Mtfedi* CfttLVol. 1«. IO*-JlB.-indff>*r4fnt cell grnvyth and colony formation in aoh agar. Ttwt*? ejipetanents reveal a novel FAAFK-mediated, negative feedback nnechsnrani lor control of signaling pathways that ate dependent on FP52 jnd a mechanism far heterologous control ot signaling vie FGF receptors. hwtroduclion Fibroblast growth factors OFGFs} constitute a large family ot growth tactdrs tfidt play irnpcirtint rolei In multiple csMar processes critical for me normal functions of Niuiy'dmum In both InyerteUratrs and uartehralBiitB-viewed in Goldferbv 19B6), Garlic Hudifl* demon-alrated lha crucial role oJ FQFa- during the early fltarjes gl ambryogenesn, when embryonic polls depend on FGF signaling tor gene expression, morphological identity, migration, and aKisdcuclDpiTienHAiman at al., IJjKJtt Beddington and Robert bcci. 1699; Cirurra at al.r 1997; Craanlny and Martin, 1B95; Dnnget al., 1954; Fnldmanet al.r 1995; Maski and OrnnX 1998; Miswandsr and Martin, 1932; Sun nt al., 1B99; Yamaguchi at al., 1B94f. FGFs mediate the* pteiotropic responses by binding lb And acnvatlraoj a Family ol receptor tyrosine Urtaaas (PTKsJ designated FGF receptors (FGFRJ 1 previewed "1 Schleaflriger. £000). Many of the caMular response* -physks, MemwU tttii>Ku1iiai-L| fjuiur CiHicer, No* York, tibm Vuh 1DQS1 rPr»o«Yl Hktau: nu t*JrDil IraHlirt*, NYU Madlcd adiDDl. htovi Vah, Nuw Vert: 10016, contain mynatyl anonore and pnescnotvrosine binding [PTBl rtorriaini In Iheir N lormlnl and rtiutlipfc tyrosine nhnnp-icirylnlicin nlnn in th,-;ir C. tr^nini 1h,it nnrMn nr, binding sites lor th* adaptar pratan Qrfs2 and lha piu-tcin lyrciinn phnspholasa 5hp2 ^Kcjuhara at aL, 199^; HRdanetal,. 1993), ANboughbolh FRS£ tamily rwrnbaie ant highly honwlogous. thalt dltterantol paltun of ax-prcnsjofi points to specihe mlos fbrnach mnmbcr. While FRSfiu \i utaqtntou5*y expressed and can be detected at every dtVfttopmmttftl atigo at 9i A HUtiae-, thd aiprt*- iion FPS2h begini nt day 9 to 9.5 and ii primarily confined lo liesuas o4 neuronal ongm iMcOoogall al al., 2001; N. OtftOn at *J., rtptibfehed ctala). krfnMtd, lha neive growth ractor^NGF) end glial eel Ibie-dferivedl neurotrophic factor pGDNFl lamihae of neuraltophins also signal lhmugh I K!J2.i ;ind F FIEi2|-! and regulate the- activity of FRSfi-dependerwt signaling pathways (KjuroKawa at al.. 2001: Mtrfclhi et aL. Sffll |. Wc have demonstrelad that disruplinn cF the FF-CSZr gene results in embryoric letnalHy at approximately E .b dua Id iovnr* daFeCts n paitrulilion |Hadari at al., 2001; our unpublished datej. Expennenis wing FRSfiu-oaticieflt mouse embryo fibroblasts lM£Fs> and MEFa tortnalnjulBd with althter Wlld-typa or V-anttai FRS2n mvtentE revealed tfiet FRS2« links tne actuated FQFR witti the ftaa-MAPK cascade. (2, lias a criu-ral rnln n FGF-dcpcridenl dcII prolitcralion and migration, end (3Q functions s» a focu& cf assembly of a mulllpmlein oornptoH lhat raratrtfe (ha fiae-MAPitcaa-rade and lha PI-3 kinase-depefident survival patFmay. Upon FGForNGF stirni*Hion, FFISZrr recruits FourGrbE motecules directly and two indirectly vta Ehp2 :i i.-az 1.--el al.r 1999|l Rescue eKpennieirte uerncj vaniderrt responses. In addrtton to its role in racrurtmeiH of the mjclubdfl exchkangd Factor Soa hy tyrosine phoapho ly-Inlcd FRS^i, Grb? tunclions as a hnh batwow FFtSZir and the docking protem Gabi. FGF stimulation leads to Qabl recniilniant, which is folkJvrtd by tecmilment of F1-3 kaiasa and acllwallon or a ctM aunnval pathMTAy Oget *l„ aqqi: Hedari et al„ EWH]. The E*p2 binding sites play a pnmary rote m FGF-induced activation of MAPK, call prokleratjon. and cell migration |Hadan at al., 2D91 |l The GnbZ binding sites oF F FE2-.r are assentiaJ for activation cf PI-3 kinase (Ong at al., 2Dfl1r and tfie ubiquitin ligase Cbl (Wongetai., 20023 and nave asec-ondary role in MAPK ctanulaljoii DHadatl et al.r 2001|l In this report wo demonstrate that n addrtion In cn- rtencwienfof tyro*"*? phcsphcrylation, FGF stimulation inducee MAP kinase-depandant phosphorylation of FFtSEa on al laaat afglil threonine nsiduea reauPJng In a