Ea FGF

Characterization of the FGF Receptor Signaling Complex in Xenop us laevis during

Earl y Embryonic Development.

PaulaRyan s.se

Athesi ssubmitted totheSchoo lof Gradua te Stu die sfor thepartialfulfillmentofthe

Requirements forthe degree of Masters of Sc ience

.Faculty of Medicine Memori al Univers ityof Newfoundland

01999

St. John's Newfoundl and

Dedicated to my parents for their support throughout my education. They are, and alwayswill.bemypillarof strength. Thank-you

Abstract

A stablecomp lex is formed betweenthefibroblastgro wth factor recep tor(FGFR)and SH2-containingproteins.during early developm entin Xenopus laevis. Theresul ts presented in thisthesis demonstratethat phosph orylation ofPLCyl and its associatio nwithFGFRIoccurs during mesoderm inductioninvitro. PLCyl is involvedinintracellular signaling pathway ofFOFduringthe time mesoderminduction is occurri nginthe embryo. PLCyl isspecifically phosphorylatedduringearly-tomid-bl astula stages, yet theexpression levelof PLCyl protein is constan t throughout these stages of developm ent. Itis speculated thatPLCylisinvolvedina negati ve feedbackloop,down-regulatingtheFGFR.Thetiming of thein vitroexperiments(3D-minutetreatment ofFGF) provides evide nce that FGF maybea component of the vegetalinducingsignal. There wereseven other phosphorylated bandsco-immoprecipatedwith PLCyl or FGFR duringearly embryo development. These substrates were identified as phosphatidylin ositol 3-kinase (p!3'K).GTPase-activating protein (Gap).

SHP2 phosphatase.Nck and three nck-associatedproteins: NAP123.NAP

iii

81 and NAP65. Sonof Sevenless (50 S) andgrowthfactorreceptor binding protein2 (GRB2)were identifiedinthiscomplex. Neither 50S norGRB wasphosphorylated. 1bisreport provides evidence fortheimportanceof these substrates forFGFRsi.gnaling during earlyembryonicdevelopmenL

Ackn owledgements

[ am deeplyindebtedtomy supervisorsDrsLaura Gillespie and Gary Paterno. who have supportedmyMasten program and offered valuable supportthrough my years workingwiththemIam grateful tothemfor their guidanceandyears of enjoyable work experience.lbey are excelleor teachers, who gavemethe opportunity togainvastexperience and knowledgein the science industry.Thank-you.

I appreciate all of the help provided by the staff at the Mlcrocorcpurer/Workstation Resource Center(MWRC). for their valuable computer assistance.

IwouldJike tothank:thestudents andstaffofDr.AlanPater'sLabfor allowingmetousctheircomputer'facilities duringthewritingof thisthesis.

Iam. gratefultothefacultyandstaffoftheTcayFoxLabs forproviding afabulouswork environment. It was a pleasure to go to workwithintelligent friendswhogracefully offered their assistance.

Finally,my love and appreciatio ngo tomyhusband, Cory,forhis support and understan din gduringthedura tionof my educationalprogram.

Table of Contents

Chapter 1 Introduction Page

1.1Introduction 1

1.2Xenopus laevis:aScientific Model 3

1.3 Earlyembryo nicdevelopment 4

1.4Mesoderm Induc tion 7

1.5Mesoderm InducingFactors 13

1.6 FibroblastGrowthFactor Receptor 21 1.7 Mechanism of Actionof the Receptor 26 1.8Substratesof Receptor tyrosine Kinases 33

1.9Prospects forThesis 41

Chapter 2 Materials and Methods

2.1Embryos,Dissectionsand lnductionsAssays 42 2.2MesodennInduction Experiments 44 2.3Immo precipationand WesternBlotting 44

2.4Nck-AssociatedProteins 52

2.5Antibodies 53

Chapter 3 Results: part 1

60-70 Phosphorylation of PhospholipaseCyl anditsassociationwith theFGFReceptor is developmentallyregulatedand occursduring Mesoderm inductioninXenopus laevis.Devel.Biol(1994) 166:10 1-111.

Chapter 4 Result: part 2

4.1G1PaseActivatingProtein 4.2PI3'Kinase 4.3S11P2Phosphatase 4.4Nck-AssociatedProtein 4..5Non-AssociatedEffectors

Chapter 5 Discussion References

71 72 74 76 79 81 85 97

Appendix

118

Identificationofphosphorylatedproteinsassociatedwith thefibroblast Growth Factor Receptor Type 1 during early Xenopus Development Biochemicaland BiophysicalResearck Comp.244:4763-767.

List of Tables Page

TableIAComponentof NAM(Norma lAmphibian Medium) 43

TableIS Componentsof NAMSolutions 43

Table 2AffinitiesofVarious Monoclonal 45

Antibodiesfor ProteinAor ProteinG

Table 3 ComponentsofSampleBuffer and Triton Medi um 46

Table4Componentsof5DS-PAGE Ge ls 47

Table5 Components ofElectrode Buffer and TransferBuffer 48

List of Figures

B&:..L!TimeCoursefor theDevelopin gXenop us embry o

~Mechanism of Mesoderm Induction during

blastula Xenopusembryos 9

.B.&!J.

ThreeSignalModel 12

.B.gj"dSummaryof MesodermInductioninXenopuslaevis 20

~Sch e maticrepresentationofthestructural features

of FGFR 24

.Ei&-L§TheRas pathwa y 30

fu..11PI3'KPathway 31

Fig 1.8PLCyPathway 32

Fig3.1 Detection ofXenopusFGFRIand PLCy-l 63

~Phosph orylationof PLCy-l during mesoderm induction

byFGForbyvegetal cells 64

~Assoc iation ofPLCy-lwithFGFRIinXenopus

blastulaeandinmarginal zone cells 64

&l,...4Stage-epecific phosphorylationof PLCy-1 65 .Ei&...l,i.AssociationofPLCy-1 with FGFRIduring

early development 66

B.&:..l.QAnalysis ofpbosphoryrosylprotein s associated

with FGFRIinXenopusblastulae 66

.Eig],,:1Assoc iationof GRB2 and SOS1with FGFRI

inXenopusblastulae 67

viii

~AssociationofGAPwithRiFRlinXenopusblastulae 73

~AssociationofPl3'KwithRiFRinXenopusblastulae 75

~Association ofSHP2with FGFR 77

~Nck-Associared proteinswithin theFGFRI

complex formedinXenopusblastulae 80

~SRC 82

EiU.fiSHC 84

fl.g"j,lSummaryof Proteins Associated 86

List of Abbreviations and Symbols Used

AC

aFGF Anti-XFR AP Bek bFGF BMP-4 BSA Ca(NO,),.4H,O CML DAG DEAE--HPLC ECDG F EDTA EGF EGFR eFGF FGF FGFR Flg GAP GDP GRB2 GTP IIRP Ig-like IP IU kDa MAPK MBT M-eSF MEK MgS04.7H,tJ MJF

(animalcap) (acidicFOp)

(Anti -Xenop usFGFR)

(Anuno nium persulfate) (Bacterial expressedkinase ) (basicFGF)

(bone morphogenetic proteins) (BovineSerum Albwn) (Calciunnitrate)

(Chronic myelogenousleukemia) (diacyl glycerol)

(diethylanUnoetbyl-higbperformanceliquid chromotography)

(Embryonalcarcimoma-derived growthfactor) (ethylenediarretetraceticacid disodiumsalt hydrate) (epidermal growth factor)

(EGFReceptor) (embryonicFGF) (Fibroblast Growth Factor) (FGF receptor) (FM5--like geoeFGFR1) (GTPase-activationprotein) (guanine di-phosphate)

(Growth factorreceptor-binding protein) (Guanine tri-pbospbate)

(hydrogenperoxide) (lnununogolbulin-like) (inununoprecipitated) (Internationalunits) (ki1oda1ton)

(MitogenActivatingproteinkinase) (mid-blastulatransition) (macrophage colony-stimulatingfactor) (MAPkinase Idnase)

(magnesium.sulfate) (Mesoderm lnducingFactors)

mRNA MZ NAK NAP NAM NF200 NGFR POGF PECAM-I PI PB'K PI-4,5-P2 PIP2 PKC PLCyl PMSF l'IPases py RGB Rpm RTK 50S 50S-PAGE SH2 50S SSB 1EMED

TGFfl

TLCK Y UV VZ WASP WB Wnt Xbra XTC Xwnt-8

(messengerRiboNucleicAcid) (mar ginal zone) (Nck-Associatedkinase) (Nck-Associatedprotein) (Normal amphibian medium) (anti-neurofi1ament) (nervegrowthfactorreceptor) (platelet-derivedgrowthfactor) (platele tendothelialcelladhesionmolecule) (Phosphotidylinositol)

(phosphatidylinosito13'-kinase) (phosphatidylinositoI-4-pbospbate) (phos pbatidylinositoI4,5-bispbos phate) (proteinkinaseC)

(PhospbolipeseCyl) (pbenylmethylsulfonyfluoride) (phosphotyrosine pho spba tases ) (anti-pbos phoryros ine) (Runninggel buffer) (revolutionsper minute) (receptortyrosinekinase) (Sodiumdodecyl sulfate) (SOS-polyacrylimidegelelectrop horisis) (Soc homology2domain) (Sonof sevenless) (SDS samplebuffer)

(N,N,N,N'-Tetra-Methyletylenediamin e) (transforminggrowthfactorbeta)

(l-chloro-3-tosylamido-7-amino-2 heptane hydrochloride) (Tyrosine)

(ultra-vio let) (vege talzone)

(Wiskott-Aldrichsyndromeprotein) (Westernblots)

(Wingless) (Xenopusbrncbyury) (Xenopustissue culture) (Xenopus Wingless 8)

Chapter 1

1.1 Introduction

Cell-cell interactionsareessential for proliferationanddifferentiationof cells during embryogenesis. Receptor-ligandcombinationsthatactivatesignal transductionpathways are known mediators ofceU~1linteractions. The expression pattern of these signalin g molecules andreceptors determines which cells can exchangeparticulartypesof information. Oncethetarget cell has receivedthesignalthere isan alteration of biochemical pathwaysthat controlgene expression,the cell cycle, or metabolism withinthetarget cell.

