingwithArtemis,C-terminally
FLAG-tagged Artemis expression plasmid and the corresponding
empty vector were transfected into mammalian cells. Proteins were
purifiedbyanti-FLAGantibodyfromboththeArtemis-transfectedand
themock-transfectedcells.Inthiscase,Artemiswaselutedfromthe
immunobeads(see“ExperimentalProcedures”)tominimizecontami-
nationfromtheagaroseresin.Bothproteinpreparationswereassayed
forhairpinopeningactivity(Fig.2B),andonlytheproteinpreparation
fromArtemis-transfectedcellsshowedsignificantexonucleolyticactiv-
ity(Fig.2B,comparelanes5and7),suggestingthatArtemishasintrinsic
exonucleolyticactivity.
Phosphatase-treatedArtemisHasIncreasedGelMobility—Although
phosphorylationbyDNA-PK isnecessarytoactivateArtemisasan
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endonuclease,wewereinterestedinwhetherbasalphosphorylationat
thethreesitesidentifiedbyMSplaysaroleinactivatingArtemis.To
addressthisquestion,wefirsttestedproteinphosphatase-treatedArte-
mis. Immunobead-immobilized Artemis-myc-His was first mock-
treatedorphosphorylatedbyDNA-PK .Thenafterextensivewashes
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withahighsaltbuffer,eachsamplewasdividedinto5aliquotsandwas
mock-treated, treated with (cid:2)PPase, or treated with CIP (Fig. 3A).
Finally,thephosphatase-treatedimmunobeadswereanalyzedbySDS-
PAGEfollowedbyCoomassiestaining.
Asobservedpreviously,untreatedArtemisappearsasadoubletafter
resolutionbySDS-PAGE(Fig.3B,lane1).However,afterphosphatase
treatment, the Artemis protein appeared less diffuse on the gel and
migratedfasterthantheuntreatedprotein(Fig.3B,lanes2–5).Com-
FIGURE2.InsectcellpurifiedArtemishaswild-typeDNA-PK -dependentendonu-
cleolyticactivities.C-terminallytaggedArtemiswasincubatedcwsiththesubstrateinthe paredwiththeuntreatedArtemis,DNA-PKcs-phosphorylatedArtemis
presenceandabsenceofDNA-PKcs,andthenthereactionmixtureswereresolvedby12% migratedevenslower,andtheresultingband(potentiallyconsistingof
denaturing PAGE. A, Artemis-FLAG immunobeads prepared from baculovirus-infected
insectcellshasexonucleolyticactivityandDNA-PK -dependenthairpinand5(cid:1)overhang multiplebands)appearedevenmorediffuse(Fig.3B,lane6).Phospha-
cs
processingactivities.B,solubleArtemis-FLAGpurifiedfrommammaliancellshasintrinsic tase treatment of DNA-PK -phosphorylated Artemis restored the
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exonucleolyticactivitycomparedwiththemock-purifiedprotein.LaneMcontainsanoligo-
sharpnessoftheproteinbandandincreasedthemobilityoftheprotein
nucleotideidenticaltothefragment5(cid:1)tothehairpintip(26nt)ofthesubstrate.Thesizes(in
nts)andtheschematicrepresentationsoftheproductsareindicatedontheright. inthegel(Fig.3B,lanes7–10).
FIGURE3.Artemiscanbephosphorylatedby
DNA-PK and dephosphorylated by protein
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phosphatasesinvitro.A,flowchartofphospho-
rylation/dephosphorylation of Artemis-myc-His
immunobeads.B,SDS-PAGEanalysisandCoomas-
siestainingofDNA-PK -andthenproteinphos-
cs
phatase-treated Artemis. Mock-treated or DNA-
PK -phosphorylated (3 pmol of DNA-PK )
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Artemis-myc-His (15 pmol) immunobeads were
washedanddividedinto5aliquots.Eachaliquot
wasthendephosphorylatedbydifferentamounts
of(cid:2)PPaseorCIPasindicated.Positionsofauto-
phosphorylatedDNA-PK ,CIP,(cid:2)PPase,immuno-
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globulinheavychain(IgHchain),andlightchain
(IgLchain)areindicatedbyarrowheads.Theposi-
tionofArtemischangeswithitsphosphorylation
statusandisindicatedbyabracket.Thesizesof
proteinmolecularweightstandards(laneM,in
kDa)areshownontheleft.