largo shih in its dcctrciphorelic mnbiiVty. Threonino phosphorylation of FflSSi n accompanied by reduced tyrosma pnoaohorylatiofl el tie docking protein, decreased recruitment crGrbiL and attenuation or the MAP kinase response. A similar FRSSr bhreonine phosphorylation n induced in response; to PDGf r insulin, or EGF treatments, growth lactate that do not induce tyrosine phdspliorylatJori ol FRS2n and do riot ATumubJa the b>o- log^al reflpoaaes ol FGFa. Tl*ew experiments, deinon-stMtn Ihfrt in flcMrSon to Ha major rnln an a rriadlaloruf etgrralvig via FOFfla, FBSSn participates in a negative leedhnck mechanism and an interraceplor ronlrnl mechan-sm for regulation ol FOF-receptw smnalma We have prcvioesly dcrpjrflslijJDd Lhol in addition Id lyrosinapmspnoryklicin, FBS&r ufidenjoassn eleclro- ptioraCic mobdHy shift thai be probably c aused by phos- pftorylaUon of FFtSSii on senneAhrewiw residues in response to FGF sflHmdalinn jKculiara at Hr37; Fiym 1A), Indeed, Hie FGF-induced etectrophoretoe mobility ehrfl was- reversed- by Irtartrnerrl at FFtSZn orinmjnoprt-cipffatflt with alkalin* p*io*plw.1sw JFigura l-flj. In con-traalr IreafanerYL ol Ihe telle Arih Qkadak-acld-iOroteiii pfiovphntfaw inhibitor, induwd * pmnowicarJ eleclro' pliorttlc mobility ahlrl-ol FfiS&s that Is- stranger lhan that induced by FGF stimulation (Frgiffc- i AJ. Tak*n togrfhw, these e*p«BiiMi1e ftuogeat that me thrift in Ihe alectro-plHjtKtic riKsbilinj ttf -FpSSit mdcrcpd by FGF stimulation ha caused by protein phosphorylation. EltcfroptKHVtK Moiilily Shift vf FH52k to CmwwI Primarily by MAPK-Mwfcated PhnouliDrvJatiDn rf FBSitt To examine trie po-gei^rty thai the mobility rtiitt od FBS^r is. causad by rtinawfsr tying downstream ot Ras, we hivt ex&nHted the effect ol ovarexriraeaion of a dominant intcHonn^ rrarlant ol F2as-Flaa N 17 {Rur. HJNj on FGF stimulation ol FFtSSn ofectraphoratic mobility shift. The ejcporimcnI presented in FigurolB shows thai expression ot Ras UN almost entirety prevents-ffie mobility shril at FFtSZc Biduond by FGF slunulalrai. TTin expression of Ras 0*1 am inhtt^dtfroacliralion ol MARK rfi FGf-allmuUad alls ^Igui* 1 6> and turreependlngFy mcreaped 1 he tyre*i rn* pti nfcxylh|tK;^i nr FrtSErcand rt& inleracb&n iwUi GrbS, even m iBiabmutalsd cells (Figut IB^TlwelffrutofRBvDN on th* mobility slwfa ol FRSa.t mtkiced by FGF stimulation *a* Ineomplate because lyicainfl phosphorylation a|» tjuw 4 >matl. but delectable, electroplKsratic mobtMy shift of FftSSa. Baaed I" esc result:;. we proposo that Ihn h/uH. cF [he □Irjclrn- t^n^Qti^ rct*iinv ^h.n -.'i n?aa:. «oui$ ^ ;i rus-ui: a region thef &iks Ihe PTE damain to the rest ol the- protein. The second cluxjarjairlino «Ctdhf 3-r**-3ir 7) cgntaming- a singka polfln-Ual MAPKphoBpHOfyialjori wle iPKIVUft located' toward ■ho C tflrrrmysi oF thw pnjt«n. The Ihird clunerqontaiiirB loui poteoual MAPK pttoaphorylstion sites (ammo ec*ds 4F3CMC4, P□TPKTPTTPLPQTV) is Flanlmd by Ihe tiro SlW-StMr^ino&rte&infltoCtemitfiusol FRS3n. Tostudy ■hair rundioiul rate in FRS2nn yib have \-:..- !" . Ihreor—ts residues, ol the PKTP clusters wrtfi vshne resi- dWrasurirtflallo^ltocirfminagdrtesis m Vanitis ttmW- FŮF. OHaoalc Acid: IB; FRSSo: + + + ] FFtSaa IP. FR53« FGF: Dex: l6:pThi - + + + + - J- pT- FFtSĚn IB: isTvr IB: rP5^'^|tŠM^M**3 FRSaa IBiGrtjE — J-Grba IP: FFlS2vt FGF: + + IB: Raa IB: pMAPK iSrlWAPK 3- RasN17 y pMAFK ]- MAPK TCL PhJiMWT. r J llrtitM EfcurjvutHJriflK: **WfPtj SNn PFHSři 4 WřRSř, 1 WE Ft- w^iHQ ^lii-iyfrn ?ňž> .wt f*m« M ijft-lr«*«l w1i*í»«1 Bríwah* m >aů-iW eí^aiste acw.iyůalwrpoín UrrStttftJWSd 0f PCř-SltHUrBMíl C*« #««t4D4K«droi«W4fH»- 5 ^fUHfW ůí J)lv9Vrt*fflWpriít#*f 4*P) 1 hf 9W ST 1C bJÍtklí rublunliq *irh uiTt-FTt£:v. jimbuljďin. IPl PC1 ř r.