Fibroblast growth factors(FOPs)are responsible for a mechanism importantfor cell-cell interaction.. The FGFfamilyconsists of about twenty familymembers(KlintandClaesson-Welsh, 1999). FGF-l and FGF-2,also called acidic FGF (aRJf) and basic FGF (bFGF), exhibit the widest expression among cells (McKeehan, 1998). FOFs act through the FGF receptor (FGFR) where activation leads to tyrosine autophospborylation ofthe receptor (Ullrich and Schlessinger, 1990). Tyrosine phosphorylation sites serve as high affinity binding sites for SRC homology (SH2) domain-

containingsignal transduction molecules(review edinIsaacs, 1997). 1bese moleculesinitiate acascadeof events, whicheventually resultinbiological responses,often involvingchangesingene transcription.

FGFsare regulatory peptideswhichfunction in tumorigene sis,repairof tissue injury, and embryonicdevelopment (Godspodarowicz,1991). bFGF is importantinthe field of vasculogeaesis and angiogenesis where it wasthefirst identified growth factor for vascularendothelial cells and serves as an autocrine growth factor, controlling the proliferation, migration, and differentiation of that cell type (reviewedinGodspodarowicz,1990). RJFs have various physiologicaleffectsdepending on the specificcell type. They caninducemitogenesis,support cellsurvivalandeither induce or inhibit cellular differentiation (Godspodarowicz , 1990). Tn vivo,FGFshave been shownto participatein mesodenninduction and tobeimportantfor normal gastrulation movementsinearly Xenopuslaevis embryodevelopment(Slacket aL,1987, ReviewedinSlack. 1994). InappropriateFOF expression or altered FGFsignaling may contribute toa number of pathologies ,suchascancer, diabetic retinopathy,rheumatoid arthritis,andarteriosclerosis (Chabrier,1996, Coffin et al; 1995). MutationsinFGFR lead to skeletaldisorderssuchas Achondroplasia (dw arfism) or Crouzon syndrome. However, the exact

mechanismsthatgiverisetoallofthesediseasesremain largelyunknown. A fullertmdetstanding of thebiology of thevariousFGFs andthe FGFR mechanism.mayenablethe developmentoftberapeutic approachesthatcan alterreceptorfunction.

1.2 Xenopus laevis:

a Scientific Model

Xenopus laevis,theAfricanclawedfrog. was utilized for studying signalingmechanismsof tbeFGfR.'Theadvan~gesofthissystem are:

I)Eggs develop outside thefemaleand are large enou gh(1-2mm) that microinJectionsanddissections

are

possible.

2)XOIOpUScareandmaintenanceisminimalinthelaboratorysetting.The oocytescan beobtainedbya simple injection ofchorionicgonadotropinand large nwnbersofrelativelybardyeggscanbe obtained.

3) Xenopus development is well cbamcterizedand documented thus standaIdizingtheclassificationof the developmentprocess.

4)lbeembryos ofXenopusareextremely baldy and are highly resistantto infectionaftermicrosurgery.

5)In vitrofertilizationoftheembryos allow sthepopulation of eggstobe at thesarre stageand gives accessibilityto allstages.

6)Everycellintheembryo containsyolkthatserves asa nutrientsupp ly.

'Ibis nuuiemsupply enables fcagI:rentsof the embryo to beincubatedin minimalsaltmediumduringexperimental conditions.

7)The overallstructure of frogembryos andtheir developmentisvery similarto thatof highervertebrates.

1.3 Early Embryonic Development

Before fertilization.,Xenopus eggs have an animal-vegetal polarity, whichisvisible: as a pigmentedanimalbalfandan unpigmentedvegetalhalf.

lbe animal hemispherecontains small and medium sizedyolkplatelets, whereasthe vegetal hemisphere contains large yolkplatelets. 'Theunfertilized egg isenclosedinaprotective vitelline membrane.whichisembeddedina gelatinouscoat (Wolpe rt.199 8).

After fertilization,. the vitelline membraneliftsotTtheegg surface and within fifteen minutestheegg rotateswithinit andthepigmentedanimal regionpointsupwards. Shortlyafter fertilizationtheegg cortex rotates 30%

relative totheinnercytoplasm. Cortical rotationdeterminesthe futuredorsal side oftheKenopusembryo,which developsopposite thesiteofspermentry andisca1ledthegraycrescent(Gerhart etal.,1986 ;Vincentetal.,1986).The Nieuwkoop centerisbelow thegraycrescent onthedorsal side. This regionis able to exert a special influence on surrotmding tissue and can determinebow thecells willdevelop . 'Thesperm provides aspatial cue that orients the rotation of the cytoplasm, which is essential fordorsal development. If subco rtical rotationisblocked, for example by ultra-violet(UV)irradiation of the embryo.then proper axisformationisinhibited(Vincent and Gerhart, 1987). These'rotationally challenged' embryos are rescued whenembedded inagar tomimictherotation (VincentandGerhart, 1987). 'Thedirectionof cytoplasmi cmovement determinesthefuturedorsal and ventral sideofthe embryo. TIlls is the first step in pattern formation during embryonic development.

First cleavage occurs about 90 minutes after fertilization at room temperature and dividesthecell throughthe animaland vegetal hemispheres,

separatingtheegg intoright andleft halves.1besecond cleavageisatright angles tothefirst cleavage .and separatesdorsalfromventralside."Thethird cleavageisalongtheequator separatingtheanimalfrom thevegetal half.

Subsequentcleavages occur at approximately3Q..minuteintervals,atroom temperature, until blastulastage(Nieuwkoop and Faber.1975).

Asdivisioncontinues.acavityor blastocoel fonnsin thecenterof the embryoandissurrounded by a thin layerof ectodermal cells and a deeper layer of endodermalcells. At blastula,theembryo existsasa hollow sphere (six hoursof development, 5000cells) (NieuwkoopandFaber.·1975).

Maternal contribution of proteins andmessenger RNA (mRNA) accumulatedduringoogenesis are required for nonnal developmentofthe embryoto themiddleof blastula stage. Transcription oftheembryonic genome begins during blastula stageatapoint known asthemid blastula transition(MB'D(NewportandKirschner,1982).Atstage 10.ninehours after fertilization. gastrulation begins. During gastrulation thereisan involutionof presumptiveendodermandpresumptivemesoderm tothecorrect positions for furtherdevelopmentof these body structures (Wolpert,199 8) (See figure 1.1).

1.4 Mesoderm Induction

Theblastula embryo canbesubdividedinto different regions on the basis of the cell type each regionwilldevelop(fatemap). Theanimal cap (AC) or presumptive ectoderm comprises ofthe upperhalfof the blastocoel roof.The region aroundtheequator,ormarginalzone(MZ)isthepresumptive mesoderm and thebottom of the embryo consists of the yoIlcy vegetal zone(VZ)or presumptiveendodenn (SeeFigure1.2). Atblastula stage,theequatorial cells, which contact boththeanimaland vegetalcells inthe equator,differentiate into mesodennIfthe stage-S embryo isdivided into explants ofanimal marginal,and vegetal cells and culturedinsalt solution,theanimalexplants willdevelop into epidermis;marginalzone explants primarily into mesoderm tissues;andvegetal explants into endodermal cells.Howe ver .animal capswill also form mesodermiftheyare combinedwithvegetal pieces ortreatedwith FGF (Nieuwkoop,1975)(See Figure 1.2B). These results suggestthatthe marginal zone cells developinto rresoderm because of their proximitytothe vegetal cellsthatsecrete aninducer.Inthenormal embryo,thecellsinthe animalhemisphere are thoughttobetoofarfrom the source oftheinducerand differentiate into ectoderm instead of mesoderm. 'Thesuggestedinducer probablyinvolves a member ofthetransforminggrowthsfactor beta (fGfl})

0

. : " ':':"·"1

· _-

. . . ..

Flgure 1.1Tune Course fortheDevelopingXenopusEmbryo

"-" ' - -- -1

1.2d.

~Thisfigure:represents stagesofthedeveloping Xenopus embryo showing cleavage furrows anddevelopmentalproces ses. The timeafter fertilizationthatisrequiredfor embryos to reacheachstageisrepresentedin days(d)or hours(h)(NieuwkoopandFaber.1975).

