Characterization of a peptide inhibitor of janus kinase 2 that mimics suppressor of cytokine signaling 1 function

2004·3.6 MB·English

by  Flowers, Lawrence O

CHARACTERIZATIONOFAPEPTIDEINHIBITOR
OFJANUSKINASE2THATMIMICSSUPPRESSOROF
CYTOKINESIGNALING1FUNCTION
By
LAWRENCEO.FLOWERS
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2004
ACKNOWLEDGMENTS
I amextremely grateful to my research advisor, Dr. Howard M. Johnson, for
giving me the unique opportunity to train in his laboratory. His superb mentoring,
motivation,andguidancehavemadeaninvaluablecontributiontomylife.Iwouldalso
—
liketoexpressmygratitudetothemembersofmydissertationcommittee Dr.Edward
M. Hoffmann, Dr. Janet K. Yamamoto, Dr. Ayalew J. Mergia, and Dr. William W.
—
Thatcher fortheirexpert technical assistance, critical review ofthe manuscript, and
patienceduringtheproductionofthiswork.
Moreover, I express my gratitude to thank Dr. Prem S. Subramaniam forhis
expertadvice,support,andhelpfulassistancewiththeexperimentalprotocolsusedinthe
current studies. I wish to thank Dr. Mustafa G. Mujtaba for his excellent technical
assistance. IalsoacknowledgeMr. MohammadI. Haiderforthepeptidesusedinthe
currentwork.SpecialthanksareofferedtoMr.TimothyH.Johnsonforhisassistancein
orderingresearchequipmentandsuppliesformystudies.Furthermore,Iamthankfulto
Dr. C. M. IqbalAhmedandMs. MorganR. Ellis.Ms. Elliswasveryinstrumentalin
performingthebindingassayspresentedinthisstudy.Thisworkwassupportedbythe
UNCF/MerckGraduateScienceResearchDissertationFellowshipgrant. Theauthoris
deeplyindebtedtoDr.BhavnaBhardwajandMr.NealA.BensonattheUF-ICBRFlow
CytometryCoreLaboratoryfortechnicalsupportduringtheflowcytometricexperiments.
The support of fellow graduate students in the Microbiology and Cell Science
Departmentisalsogratefullyacknowledged.
n
Additionally,Iacknowledgevariousmembersofthefacultyandsupportstaffin
theMicrobiology andCell ScienceDepartment. Iwouldalso liketo thankthe many
nameless mentors that have guided me throughout my educational journey from
elementaryschooltograduateschoolfortheirfriendship,insightfuladvice,andguidance
throughoutmyentireeducationalcareer.Lastly,Iwishtothankmyfamilyandspecial
friendsfortheirnever-endingencouragementandtirelesssupportthroughoutmylifeand
educationalendeavors.
111
TABLEOFCONTENTS
pane
ACKNOWLEDGMENTS
ii
LISTOFTABLES vi
LISTOFFIGURES vii
ABSTRACT ix
CHAPTER
INTRODUCTION
1 1
ProteinTyrosineKinases 1
Cytokines 2
InterferonGamma 4
Interleukm-6 7
TheJAK-STATPathway 9
JanusKinase(JAK) 12
SignalTransducersandActivatorsofTranscription 15
NegativeRegulationoftheJAK-STATPathway 18
SuppressorsofCytokineSignaling(SOCS)ProteinFamily 21
JAK-STATPathwayandCancer 26
ProstateCancer 29
TargetedApproachtoCancerTherapy 31
SignificanceoftheStudy 33
2 MATERIALSANDMETHODS 35
Peptides 35
CellCultureandViruses 35
BindingAssay 36
CompetitionExperiments 37
InvitroKinaseAssays 38
AntiproliferationAssay 40
ImmunoblotAnalysis 40
FlowCytometry 42
AntiviralAssay 43
Immunoprecipitation 44
CellCycleAnalysis 45
IV
3 RESULTS 47
TyrosineKinaseInhibitorPeptide(Tkip) 47
TkipBindstotheAutophosphorylationSiteofJAK2 48
TkipInhibitsKinaseActivityofJAK2 50
TkipdoesnotInhibitKinaseActivityofVEGFRorc-Src 51
TkipInhibitsKinaseActivityofEGFR 51
TkipInhibitsKinaseActivityofJAK2andEGFRinaDoseDependent
Manner 55
TkipPreferentiallyBindstotheActivatedJAK2AutophosphorylationSite 55
TkipInhibitsSTATlaActivationNotActivationofVEGFR 56
TkipInhibitsAntiviralActivity 60
TkipInhibitsMHCClassIUpregulation 60
EffectsofTkiponSTAT3ActivationinHumanProstateCancerCellLines 63
TkipInhibitsCellCycleProgressioninHumanProstateCancerCellLines 63
TkipInhibitsAntiproliferativeActivityofPC-3ProstateCancerCells 64
4 DISCUSSION 68
REFERENCES 74
BIOGRAPHICALSKETCH 83
v
LISTOFTABLES
Table Eige
1 HostDefenseProteinsInducedbyInterferongamma 7