ňfc, isúiitlffMid wl|ti ad«^iM*uťUHi-lii<]iu:M] rtu H-17 MfB U< umotitd ar «ora trutbd cnadnlqric Z p.M [ÉMDmDTtiarHHU |DiHj. Lyums turn undlmidalDd cm :í!F.::nv blud cdtlt ikona taHknafvaclprlalad allh jrvU-FFie^k dnUbcdtaí; relkm«J by SP5-PAQE ImtnuKbUlhg mw vartaur, .mbbodns □i IndtBůd. /.ho iho*n, ImrriJKtJoli xlltt antirtas, antJ-MAPK, and arA-p-KUPK. arittumUů. ol talal odl Kintas |TTCL|. naletíšioAíh chístffrisnumbored accordingln itsorder ol appearance along 1J» FFiSÍt molecular, oř si sighl cambinod íFFISJu B\Tj. The aHnd nt Ihc subslilutienE on 1he eleotroptiaretio mot>*ty ol líie FFlSii mutants was dtrinrmlnnd by SOS-PAGE analysis.FF52n irranj-nopreciMtsteB. isolated rnom tysate& ol uiiatMnulatsd or FGF etlrnulaled! 293 c^lla ejipraasiny cdhnr iMdd-type f11 ■ * I-4 YH1I >jH?l * * . H »m |ti3d' I tan Pt Ft' Pl'l ....... prri' p1tf M-.l RS-" iniP i n□c lt r PJr.--.--tP H-- J If pq-pifPT-pipg. r* F □ H P 1 5 FO. it ■ P T T II P p D-i i k i p t r pi p-a - rr P n-1 P K 1 P T ■ pi PC -TF FRSZa: Ml HI U] M1ft M2.0- BV VrT IB fRS&iL iF>HHiai fresh n^fr BV1 *jf iv"1 l^J:pf^^■ «.*F453u J liT-FHWlf |- PRSZa FT FFJE2II fop T^,rr:'r7rT7 B.Fbg 4 pThi ]-FK3d I ..r.niF.: W,: UEHMAPK- AC nT * rush _) 3- MEKMVW to. V- '.".Ft e Ffit*i ic-FKiiii ]- a.pThi ■■■■ P.■■■:> 1} Fhjurr a N".' Ot<;irt>ulnjnfllS MottUr Shhl * fcpwwd brr PtiC*vliCfviallOii 0< fJuht TlifeoMnO FMWOwM or* fWSii By MWK |fl * idwrWfUc p«nHkFl & IWuft* KffiUn KllHlftfl lhu Irrviflir-jHKMiHrorrHaikXI sTlm ai>il1h*|HilatJY* PA*PK rrrtHyiHr (Till} u"(nehc*yly- Itatt Mm rAnrrr are wifjansmit ot me *«» KM sequence* opt In? mr*.' ckMnt OMlartrid The puurrre bihxppnf jHKM|»K>nrt3lk)ii Oi HBk j*J tent p«<« win**"* hHrtftocted w Ac fflSiss-Vrr w«rto#j rasid imunts w wNcn Die (Hiialu* M^i* TnimHime priWPtKfYWW* «» h PW WW i*1»r$4* fllrtlwa -Mr imWhri «WrllUHy to wfllkrH fM1, MS. UJJ Of *l CWlOf^ri Ot/l LytMM HOT! in~i^|rT>pl*^riuiofr mtn iuiH-FRS?,. ^pririochw Ihfkwhm] riy |rrunirfitt»*1|rfl *nn lUlH-fRMh jmwUw- rfl HEK ifiJ t* 1rerHf«;|«p urn FTtaan-WT or wffli flw FRf^,-FjV- rihimiit nne FflJi:, ■ MFJf* «4XW(tnQ FFuKurWT W tt* FftS^JW rruiLam-bmu slInriiriJMd bIIIi F^Fwmuu:«(i iiiiriitTi Jnhiil FTtj^-.h |piuiHmrifH«clplrur« Hre Rnrnup^iLnlMF nrai jnu-p-Thr, jmi-FFliii.r, onn-Cjfc$ ArrilbDdAL (P| HEjK jfd ultaL ViMd tranglMMd vw* ilu> IcoVhw^ FUAiCtugguJ pevtaipa: *|ld^pfi FHR-Ji, |kj, ^Ud-1yfM FFliS>-; a LihJiTkMk: pMlAln In HMch ew myclttcuBon ilgrul brtflBH FIE panuln a Fn±K, iini mtocImtJ m huulal FBfiEj'i (FHfiSi ind a chlnuwia Linvuud irf Ths nLyri&inlarlQri d^vul and PTE [uibiln cri FFtfiBy ItiLid iu itw uti ol FRfilii |FFI5£|.Vi.p. Lywuu or traduudjtmt w FeF-STtnnAU«d id: **h Irnrnunc^m^prtiiacI wlih anu-FLACi jmboalH lotawMl bj Iriuiuinobtetlng rtWi prnl-FLAin. jrvu-p-Thr, dt antlp-Tjr unUndaa (E^ HEK 2t£ cafe: ■Aaiu-Dulrarisracbad witti FTrti.-. and u4n cttharon upnulon idcborlar actlifltad FJ^c IspoAd MEK-MAF* of wetti akjHBCslort vbdnr for sUd-typa 41 ^-MEf^-MA[WTj. Oall tfsaUa ct unsllrnuytod or FQF.xUmiilalDd cols vimut rlrst ImmunDprrclprrjItecl ard ■r-:r "i"ij^9!il9lLcdrt1U" ntll FFIEI.. 3JilfboilMi k:^jiII-t,^-^!^! trnmumblnl cl-kitaJ c-dH h^'ulm ITiIL'l, to wjiuJ tturukpreukvi tovisl trf thfl activated and vrtJ-rppe: UEK-MAPN pnrtsHnr, |F1 l"i iMzn pho 74wr,rUib:fi of F R E-2n purtTied 3dlvat«d tAAPK. FREib ^nd tta FR&2icWJ mu\mtvantrvnw3iiiw\&\ulrifc^un5\\m\ikt.&l a FuF-iBmul Jlod MEFi. The Immunocrwapblas urar* washod enca vrth a khiaso t'jflor and trum dlhcr loll itrrlrutad or incubated wr.i acllvjtod ET4U tor 30- rrin at S&C TTm samplK mhv aruly^Dd try orn™ j-olll:i:| will anllpThr or irt FRKlc anllbcclDS. Aohauxl ERK2 vus ImmunDblcrlDd MBi anllip-MAFK anUbccbe. or Maiiuufl. FfiSSn mularrta. Only niuteltien cd all eigfil pcrtatiwe MAFK phcophorytaiion sites at the ihw dm-(era preverHed the etecttoplwwlic mobility Bhih ot FBa?i3 induced by FGF slirTMaaJinn JFigure 2SJ. Similar results were obtaiied when all eioM threonttnes were substituted by aoaiiino rcsiduox (da I a not shown]. In addition, by itaina errhbodies raieeoj aosin&t the canoiwal MAP kinase p-Trar mles (anb-p-Thr alrabodiesl For imrnU- noblottinfli fljnll-FRS2.i pnmunorjrecrpitates Iroirt rysaCea of Z93 golf or from fysaies ot FFSlr 1 MEFs eiprw irm either FFlSii-WT or the FRS2a-0V rnutent, we dsm- □nstratad thai FGF stinHdaticn enhances threonino phoBphorylalian of FfiSS™-"VT hurt not threonine phosphorylation ol FR52ii-flV (Figure ZCj. m order to shed lurther