B

~

_saltsolution

~

_FOF

"e getalzone

iii)

V" g etal pieces

Figure 1.2 Fate MapofaXenopusembryo A)Showsa blastulaembryo subdivided intothree regions. 1)presumpti ve ectodermof theembryo(ligh t stippling)2)presumptivemesoderm (black area)is located around theequator.

which will developinto mesodermal tissueas indicated and3) presumptive endoderminvegetal hemisphere (white area).

B) Mesoderm Induction Assay. Tissue from the animal pole (presumpti veectodenn) is surgically removedincub ated (i)insalt solution or (ii)saltsolution and FGFor(ill)incontact with a vegetal piece.Themodel derivedfrom this experime nt isthat a signal isemitted from thevegetalzone duringmesoderm ind uction that mayinvolveFGF.

family,but FGF hasto be presentinthe marginal and animalcap cells to make them responsive tothe induce r (Dale,1997).

At aboutthe 32-cellstage(stage 6) thedorsalmarginalzone cells begin tobeco me dorsal mesoderm (Gimlich, 1986).DaleandSlack(1987) found that inisolatio n,dorsalmeso dermformsmainlynotoc hord,whereas ventral mesoderm givesrisetobloodbut no muscle is for med. When they areincubated together,howe ver ,theventral mesoderm also forms large amountsof muscle. They suggestedathree-signal modelfor mesoderm induction to explain theirresults(SeeFigure 1.3).The first signalorigi nates in thedorsal side of the embryo, whichinduces notoc hord,anda small amou ntofmuscle. The secondsignal, fromthe ventralsideofthe embryo induces blood. A third signalcomes from thenewly induce d dorsal mesoderm. or'Spemannorganizer'.Thissignal causes the ventra l mesod erm.

adjacent todorsal mesodermto form muscle instead of blood. Spemannand Mangold (1924) identified the dorsal lip regio n ofanembryo as the organizerregion. This regionin thedeve lopin g embry oinvol utesduring gastrulatio n. TIleSpemannorganizerhastobeinduced itself first throu gh the Nieuwkoopcen ter(locate doppositethesiteof sperm.entry belowthe 10

graycrescentontb:dorsal side)(Wo lpertetaL,1998).1be'organizer' was ableto induce a wholesetof dorsaland lateralstructures when transplanted intotheventral side of an earlygastrulaembryo.

The prospecti vemesodermdifferentiates intocardiac andskele tal muscle,notochord. boneand cartilage, blood. kidneyand mesenchyme.

Recent evidencesuggests thatfactorsreleased bythe Spemannorganizer inhibitthe induce r(ReviewedinHeas man,1997). Inthe regionclosestto theSpemannorganizer,inhibitio n ofventralmesoderm isthe greatest.as a result thereismo redorsal mesoderm induced.The furtherthedistancefrom theorganize rtheconcentrationgradientofthese factors is decreased causing lessinhibition.Thisgivesrise tomoreventral tissuesinduced by the vegetal factor .Factorssuc hasnogginandcbordin.dorsalizetheembryoorfact o rs such as bone morpbogeeetic protein (BMP-4) and Xenopus wing/us8 (X wnt-&)ventralize theembryo (Wolpertetal.,1998 ).

It

Q@

^{¥V 0}

^{-+ VII}

^{¥V 0}

^{0 - +}

~TheThree Signal Model. Two mesoderminduction signals are assumed toderive from thevegetalregionof the earlyblastul aintothe animalcap regi on (A). The dorsal-vegetal(DV) sign alinduces dorsal mesoderm, or'organize r'tissue (0 ), while theventralvegetalsigna l(VV) induces generalventra l mesoderm(VM).The organizer produces a third signal probabl yduringgastrulati onthatinduces additio nalmuscle.Onlythe mostventralcells fonnblood-formin gmesoderm (M3). Thereismore dorsalmesode rm (MI) formed ne xt tothe organiz er region (DaleandSlack, 1987).

12

1.5 Mesoderm Inducing Factors

Thecomponents of the mesoderminductionsignal areunknown. Therehave beena number ofstudies to identifythese components. Ifanimalcapsare cultured in XTC (Xenopus tissueculture) media, the animal cap will differentiateinto mesoderm (Smith. 1987). The XTC cell line secretes Mesoderm Inducing Factors (MIF).Ithasbeenshownthattheseinvitroactive factors are actuallyknown cytokines orgrowthfactors (Slack,1987 ). MIPs fallinto two categories: those belongingtothe TGF/3 superfamilyand those belongingtotheFGF family.

The membersofthe TGF13 superfamilyexist as dimeric proteins linked bydisulfide bridgesandare initially synthesizedaslongerprecursors andlater processed (Massague,1987). Membersof the superfamilywithMlF activity areTGFJl 2and 3.activin (Smitherat.,1990) and certain bone morphoge netic proteins(BMP-4)(Kosteret al.;1991;Jo neset al.•199 2). A homologue of activinAwas identifiedastheactive compone nt inmesoderm inducing XTC medium(Smithetai.,1990).Studiesinhibitingactivin activity with follistatin didnot preventmesodennformati on in earlyembryos(Schulte-Meckeetal.,

13

1994).Because of thesefindings, activin wasnot believedtoplay arolein mesodermfoonationinvivo.

Recent evidencehasnow implicated activin , ora closerelativethathas yettobe described, tobeimportant in development(Dyson and Gurdon.

1997 ). Twomembe rsoftheTGFl3familyshow ntobeexpressedmaternall y and tohave mesoderm-inducing activityareVgl and BMPs (Hyattet al., 1996).Vglisavegetally localizedmaternal mRNAthatbecomes distributed inthevegetalhalfof the embryo afterfertilizati on. It is speculated that Vgl is importantin dorsalaxisformation,left-right axis determina tionandendode rm specifica tion. DysonandGurdo n designed anewdominant-negative receptor

lackingthetransmembranedo~selectively blocking activin functionbut

not Vglfunction. Embryos injected with this construct lackedbead structures, suggestinga deficiency in anteriordifferentiati on. Analysis of theseembryos showed thatblockingactivin signaling reducestheinitialinductionofXenopus brachyu ry(Xbra)to20%ofthewild-type leve l.Xbraisan immediate-early (independent of proteinsynthesis) pan-mesodermal gene marker (S mithetal., 1991 )and a reduction inXbrasuggests arole for activi n in initial mesoderm ind uctio n.

FGFsareheparinbinding growth factorsthat havealso been foundtobe importantinearly embryo development(Slack et al.•(987).Thename FGF wasderivedfromtheassayusedtoidentify theirgrowth promoting activity.

stim ulationof DNAsynthesisinfibroblasts (Slack, 1994). Subsequently.it became apparentthatthese growthfactorswere ableto promotethegrowthof mesodermaland neuroectodermalcells bothduringembryogenesisandindie adult(FemigandGallagher.1994).

ThreetypesofFGFhave so farbeen clonedfrom Xenopusthat are direct bo mologues of each mammalian type:FGF -2.FGF-3andembryonic FGP(eFGF) (Reviewedin Slack, 1994).Allthreetypesof Xenopus FOF are expressed in earlydevelopment FGF-2 and eFGFareprese ntintheoocyte andfertilized eggand are thereforeavailableatthe rirre ofmesoderm induction.FGFhasbeenfoundtoberesponsible forthe maintenanceof Xbra andXbraactivates expression of FGP(Schulte-Merker andSmith. 1995).As mentionedearlier.Xbrais an immediate-early pan-mesodermal genemarker whoseexpression decreases withtheinhibition ofactivin. Overexpression of Xbrain presumptiv eectodermafter injection of itsmRNA cause sformation of ventral mesoderm (Schu lte-Merker and Smith.1995 ).When eFGFisinhibited

"

by the dominantnegative FGFR,inXenopus,there is achange of dorsal mesodermsuch thatit moves around the blastoporelip instead of elongating in an antero-posteriordirection(Isaac setal.;1994).In these embryos thereisa reduction inXbra expression during gastrula tion. eFGF andXbra are ableto activate the expressionof each other, sugges ting that they are componentsof an autocatalyticregulatory loop (Isaacset al .•1994).Iftheactivityof the FGF familyis inhibited by overexp ressionof a dominan t-negativeFGFR,there isa reductionin mesoderm formation, abnormalities arisingfrom and inhibition of normal gastrulationmovementsand defects in the formation of theposterior pans (Isaacs et ai.;1996).The authorsspeculatethat the mesoderm formation andcellmovementdefectsare attributab letoloss ofXbraexpression,andthe posteriordefects were due to a lackof posterior geneactivity.Overexpression of the eFGF givesrise to a posteriorized phenotype,inwhich posteriorgenes are expressedina more anteriorposition (Isaacset ai.,1996)

It isno t clearexactly how these growth factors work together in mesoderm inducti on andaxialpatterning.There are various reportsto identify theexact compo nentsofthesignalin thethree-signal modelexplainedearlier, Fromthese experiments.there have been modificationsof the originalmodel

UV-irradiatedembryos haveprovidedan insightinto themechanis mof dorsalpatternin g ofmesoderm. UV-irradiated embryos formmesoderm that does not contain muscle tissue or notochord,butcontains mesenchyme.

mesothelial tissueandblood tissue(Smithand Slack.1983).Theembryos lack do rsal/ventraland anterior/pos terioraxes. UV irradiation is effectiveat blockingthis dorsal pathway whenitis applied tothe vegetalpoleoffertilized eggs shortlyafterspermentry,or tothe vegetalpole offull-grownoocytes (Holwil letal.,1987).This suggeststhata componentofthedorsalpathway mustbeestablished during oogenesisand localized tothecortex of the vegetal polebefore maturation. Inembryosirradiatedafter fertilizati on. Gerhart (1989) found that the UV disrupts corticalcytoplasmic movementsby disorganizing microtublues andpreventing thenormal displaceme nt of a dorsalizingactivity tothe dorsalsideof the embryo.Theexact nature ofthis dorsalizingactivity is notfullyunderstood. One approach usedto ide ntify moleculesimportantin thismechanismisto determinewhich molecule will rescuethese UV-ventralizedembryos(Slack. 1994). Another experimental approachto identify molecules important inpattern forma tion uses the

17

depletionof mRNA from the oocyteand studiestheeffectthat depletionhas on development (Heasman erai., 1994 ). This method uses antisense oligodeoxynucleotides to target-specificmRNAs,whicharethen cleaved by endogenousRNaseH. Combinations of these twoexperimental approaches haveled to advancementintheunderstanding of the dorsal! ventral pathway.

