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1、THEANATOMICALRECORD252:194±204(1998)InVivoandInVitroAssessmentofMitogenActivatedProteinKinaseInvolvementDuringQuailSecondaryPalateFormationB.M.HEHN,1M.F.IZADNEGAHDAR,1A.V.YOUNG,1J.S.SANGHERA,2,3S.L.PELECH,2,3ANDR.M.SHAH1*1DepartmentofOralBiology,FacultyofDentistry,TheUniversityof
2、BritishColumbia,Vancouver,BritishColumbiaV6T1Z3,Canada2DepartmentofMedicine,TheUniversityofBritishColumbia,Vancouver,BritishColumbiaV6T1Z3,Canada3KinetekBiotechnologyCorporation,Vancouver,BritishColumbiaV5Z1A1,CanadaABSTRACTSpatiotemporallyregulatedcellproliferationanddifferentia
3、tionarecrucialforthesuccessfulcompletionofmorphogenesisofthevertebratesecondarypalate.Anunderstandingofthemechanismsbywhichthesecellularphenomenaareregulatedduringpalatedevelopmentinvolvestheidenti®cationofthevarioussignaltransductionpathways.Inthepresentstudy,thepresenceandactiv
4、ationofmitogen-activatedprotein(MAP)kinaseswereinvestigatedduringthedevelopmentofquailsecondarypalate.Thepalatalshelvesweredissectedondays5±9ofincubation,homogenized,andcentrifuged,afterwhichthesampleswereseparatedbyanionexchangefastproteinliquidchromatography.Thefractionswereana
5、lyzedformyelinbasicprotein(MBP)phosphorylation.Inaddition,primaryculturesofquailpalatemesenchymalcells(QPMCs)weretreatedwithepidermalgrowthfactor(EGF)andpreparedforMBPphosphorylationassays.Atemporallyregulatedpatternofphosphotransferaseactivity,characterizedbyathree-foldincreasei
6、nphosphotransferaseactivitytowardMBPbetweendays5and8ofincubation,wasobservedduringquailpalatedevelopment.Westernblotting,usingMAPkinaseantibodies,demonstratedthepresenceofa42-kDaisoformbetweendays5and9ofincubation,duringwhichthelevelofproteinremainedconstant.Antityrosineimmunoblo
7、ttingwith4G10alsodetecteda42-kDaprotein.Phosphotransferaseassays,usingeitheraMAPkinase-speci®csubstratepeptide(S5)oraproteinkinaseCinhibitor(R3),furthercon®rmedthepresenceofaMAPkinaseinthedevelopingpalateofquail.Becausediversebiologicalprocessesoccurconcurrentlyduringinvivopalate
8、morphogenesis,theinvolvementofMAPkinasew
