Identification of a Mutant Kinase/ATP Analog Pair_蚂蚁淘，【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城

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Identification of a Mutant Kinase/ATP Analog Pair

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IdentificationofaMutantKinase/ATPAnalogPair

ScottT.Eblen,N.VinayKumar,andMichaelJ.WeberDepartmentofMicroBIOLOGyandCancerCenter,UniversityofVirginiaHealthSystem,Charlottesville,VA22908ExcerptedfromProtein:ProteinInteractions,SecondEditionEditedbyEricaA.GolemisandPeterD.Adams

ABSTRACT

ProteinKinasecascadesregulatemanyaspectsofcellularbiochemistryandphysiology.Determiningthedirectsubstratesofproteinkinasesisimportantinunderstandinghowthesesignalingenzymesexerttheireffectsoncellularprocesses.ArecentdevelopmentinthisareatakesadvantageofthesimilarityintheATP-bindingdomainsofproteinkinases.ConservedresiduesintheATP-bindingsitecontainlargesidechainsthatcomeintoclosecontactwiththeN6positionofboundATP.Mutationofoneormoreoftheselargeresiduestoalanineorglycinegeneratesa"pocket"intheATP-bindingsitethatallowsthemutantkinase,butnotthewild-typekinaseorothercellularkinases,toutilizeanalogsofATPwithbulkysubstituentssynthesizedontotheN6position.KinasereactionsperformedwithamutatedkinaseandrADIolabeledATPanalogsinacellularlysateallowspecificlabelingofdirectsubstratesofthemutantkinase,whichcanlaterbeidentifiedbymassspectrometryorothermeans.Oncepocketmutationshavebeengeneratedinthekinase,itisnecessarytoscreenATPanalogsfortheircompatibilitywiththekinasemutant.Thissteprequiresanactiveformofthewild-typeandmutantkinase,eitherasrecombinantactivatedprotein,orasproteinthathasbeenimmunoprecipitatedfromtransfected,stimulatedcells.Inaddition,aknowninvitrosubstrateforthekinaseisalsorequired.ScreeningthemutantkinaseswithATPanalogsisbestperformedinatwo-stepprocess.ThefirststepinvolvesassayingtheABIlityofanATPanalogtoinhibittheincorporationofradioactivephosphatefromnormal[y-³²P]ATPintoaknownsubstrateinaninvitrokinasereaction.ThismethodallowsalargenumberofATPanalogstobescreenedeasily,withouttheneedforcreatingnumerousradiolabeledATPanalogs,savingtimeandbypassingtheneedforunnecessaryradioactivework.Moreover,quantitativecompetitionstudiescanbeperformedtoidentifyanalog-mutantpairswithhighaffinity.ItisimportanttokeepinmindthattherearetworeasonsanATPanalogcaninhibitincorporationoflabeledphosphateintosubstrate.ThefirstisthattheATPanalogisagoodATPsourceforusebythekinaseandcompeteswiththe[y-³²P]ATPasasubstrate.ThesecondisthattheATPanalogissmallenoughtointeractwiththeATP-bindingsiteofthekinasebutisunabletobeusedbythekinaseasanATPsourceandisthereforesimplyblocking[y-³²P]ATPfromtheATP-bindingsite.Todistinguishbetweenthese,itisnecessarytodirectlytesttheabilityofthemutantkinasetophosphorylateasubstratewithanalogATP.DoingthisrequireseitherradiolabeledATPanalogsor,preferably,aphospho-specificantibodytothephosphorylationsiteontheknownsubstrate.Inourexperiments,weusedacommerciallyavailablephospho-specificantibodytotheERK2substrateElk1.Aphospho-specificantibodyallowsalargenumberofATPanalogstobescreenedwithouttheirhavingtobemaderadioactive.Inthisassay,activewild-typeormutantkinaseismixedwithATPanalogandsubstrateinaninvitrokinasereaction.Thereactionisthenanalyzedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE),transferredtonitrocellulose,andeitherstainedwithPonceauSandcountedbyCerenkovcounting(if[y-³²P]ATPanalogisused)orimmunoblottedwithaphosphospecificantibody(ifavailable)todeterminetheextentofsubstratephosphorylation.Thekinase/ATPanalogpairthatprovidesthebestphosphorylationofthesubstrateisthenusedforfutureexperiments.Onlythoseanalogsthatcannotbeusedbythewild-typekinaseandothercellularkinasesarechosen.