Dorsalaxis formationinXenopusappearsto depend upon cytoplasmic componentshomologoustothose of a signaling pathwayinDrosophila.which is initiatedby wingless(Wnt),a secretedprotein. This wingless-initiated pathwayhasbeen shown tobe ofcritical importanceinthe dorsa-v entral patterningofDrosoph ila.Several Xenopusmembersof the Wntfamily(X wnt genes) are able to rescuetheventralizedphenotype of UV-irradiatedXenopus embryos:Xwnt~8(SmithandHarland.1991 ), Xwnt-8b(Cuietal.,1995)and Xwnt11 (Ku andMelton,1993).

BMPshavebeenimplicatedin thedevelopmentof the ventral pathway. Suppress ionofBMP4 acti vityinthe UV-irradiatedembryo leads to a restorationof axial structures,although nothead structures(Steinbeisseret

al.,1995). Thissuggests that theove racti viryoftheBMJl pathwaymaybe partlyresponsibleforthephen otypethat resultsfrom UV irr-adiation,

TIlepatterningmechanismthat basemergedin receenyearsconsistsof an interactionbetweendorsal andventralcells (S mithaodHar land. 1992).

The timeatwhichthisinteractionoccursisnotyetknown, Inadditionto signals inthe previo usmodel.a fourthsignalhasbeenspeculated,establishing fwtherpatternwithinthemesoderm,by interactingwiththethirdsignal.The secre teddorsalizin g molec ules. suchasnoggin andcho r<l.inare productsof genes transcribedinthe Spemannorganiz er.1beyconvertventralmesodermal tissue to dorsal mesodermaltypeswhentheyare ove rexpressedon theventral sideof the embryo (Smith andHarland, 1992) (SeeFigurelA).

I '

(gene~I~:u~g\ignal) /nd\~~~

^{_. __}

eg.Vgl,ACtivin / ~

Ventral mesoderm Dorsal mesoderm

\

Spemannorganizer

ven~~:~~

_BMP-4.Xwnt-8

! ~,,~:s~g~~.

noggin orchordin Patterning of Mesoderm.

VegetalRegion- - - - -- - - -- _ _

Figure 1.4 Swnmaryof Mesoderm. induction inXenopus laevis (Wolpert etal.,1998)

1.6 Fibroblast Growth Factor Receptor

The apparent importance of FGFin mesoderm induction triggered various studies to determine the mechanism of action of this growth factor.

FGFhasbeen shown to act throughthe transmembrane FGFR. Four FGFR genesare known to existinmammals (Rutaet al.•1988; Dionneet ai.,1990;

Keeganet al .•1991;Machael et al.•1992).The known forms are:

1)FGFR. Fig (fms-like gene FGFRl) (Rutaeral., 1988):This was the first FGFR tobecharacterized. Itwas purified from chicken embryoextracts incubated with biotinylated FOPinthe presence of heparin. The 92kDa protein contained a single membrane spanning region. an amino-terminal signal peptide, andthreeextra-cellular immunoglobulin-like domains (Ig-like).

Between the first and second Ig-like domains the receptor contained a unique domain that was not present in other known growth factor receptors. This domain consists of eight consecutive acidic residues andisreferred to as the 'acid box', This is a unique feature of the FGFR and maybeimportant for stabilization of the protein configuration during ligand-receptor interaction (Leeet al .•1989).

21

2) FGFreceptorBek(FGFR2) bacterialexpressedkinase (DionneetaL.,1990) This clone was obtainedby screening a mouseliver cDNAexpression library withanti-phosp hotyrosineantibodies (Kornblu thetaL.,1988).

The amino acid sequenceof the bek cloneandthe correspondingregion of fig are 85% identical.

3)FGFR3 (Ke eganetaL.,199 1)

To isolatethis gene,a cDNAlibrary from thehuman chronic myelogenous leukemi a(CML) cellline was screenedwiththe chicken v-scagene, areceptor-liketyrosine kinase. TIllsclonehasthreeIg-like regions andisactivated by both aFGF and bFGF.

4) FGFR4 (MachaeletaL.,1992)

FGFR4has beencloned fromhuman k-562erythrol euke mia cells.

FGFR4 bindsto aFGF, butnot bFGF.TheFGFR4 proteinis somewhat unique

22

inthat it contains a core of onlyfour acidicresidues inthe acidbox domain.

comparedto eightacidic residues inFGFR1.

The FGFRI openread ing frame consists of 8QO..822 amino acids.with a 90kDa protein formed. The estimated mass ofthe FGFR isapproximately 110-150 kDa, with 6-9 potential N-linked glycosylation sites in the extracellu lardomain. The openreading frameconsists of an 18-24amino acid signal peptide. an extracellular domain of 346-356 amino acids. a transmembranedomain of 21amino acids.anda cytoplasmicdomainof 410- 425 aminoacids. Theextracellulardomain of theFGFR tyrosinekinases contains two orthree Ig-Iikedomains.The cytoplasmicdomain consistsof a juxtamembraneregion.the kinase domain, which containsa14 amino acid insertion, and a C-terminaltail(Jaye et al.,1992) (See Figure 1.5).

The only forms identified todateinXenopus are FGFRIand FGFR2 (Cornelletal.,1995 ). Studies of distributionshow that FGFRImRNA is present throughoutthe earlystages of development(Slack,1994 ).The mRNA ismore abundantinthemarginal zone andanimalhe misphere. FGFR2 is not

2J

expressedinthe blastula but isfirstdetectedin the anterior neural plate during gas trul ation (Freisel and Brown, 1992). Theexpression of FGFR2 suggests that it is not involvedintheestablis hme nt ofmesoderm

Figure1.5 Schematic represe ntationofthestrudural features ofFGFRl.

Sequence analysis predicts a protein with a hydrophobicleader sequence (black box),threeextracellularIg-likedomainsU.

n

andllI)with disulfide linked cysteinresidues (5), and a short acidic sequence (black box between 1 andIl).Thisisthe extracellular ligand-bindingdomain.Thereisalsoasingle hydrophobic transmembrane domain(TM).Thecytoplasmic domain contains ajuxtame mbcane region (believedto be important in regulationof thesignal) and an intracellular tyrosine kinase domaintophosphorylatetyrosine residues.

Tbetyrosine kinase domain is splitby a l4-amino acid insertsequence(KI).

TheFGFR field becamemore complex.with thediscoverythat there are variousalternativelysplicedmRNA transcripts from at least FGFR I and FGFR.2 genes. Alternative splicing results in either 1) the inclusion/exclusionofadd itionalaminoacidsor2)the use of alternated coding exons withnonetgainor loss of aminoacids . Ineithercase,theresulting proteins arestructurally different (Jo hnson and Williams,1993).

Thesignificanceofthelargenumber ofisofonns is not completely understood;howeverit isknown that alternative splicing intheextracellular domain of thesereceptorsresultsin altered ligandbindingspecificity. There are reports oftheisolation of cDNAsencodingFGFR I proteinsthatare missing IgdomainI (Johnson et al.,1990) . Analysisof thisFGFRI ge ne revealed the presenceofanintrcnseparating IgdomainI from the remainde r of theFGFRIcodingsequence(Johnso netal.,1991).Thusit appearsthatthe presenceor absence of the Igdomainismediated byalternative splicing.The predominantform ofthe receptor expressed during embryogenesisisthethree Igfonn(Johnson et al., 1990). The third Igloop can alsobespliced toform variants resultin g inother FGFR forms:IDa, Illb,orIllc. It has been demonstratedthat the replacementof theIllcexonbytheIIIb exonreduces the relativeaffinitiesofthereceptor forbFGF approximatel ySO-fold(Werner er

25

ai.,1992). Thisfindingsuggests thatthesecond half of thethirdIg-like domain confersthespecificityforbinding tobFGF. Differential splicing withinthethirdIg-Iikedomain providesamechanism tochangethe affinity of the receptor fordifferentligands (Werner et al.,1992 ) and introdu cessome degree of specificity.

