同位素法测定底物磷酸化活性方法 Phosphorylation of Substrates相关交流-蚂蚁淘在线

PhosphorylationofSubstrates

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

ABSTRACT

Ideally,onewouldliketobeabletodirectlyphosphorylatesubstratesinanintactcell.ThiscouldpotentiallybeperformedbyintroducingATPanalogintoliveorpermeABIlizedcells.However,cellsareimpermeabletoATP,andtheadditionoflabeledATPanalogtodigitonin-permeabilizedcellsresultsinthehydrolysisoftheanalogATPwithin1min(Chaudharyetal.2002).Therefore,wehavechosentoapplythistechniquetocelllysatesorfractions.Herewedescribetwoapproachestodetectdirectsubstratesofaproteinkinase.Thefirstapproachinvolvesidentificationofkinase-associatedsubstratesbyimmunoprecipitatingataggedformofthemutantkinasefromtransfectedCOS-1cellsandperformingakinasereactionbytheadditionof[γ-³²P]ATPanalog.Thesecondapproachinvolvestheadditionofrecombinantmutantkinaseand[γ-³²P]ATPanalogtocelllysates.WehaveusedthesetechniquesforthephosphorylationofERK2substrates;however,thismethodologycanbeappliedtootherproteinkinases.

MATERIALS(forApproachesIandII,asIndicated)

Buffers,Solutions,andReagents

Recombinantkinase(II)
[γ-³²P]ATPanalog(I+II)
SepharoseCL-6Bbeads(I)
Lipofectamine(I)
AgoNIST(I),e.g.,epidermalgrowthfactor,serum,platelet-derivedgrowthfactor
FLAGM2agarosebeads(I)
SDS,20%(I)
M2lysisbuffer(II)(seeProtocol1)
PBS,roomtemperature(I)andicecold(I+II)(seeProtocol1)
Serum-freemedium(I,e.g.,Dulbecco"smodifiedEaglemedium)
FLAGpeptide(5mg/mlinhypotoniclysisbuffer)(I)
Kinasebuffercontainingbenzamidine,10mmand100mmNaCl(II)
2xLaemmlisamplebuffer(I)
Hypotoniclysisbuffer(I)
20mmHEPES(pH7.4)
2mmEGTA
2mmMgCl₂

Cells

COS-1cells(I+II)

Plasmids

Maxi-prepplasmidDNAscarryingepitope-taggedversionsofthewild-typeandmutantformsofthekinaseofinterest(I)

SpecialEquipment

Tissueculturedishes(I+II)
Hypodermicneedle,11/4"",27gauge(I)
MiniprepColumns(I)
Dialysiscassette,10,000molecular-weightcutoff(II)
SDS-polyacrylamidegel(I)
Rotator,setupinacoldroom(4°C)(I)
Boilingwaterbath,presetto100°C(I)
Incubator,presetto37°C,5%CO₂(I+II)
Incubator,presetto30°C(I+II)
Microfugecooledto4°C(I+II)

METHOD

ApproachI:PhosphorylationofKinase-associatedSubstratesItisimportanttostandardizetheprocedureinsmall-scalereactionspriortoscaling-uptheproceduretoidentifynovelsubstrates.