MATERIALS

Buffers,Solutions,andReagents

M2Lysisbuffer(cold)[II],freshlyprepared
50mMTris-base(pH7.4)
150mMNaCl
10%glycerol
1%TritonX-100
0.5mMEDTA
0.5mMEGTA
50mMNaF
40mMβ-glycerophosphate
5mMtetrasodiumpyrophosphate
0.1mMsodiumvanadate
10µg/mlaprotinin
5µg/mlleupeptin
2mMphenylmethylsulfonylfluoride(PMSF),fresh
Aceticacid(1%)
10xKinasebuffer
250mMHEPES(pH7.4)
100mMmagnesiumacetate
10mMdithiothreitol(DTT)
KinasereactionbufferA(fortheanaloginhibitionassay)
1xkinasebuffer
10µMATP
100µMATPanalog
10µCi/reaction[y-³²P]ATP
1-5µgofaknownsubstrate/reaction
KinasereactionbufferB(forthesubstratephosphorylationassay)
1xkinasebuffer
100µMATPanalog
1-5µgofaknownsubstrate/reaction
10µCi/reaction[y-³²P]ATPanalog(requiredonlyifaphospho-specificantibodytothesubstrateisnotavailable)
1xPBS(cold)[I]
25.6gofNa₂HPO₄&dot;7H₂O
80gofNaCl
2gofKCl
2gofKH2PO₄
Bringto1literwithdistilled,deionizedwater;autoclavefor40minat121°C.
PonceauSsolution
0.5%(w/v)in1%aceticacid
2xLaemmlisamplebuffer
100mMTris(pH6.8)
2%SDS
20%glycerol
4%β-mercaptoethanol(addedfresh)
Dulbecco"smodifiedEaglemedium(DMEM)
Fetalbovineserum(FBS)

CellsandAntibodies

COS-1Africangreenmonkeykidneyepithelialcells(ATCC)
Antibodiestotheepitopetagontheproteinkinaseofinterest
Aphospho-specificantibodytoaknownsubstrateofyourkinase(ifavailable)

Gels

SDS-PAGEgel

SpecialEquipment

Microcentrifugewithcoolingfacility
Transfertankforwesternblotting
30°Cwaterbath
Scintillationcounter
Heatingblock,presetto100°C(I)

AdditionalReagents

[y-³²P]ATP(6000Ci/mmole)
AgoNIST(I);e.g.,epidermalgrowthfactor,serum,platelet-derivedgrowthfactorLipofectamine
M2agarose(forFLAG-taggedproteinkinases)
ProteinA-orproteinG-conjugatedagaroseorSepharoseCL-6beads(forpreparationofantibody,seestep7)
BCAproteinassaykit
Hypodermicneedle1",27gauge
Syringe,1cc
Nitrocellulosemembrane
Vacuumflask
Shaker,presetto4°C
Incubator,presetto37°C[I]
60-mmtissueculturedishes[I]

Plasmids

Expressionplasmidofchoice

METHOD

CellularTransfectionandImmunoprecipitationBeforeproceedingwiththeexperimentsoutlinedbelow,allkinasepocketmutantsshouldbecharacterizedfortheirexpression,localization,specificactivity,andsubstratespecificity.Theresultsshouldbecomparedwiththoseobtainedfromthewild-typekinasetoensurethatthemutationdoesnotsignificantlyalterthesecharacteristics.