Currently,only single cDNAs have beenisolated forFGFR 3 and FGFR4.Both clonedcDNAsencode threeIg domain receptor forms(Johnson and Williams,1993). Thediscovery of multiple forms ofthe FGFR raises many importantquestionsfor futureinvestigations. Due tothedistributio nof FGFR I mRNA, this work examinesonly FGFRI and its importance in mesodenn induction.

1.7 Mechani sm of Action of the Receptor

The mechanism of action of the FGFR is called the receptor oligomerizatio nmodel (Ullrich and Schless inger, 1990) . Binding ofthe ligand. FGF totheextracellulardomaincauses a conformati onal alteration of theFGFR, which induc es oligomerizationofreceptors.Dime rization cantake place betweentwo identic al receptors(homodime rization) orbetweendifferent membe rsof the same receptor family (heterodimeriz ation) (Ullrichand 26

Schless inger,1990).Dimerization ofthe receptoractivates the internalkinase domainandenablesthe receptor to autophosphorylate on tyrosineresidues.

Phosphorylation of dimerized recept ors appears to occur via anintermolecular transphosphory lation mech anism. The phosphory lationofthe internalkinase domain enablesthe receptor to autophosphorylateon tyrosine residues.Of the five tyrosineresidues loc ated inthe cytoplasmic partofFGFR 1,not including the kinase domain, tyrosine776 (Y766)appears nottobephosphorylated, whereasY463,Y583, Y585andY766 are potentialphospho rylation sites (Mohanunad i et ai.; 1996).Three phosphorylationsites havebeenidentifiedin theFGFRI kinasedomain .Y653,Y654 andY730.Y653andY654 appear to beinvolved inregulationofthe kinase, sincemutationofY653 and Y654 to phenylalanineresidu esleadstoloss of kinase activity (Mohammadi et al., 1996 ).1bephosp horyl ationoftheinternalkinasedomain enables thereceptor tophosphorylate othe rcytoplas micmolecules. The semolecule s transduce biologicalresponses, often involving changes ingenetranscription.

Phosphorylation ontyrosine, withintheFGFR,acts as a switch[0allow thebinding of cytoplas mic proteins containing SH2 domain s (Fengand Pawso n. 1994 ). SH2 domain s are approximate ly 100aminoacidsinlength.

They acenon-catalytic regions,conserved amonga series of cytoplasmic

signaling prote ins. SH2containing proteinsbindtophosphorylated tyrosine residuesonthereceptor. The intracellularkinaseactivity ofthe FGFR phosphorylates these substrates. Various SH2 domains have different preferences for differenttyrosineresidues.addingspecificity tothis interaction (FengandPawson,1994).

Oneapproach used to study thefunction oftheFGFR inXenopus developmentwasthe construction of adominant-negativeform ofthereceptor.

This construct involvesdele tingtheFGFR intracellular kinasedomaincreating anon-functionalmutant of theXenopusFGFR. WhenmRNAscodingfor these mutants areinjected intotheearlyembryo they aretranslated,andthe resultingmutantproteinsfonn non-productive dimers withendoge nouswild- typereceptor.Thefunctional response of the receptoris abolished. Animal capexplanrs cut from embryosinjectedwiththe mutan tfailedtoinduce mesoderm whenincubatedwith bFGF (Amayaet ai., 1991). In whole embryos, the injection caused specific morphological defects during gastrulation and posterior developmen t The absenceofFGF signalingcauses a reduction,although not atotal depletion ofmesodermformation(Slack, 1994). Thereisalso a severeeffectonaxisfonna tioninwhichformationof the posteriorpartsisreduced. As a consequence, thereis inhibition of

invaginationandelongationof thedorsal mesoderm (Slack, 1994). 'Ibis suggeststhat FGFsignalingisnecessaryfor normal gastrulation movements, particularlyin the dorsal mesoderm.

The activation of theFGFR resultsin thephosphorylation of various intracellular proteins leading totheactivationofvarious downstream targets (lsaccs, 1997). Signal transduction pathwayslead toa varietyof physiologicaleffects. The mechanism through which the various FGFRs signal specific genesto produce different physiolo gical effectsisnotcompletely understood. A better understandingof the mechanismthrough whichthese pathwaysinteract will helpgive an insightin the aboveprocess. The stimulatedFGFRis able to activatea numberof known pathways. Some of thesepathwaysinvarious cellsystems are: 1) the Ras pathwayand the Ras- dependent activation of MAPK(Figure 1.6)2) the activation of the PI3'K pathway (Figure 1.7),alsoshown toactivate the MAPK pathwayand3)PLCy pathwayand the Res-independent activation of MAPK (Flgure1.8).

29

FGFR~G F. rn::;" ...

"'a"''''',,/-A'''C'''bV_.. . .

Grb OTP

50S

1

, /

Otbercffccton Other

-====~

.ff••'on

+

Figu re1.6Signaling throughthe FGFRtoActiva te theRas Pat hwa y.

FGFR isactivatedwhen FOF binds. Thisleads to the dimerizationof the receptor.activating the internal kinase domain. Autophosphorylationof the receptoroccurs,providing binding sitesfor

sm

containingadapter proteins.

One such protein, GRB2, complexes with the Ras-guanine nucleotide exchange factor Sonof Sevenless (5 0 S). The recruitmentof this complexto theplasma membrane is believedto bring Sos into close proximitytoRas .thus allowing itto activate Ras bycatalyzing the exchangeof GOP(inactive form) for GTP (active form).The activity ofRasisincreasedbyGlPase Activation protein (GAP)leadingto an increase of hydrolysisof GTP to GDP. Once activated, Ras isableto interact with andactivate othe rproteins,whichinturn stimul atessignalin g cascades that inducea variety of cellularresponses. Ras activation culminatesinthe activation ofRat,which phosphorylatesprotein kinas e(MAPK or ERIC) (Smithet ol.,1995).

Figure 1.7 PI3'K Pathway FGF+ FGFR

PI3'K

1

PI ...---.... PI3P ----+PI3.~2---lll>PI3.4.S,P3

Figure1.7 SignalingthroughanRT Kto activate Phosp ha tidylin osit ol 3'·Kinase Pat hway for inosi to l phosph a te metabolism.FGFactivates the internal kinasedomainoftheFGFR. This kinaseisable to activate other cellular substrates.One ofthesesubstratesisPI3'K. PI3'K is importantin thebreak down ofphosphotid ylinositol(PI) into phosphatidylin ositol 3- phosphat e (PI3P). ThisisthebeginningofthePI3Kpathwa y,whereothe r products are produced for example:PI3.~2.phosphatidy linos itol3,4- bisphosp bate:PI3.4,5.P3.phosphatidylinosto1 3,4.5-tri sphosphat e.These chemicalsare importantsecond messengers , whichare involved in a large number of differentbiologicalpathways.Thispathwa yandother related inositolphosphate pathwaysformthebasis ofvari ous transduction mechanismsnow knownto regulate a largearray of cellular processes, includin gmetabolism,secretion, contraction,neuralactiv ity andcell proliferation(Heldman,1996).

31

Figure 1.8PLCyPath wa y

FGF+Receptorl---'"PLCy ----+DAG ---...PKC

Grb;SOS

!pi

^{Ca'· /}

~

+RAs----MI

MAPKinase

~

Mitogenesis

l

Figure1.8 AModel for mitogeni c signal transducti onafter growthfactor stim ulati on(Huang, 1995). TIllsshowstwo pathwaysused totransduce signals fromtheFGFR. a Ras independentand Res-dependent activation of Raf. The PLCy pathwayproduces diacylglycero l(DAG) and/orinositol triphosphate (lF3)upon activationof the receptor and phosphorylationof PLCy. IP3 releasesstores of intracellular calcium (Ca

2"1

acting assecond messengerto activate PKC. DAG also activates PKC.which is known (0 stimulatedRatand the MAPKpathway. 1beRas-dependent pathway involvestheRaspathway showninFigure 1.6. This figure shows that the various pathwaysare able to interact within the cell(Huang, 1995).

"

1.8 Substrates of Receptor Tyrosine Kinases

Despitethestruc tural diffe rences between recept or tyrosinekineses (RTK) suchas Epiderma l growthfactor (EGF) .FGF orPlatelet derived (pDGF),theyallsharea commo n mechani sm for activation. FGF probably acts through thesame system asPOOP and EGF-althoughinsomecasesitis notcertain whichexactmoleculesareinvolved in vivo. Some of the other substrates.acti vate dinRTKsyste ms,are:

1)Phospholi paseCrt (PLC-yl )One of the PLC isoforms cleaves the phospholipid phosphatidy linositol 4,5-bisphosphate (PIP2) to the seco nd messengersdiacyglyceroland inositoltriphosphate,whichintumactivates protein kinaseCandraises the intracellularcalciumlevelrespectively(Rhee et al.,1989) (See Figure 1.8). PDG Fstimulates PI turnoverincellswhere PLC- ylistheprincipalPLC isoform,andoverexpressionofPLC-ylenhances the accumulationofinositolphospbatesinresponsetoPDOP(Margolisetal., 1990 )

"

2)SHCisexp ressed as two proteinsof 46 and 52 kDa. eachcontaininga Ccterminal SH2domainandan N-terminalglycine/richsequence. Although no enzymatic activity has yet been described for the SHC proteins.

overexpressionofSHChasbeenshowntoresultin cell transformation.SHC proteins become tyrosine phospbory latedby activatedtyrosinekineses. In addition,SHeproteinsform complexeswithGRBlsem5gene productthat linksreceptortyrosinekinesesandp21'",suggesting a role forSHCproteinsin theRaspathway(Richard.1995).