33842 JOURNALOFBIOLOGICALCHEMISTRY VOLUME280•NUMBER40•OCTOBER7,2005
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
FIGURE4.DephosphorylatedArtemis-myc-His
immunobeadsstillhaveDNA-PK -dependent
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endonucleolyticactivities.Hairpinopeningand
5(cid:1)overhangprocessingactivityassay(A)and3(cid:1)
overhang processing activity assay (B) of the
dephosphorylatedArtemisareshown.Approxi-
mately 1 pmol of untreated, (cid:2)-PPase-, or CIP-
treatedArtemiswastestedinanucleaseactivity
assayintheabsenceorpresenceof0.5pmolof
DNA-PK .Positionsandsizes(innts)ofthemajor
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productsareindicated.Theexonucleolyticprod-
uct(1nt)isindicatedbyexo.Diagramsadjacentto
thearrowsaredepictedtoemphasizethecleav-
agepositionsinthesubstratethatresultinthecor-
respondingproducts.Thestructuresofthesub-
stratesareillustratedonthetop,withanasterisk
indicatingthepositionoftheradioactivelabeling.
WehavereportedthataftertreatingtheArtemisimmunobeadswith
DNA-PK andextensivewashing,thephosphorylatedArtemisisnot
cs
abletodemonstrateanyendonucleolyticactivities(10).Wehadsug-
gestedthatthiswasbecauseDNA-PK ,whichpossiblyrecruitsArtemis
cs
to the substrate, was washed away; the same washing buffer used in
purifying Artemis by immunoprecipitation was sufficient to remove
DNA-PK . We have now found that DNA-PK remains associated
cs cs
withtheArtemisimmunobeadsafterthephosphorylationandstringent
washes(Fig.3B,lanes6–10).ItispossiblethatDNA-PK orautophos-
cs
phorylatedDNA-PK hasahigheraffinityforphosphorylatedArtemis.
cs
Phosphatase-treatedArtemisRetainsDNA-PK -dependentEndonu-
cs
cleolyticActivities—Inordertostudytheroleofbasalphosphorylation
ontheactivationofArtemisasanendonuclease,untreated,(cid:2)-PPase-
treated, and CIP-treated Artemis immunobeads were prepared as
describedinFig.3A,andanaliquotofthesebeadswassubjectedtoanin
vitronucleaseassayinthepresenceorabsenceofDNA-PK .Mock-
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treatedArtemisdemonstratedDNA-PK -dependenthairpinopening,
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5(cid:1)overhangprocessingactivities(Fig.4A,lanes1and2),aswellas3(cid:1)
overhangprocessingactivity(Fig.4B,lanes7and8).Mostinterestingly,
FIGURE5.TriplebasalsitemutantofArtemis(ARM49)caninteractwithDNA-PK .A,
both(cid:2)-PPase-treatedandtheCIP-treatedArtemiswereabletodemon- ARM49interactswithDNA-PK atwild-typelevel.Emptyvector,wild-typeArtemisc-sex-
cs
pressing,orARM49-expressingplasmidswastransfectedinto293Tcells,andMyc-
strateDNA-PK -dependentendonucleolyticactivitiesatlevelscompa-
cs taggedArtemiswasimmunoprecipitatedinthepresenceorabsenceofananti-Myc
rablewiththeuntreatedprotein(Fig.4,AandB,lanes3–6and9–12). antibody.B,ARM49wasexpressedatthewild-typelevel.Artemis,co-immunoprecipi-
TheTriplePointMutantofArtemisattheThreeBasalPhosphoryla- tatedDNA-PKcs,andtheloadingcontrolSV40largeT-antigenweredetectedwiththe
correspondingantibodies(see“ExperimentalProcedures”)byWesternblotanalysis.The
tion Sites Has DNA-PK -dependent Endonucleolytic Activities—In
cs sourceofproteins(fromimmunoprecipitatedmaterial(IP)orthecelllysate(input))is
ordertoconfirmourphosphatasetreatmentstudiesofArtemis,atriple indicated.