GrowCOS-1cellsinDMEMsupplementedwith10%FBS.Platecellsthedaybeforetransfectionat4x10⁵cellsperdishin60-mmtissueculturedishes.Incubateat37°Cand5%CO₂overnight.
Transfectcellswith2µgofexpressionplasmidusingLipofectamine,accordingtothemanufacturer"sspecifications.Wehavefoundthata1:4ratioofDNAtoLipofectamineisoptimalfortransfectionofCOS-1cells.
Incubatethecellsfor24-72hoursat37°Cand5%CO2.Therestofstep3andstep4areoptionalandcanbevarieddependingonthekinaseofinterest.Formitogen-activatedkinases,serum-starvetransfectedcellsbywashingtwicewithroom-temperaturephosphate-bufferedsaline(PBS)andplacinginserum-freemediafor4-12hrpriortoharvest.
Stimulatethecellsfor10minbeforeharvestwithanagonistthatactivatesthekinaseofinterest.Harvestthecellsat24-72hrposttransfection.
WashthecellstwiceincoldPBSonice.Drainthedisheswell.Add0.5mlofM2lysisbufferandscrapeeachdishofcellsintomicrocentrifugetubes.Vortex.Letthecellslyseonicefor15min,vortexingoccasionally.Centrifugeat15,000gand4°Cinamicrocentrifugefor15min.Transferthesupernatanttoafreshtube.
PerformaproteinassayonthecelllysatesusingaBCAassaykitfromPierce.Wetypicallyobtain0.5mgofproteinper6-cmdish.
Preparetheantibodyfortheimmunoprecipitation(HarlowandLane1999).WeuseM2agaroseforFLAG-taggedERKs.Iftheantibodytotheepitopetagontheproteinisnotpreconjugated,prebindtheprimaryantibodyforyourepitopetagtoproteinA-orproteinG-conjugatedagaroseorSepharoseCL-6beads.Ifnecessary,useasecondaryantibodytolinktheprimaryantibodytothebeads(HarlowandLane1999).Theamountofantibodytousewillbeantibody-andprotein-specific.Forthesmall-scaleimmunoprecipitationsdescribedinstep8,0.1µgofantibodyperimmunoprecipitationshouldbesufficient.Batchprebindingcanbeperformedwithenoughantibodyandconjugatedbeadsforallofyourreactions.Prebindingshouldbeperformedin1mlofM2celllysisbufferfor1hrat4°Cwithconstantagitation.AdditionalunconjugatedSepharoseCL-6beadscanbeincludedintheprebindingtoincreasethebeadvolumeforeachkinasereaction.Wefindthatatotalof30µlofbeadvolumeisoptimalforeachreaction.Afterprebinding,centrifugetheantibodymixtureat15,000ginamicrofugefor30sec.AspiratethesupernatantandwashthebeadstwiceinM2lysisbuffertoremoveunboundantibody.Aspiratethewashbuffer.ResUSPendthebeadsinanequalvolumeofM2lysisbuffertomakea1:1bead:bufferslurry.
Aliquot50µgofcellularprotein(asdeterminedinstep6)intofreshmicrocentrifugetubes,usingonetubeforeachreaction(usemoreorlessprotein,dependingontheabundanceandactivityofyourkinase).Wetypicallyperformkinasereactionsinduplicatetocontrolforvariabilityintheassay.AddM2lysisbuffertoatotalvolumeof750µl.
Aliquotthepreconjugatedantibody(60µlof1:1bead:bufferslurryfromstep7)intoeachofthetubescontainingcelllysate.Incubateat4°Cfor2hrwithgentlerocking.
Centrifugethesamplesat15,000gat4°Cfor30sec.Aspiratethesupernatant.Washthebeadsthreetimeswith1mlofcoldM2lysisbuffer,followedbytwowasheswith1xkinasebuffer.Aspiratethefinalwashbufferdowntothebeads.Keepthesamplesonice.
Justbeforestartingthekinasereaction,aspiratetheresidualkinasebufferwitha27gaugeneedleattachedtoa1-ccsyringeandavacuumflask.Keepthebeveledsideoftheneedlefacingthewallofthetubetopreventaspiratinganybeads.Usethisimmunoprecipitatedproteinfortheanaloginhibitionassayandthesubstratephosphorylationassay.
Analoginhibitionassayandsubstratephosphorylationassay:2x50-µgaliquotsofcellularproteinarerequiredfortheanaloginhibitionassayandafurther2x50µgarerequiredforsubstratephosphorylation.
1. Add40µlofkinasereactionmixturetoeachtubeonice.(Note:KinasereactionmixtureAisforanaloginhibitionassays.KinasereactionmixtureBisforthesubstratephosphorylationassay.)Mixgentlybyflickingthetubewithyourfinger.
2. Incubatesamplesina30°Cwaterbathfor10min.Placethesamplesoniceandimmediatelyadd40µlof2xLaemmlisamplebuffertostopthereaction.Vortexthesamples.
3. Heatthesamplesfor4mininaboilingwaterbathorheatingblock.Centrifugethesamplesat15,000gatroomtemperaturefor30sec.
4. LoadthesupernatantonanSDS-polyacrylamidegelandelectrophorese.
5. Transferthegeltonitrocellulosemembrane.
6. Placethemembraneinaglassdish.Stainthemembranefor5minwithPonceauSsolution.Removethestainanddestainthemembranetwice,for5mineach,with1%aceticacid.
7. If[y-³²P]ATPor[y-³²P]ATPanalogisused,cutthesubstratebandsoutofthegelandcountthemindividuallybydryCerenkovcountingonascintillationcounter.Ifaphospho-specificantibodytothesubstrateisavailable,leavetheblotintactandimmunoblotwiththeantibody(HarlowandLane1999).
8. Fortheanaloginhibitionassay,calculatethepercentageoflabeledphosphorylationfromeachreaction.Thecpmfromduplicateassaysshouldbeaveraged,andthevalueobtainedfromthekinasereactionsthatdidnotcontainATPanalogshouldbesetto100%.Thencomparetheaveragecountsfromtheotherkinasereactionstothoseofthecontrolreactionandexpresstheratioasapercentage.
9. Forthesubstratephosphorylationassay,usethemutantkinase/ATPanalogpairthatgivesthehighestphosphorylation,eitherthroughimmunoblottingorradioactivemeasurements,forfuturestudies.