3)Ras andrelatedproteins oftheRassuperfamily playcritical rolesin thecontrolof normaland abnormalcellproliferation (reviewedinMilligan.

1992).Inmammalian cells therearefour trueRas proteins (encoded byHa- Ras. N-Ras.Ki-RasA,andKi-RasB)which.upon mutational activati on, can function asindependent oncogenes. 'These proteinsrelay signals fromtyrosine kinasesattheplasma membranetoanetwork ofserine/threo ninekineses whichsubsequently leadstothenucleus.Thep21Rasproteinisactiveinits GTP-boundstate.TI1isformis slowlyconvertedtotheGOP-bo undformby theintrinsicGTPaseacti vityof Ras. Thisactivityisgreatlyenhancedby GlPase-activating proteins(GAPs)which subseq uently lead to hydrolysisof

GTP moleculetoform GOP(See Figure 1.6).Maintenanceof Resinthe GTP formcan lead totransformati on.OneclassofRasmutations.conunonl y found inhuman tumors,results in anaccumulatio nofRas-GTP (Milligan.199 2).

4) Phospha tidylinos itol 3'-kinase (pI3'K) is a lipid kinase that phosphorylates the03position of pbospharidylinositot,phos phatidylinositcl- 4-phosphate, or PI-4.5·P2 (Whitmanetal., 1988) (SeeFigure 1.7). PI3'K exists as a heterodimericcomplex thatcontainsp85 andaIi 00kD protein.The purified p85subunit hasno detectable PI3'Kactivity.butbinds tightlyto activatedPOGfRorEGFRinvitro. suggestingthat p85 acts as theregulatory subunitandpHD isthe catalyticelement (Ots u etal.,1991). Activated mammalian PI3'Kcontrols several important cellulae functions. including cytoskeletalorganization, ceU divisionandsurvivalmaintenance (Roc heetaL 1994)."Theexactrole PI3'Kplaysinsomany different ceUular responsesis not yet known, but itisbelieved that PI3'Kactivation leads to SHe phosphorylati onresultinginrecruiting more GRBISOStothe membraneand as a resultincreasing the mitogenenicresponse(llasaca eta/.,1997). The authorsoverexpressed PI3'KinCos cells and foundincreased MAPKactivity.

Thisstimulatio nwasabolishedwithan inactive mutantof PI3'K.

"

5)Rat is a proto-oncogene thatencodesa 68 kDaserine/threonine kinase thatisphosphorylatedand the kinase activityis increasedafter PDGF stimulation(Kelch.1993),EGFR (Hockeretai.•1998)and FGFRstimulation (Miyamotoet al.,1998).Therearedifferent forms of theprotein ;A-Rat.C.

Raf and V-Rafshow ing differentexpression patterns (Storm and Rapp,1990).

Rat interacts directly with Ras to activated the MAPK pathwa y and mitogenesis (reviewed in Moodie.1994)

6)NCK and Growthfactor receptor-bindingprotein(GRB). These area uniquegroup of associated moleculesdue to theirfunction. They act as adapterorlinking molecules. Nckisa 45-kDa protein consisting ofoneSH2 domain andthreeSH3 domains(Lehmannetai.•1990). SH3 domains are thought to mediateassociations withadapter proteinsor target proteinsthrough recognitionof proline-richsequences (Pawson and Schlessinger.1993).It has been shown that Nckisacomponentina complexwiththeactive PDGFR and theEGFR. through the SH2 domain(Yiet al.;1992 ). Nckhasalso been shown to interact with aserine/threonine. Nck-associated kinase (NAK), specificallytothe secondof Nck'sthree SH3 domains (Chou and Hanafusa, 1995). A numberofother Nck-associatedproteins have been foundin other

30

systems of various molecularweights(this willbe describedindetail in Chapter 5).

ORB is the mammalian homologue to Sem-5 gene product inC.

elegans. Sem-Sfunctionsbyactivationof Ras through asignal transductio n pathwaythat is initiatedby the let-23 RTK.,leadin g to vulva induction(C lark.

1992). Recently,a ORB homologueknown as DRK was identified in Drosophila .DRK. is involvedinSevenlessRTKsignaling duringspecification of the R7 photoreceptorceilinthe developingDrosophilacompound eye (Olivier et al.,1993;Simon et al..1993). DRK, like GRB,hasbeensho wn to bind toSOS.a guanine nucleotideexchangefactorfor Ras. The 24 kDa structure of GRBconsists of one SH2 domain flanked by twoSH3 domains (Matuoka et al., 1992) . GRB associates spec ifically with the autophosphoryLated EGF or PDGF receptors via its SID domain. The receptor-GRBcomplex binds the guanine nucleotide exchange factor.SOS through its SH3 domains. SOS promotes the conversionof GDP_p21tWto GTP- p2 1>41,allowing the tyrosine kinase to modulateRasactivity (Galeetal., 1993) (See Figure1.6).

37

7) SHP2Phos phatase(also calledSyp,PTPID.and PTP2C) structure consistsof rwoSIDdomains. (Reviewed in Feng and Pawson.1994). It appears thatSHP2 maybe substrate-selectiveand that both thecatalyticand 5HZ domains may contributetothis selectivity (KlinghofferandKazlauskas.

1995). Furthermore.itseems that SHP2 playsa roleinnegativefeedback.in thatit dephosphorlylatesthe PDGFR. When FGFR isactivated during differentio n of PC12cells. SHP2 activation appears tobeimportantinMAPK activation.througha linkage protein calledFRS2 (Hadari,1998).The authors interrupted aco mplex formationbetween FRS2 and SHP2 byexpressing a Y436F mutantFRS2inPC12cells. The expressingcells allowed transientbut not sustainedactivation of MAPK,leading to adecrease FGF inducedneurite outgrowth.

8) Src is onemember of a family of nine tyrosinekinases thatregulates cellular responses to extra-cellularstimuli. Members of the SRC family containanSH3 and Sill domain with a catalytic tyrosinekinasedomain.

(Cooper, 1993). In vitro experiments show that changes in Src phosphorylationinduce changesinits activity. Ithas beenshown that C- te rminal phosphorylation is inhibitory and kinase domain phosphorylationis

38

stimulatory(Kmiecik.et01.,1988).Keeping Src inactiverequires extensiveC- terminal phosph oryt arion. Sofar,Csk (C-terminalSrckinase)istheonly known kinase thatspec ificall yphospborylates Srcatits C-terminus(Reviewed inBrown.1996).

9)FOF receptorsubstrate 2 (FRS2)isarecentl y characterizedadapter moleculeof 90KDa(Kouhareetal.,1997).FRS2structureisuniqueinthat it lacks an SH2 domain,but contains aphosphoryroslne binding domain which mediates phospho tyrosine -independentinteractionwith amino acid residues 407-433in FGFRI,as examined in ayeas t twohybrid assay (Xueta/.,1998).

Activationof FGFR I leadstotyrosinephosphorylati on of FRS2at Y 196, Y306,Y349andY932 allowingfor binding ofGRB(KouharaetaL,1997).

GRB,as mentioned earlier,linksSOS to

me

activationofGTP- binding protein .Ras.Inaddition phosphorylation ofFRS2atY436potentiall yallowsbinding of

me

tyrosine phospha taseSlfl>2(Hadariet01.,1998). Thusfar FRS2has been shown tobeutilized byFGF Randnervegrowthfactor receptor(NGFR), butnot by other typesof RTK(reviewed in Klint andClaesso n-Welsh,1999).

This would possiblyimply that FRS2 hasa uniquesignaling function, however, GRB and SHP2 operate downstreamof both PDGF and EGF.

"

WhetherFRS2hasaparticularroleinspecific FGFRmediatedbiological responses remainstobesho wn..

1.

9 Prospects for Thesis

Thegoalofthiswork.wastofurtherthe understandingofthe FGFR transductionpathwayduring mesoderm.inductionintheXenopus embryo.

FOFisinvolvedinmesoderm inductionlhroughactivationof the FGFR(0

differentiallyexpress specific genes involvedinearly embryo development.

Thehypo thesis for thiswork. wasthatFGF isinvolvedinmesoderm. induction through the phosphorylatio n of moleculesleading tothe transcriptional activationofspecific molecules.Myfocus was[0identifythecomponents of thissignal,andwiththisinformationthere maybea better understandingof

howtheFGFRisinvol vedinmesoderm inductionintheXenopus embryo.