light on ttw rots pMiyedby the three threonlne-Hdn clusters In medlabEng the cellular MWéulv Oil 71Ě nasponae ol FftSSa, have Generated chimeric FRS2 pioleins in which ttiecai^oxytenniniol FRS&t (contains a PXpTP MlaftJ and FRS£p 1 lack a nauihus MAPK phosphorylation silosf wore swapped. The chimmc FFS2 proteins are designated FH£2Wp or FTB2|y«. Next, 293 DtHb were Crams Fee ted wilh MpnHmn vectors lhat direct the synthesis-otFGFfll tcgelherwithexpres-SK^vecb3rar«FIJVG-LiggBdFR52itiFn&2^, FRSZtJ^a FflS2rJAr. The experiment presented in Figure £D depicts SDS-PAGfc analysis or lyislies IrOrn unstmutited :peri-menl demonstrates that endowment of the amino temml-nus of FR320 with the cartway tttrw of FUSScr «n-al^efttliemolwxjIalobKonwtn^nMwp+iofliJtio^laljsd and its mobility shifted n SD3-PAGE. By contrast, tfw Ffl£3«/U protein was not pho&pn orylaled on threonine residues nor was the-cloclroplionctic mobility ol the chimeric proteai altered in response 1& FGF slirnuJslion. Tho integrity or both cremate FFSffu/lfl and FR52|-!rii pioteins was nmilBW to Wat ol FftSSn: and FRS£p since bolFi proteins werL-tyrosine cliosphurylated in rcspuu:^ lo FGF stimulation (Figure 2Dt. These results further export the notion thai Ihe three thraonlne-ritti elusion in FR52u aro the targets of MAP kinase-dependent threonine phosphnrytellnn. To further confirm thai the e I eclropti orotic mobility shrfl of FRSZu is caused by MAPK phoaphorytalion. 2ftt cells were cohan sleeted with an npmwiosphorylatiori. is enhanced relative to tyrosine ptios-phoryfalion ol wild-typa FFtSZtc. Moroovcr, tyrosine phosphorylation of FRSíi-BV is detected even m un-slirnuLMod liKFs. Cůfccllvely, ttieoe ůipertrnents ndi-cate that MAP itinBwe-dependeril phoophoryistion ot FRF*2ft may lurullon as a ewrtch tttal ne^atrvety regulates FGF-dependent signafag pathways that aro mediated by FRS£n. Indeed, the experiment preaemed In Figure iA shows that the dLration of tyrosine phosphor-ytaiion ol Ihe FRSfin-SV mutant is prolonged as oorn-parod to that ol wilďtype FFtS2fTDfw*i*i*Pftwtftny- UtJafl Wl E.lť£tdty3hťr All(: Uí rfk|fty Sfi|t1 fcft- and Cornpta* FqhiuíiIími *tth ůrbS. knwtimJiíM] o FCF-iniTMJHlfloUEň lh-praulng FnSři.-WT oř rtu Fnfiř.v-e1; n-uu*n vturabn uufiQiDd of třUfUfd *rrh itu HEK Inhlbhjr U013S. Cbl Ijtuuů; mu ~iir)nrerťl Id Immuraprvtrip rial km vunJi jtjU-FTIEůe onn-bod Iíů rol \a m od tiy I mm u noUotlhg wtBi an U -jrrllpltr, ariU-p-Tyr. Df jno-Cnba CBJtJfeHrriulalBdiíiFůF-sIbrráJatBiJMElftíM-pnmlnrj FRE2i£-WTar rtu FHEŮr-lW mubinl ■junralatl untniJlBd-CT IrůitedYillh a MEK. In ráMtcr [UDÍ in, PKC srHMtor JGF100303X1, C¥ R-l rtUHlCT [WtflrrunnlrO.CíHl t ■jt« winisublDclDdhi-tnnmnDpnKtirtallafi wttfi anti.FFSaa anllndkK kAmU In/ Im-■MlNumij wlUi anll-p- Tít anbtxrltoG. threonine phosphorylation, it foiowo thai FtiF-depen- dent signahng pathways UuE m dnpnrpdrjil upon FAS2n will 0s potwUisted in MEFs. expresana the FR52a-BY íťiUtáťil. Indotd. the MARK rtípůnrtů Is hnúrt aistiiinorl m FRS2n-flV eKprewing ceils a* compared With MAPK KlimuEaJlůn in Ďéft-i mortising lAnM-ty-po FFS2y? Figuro ^Bf. By contreit, tyrM»»* phosphoryia.-nui i of printeoliL>iipfr££ CY wi^s swiilar In f ASSa-V/T and FflSa^^BV eaprewing cefe figure 4Cp, EiJltóňtůtf MilůOMUt Rfrspůiiíe at Cella Ej< pra stung lha FRSSrt -9V Wutairi We next Mammed Ihe biolofjicaf consequences, of the disruplFon of FR52.ri Ihrmnaui ph ascii ary I alien in FRS2n"' MEFs exprassirrg erttwr wild-type or miitanl F-H52i3. Using Ihe Boydnn cEiarabur assay, woobsnrvcd that 1he mobility ni unstimulated c^fs expressing f FtSin-UV iu íinhaniíjod, arid that FGť-ítlmUlatiůn ira-dknéd stronger ob9 n*tjf3lKjn of UEFs expnw&ing FREifi3-JJV Ha campaiftsd Id MEFs úíiprrtílng rtJU-typo FRSín (HopiFie !