Plate1x10⁶COS-1cellsonto100-mmtissueculturedishes1daypriortotransfection.Incubatethecellsat37°Cin5%CO₂.Transfectthecellswith6µgofplasmidDNAcarryingepitope-taggedversionsofthewild-typeormutantformsofthekinaseofinterest,and24µlofLipofectamine,accordingtothemanufacturer"sinstructions.
24-72hrposttransfection,washthecellstwicewithroom-temperaturePBSandserum-starvetheminserum-freemediumfor4-12hr.Thenstimulatethecellswithagonistfor5-10min.Dependingonwhichkinaseisbeingstudied,differentstarvationtimesandstimulantscanbeused.
WashthecellstwicewithcoldPBSonice.Addhypotoniclysisbuffer(0.5ml/100-mmdish).HypotoniclysisbufferisutilizedtopreserveERK2interactionswithassociatedproteins.Foridentifyinginteractingsubstratesthatformstableassociations,M2lysisbuffer(seeProtocol1)oranotherbufferwithincreasingconcentrationsofdetergentorsaltscanbeused(HarlowandLane1999).
Scrapethecellstogetherandtransferthemtoa1.5-mlmicrocentrifugetube.Donotvortex.Lysethecellsbycentrifugingat15,000ginamicrocentrifugefor20minat4°C.
Transferthesupernatants(~1mgofprotein)intofreshtubes.Keepsmallaliquotsaside(20µg)tochecktheexpressionlevelsoftheexpressedproteins.
WashtheappropriatequantityofSepharoseCL-6Bbeads(30µl/sample)withhypotonicbufferandmixwithFLAG-M2agarosebeads(10µl/sample),orbeadsconjugatedtoanotherantibodytothechosenepitopetag(~1µgofantibodyper1mgofproteinlysate).
Washthemixtureofbeadstwiceinhypotoniclysisbufferandthenaddthemtothelysatespreparedinstep5(40µlpersample)forimmunoprecipitation.Incubatethebeadsinlysatefor1-2hrat4°Cwithconstantrotation.
Washtheimmunoprecipitatesthreetimeswithhypotoniclysisbuffer(1mlperwashforeachsample).Afterthefinalwash,aspiratethefewremainingdropsofbufferusinga11/4inch,27gaugeneedle.
Performkinasereactionsinatotalvolumeof40mlcontainingkinasebufferand10µCi[γ-³²P]ATPanalogfor3-15minat30°C.Wefindthatshorterkinasereactiontimesarepreferable,butthetimeofincubationshouldbeoptimizedforeachkinaseandcelltype.Weusepilotreactionswithlabeledanalogtoidentifyconditionsforwhichthereactioncanbescaledup.Scale-upreactionsareperformedwithunlabeledATPanalogandtraceramountsoflabeledATPanalogontheimmunoprecipitate.Eventually,reactionsshouldbescaleduptogivesilverorCoomassiebluestainableamountsoftheproteinofinteresttoallowidentificationbymassspectrometry.
Poollabeledandunlabeled(whenscalingup)kinasereactionsandadd80µlofFLAGpeptide(5mg/mlinhypotoniclysisbuffer).EluteFLAGERK2-QGanditsassociatedproteinsbyincubatingonicefor15minfollowedbyincubationat30°Cfor15min,withoccasionalvortexing.
Resolvethesamplesin1xLaemmlisamplebufferona1-dimensionalSDS-polyacrylamidegel.
ToperformisoelectricfocusingpriortoSDS-polyacrylamideelectrophoresis,add20%SDStothesamplestoafinalconcentrationof2%andboilthesamplesfor4min.RemoveSepharoseCL-6BandM2agarosebeadsbyloADIngthereactionintoaminiprepcolumnplacedinamicrocentrifugetubeandcentrifugingfor30secat15,000g.

ApproachII:PhosphorylationofSubstratesinCellLysateswithExogenousKinaseThisapproachusesrecombinantproteinkinasetophosphorylatesubstratesinacelllysate

Platecellsandincubateat37°Cand5%CO₂untilthecellsare80%confluent.WashthecellstwiceonicewithcoldPBS.Drainthedisheswell.Add0.5mloffreshM2lysisbufferper100-mmdishandscrapethecellsintomicrocentrifugetubesonice.Keepthesamplesonicefor15min,vortexingoccasionally.
Centrifugethesamplesat15,000ginamicrocentrifugeat4°Cfor15min.Transferthesupernatanttoafreshtubeonice.
Poolthelysatesanddialyzethemagainstkinasebuffer(withoutATPorDTT)containing10mmbenzamidineand100mmNaCl.Dialyzetwicefor2hreachin1literofbufferusinga10,000MWCODialysisCassette.ThiswillgetridofcellularATP.Performaproteinassayonthelysate.Dialyzedlysatescanbestoredat-70°Cuntiluse
Addpurified,activerecombinantmutantkinaseto100µgofcelllysate.Weuse10-100ngofactivatedERK2,butthismustbedeterminedempiricallyaccordingtothespecificactivityofthekinaseofinterest.
Add10µCiofradiolabeledATPanalog.Mixandincubateat30°Cfor3-15min.Wefindthatshortreactiontimes(3min)areoftenpreferable.Thisismostlikelyduetodepletionoftheanalogovertimeandtheabilityofphosphatasesinthereactiontodephosphorylatesubstrates.

REFERENCES

ChaudharyA.,BruggeJ.S.,andCooperJ.A.2002.Directphosphorylationoffocaladhesionkinasebyc-src:EvidenceusingamodifiednucleotidepocketkinaseandATPanalog.Biochem.Biophys.Res.Commun.294:293-300.HarlowE.H.andLaneD.L.1999.UsingAntibodies:ALaboratoryManual.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork.

Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:Protein:ProteinInteractions,SecondEdition:AMolecularCloningManual,editedbyEricaA.GolemisandPeterD.Adams,©2005byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.457-461.

Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission.

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同位素法测定底物磷酸化活性方法&nbsp;Phosphorylation&nbsp;of&nbsp;Substrates

同位素法测定底物磷酸化活性方法 Phosphorylation of Substrates