Chapter 2

MAT ERIALS AND METHODS

2.1 Embryos, Dissections and Induction Assays

Xenop us embryoswere obtained byartificialfertilization.handled and dissected as previouslydescribed byGodsa ve et ai.,(198 8) and staged accordingtoNieuwkoo p and Faber (1975). Female Xenopus laeviswere induced[0lay eggsby subcutaneous injection of500 LV. of humanchorionic gonadotrophinin0.5 mlH20. Theeggsthatwereobtainedwerefertilized usingXenop ustestes, storedinIxNanna! AmphibianMed ium(NAM)(See Tablel; Slack and Forman 1980), then dilutedand broken-apartinH20. Once the eggswere fertilizedand rotation occurred,tbejelly coats wereremoved using 2.5%w/vcysteinhydrochloride(SigmaChemical Co.)pHadjusted to 7.8-8.2 withsodium hydroxide (NaOH) (Fisher). Theembryoswerewashed thoroughlywithabout1literof water and allowed to developinpetridishesin lnONAM.

42

Duringdissec tionsthe embryosweremanipulatedinNAM .NAM/2+ ImglmLBSA (BovineSerum Album:SigmaChemicalCo.)wasutilizedto culturecutexplants.

TABLElA.Com po nentsofNAM

NaC I KCI Ca(N°3h.4H20 MgS04.7H20

EDTA (0.5M,pH8.0) Hepes(IM,pH7.5)

glL(inlOx) 65 1.5 2.' 2.5 2mL 100mL

mM(Finalinlx) 110

I 0.1 10

Table18 Compon entsof NAMSolutions ForlOOmLofIxso lution. add:

NAM NAM12 NAMI20

lOxNAM (from TableIA) IOmL 5mLs O.5mLs Gentamycin (lOmglmL) 0.25 mL O.25mL O.25mL NaBicarbonate (0. 1M) 1.0mL l.OmL

SterileH20 88.75mL 93.75mL 99.25 mL

43

2.2 MESODERM INDUCTION EXPERIMENTS

The FOFusedinthisstudy wasrecombinant Xenop usbFG F. expresse d and purified according toKimelmanet at.(1988) andstoredat-20°C.Animal poleexplants were dissected manuallyasdescribe dinGodsaveet aL(1988).

One hundredexplants were inducedeitherbyculturinginthepresence of lOOngfmL Xenop us bFGFdilutedinNAM+BSAor incuba tingtheanimalcap directlywithvegetalcells for30 minutes.The control explants wereculture d inNAM +BSAalone.After incubatio nonice,the explantswere solubilized andprepared forimm unopreci pitatio nasdescribedbelow forwhole embryos.

2.3 IMMUNOPRECIPITATION BLOTTING

AND

WESlERN

To study the FGFR complexes formed duringmesoderm induction . immunoprecipitations were performedusin g150 stageeightembryos. The embryos were hom ogenizedinlee-coldsolubilizationbuffer.which contained 1% Triton. 10 mM Tris. pH 7.5,10 mM EDTA, 1 roM PMSF (phenylmethyls ulfonyl tloride), 25JlM1mL aprotinin, 25mglmL Ieupep tin, 5mgl mLTI.CK.l00J.,LMsodiumortho van adate, 100mMNaP, lOmMsodium

pyrophosphate. After 30minutesofsolubiliza tio n.on ice .theextrac twas centri fugedat4"<:and1O.0CX>rpmfor5minutes.toremovetheinsolubl e material. Thesupernatant wasremovedand placedintoa newtube. The app ropriate antibod y was added. at the required conce ntratio n for immunoprecipitation (alistof antibodiesareincluded in Section2..5). The solution was incubated.rotating.at40Covernight

The follow ingdaythe complexes were isolatedbyaddingProteinA-or Prote inG-Sepherose(phannacia).Theappropriate Sepharose woulddepend

onthesourceoftileantibody(SeeTable2).This mixture wasincubat ed at TABLE2Affi nities ofVarious Mon ocl onal Antibod iesfOr"Protein

AOr"Protein G

ANTIBODY

RabbitIgG RatIgG 2a RatIgG 2b RatIgG2c MouseIgG 1 MouselgG 2a MouselgG 2b MouselgG

Affinityfor protcinA ++++

+++

Affinityfor proteinG

++

+++

+++

40Cfor1hour.withrotation.TheSepharose bound to the immunoglobinsof theimmunoprecipitatingantibody. Thisisolated the antibody from solution, whichrecognized the approp riate protein,isolatinganycomplexesformed,

Once the Sepharo se bound the Immunoprecipita ting antibody, the resulting comp lex was stable. Centrifugingthe complexes for1minute separated thesolution.Thesuperna tantwas carefully removedanddiscarded.

"Theremaining beads were washed three timeswithImLof extractionbuffer (lOmMTris pH7.5,lxTriton,10J.tmvanadate,LmMsodium pyropho sphate, and1Om..~NaF1,andthenwashedtwicewith lSOmMNaCl After thefinal wash,thesupernatant was carefully removed and 35mLsof 1.5xSamplebuffer (SeeTable3)was adde d..

Table 3Com po nentsofSam ple Buffer and Triton Media lOxTriton

1OOm1\lTris.pH7.5 10%Triton 100mMEDTA 0.2%Sodium- azide Sterile Water

2x SSB O.lM TrispH6.8 4.5%SDS

3.2 M!l-Me=ptoelhanol(jl-ME) 22.2%Glycerol

0.1%Bromophenolblue

Table4 ComponentsofsnS-PAGE Gels

8% 10% 12% 14%

30% O.9mL Ll3mL L36mL L58mL

acrylami de*

35.8% L5mL L87mL 2.24mL 264mL

acrylamide

RGB** 3.8mL 3.8mL 3.8mL 3.8mL

Wate r 3.6 mL 3.0mL 2AmL L8mL

2O%SDS 401'1 401'1 401'1 40111

TEMED*** 51'1 51'1 5111 5111

IO%AP**** 831'1 B31'1 B31'1 B31'1

*30%acrlyamide (19-1acrylamide-bisacrylamide;Bio-Rad)

**ROB:Runnin g GelBuffe r(O.5MTris,pH7.5)

**.-rEMED;N,N,N' ,N'-Tetra-Methylethylenediaminutese(Bio-Rad)

****AP:Ammonium persulfate (Bio-Rad)

To dissociate the proteins present in theFGFRIco mplex. thewashed immunoprecipitate was boiled for5 minutes insample buffer (IxSSB, l%SDS ), whic hdissociatedtheantibody fromthe ProteinAlG.The complex wasrunonSDS-P AGEelectro phoresis to separateallof the componentsof the complexaccording to size . An8%gel wasusuall yused toseparatethe appropriate mol ecul ar weight range,but depending on the particular size of the

proteinof interest, other percentagegels were prepared(as specifiedin Table 3).

TheSOS-PAGE was runinelectrode buffer at 30mAfor approximately 1.5 hoursthen soakedintransferbuffer(SeeTable 5),to remove SOSfrom the gel(SDS willdecreaseme transferof proteins).The proteins onthegel were transferred to Hybond-ECL nitrocellulose membrane (Amersham ), by assembling theWes tern apparatusas specified bythe protoc ol supplied by the supplier(Bio-Rad). This assemblywaspreparedwhilesubmersedintransfer bufferto prevent bubb les,asbubbles inhibittransferinthat area duetothe fact that the voltagecannotflow through. Transfer of the proteinoccuredfrom the geltothenitroce llulose toward the positiveelectrodewith60volts of currentforI 1/2hours.

Table 5 ComponentsofElectrodeBuffer and Transfer ButTer ELECTRO DE BUFFER

50mM Trisbase 383mM Glycine 3.0mMSDS

Tris base (FisherChemicals) Glycine(Bio-Red, electrop hores isgrade) SOS (Bio-Rad,electro phoresisgrade)

TRANSFER BUFFER 25mMTris pH8.3 192mMGlycine 20%methan ol

48

Thenitrocellulose membranewasplacedin Blotto (20mM TrisbasepH 7.5,137mMNaCL5%proteinpowder (powdered.milk;Carnation),0.2%

azide(anti-fungal agent),0.1%Tweeo-20(Detergent;Bio-Rad Chemicals) for one hour with rockingmotion.Thisblockedallthe non-specificbinding sites 00thenitrocell ulose.Theappropriate primary antibody wasthen added tothe membrane at the requiredwestern stainingconcentration, diluted with Blotto.

Thiswasincubated overnight,at roomtemperature, withrocking movement so that completecoverage of the membrane was obtained.

The following day,the western staining wouldbecompleted. The membrane wastransferredto alarge container and washed 5 times withexcess volumes of TBS-T(20roMTrispH7.6,137mMNaCI, 0.1% Tween). The secondary antibody was incubated forone hour. The secondary antibody depended on the particularstaining antibodyused. Thiscould be:1)HRP (hydrogenperoxid e)-labeled goat anti-rabbit(heavy chainand light chain, AmershamCo.) 2)HRP-Iabeledgoat anti-mouse (lightchainspec ific,Cedar Lane LaboratoriesLtd.)or 3)[fa biotinylatedprimary antibody was used,a streptavidin-HRPconjugate(Ame rsham)wasusedinsteadof thesecondary antibody.Themembrane wasagainwashed 5 times with excessvolume s of

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TBS-T. The ECL (Amersham) staining involved the incubation of conunercially purchasedreagentsusing a 50-50 mixture of solutionA and solutionB. This was inc ubatedfor I minute covered with a plasticfolder,and immediatelyexposed toEeLHyperfilm (Amersham).The exposure time ranged from 30 seconds to 20 minutes.

