MMB 233 Protein Kinase C Protocols

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1、Hoang-DungTRANandfriendsMethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME233ProteinKinaseCProteinKinaseCProtocolsProtocolsEditedbyEditedbyAlexandraC.NewtonAlexandraC.NewtonHoang-DungTRANandfriendsInsandOutsofProteinKinaseC31TheInsandOutsofProteinKinaseCAlexandraC.Newton

2、1.ProteinKinaseCTheseminaldiscoveryofproteinkinaseC(PKC)byNishizukaandco-workers(1)inthelate1970sprovidedthefirstchapterinthestoryofoneofthemoststudiedenzymesinbiology.ThesubsequentfindingthatPKCtransducessignalsthatcauselipidhydrolysis,followedshortlythereafterbythediscoverythatP

3、KCwasthelongsought-afterreceptorforthepotenttumor-promotingphorbolesters,catapultedPKCtotheforefrontofresearchonsignaltransduc-tion.Morethan35,000researcharticleshavebeenpublishedonPKC.Anabundanceofreviewsdescribethestructure,regulation,andbiologicalfunctionofPKCandtheinterested

4、readerisreferredtotheseforamoredetailedoverviewthanthebriefoneprovidedbelow(1–11).ThegoalofthisvolumeistopresentalabmanualonclassicandnoveltechniquesthatarecurrentlyusedforstudyingPKC.2.PKCPrimerPKCcomprisesafamilyofserine/threoninekinaseswhosemembersplayapivotalroleincellsignal

5、ing.Specifically,PKCisozymestransducethemyriadofsignalsthatproducelipidsecondmessengers.ExamplesofphysiologicalstimulithatactivatePKCaremitogens,whichoperatethroughtyrosinekinasereceptors,orcatecholamines,whichoperatethroughGprotein-coupledrecep-tors.PKCcanalsobehyperactivatedbyt

6、reatingcellswithphorbolesters(seeChapter34).ThesemoleculesbindPKCovertwoordersofmagnitudemoretightlythandiacylglycerol,andcoupledwiththeirresistancetometabolism,causeessentiallyconstitutiveactivationofPKC.From:MethodsinMolecularBiology,vol.233:ProteinKinaseCProtocolsEditedby:A.C

7、.Newton©HumanaPressInc.,Totowa,NJ3Hoang-DungTRANandfriends4NewtonTherearethreesubclassesofPKCs,whichhaveincommonanamino-terminalregulatorydomainlinkedtoacarboxyl-terminalkinasedomain(Fig.1).Theregulatorydomaincomprisesatleastonemembrane-targetingmodulethatdirectsPKCtothemembrane

8、inresponsetogenerationoflipidsecondmessengers(s