2 CytokinesandtheJAK-STATPathway 10
3 SequenceComparisonoftheMammalianJAK2AutophosphorylationSite 14
4 SequenceComparisonoftheJAKFamilyAutophosphorylationSite 15
5 AminoAcidSequencesofthePeptidesUsedinthisStudy 48
6 EffectsofTkipontheCellCycleofDU145ProstateCancerCells 66
7 EffectsofTkipontheCellCycleofLNCaPProstateCancerCells 66
8 InhibitionofPC-3CellProliferationbyTkip 67
vi
LISTOFFIGURES
Figure page
1 OverviewoftheIFNySignalingMechanismThroughtheJAK-STATPathway 11
2 GeneralDomainSchematicoftheJanusKinase(JAK)ProteinFamily 15
3 GeneralDomainSchematicoftheSignalTransducerandActivatorof
Transcription(STAT)ProteinFamily 18
4 GeneralDomainSchematicoftheSuppressorofCytokineSignaling(SOCS)
ProteinFamily 23
5 BindingofTkipbyJAK2AutophosphorylationPeptide,JAK2WT 49
6 KinaseInhibitorySpecificityofTkip 50
7 TkipDoesNotInhibitAutophosphorylationofVEGFR 52
8 TkipDoesNotInhibitTyrosinePhosphorylationActivityofc-Src 53
9 TkipInhibitsAutophosphorylationofEGFR 54
10 PositiveControlforInVitroKinaseAssay 54
11 DoseResponseofTkipInhibitionofJAK2,IFNGR-1,andEGFR
PhosphorylationInVitro 56
12 BindingofUnphosphorylatedJAK2WTPeptideVersusPhosphorylatedJAK2WT
PeptidetoTkip 57
13 TInkhiipbiItnhViEbiGtsFIIFnNdyucIendduAccetdivSatTiAonTo1faVAEctGiFvaRtiionnBionvWiIneSHAorCteilclsEBnduotthDeoleisalNoCetlls
(BAECs) 59
14 TkipInhibitstheAntiviralActivityofIFN7AgainstEMCVirus
onWEHI-3Cells 61
vi1
Figure Eage
15 DofowMnHreCguCllaatsisonIoonfIWFINS7HInCdelulcsedUsCienllgMTekimpbraneExpression 62
16 EffectsofTkiponHumanProstateCancerCellLinesDU145andLNCaP 65
17 ProposedModelofInhibitionofIFN7MediatedSignalingbyTkip 70
18 AlignmentoftheKIRofSOCS-1andTkip 73
vni
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
CHARACTERIZATIONOFAPEPTIDEINHIBITOR
OFJANUSKINASE2THATMIMICSSUPPRESSOROF
CYTOKINESIGNALING FUNCTION
1
By
Lawrence0.Flowers
August2004
Chair:HowardM.Johnson
MajorDepartment:MicrobiologyandCellScience
Tyrosine kinases are extremely important biological molecules involved in
numerouscellularsignaltransductionevents.Fidelityofthesesignaltransductionevents
is essential for normal cellular functions. Many diseased states such as cancer and
autoimmune diseases are associated with defective tyrosine kinases. Positive and
negative regulation ofcytokines such as gamma interferon (IFNy) is key to normal
homeostatic function. Negativeregulation ofIFNy in cells occurs viaproteins called
suppressorsofcytokinesignaling(SOCS) 1 and3. SOCS-1 inhibits,IFNyfunctionby
bindingtotheautophosphorylationsiteofthetyrosinekinaseJAK2.
Ashort12-merpeptide,WLVFFVIFYFFR(Tkip),wasdevelopedthatboundto
theautophosphorylationsiteofJAK2,resultingininhibitionofitsautophosphorylationas
wellasitsphosphorylationofIFNyreceptorsubumtIFNGR-1 tyrosine440.TheJAK2
tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine
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autophosphorylation of vascular endothelial growth factor receptor (VEGFR) or
phosphorylationofasubstratepeptidebytheprotooncogenetyrosinekinasec-src.Tkip
alsoinhibitedepidermalgrowthfactorreceptor(EGFR)autophosphorylation,consistent
withthefactthatEGFRisregulatedbySOCS-1andSOCS-3,similartoJAK.2.Although
TkipbindstounphosphorylatedJAK2autophosphorylationpeptide,itbindssignificantly
bettertotyrosine1007phosphorylatedJAK2autophosphorylationpeptide.SOCS-1only
recognizestheJAK2siteinitsphosphorylatedstate. Thus,TkiprecognizestheJAK2
autophosphorylationsitesimilartoSOCS-1,butnotpreciselythesameway.
ConsistentwithinhibitionofJAK2,Tkip inhibitedantiviral activityandMHC
classIupregulationoncellsataconcentrationofapproximately10uM.Thisissimilarto
theKdofSOCS-3fortheerythropoietinreceptor.Thesedatarepresentaproof-of-concept
demonstration ofa peptide mimetic ofSOCS-1 that regulates JAK2 tyrosine kinase
function.
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