TheECLdetection systemworksontheprinciple ofluminesce nce : emissionoflightresultingfrom the dissipation of energyfrom a substancein an excited state. The chemicalreaction occured viahydrogen peroxide catalyzingtheoxidationof lumina linalkaline conditio ns. After oxidation,the luminal wasinan excited state, which thendecayedto groundstate emitting light.Increased chemiluminescencecan be achievedby oxidation of lumina l inthepresenceof chemicalenhancerssuch as phenols. This would increase thelight emitted 1000 foldandextendthetimeof light emission. Thelight producedpeaked after 5-20 minutes and decayed slowly,witha half-lifeof approximate ly60 minutes.The maximum lightemissionwas a wavelength of 428nm(reviewedintheEe Lprotocol book).

The EeL westernstaining system has a number ofadvantages forusein this experimental proced ure:

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1)Incre ased sensitivityanda non-radioactivedetectio n. Thiswas saferthan using radioactive materialsandthe detection of lessthanIpg of antigenwas proposedto be detected;thiswas at leas t Hlx more sensitivethan other non- radioactive methods.

2)Ahigh ratio of signaltobackground,resultingin cleanerblots.

3)Resultswere obtainedinshort periods oftime

4) Stripping andreprobing membranes withdifferent antibod ies waspossible and enabledmore information to be obtained fromthesamemembrane.

To restain the same membrane with different antibodies,thecomplete removal ofprimaryand secondaryantibodiesfrom membraneswas required.

This was achievedbyincub ating the membraneinstripp ingbuffer(lOOmM2- mercaptoethanol,2% SDS, 62.5mMTris-HC1,pH 6.7)andincubatingat 50°C for 30minutes withoccasionalagitation. Themembranewas washedwith excessTBS-Tat room temperature foraboutIhour. TIlestripped blot was then incubatedinBlotto for Ihour andthestaining was completedas describedabove.The protocolsuggestedsequentialreprobingof membranes was possible.several times withthe membranesstoredwetat4°C after each immunodetection.Ifoundthatstripping membranes resultedinan increase of background.

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2.4 Nck ASSOCIATED PROTEINS

The Nck experimentwas carriedoutas describedabove with a few exceptions. The FGFR complex was immunoprecipatedasdescribed. Tbe pro tein spre sentinthe complexwere dissociatedbyboiling the beads twicefor 5 minutesinthe presence of1%50S. The supe rnatantcontaining the denaturedFGFRcomple xpro teinswasdiluted[0afinalconcentrationof0.1% SDSinsolublilzatio n buffer.Thisstepwas include d becausetheNckantibody required non-denaturin g conditions for immunopreci pation. The diluted supernatant wasincubatedfor I hour with anti-Nck. TheNck-antibody co m plex was isolatedwith pro teinAas describedabove. Thisthen gave us a Nck- pro reinAcomple x toincu bate overnig htwith the supe rnatantof anFGFR void extract.Anydifferences or increasesinphosphoryla tedpatternshould ide ntify Nck-ass oc iate dprot ein s.An FGFR-void supe rna tan twasused. sothat theNckantibodywould not bind totheintactFGFRcomp lex andgivethe samepattern asthephospho rylatedreceptor.

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2.5 ANTIBODIES

1berewereanumberof differentantibodiesusedfoetheseexperime nts:

1)Anti-PLCyl .used forimnwnoprecipitation (Upstate Biotechnol ogyInc.), wasa polyclonalantiserumprepared againstafusionprotein containing amino acids1064-1291of bovinebrain PLCyl. This regionisloca ted at theC-tenninus ofPLCylgeneproductdoesnotinclude the SH1JSID domains. If thesedomains wereimmunizedfortheproductionofthis antibody.itmaycross-reactwithNck, due to seq uence homologyin theseregions (concentratio nl~).

2)Anti·PLCyl .used forwestern blottin g (UpstateBiotechnol ogyInc.),was a mixture of DEAE -HP LC purified monoclonal antibodies directed against purified bovine brainPLCyl (concentration 1.0J,lg/mL). Two PLCy antibodi eswere utilized becausethemonoclonalantibodywould givelessback ground whenstaining thewestern blot. Ifoundthatifthe antibody used toimmunoprecipateand.theantibodyused tostainthe

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membrane were from twoseparatesources the stainingoftheheavy chain wasdecre ased ,resultinginacleane r result.

3) Anti-FGFRl, (Upstate Biotechnology Inc. ), was an affinity-purified monoclonal antibody raisedagainst a fusionproteincontaining amino acids 22-325 of theextracellular domain ofthehuman~FGFRI(jIg) (co ncentration used was 1OJ.1g1mL for immunoprecipitaticn and 1.0J.1g/mLforWesternblotting).

4)Anti-phosphoryrosine(Anti-PY)(UpstateBiotechnologyInc)willrecognize allphosphategroupson tyrosine. Anti-FY monoclonalantibody (from clone 4G10) wassuppliedas aProtein A-Sepharose purified IgG or IgG-biotin conj ugate (pY-b). Conce ntratio n used forstainingblots:

l.O~g/ml.

5)And-Nck (Transd uctionLaboratories)was amonoclonalantibod y raised against amino acids 279-377 ofhuman Nck.

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antibody cross reacted withtheXenopus prote in anddid notdispla ycross-reactivity with PLCy l. 1beprimaryantibody concentra tionwasO.5~mLand the immunop rec ipation concentratio n was 4JlglmL 1bis antibody showedcross-reactivitywith thefrog.

6) Anti-GRB2 (Transduction Laboratories ),was a monoclonal antibody producedfrom purifiedGRB2 proteinfromrat brain(concentrationused forimmunoprecipati onwas 4JlglmLandstainingwasO.5J,J.gfmL).

7) Anti-50S (Ups tate Biotechnology Inc.), was a polyclo nal antibody manufactured byimmunizin g rabbitsagainstarecombinan t GST-fusion proteincorrespondingtotheC-terminalregionofthemurine Sonof Sevenle ss Iprotein (concentrationusedforinununoprecipation was 5.0JlglmLand to immunoprecipete51lgpersamplewasutilized).

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8}Anti-Xenopus FGFRI (produced inlaboratoryby Gillespieand Paterno).

wasprepared by inunmizing rabbits with asynthetic RJfRl peptide (MultiplePeptideSystem)conjugated tokeyholelimpethermcyanin (Pierce). Tbe RJFR Isequence(CLP KYS NGGLKKR)corresponds to theC-terminal 13amino acidsoftheXenopus FGFR I(Frieseland David,1991). Anti-XFRwaspurifiedusinganFGFR I peptide-agarose columnprepared withtheProt-on kit(MultiplePeptide Systems).

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antibody had theabilityto immunoprecipate at15~mLandwesternblot ata concentrationofand1.0~WmL

9)Anti-SHC (Transductionlaboratories),waspreparedbyimmmizingrabbits witha C-terminal peptideof theSHeproteincorresponding to positions 3594 73of the intact protein. Thepolyd onalantibodywas affinity purifiedon a column of the immmogen coupled to agarcse. To stain western blots a dilutionof1:250 was used. To inununoprecipa te4fJ.g of antibodywas added tothesupernatant

10) Anti-SHP2 (Transduction laboratories) A 20kDa protein fragment corresponding to residues 1-177 of human SHP2 was used as an immunogen. This monoclonal antibody was purifiedfrom mouse ascites bychromatographictechniques. Adilutionof1:2500wasused tostain westernblotsand 4jlg for irnmunoprecipita tion.

II) Anti-humanGTPase Activationprotein (GAP)(Upstatebioteclmology Inc.). This[gO antibody wasproducedusingfull lengthhumanGAP producedin baculovirus. Thismonoclonalantibodywaspurifiedusing protein G chromatography. Toirnmunoprecipate5Jlgofantibod ywas added tothesupernatantandadilution of 1:1000was used tostain.

12)Anti-ratP13'Kinase(UpstateBiotechnologyInc.).Theimmunogenusedto produce the antibody were glutathine-5-transferase fusion proteins containing(1)the NeterminalSH2domain of ratPI3'kinase and (2)the full-length85ksubunitof ratPI31dnase. 'Theproductproducedwas a

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mixtureofequal amountsof rabbitantiserumrecognizing p85 subunit of PI3'kinase. To immunoprecipare51lgwas added to the supernatant

13)Anti-humanPI31.cinase,monoclonal(UpstateBiotechnologyInc.). Two antibodies agains t PI3'kinase were needed because the monoclonal antibody gaveacleanerblot with lessbackgroundandthe amountof heavychain identifiedwas smaller. 1hemonoclonal antibody was immunized usingrecombinanthuman p85 alphaexpress edin Ecoli.

lugzml of, protein A Sepharose affinity chro ma to gra phy purified antibody wasusedtostaineac h western.

14) Anti-human SRC (SantaCruz Inc.). Thisantibody was an affinity- purifiedrabbit polyclonal, raised againsta peptidecorre sponding to residues 509-53 3 withintheCOOH-terminal region of the humansrc product. To immunoprecipatel.OJ..Lgwas added totheemb ryoextract Tostain awestern blotO.llJ.gfmlwas used.