\ftr, In arJtřrkín, MEFi sMprewing F FISin-B-V cirt-w luatL- rhm: w'lld-lvfjif WLF a uid caI- U-ÍM^igfror*s»-«kfwndentyow«h iniwponwtfl FGF aliniiífliion ifiotireéfl). Ttwlra(tbrůfm*d-likií(flBrií)t^|M vt t,\t* fRSEťt-BV cat? ww ol» i4f1o;i9d in ihe *ilirj of ttiom oůIIs 1o form «>HV4S in sort aaar Iťiguro 54). This- rtnchnra^c-Didrpandnnl qrťirfh and Ihc ability □! MEFa axprassinfl trie FRSSn-SJ fiiutafil to fjraw =n eotl agu/ demonslra[DS \t\ul lhc Ttfonc|ef [nilngaruc agvul eaarted by Iho PPĚ2n-9V nwlBnt protein i& capsHo of IrtlUiriMg iůrttoi hallhiirki ůJ cd trMfflbrtnillůťi. MůnS- dvw, íheas experimente demonstrate rhat FQF-indu&ed thmninn phcisphnrylaiiDn □[ FR52ic nngalively rogu- laUfl t^roBme přiosptorvlartnoíi of tu* dockinrj protein, a nlup ůrutU rťn rttnrtlrnefil and atllvalinn Ol multiple Hflnal tFansduction pOmwayE. hMtJpla flrawlh Fůůtcrfa Induce HAPK-Dep*ndanl Thrflwm* PhPsplwrylBitipn of FUSIji Tú 1urtl»r UndůralartJ (he EOKlIlHty ůí FRS2n tílrtoťilňe phmptiwyMicin in smrth fflctw sigrufcigL wo studiad Hi* phosphůryhaíion prolili of FRSSn mi i'eeportw to diílcri-ni grnwlh fad«s. TThí cxpcrímcnl pminnhul in Figura 6 sho*a Hnát Ihe eleclrepíiůrůlic mobility eriilt and I hríiíininr phoaphorytaliDn al FfíS2n aru inducod in r«ponw to insulin, E&F, or PtMF stnnulatKm- These ■ .■ k' r;l::■.:111jI;l: slanuliriDnat mckice tyrosine phusp^ioryln.-tton ot FHESo and, ho Iv, ftwe is no evidence ttiet FR52n pldPHír a lůfc in medtíJlIng their ÁfliacaMa]- rt-sporoos. The elect rop ho r-etie rnoMrty E#ii*t of FflS&r IndUrtťf by EtiF AjUmidallún WůA blocked by eJtpttssolún of « dominant ^rfwins nwtant of Ftes [Ffbl N 17}. thus conllrTWigUiat kaiaacĚ thai art down sira am al flaaare mapansihio For lhc EGF- indiírod nnpqni* fFíg^na Wr- AddifJonflíVn unmunobJoling of anli-FRS2u invnunopra-cifijtíUíití wilh anli^i-Thr Brrbbodiei shewed Ihtrt ECF-induced Ihroomiia phosphorylation ol FFtSĚn i&etrociory rnrdíhrcd by a»picssicn ol FlasN-17 m PC12 cct-n. Sinv- larty, tl»elefftll[Wi PikBptaŕiwľiou of Fnefc< ňd tw>K » HJKrt py TIV»urtíW Ptw*pfK¥yl«f«i 4i tfw PMíínflPUŕtuln ^; Tím HlhUliJ, uť FCFhMOuCLů tylftün* (KHM(Hkiŕ|laiton cň Fftů^-VlT ĺv HE^i-BV ď* kraden al FQF-Iii(1uľ«1 canpbi Jor-(natton batwaan lita jrcl FRSír.-WT cm FR33h-B(ŕ. Cd ^uAu ií»( r^jijM^M] in ht-rnuncpralrtfaBOT *rHiarrrJ-fra3s arvttiorJ. les totcŕ/tul Im m u nóbl cti hg *Hh Jrtl-p. Tjr, anU-FH33*i. or m*Orba arrnbocta. ■|[Ji Th*+jnüür-i cl PQFIndJccd MAP khau mlaHan In MEFs cxiircír. rq FTlEikr-WT or f Fffian-HV. Iota carl Marce rTEĽ| warn irmnoWottod Htth anU«flPK or aratp-MflfK anrJtocbes. (C) 1aa HthOcs oj PGF-nduc«d tyrorclnn prnsprKHvtottori al phoearrolfeuui CtíFLC-J *iMEFruiprasilns FB62inWT or FR83a-flV. Ool hrcalw cutflpclDd to nmuTcprc-apMnn wTh ar* PLCT anflbodfcK ľcHltJuUfr ťy ŕ^numrtjKjiana; wnii jnü-PVjCrtKart-p- TCL C rGFiniinl: Q í IB: pTyi [§| IB: PLCr|; ■a- i S 15 3D BO l3f* * 1 5 IS 30 60 130 lP:P|jCr I PLC-/ WAP IjincöSTjspendim ptosphoryiaiion of FFtSSJu is en-haiw*d. br slimuli títaH tftduca tywoaine ohoiipiiurylaliun tJ FFS2(t fijfc, FGF) and by stimuli ihwt tk? rwt indue* tyrosine phsBpHoryfaiion of (ha doctjnrj protein (Le., aistán, ÍGF. and PDGF|l Černotu FrjrmarirjFi balwrtn FRSZii ahd HAFK m FGF ílimulBrtr*d Calls We Wei* ÉhrlBfHílŕd In thn iviichmiism úi MAPK-depen-chrflptwsphorylslirjn-öf FFSZVj ar^howttaHttenualiori ol lyreeint plKrspriorytalIcin ie cůjueeů by ihraufw» ptiosphory1n.ttQn -pf Hhia dwhkíg proriqin, In wäpmwpn- ťWMipi!atBrt-í-jennnent5, lis™ rtoliced compter loľ-maiictn twtvHNn MAPK sntf FFBE& mpern FGF írmrtnwnt ŕFifjure 7A]. We enbasquwUly anatyssd Ihe «i mívo asao- ciabori holwo™ HAFK and FHE2Vi in FHS3n 1 MEFx ..■«un-^Mifj eiUMr FRS2n-W7 w ma FRS2n-SV fiiutarrt. bi this axpetBi wiilL kysates Imm lonnrlanulalod nr FGF simulated; mite-wwe 5«t4ec[ed 1» imrwnopreciprtation With inH-FRS2 intibůtHůa lůllúWéd by IrninLmĽfckiLlitirj wihi ňrto MAPK smihndirs. ar,ti p PK. nr anti p Ty r aiftttodltä. Tlie eRfjejT-jneirt rrfrSMllad m fw^ufů 76 ^ow&^rjrTif^kNfonnBlK¥>bfltw»n FFl32n. and Ihe Bcti-v:ľ.L-d kimi úi MAPK \ň fy*ar*4 Erorti FGF-nlimJalBd (Müs, whHs compleot f-ormation íwiwaen the Ffiiix-tiv rTHTlani and MAPK was nLrDngly rDdiKod. To iurthw aríflJyze Ihe mtenacliofi b#twa«i F-HSí-i and MAPK. 293 c úle wen pofcrafivfwtsd: with an gHpmsian íreetůr tor FflSii toflÉnliaf wlLlt akprHalun weitor*1lkflJ dir«ťt (tw «Kprewieni of ajctwvtodj HAFK or wU^vP« MAFK chlmarie ptotelns. Ly^artcl h-cirn TMtíUmUlated. ůr FGF-nlinHilatod «II& war« «lAjactad to «nmunopr^cipi-talicxi vjŕlh anli-MAPK anlibod»« ľcfcwod L7y immu-noblüttiRfl weih anft-FRSÍn arrtftodisa. Unity ihn? acti- vaEcd larm of MA.FK Inmicd a enmpicx wHh FFtSSir iŤn*jrť" 7C>. In addiliůn, complait famuliori btlwewi FFIS?