Calcium-dependent protein kinase : activation mechanism and interacting proteins

140 Pages·1997·5.2 MB·English

by  Yoo, Byung-Chun, 1964-

CALCIUM-DEPENDENTPROTEINKINASE:
ACTIVATIONMECHANISMANDINTERACTINGPROTEINS
By
BYUNG-CHUNYOO
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARHALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPIHLOSOPHY
UNIVERSITYOFFLORIDA
1997
Tomyfatherandlatemother
ACKNOWLEDGMENTS
Iwouldliketothankmyadvisor.Dr.AliceC.Harmon,forintroducing
metooneofthemostexcitingfieldsinplantscience,andforsupport,
guidance,andencouragementduringmyPh.Dprogram.Iwouldalsoliketo
thankthemembersofmycommittee,Drs.RobertJ.Perl,WilliamB.Gurley,
DavidA.Jones,andDonaldR.McCarty,fortheirscientificadviceand
discussionthroughoutmystudies.Iamgratefulformypastandpresentlab
members.Specialthankstothepastlabmember,CandaceMcCafferyforher
assistance.
Iwouldliketoexpressmydeepestappreciationtoallofmyfamily
member,especiallymyfatherandlatemotherfortheiremotionaland
physicalhelp.Mywarmthanksgotomyson,Jaewoong,forgivingmea
joyfullife.Lastly,Idonotknowhowtothankmycolleagueandwife,Jung-
Youn,foreverythingshehasdoneforme
111
TABLEOFCONTENTS
ACKNOWLEDGEMENTS
iii
LISTOFTABLES vi
LISTOFFIGURES vii
ABSTRACT viii
CHAPTERS
1. LITERATUREREVIEW 1
Introduction 1
StructuralFeaturesandSequences 2
BiochemicalPropertiesofCDPK 8
PhysiologicalRolesofCDPK 11
24.. ACTIVATIONMECHANISMOFCDPKa 18
Introduction 18
ExperimentalProcedures 20
Results 27
Discussion 47
3. IDENTIFICATIONANDCHARACTERIZATIONOFINTERACTING
PROTEINSOFCDPK 55
Introduction 55
ExperimentalProcedures 57
Results 66
Discussion 88
REGULATIONOFSERINEACETYLTRANSFERASEBYCDPK 94
Introduction 94
ExperimentalProcedures 96
Results 100
Discussion 107
IV
REFERENCES 112
BIOGRAPHICALSKETCH 129
V
LISTOFTABLES
2-
T3-able page
2-1. KineticparametersofCDPKandthechimericenzyme,CDPKa(l-328)-
VU-1 28
2. EffectofCLDontheactivityoffull-lengthandmutantCDPKa 44
1. CharacteristicsofcDNAclones 70
3-2. cDNAclonesencodingproteinsthatshowsequenceidentitiestoknown
genes 71
3-3. SummaryofphosphorylationofGIPsbyCDPKs 84
VI
1- LISTOFFIGURES
2-
Figure page
1. StructuralorganizationofCDPK 4
1. Strucuralfeaturesofrecombinantproteinsusedinthisstudy 29
2-2. Purityofrecombinantproteins 32
22--3. BindingofbiotinylatedCLDbyfull-lengthandtruncatedCDPKa 36
3-
2-4. ComplexformationbetweenCLDandsyntheticpeptides 39
2-5. EffectofCLDoninhibitionofGST-CDPKa(l-312)bypeptide310-332 42
23--6. ActivationofCDPKaandtruncationmutantsbydigestionwith
4- trypsin 46
2-7. ActivationofHis6-CDPKa(l-398)byadditionofCLDorcalmodulin 48
8. ModelsforregulationofCDPKactivity 50
1. PurifiedclonesencodepeptidesthatinteractonlywithCDPKy. 67
3-2. NucleotidesequenceoftheGipcDNAclonesandthededucedprotein
sequence 72
3-3. PhosphorylationofGIPsbyGST-CDPKy. 80
4. CDPKyinteractsspecificallywithpolyubiquitin 83
1. Alignmentofaminoacidresiduesofsoybeanserineacetyltransferase
withknownSAT 101
4-2. PhosphorylationmakessSATinsensitivetothefeedbackinhibition
byL-cysteine 105
4-3. DeterminationofIC50forGST-CDPKpwithL-cysteine 106
4-4. sSAThasonepossiblephosphorylationsiteforCDPK 108
Vll
AbstractofDissertationPresentedtotheGraduateScliool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
CALCIUM-DEPENDENTPROTEINKINASE:
ACTIVATIONMECHANISMANDINTERACHNGPROTEINS
By
BYUNG-CHUNYOO
May,1997
Chairman; Dr.AliceC.Harmon
MajorDepartment: PlantMolecularandCellularBiology
Theactivityofcalmodulin-likedomainproteinkinase(CDPK)is
regulatedbythedirectbindingofCa2+,UnmodifiedsoybeanCDPKaanda
chimericenzymeinwhichthecalmodulin-likedomain(CLD)wasreplaced