pointmutantofArtemiswasgeneratedwhereallthreebasalphospho-
rylationsites(Ser-385,Ser-516,andSer-518)weresubstitutedbyala- thewild-typeprotein,ARM49caninteractwithDNA-PK (Fig.5A),
cs
nines (mutant ARM49). First, the ability of ARM49 to interact with anditcanalsobephosphorylatedbyDNA-PK toalevelcomparable
cs
DNA-PK wasexaminedbyanimmunoprecipitationassay.Similarto with the wild-type protein (data not shown). The mutations did not
cs
OCTOBER7,2005•VOLUME280•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33843
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
FIGURE6.TriplebasalsitemutantofArtemis
(ARM49) has DNA-PK -dependent endonu-
cs
cleolyticactivities.Wild-typeArtemisorARM49
wasincubatedwiththehairpinand5(cid:1)overhang
substrate(A)orthe3(cid:1)overhangsubstrate(B)inthe
presenceorabsenceofDNA-PK .Labelsarethe
cs
sameasdescribedinthelegendtoFig.4.
seemtocompromisethestabilityoftheArtemisproteinbecauseitwas PK existbesidestheclusterofsites(Thr-2609toThr-2647)thatthey
cs
expressedatthewild-typeproteinlevel(Fig.5B).Inaninvitronuclease identified.
assay, ARM49 demonstrated DNA-PK -dependent hairpin opening RegardingArtemisitself,noneoftheinvitroDNA-PK -phosphoryl-
cs cs
and5(cid:1)overhangprocessingactivities(Fig.6A,comparelanes2–4),as atedsitesonArtemisislocatedatanSQorTQsite.Althoughsomeof
wellasthe3(cid:1)overhangprocessingactivity(Fig.6B,comparelanes2–4). thewellcharacterizedinvitroDNA-PK phosphorylationsitesareat
cs
ARM49iscapableofcomplementingArtemis-deficientfibroblastsin SQorTQsites(suchasp53(23)),phosphorylationsitesdeviatingfrom
aV(D)Jrecombinationassay(TABLETWO).Toexcludethepossibility this“consensus”havebeenreported.Infact,three(Thr-2624,Ser-3205,
ofgeneratingaDNA-PK -independentmutant,ARM49wasalsotested andSer-4026)outofthenineautophosphorylationsitesofDNA-PK
cs cs
foritsabilitytocomplementV(D)JrecombinationinDNA-PK -defi- are not at SQ or TQ sites. The DNA-PK phosphorylation sites on
cs cs
cientV3cells.TheV(D)JrecombinationdeficiencyinV3cellswasnot Ku70, Ku86, and XRCC4 are also at sites other than this consensus
complemented by transfecting in only ARM49 (data not shown). In (24–26).Therefore,DNA-PK doesnothaveastrictpreferenceforSQ
cs
summary,thesedatasuggestthatthemutationsatthebasalsites(Ser- or TQ. It has been suggested that DNA-PK prefers serines and
cs
385,Ser-516,andSer-518)didnotabolishtheabilityofARM49tobe threoninesfollowedbyahydrophobicaminoacidinvitro(18).Despite
activatedbyDNA-PK invitroandinvivo. studiesofotherproteins,Artemisisamongthefirsttoillustratethat
cs
DNA-PK phosphorylationcanhaveaphysiologicaleffectonthefunc-
cs
DISCUSSION tionofaprotein.
Artemis Is Phosphorylated by DNA-PKcs Only in the C-terminal Furthermore, the DNA-PKcs phosphorylation sites and the three
Domain—InlightofthefindingthatthekinaseactivityofDNA-PK is basalphosphorylationsitesofArtemisarealllocatedintheC-terminal
cs
necessarytoactivateArtemisasahairpinand5(cid:1)and3(cid:1)overhangendo- domain (Fig. 1B). Considering the crucial role of DNA-PKcs kinase
nuclease, we sought to map the DNA-PK phosphorylation sites in activityinactivatingArtemisendonucleolyticactivities,theC-terminal
cs
ArtemisaspartofanefforttostudytheregulationofArtemisactivities. domainmaybeanimportantregulatorydomainofArtemis.Mostlikely,
WealsomappedthephosphorylationsiteswithinDNA-PK itself.A theC-terminaldomainhasaninhibitoryroleintheactivationofArte-
cs
total of nine phosphorylated serines and threonines are detected in mis.Inlinewiththisthinking,ithasbeenshownrecentlythatthe“core”
autophosphorylatedDNA-PK .Sixofthesesiteswerereportedbefore Artemis (metallo-(cid:1)-lactamase domain/(cid:1)-CASP region) can comple-
(19),andthreeofthemwerepcsreviouslyunknown,indicatingthatour mentArtemis-deficientcellsforV(D)Jrecombination(14).4
DNA-PK invitrophosphorylationsystemissimilartowhathasbeen TheRoleoftheC-terminalDomainofArtemis—Artemisisastruc-
cs
usedbyothers.Inagreementwithourfindingofthethreenovelsites, ture-specific endonuclease and recognizes single- to double-strand
Lees-Millerandco-workers(incollaborationwithothers)(21,22)have
recentlysuggestedthatadditionalautophosphorylationsitesofDNA- 4U.PannickeandK.Schwarz,unpublisheddata.