« ami MAPK wfl» disnipHad by Ireming Thí pbI? wíUi lhů MEK MhlbHůr UOiáfi Pn^im 7Dr. CollMrlIuuiy, th>o*5 íbr/w that aclFvatedi MAPK forme a «m- plaii With FRSZn \ň rtotpůftia tu FGF ctúnUlahůn and Hi -/- WT *V 3 TO Flyura 5, BfrfaKMi Cti\ Mufllry JJ,d Colony Rvmuttun In Butt Aijii uf Oris Expitts*^ llw FRalL-i-iV Uut^d (A) UMlfnHJDtod or FOF-sariijlM3 HEFi aapniuic^ FftHi-ViT FRMr.-flu' mud iiibfacivd nj Lvti iniuuiriof, juj^ uiIihj itw Eupduii chamtur UftsBrrkJalBti ijopun squajmi; FGF ttroJated Klcowi souir-osi colts. IT* raiJti are Itu a/tmjja d Ihru iupfrirptrrii (HI FOF-lnduc&d idl prdirnnHSor, c4 MEFs Hum RftaSa ' (tk&sd dictosfc, MEFs upra^ HQSa-WT bipw squoresl, or Da FR&Zi-BY mubd Jofossft squajirt]. Ccl numbers, worn onintud anjr lha coursa nf In aa^s. TTk mulls it: Ire j-hwnna or Sho espflrhncncs ptrlnrcn&d In rttpllcalBi. [CI Itw growth at FFIEKii ' ME Fi and FFr53*i ' UEFSi EUfxnsilng FR92b-WT or at* FEES™-BY mutint altKhdd Id mwxjtipi^; af hi D.3K soft agar. Anchorage-MspwKbfTt growth ol macroscopic tolcnlnsi was scored jflnr 14 days ol FGF stimulation. Thn oLinnrn darlcls qunHtiOJin nfflia Datfrnr Imrrtnn flfflclancr al Bno FRE£:i ' MEFs- of Inn MEFs- copras Ing FREn^WT ow FHE-in-BV mutant. is Scaly pflsjwraiMe for tiwaLminu phosphoryWiwi ol PR Sib and perhaps also kir lhs attenuation of tymuaw p^g^jhfi*ylartK*i gl 1hc rjgtiing prnlcm. Duguiajicm The dncksuaj pmlnn FRS2n ^s a 1icy modiartw gF signaling wa FGF receptors as well as via members, ot the HQF GUNF Families of ndutchpsfJuc factor rc-captors (KtKihara et af., 1W7; H&dan el bJ., 1906; 2001; Ong el ad.. SOOi), 2M1; DhaJIUln at Jd., 2000; Yan ct ai.F 2002J. In ttiis report we derhcmstraie thai ■> sAtrhon to rts posnurva r'&rjUldkl4ry rota ft, FTtSZu lunrinonE ai a toy cenrrt-ponont gl a. MAP l< in aw dependent negjaliw fsedborJi rrtechtflnicih I hat enwrap iiiilf oll&d donagfrciJ ingnaling via. PQF rwwpton (Figure ?Ej, Furlhnrnngrg, lha ability QlaTauiini. EOF. wPDUF toil dues Ihreoniiw phLiBp'-..--y-iBtJqn g| FRS&n FIW add an acjcjitigpal laysir cjf nagutaligp h> tiiia oyatwn ti^ ptiovirJirifl a nwchaflBm lhat ensures [hczhdanccHactivaltonicyl iccnunnn signaling pattiwary that it bliriMUsted bv muatipb recaplor tvr-OBine hmaaes i! ■:;:[!■ ■:: Tl.j tinoi:- lyr;>:;inL- iiliviriliurv^i'iun i:; cm;: :il 'ui r&cfUFlnwif and acftvErtraf* of FriSaii-dspandent Brs"ial- ii m ^j.1hwiV^. it in: capeiittd thai IWrtiainci ptluonhGryla- ttw* oP Ihe dodcing protem wil be tipder light rontrnl Mp«ci&lry In a sjlualwci when lha key mediator 41»-. FRSS^f*1 gannitutwaly asweiated wilh ih= FGF pawn-tor (Ong elal, To oreveol inadvertenl aclitfation □F FGF-dopandnrat signaling pathways, by randam,. ncn< liuand-depeixlerii dinwnzsJion of FQFR mal«cu4ee ISchlassingRf. 2 DOG}, addrlicnul conHrcH mm h ami ami g[ key cirtermediates are esaenlial. Experiments preesnted In this mpcrl dcmgnslnate Uial FR5-2n e a targot gl MAPK phObp*iorvlatian. activated by FGF or by aJhar grCvrth rajStoriPhdSplWfylaMrom at FRSEn by MAF* dc-ors *T eight specific ttueonine nes»di«s iwttiin oofkseo-sua PXIP mdils) IhaL ar* dunttrtd In three iHpaftJn rpoV»i* of ttie FRSfiii rwtecule AHEiough thyronine phbsptioryiMJnfi m FflSin tun tut clrearu&d m qinsUmu-janqd cplrs^ gruudh laclgr Si1in«Mslign twtlHr anhancw Hfera pnoc&bA. Conver&slu. ulocHing threonine phGavlnr-ylacign gJ FR53«i touHs ~t enliangod h/nwirKt ptig*ffl*5r' yiation py-ffisao J- pT-FR5Ě a ] Giľ2 iP: frs2 n LŮfl^ťJ- - I E P F - I E P F U012fl: .....+ + r _ _ IS: FftSĽ IB: pTyr □B: L.l ,■ C: Crb? 1 ■■IM1 pV-FP.SE FRĚ2 IR- FflSSm TCL LJgand: - E F - E F 16: Ras ]-ŤlaBNl7 IBilíAFK -—r ■■__~y UAPK iFľ pMAPK ] } pMAťK IB: phUPK E P F ■ I e P F ■ ■ ■ 4 + 4 4- 4- ID MAPK } pMAPK ] MAF+í T Ol RuuIhiě Muůftu Cvni™ Fuctin«duu~nti<ü4i>i4>thN>Ui^'i Ji\u£*á3ii>\fiíi4U: Mi^dlty Ettrt urFHtSi:-, (pi) PC1S cob upTM^Ing Fin; W-17 |n m fa)iKftb war.ta uh» tnuhM Mih munuľttuuuHí^ |T>n| or iw« l«tt lUiirejucd. Cotta *oro 1h*n i ti mü [JÍLUJ *hrh FCF (F) U FCF (FlTillfcitaüäri^ Ľl4 üHühJLiJTijJi. C All lyunftf, n m-ti f/iljlLh*l ňj hiiiiiniiipIGCipImtfji'. mill- .j .li-FrlLl'.