byVU-1calmodulinhadsimilarvaluesofVmaxgpp(3.19,3.46and3.60,3.93
pmol/min/mg,respectively),andeachwasactivated30-to70-foldbyCa2+.To
askifactivationresultsfromthebindingoftheCLDtotheautoinhibitory
(junction)domainofCDPKainamanneranalogoustotheactivationof
calmodulin-dependentenzymesbycalmodulin,recombinantCLDand
truncationmutantsofCDPKawereexpressedinbacteriaandhighlypurified.
Inblotoverlays,biotinylatedCLDboundtomutantscontainingresidues312-
328ofthejunctiondomain.InanelectrophoreticmobilityshiftassayCLD
boundsyntheticpeptidescontainingresidues318-332inacalcium-dependent
Vlll
manner,providingdirectevidenceforbindingofCLDtoasiteinthejunction
domain.MutantsofCDPKafromwhichallorpartoftheCLDhadbeen
deletedwereconstitutivelyinactive.Additionof20 CLDtothesemutants
inthepresence,butnottheabsence,ofcalciumstimulatedtheiractivities,but
tovariousdegrees.His6-CDPKa(l-328),whichcontainednoneoftheCLD,was
activatedonly5-fold,buttheactivityofHis6-CDPKa(l-398),whichretained
nearlyhalfoftheCLDinitssequence,wasstimulated64-fold.Thelatter
activityapproachedthatofunmodifiedCDPKaandwashalfmaximalata
CLDconcentrationof7jxM.OurresultssuggestthatbindingofCLDtothe
junctiondomaincontributesto,butisnotsufficientforactivation.Although
calmodulinsupportedfullactivityofthechimericenzyme,itsadditionto
His6-CDPKa(l-398)resultedinactivitythatwasonly6%ofthatofthe
unmodifiedenzymeandwhichwashalfmaximalat20 Arabidopsis
calmodulin.Theseresultssupporttheconclusionthatsimplebindingofthe
calmodulin-likedomaintothejunctiondomainisnotsufficientfor
activation.
ToidentifytheinteractingproteinsofCDPKs,interactioncloningwas
employed.GST-CDPKa,p,andywereautophosphorylatedandusedasprobes.
Twelveinteractingcloneswereisolatedandcharacterized.Eightoutoftwelve
wereidentifiedfromadatabasesearch.Fourofthemwerephosphorylatedby
CDPKs.Oneoftheinteractingproteins,soybeanserineacetyltransferase
(sSAT),whichisinvolvedinL-cysteinebiosynthesis,showedthatthe
phosphorylationaffectedtheallostericregulation.Phospho-MBP-sSATwas
insensitivetofeedbackinhibitionbyL-cysteine.Thestoichiometrywas0.96
molofphosphate/molofMBP-sSAT.Thesedatasuggestthattheplantmay
haveyetanothermeanstoregulateL-cysteinebiosynthesis.
IX
CHAPTER
1
LITERATURE REVIEW
Introduction
Calciumhasbeenwellrecognizedasanintracellularsecondmessenger
inplants.Transientchangesinintracellularfreecalciumconcentrationshave
beenconnectedtomanysignaltransductionpathwaysinresponsetoa
numberofphysiologicalstimuli,includinglight,touch,wind,pathogenic
elicitors,cold-shock,gravity,andhormonessuchasabscisicacid,auxin,
cytokinins,ethylene,andgibberellicacid(Bush,1993,1995,Gilroyetah,1993,
PoovaiahandReddy,1993,RazandFluhr,1992,TrewavasandKnight,1994).
TherestingleveloftheintracellularCa^+concentrationismaintained
attheverylowlevel(30-100nM),whileduringstimulationtheCa^+
concentrationinthecytoplasmincreasesashighas10(j.M,dependingon
stimuli(TrewavasandKnight,1994).Althoughtheprecisemechanismthat
linksthesestimuli-inducedchangesincytosolicCa^+concentrationtothe
physiologicalresponseshasyettobedefined,understandingofcalciumsignal
transductionpathwaysinplantshasbeenfurtheredbythediscoveryof
proteinsthatserveasthetargetsofcalciumregulation.Amongtheseare
calmodulinandCDPKs(calmodulin-likedomainproteinkinaseorcalcium-
dependentproteinkinase)(RobertsandHarmon,1992).Thesetwoproteins
appeartobepresentubiquitouslyinplants.Calmodulindoesnothave
intrinsicenzymaticactivity,butuponbindingtocalcium,itmodulates
activitiesofmanyotherproteins.CDPK,ontheotherhand,hasprotein
1