33844 JOURNALOFBIOLOGICALCHEMISTRY VOLUME280•NUMBER40•OCTOBER7,2005
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
TABLE TWO
V(D)JrecombinationassaysinArtemis-deficienthumanprimaryskinfibroblastscomplementedwithARM49
TheexperimentswerecarriedoutasdescribedforTABLEONE.T.E.indicatestransfectionefficiency.
Controls
T.E. (cid:2)ARM49
(cid:1)RAG2 (cid:1)wtArt (cid:2)wtArt
Artemis-positivefibroblasts
Exp.1 61.54 0.09 2.17 4.54
Exp.2 58.37 0.05 6.08 6.69
Artemis-deficientfibroblasts
Exp.1 66.43 0.02 0.03 2.75 0.99
Exp.2 67.96 0.01 0.05 6.66 4.49/4.04a
aTwoindependentvalueswereascertained.
FIGURE7.ModelfortheactivationofArtemisby
phosphorylation.A,Artemiswithoutphospho-
rylationbyDNA-PK isinactiveasanendonucle-
cs
ase.IntheabsenceofDNA-PK ,thecatalyticcen-
cs
terofArtemisismaskedbytheC-terminaldomain.
B,onceArtemisisphosphorylatedbyDNA-PK ,its
cs
catalytic center is exposed. Artemis with this
“open” conformation is active endonucleolyti-
cally.Darkgraydiskrepresentsthemetallo-(cid:1)-lac-
tamaseandthe(cid:1)-CASPdomain(catalyticcore).
Theopentrianglewithinthedarkgraydiskindi-
catestheactivesite.Lightgrayovalsymbolizesthe
C-terminaldomainofArtemis,andthearchcon-
nectingthecoreandtheC-terminaldomaincorre-
sponds to a possible hinge region. The circled
minussignsontheC-terminaldomainin(B)denote
phosphorylatedserinesandthreonines.
transitions.IftheactivityofArtemisisnotproperlycontrolled,itcould ARM49 mutant was indistinguishable from wt Artemis regarding IR
potentiallycausegeneticinstabilitybecausesomeDNAmetabolicinter- resistance.ThisisconsistentwiththefactthatARM49demonstrateda
mediates mimic Artemis substrates. For example, DNA replication, mobilityshiftinamannerthatisindistinguishablefromwtArtemisin
DNAtranscription,andDNArecombinationallinvolveseparationof responsetoIR(supplementalFig.S2).BecauseIR-inducedphosphoryl-
thetwoDNAstrandsandgenerationofDNAsingle-todouble-strand ation of Artemis is lost in A-T cells (29), these three basal sites are
transitions.TheroleoftheC-terminaldomainmaybetoinhibitArte- unlikely to be the phosphorylation sites of ATM (or of ATR) upon
misintheabsenceofDNAdouble-strandbreaks.Onceadouble-strand exposure to ionizing radiation. Perhaps a common kinase present in
DNAbreakisgenerated,DNA-PK isactivateduponassociationwith vertebratesandinvertebrates(Fig.1B)phosphorylatesthesethreesites
cs
DNAends,andthenArtemiscanbeturnedonbyassociationwithand invivo.
phosphorylation by DNA-PK and function in V(D)J recombination OnegrouphasshownthatSer-645ofArtemisisphosphorylatedin
cs
andNHEJ. cellstreatedwithIRinanATM-dependentmanner(31).Wecreateda
The Role of Basal Phosphorylation—Phosphatase-treated Artemis S645AmutantandfoundthatcellsexpressingthisArtemismutantwere
andArtemisthathasmutationsatthethreeMS-identifiedbasalphos- indistinguishablefromwtintheirsurvivalinresponsetoIR(supple-
phorylationsites(ARM49)stillretainDNA-PK -dependentendonu- mentalFig.S1).Therefore,althoughthissitemaybephosphorylatedin
cs
cleolyticactivities(Figs.4and6),andARM49cansupportV(D)Jrecom- responsetoIRinanATM-dependentmanner,itdoesnotappeartobe
binationinaDNA-PK -dependentmanner(TABLETWOanddata functionallycritical.