- AAbtĽdkä raided bp niinjp:*+jnliitf «Ith ajill-FFflili., jiH-p-Tyr, anfl-p-Thr, jh onU-uítiS DiirtbodiK.. Alu ioohii, jniRu!., jjid-MATK, j'd jn*-e-MAPC tiiTuJFíťbifiK DdDU CiHi hrfJHÄ (TCLJ, (B| ME F; ií4HBiing FIÍ3Ji.-WľT *m krti j-dninhJd ůf lT*iíůd >ltti HM MEK Witttar U012Ě, Trm cftJr. im (tum ŕillniiilalťwl wltti InsUki |l|, EQF (EJ, FOGF [F^of FQF (Fl, rmkmud fty cdl saUtiuaUon, Call I>ii1ni *uo ubfotriMl ta rmMK«™i|illaUoii wBi anllFftaSn jntitodbs fcftmrcl bp ImmiinnhJDtlhg «Ith ann-FFnrai, arrtip-Tyr anll-p-Thr, dt d-Oli! ůrtttodlnSL. Abo stu.n, jn d-IHRK and antJ-f-MAPK Im m m mit J cti ü1 tuĽil cdl lysolre rrCL|. nscflwaryfo'P'w^l'nB^öwwlsiTttyroj^pfcwptior' Vlauüri ü1 FfiSSn In tuJi(jSüeJi1 cöllft. Thüsr -in FGF aHmu-Litnd CEfflü, throaninn ptiosphcTVlBft™ aF FFI52ii func-lions, as. part ol a negatwa leedback mectisnianL Tbo second nvnmbor aF ftw FPS2 Family cF docking proteina, FHSSfl, is also tyroame phoBpnoryfaled in response In FGF stimulation. Hcwnvur, unlike FR32n, tho closely related FRS2p protein lacks the putative MAP« canHinsus phosphorylation sibes; ä b nül phospfoory-lated on threonine readuee and does not undergo an «I»c1rt0ti4rer.it mob*ly stillt lh nsspänftdte FGF stimulation. Thai«* of MAPK F>bwpte^atKKi sH*js <>i FfiSap maVWDicalfrlhal FR5Ü^ iü subieL-Lüd [DdiHomH rL^jb-lgry pmocssw* » cgrnpaned to FFtSESn. Whihs Ihe rwi-rnnall^ realrieLrf a wwaaJon patlam of may sug-Bnt^tf in'vorwfiwrTt in *iBnrfn*g by noiwirfwafztoic fEioimi FRSän, on the other hand, txhibit&a bnoader eupresmöM pettont (indwAig «HprvffioD n tlw nsTvuu* s^tvn) hiahhahtinq its more plwtroptc mvorMement in growth J;:::y:r signaling. In jdHrlmn. andysis nt FH3?ii ' mine dtemortstfaJed that FR52a otaya ä cnücal rote in emfay-onic Jcvefaf]inQrtl iE a iupf mDdlator nt FGF rDDCplnr signaling. FFtS2tr i& hKe>y to function aa a *ey elemanT m !h* GüntrOl hjT lüpfHÜuiö by both FGF ind naurtrtrtfAic '■sclurs in rm*ip*(f will, tissues, w>d organa. The clou aanůíkitiofl »f FRSŕa whli Inadlw FGF receptúre may naquim stringofH contfcrf nwchsniííriř hr nafluu^Hm at tyrastftů p ho aph orylali o n ol f BS 2o hi i*istmulatB ]} J} MAPk ] Jlv-FKSiii J fttHPK PP FR&Jlc a- FP&:.j IB Mtc ra majii IB jTi. e: MAPn IB. pMAPK nn:r TO. WT K.I t-1 -I HBJ I »UTK f mttt } pHHPK IP ? nr.'.. rFnsÄ. -FFbäJbh RflureT Crmiptai. Frurnailon tMcvufetn FRi», ifpd Arikaiüd MAP* In RJF--BambUrt>u (A) UrtEaTMdatad or FGF-idirujlHiHjl FftfcLv. ■' Mf Ft w MF.Fsi AkfrftKcung. Fltfi^.-WT wmi Fiut>)nc«d tü hrninuiofiraciTirLtf tan hut: . anHbcckM UkiirtM ay nwfkriFabhvtttng^rrh iind-FniJi, of jrnl-MAPK afrttoorMüb (Hl UterkVHiLxIbd oj FGF-faYTiArtod MH=t oipr&ukig FReii-WT or FH5£h-flV matt, ii^fxtad to InviurHiprclpltarkkii «Ith i anmuxka roUatdatiy kflEuwbkrrdnrj»rlh jjiB-FRHii.. arrtl-MAPK. ami-p-MAPK. or ^«titI-p-Tyr jrnfaonlüc. |C| HEK SIr3 oats -*■*"*-ivltri Aipraulnn vmilorc. 1i:-r FRESi. Icgechrrr *rtti dxprcsion njitofr, for ellhar nild-lypn Myc-laggod-MEK-UAPK[WTJH-acUuälHlHpc-Lagncd-HEKHAPr;|A.C& Lysates 1romunMHriLaitaJorFQF-sfcTrxJaloQcoMr,u^nisubibclnd 1dtniuECHactprLa ttwiiwrrri ariU-FFIEin fitt>«H«lDtt:iWDd by FrrvnukcMjtf rig *Ai anllFRzlSt. oranrJ-MyG tag anllncdeL.AIsri :^rvrj-frfJUyc-'lafl IrrununoUDti dHdU Lüfl Jyutbs fTCLJ. (D) UsAndJladw PGFsrJmuLalDd MEFs eaprnsslng FRaZa-WF irart Inlt untreated or 1 reared wfli the MEK HtrAr UOIk. Cell lysales vhtu subletted to Tmucoölllnc «rill anU-FREIn. jrtJ.p-Thr, anil MAPK, cr anllp MAPK anllbcdcs. |E>A Lcton-ieücptctlnD PATJ^K.rTK^alBd ^^4>3fytÄri and ^trnu jllDfi nr =pj-^lnq via the docking prolaln FRS2n. PGF-Induced acUvallon □C FQeFR loads (a tmrirnr prraqrhorplalkm ol FFSZii [bUcmd try Qrt^Sos^THHrLAHl actwalkm ol fse. Axnvalod Re Sfcnlatw a khrai cascade ccrrpciKd ol Rai, UAFKK |MEKJ, and MAPK OERH}. MAPK acbalDd tjf FQF rocDptOT or Tl rtKpon» to reu In. POOF, or FJQF slImuLifion ohDEDfitHvlalLK FRESc on nt bis I & ttinioolnu racldorrs jvsulbng kn fccrjcod tynseno phosphorflaUon et FRE-2ii, dtnainhad rccr.i Lninil o' 0rj2. olhm c"m Im proltvps '.v rurcUcr 'lj\ ' --SI . ncoarlivcly rogukiL« FGFFI-mBclialed lymsinD plLosphnry-lalion or tr» dcckinfj pratan. Doas. ttlreaiirw ptJospnorv-lafion nnahD FBSft* a poorer sirbEtrajia fa ar^tjvattori (l-rrusine phofrplo^lLrti^nJ, at a iMrtei biLnstiaJe lot Hiac-Irwrtion rjd^orpl*ortHrjt«ri)? 