cs
not shown). This illustrates that the DNA-PK -independent basal IsDNA-PK RequiredtoRecruitArtemistotheDNASubstrate?—We
cs cs
phosphorylationdoesnotaffecttheabilityofArtemistobeactivatedby have shown previously that after pre-phosphorylation of Artemis
DNA-PK .Recently,therehavebeenreportsthat,inadditiontoDNA- immunobeadsbyDNA-PK andextensivewashes,thephosphorylated
cs cs
PK ,activationofataxiatelangiectasiamutated(ATM)kinaseandpos- Artemis does not have endonucleolytic activities. We suggested that
cs
siblytheATM-andRad3-relatedkinase(ATR)canleadtothephos- thiswaslikelybecauseDNA-PK waswashedaway,andtherefore,the
cs
phorylation of Artemis, although it was not clear whether this is via physical presence of DNA-PK is required to recruit Artemis to the
cs
directphosphorylationorviaanindirectpathway(27–30).Weconsid- DNAsubstrate(10).However,datafromthisstudysuggestthatauto-
ered the possibility that the basal phosphorylation identified in our phosphorylated DNA-PK has very high affinity for Artemis and
cs
studycorrespondstoATM-and/orATR-dependentphosphorylation remains associated with the Artemis immunobeads after stringent
ofArtemis.Hence,inadditiontoV(D)Jrecombination,wealsoexam- washes.
inedtheionizingradiation(IR)sensitivityofcellstransfectedwithwt WorkfromothersmayshedlightonthephysicalrolesofDNA-PK
cs
ArtemisortheARM49expressingplasmid(supplementalFig.S1).The incomplexwithArtemis.Somehavesuggestedthatautophosphory-
OCTOBER7,2005•VOLUME280•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33845
ArtemisPhosphorylatedbyDNA-PKOnlyinC-terminalDomain
latedDNA-PK dissociatesfromDNAends(32,33),whereasothers 7. Rooney,S.,Chaudhuri,J.,andAlt,F.W.(2004)Immunol.Rev.200,115–131
cs
haveraisedthepossibilitythatautophosphorylatedDNA-PK simply 8. deVillartay,J.P.(2002)Curr.Opin.AllergyClin.Immunol.2,473–479
cs 9. Moshous,D.,Callebaut,I.,deChasseval,R.,Corneo,B.,Cavazzana-Calvo,M.,Le
changesitsconformation(butremainsassociatedwithDNAends)to
Deist,F.,Tezcan,I.,Sanal,O.,Bertrand,Y.,Philippe,N.,Fischer,A.,anddeVillartay,
allowthedownstreamNHEJfactorstoaccessDNAends(21,22).Thus, J.P.(2001)Cell105,177–186
upon autophosphorylation of DNA-PK in the Artemis-DNA-PK 10. Ma,Y.,Pannicke,U.,Schwarz,K.,andLieber,M.R.(2002)Cell108,781–794
cs cs
complex,ArtemiseitherdissociatesfromDNAends(alongwithDNA- 11. Rooney,S.,Sekiguchi,J.,Zhu,C.,Cheng,H.L.,Manis,J.,Whitlow,S.,DeVido,J.,Foy,
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substrate binding because of a DNA-PK conformational change.
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theDNAsubstrate. (2004)EMBOJ.23,1987–1997
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haveaninhibitoryroleinthebindingofArtemistoitssubstrate,andthe Res.30,3592–3601
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absenceofDNA-PK ,thecatalyticcenterofArtemisismaskedbythe 132–141
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C-terminaldomain.ThisinactiveconformationdoesnotallowArtemis 19. Douglas,P.,Sapkota,G.P.,Morrice,N.,Yu,Y.,Goodarzi,A.A.,Merkle,D.,Meek,K.,
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toprocessDNA.UponassociationwithandphosphorylationbyDNA-
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PK atmultiplesiteswithintheC-terminaldomain,theinhibitionof
cs 91–111
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fortheworkwithsupplementalFig.2,andtheSchwarzlaboratory,especially 1819–1828
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