0n« rxwabb fl-iplcwlion it ttiat cornplej; kjrTriatMMi beiwetn FGfR and the threonine pfKHphortfatod torm ol FR5£u interMrqe with Vie Intrinsic: catalytic aclivity dE the- fGFR prolmn tyrnsuin rOHW9i>CP7KJ domain Alten^tivigly.ttireonirrripnosphof-ytalion may inlerloro with coinplfix romnlion bclwecin FfiSfii and FGFR. wauttairj if daofeasadi lynoanw plioa-ptinrylttlion of lhr» doc*;ing protein, Frwever, we wure unable In tkiieel any channw- in ainjciphu^phDrylaticin at FGFR, lynoairw phoflplio>vlalifjri of She kdatanrttthomrtr, ori^oeph^PpitH C-j, two vwll-known sAbrrat^of FCFR. in ML? k- exprcrssing cither vjild-type or Ehc- FR5Zrr-eV mu-(arrtprotein.ttwerijlFng outtfie posajbility that Ehe intrinsic FTK activity oF FGFFl is rwojalivery regiiated by Threonine phosphorylation or FTlSZ-r. Furthermore, wd wcra ;ilao unable to detect cherroea in complex fofmation twtwiwn FGTR with either FRWu-WT orttia FRSiiir-PV mu'anL protean, ktidbcatirtg I hit the PTB-mexlialed com- plex lormaEion vnlh FGFPJis not irifludncod by trveonina phoBCnoryiation of the docking protem. tVe hme demonsTre(ad in thäs report (hal m response to FQF eliiriLildlJrjrir the activated form of MAW lorma n cornplfffl viiih FRSSn- AHhoooh The molecular details □r Ihe mode ft Intej^hcfi between jcli^aJerJ MAPKand FRS2« rflrnam to be determipeol Ihe wwnprex formed tralween acCivaLccI MAPK and FFtaV2it may inborlarc- With the capacity ol FQFR to interact with and tyrosme prio&-phonrbN FRSSoi, ThB rneohanisim rasomblos; Hie direct interaction betiveen the matirtg-Bpsclht- MAPK and Ihe phercmcirB-rwponHv* G protein tß'ti of yeast resulting in dLTfrnrcgulaaicm d 11m pimrorpunn-inchicEKl Qü-MAPK pattmay (Metotfeev et aJL, 2O02J. An addilional [wrt-mutuAly ™di±siyd powibilrty is thai pweonine phosptiorytaliun induces a ^Irurlurol rl-jnqu n: I'RS?-1 rerrdorina tt» doc4uno protem a poorer utHlrate toward the FGFR. It should be noted mat several p-Tfir srbas Ha In close proximrlv lo lho fJ-Tyr sites in Iha pnrnary Btructure of FftS3cL. Tnreorttne phoEphotylatJoin may confer b (oral change in ihe striKture erf the dwfcing ptnlttri prönlrnal lo Ihe tyrosine ptosphDrytotlort tolas. MWéulv Oil 71Ů Thietoprtlwíwith Hie Pfiůůínc*ů1 bound MAPK protein in cU?fů prawiity may mlerfare rtilh FGFF-mediaíisid tyrosine pfiůsphor^latioii ů1 FflSín. An additional non-mirlually exclusive mechanism is thai Ihrarjnirte phos-řiorviaiion 4ill ťůflír bindinfl ot pnc-1ein|&i thai bin;l 1o lhcir Icircjpl 5 in a phpsphnirylalinin-dcpcndDntrriannar by means of the* 14-3-3r WW, or FHA domains (Vaffe and Elm, 2D01; Sudnl, 19QB; Tziuian at al., HKH; Tzhricm iind Avnich, 2000). PhospiKMniwnirie-mediHted ccmplen 'umiziCiDii bVHh jli.jr pruLLi lisi may -n■_■ rI-_:r■_■ hvi'M Lid irleraclion between (he catatyťc domain ot FGFR and FflSSa, a slap neůea*ůťy lat tyrosine prtutphorylaticin (a lake place. Furthermore, rftto bound rwi>ieini*erxiow3d with intrinsic protein tym&aie phosphatase artiwly, cgrnpln Ignnstipn may raduw tyraiino pUwphpryla-lion dl FBSÍn by a phosp^olhre^iňe-dependeritphús-pticHyrosino doptiDophcrylaticn. MbMphurvlatHjn on Ser/Tnf residues *s a wall-known rncchantsiri Far 1hc regulation of admLy oF prcliiin:; involved in growth factor receptor signaling. II has been shown lhat phosphorylation nF the- EGF receptor «1 Thrí &fl &y PKG leads to modulation ot the EOF receptor liqand binding activity 4rtd ttiihjflťí in inlraceBular tral- ticKing 01 EGF raceplorfoaowinfl endocytosis fj&ochet at al., 1BW; Livnah at aJ., 198BK hi addition, phusphory la-liwi of iftSi (insulin receptor subsirat* 1) by PKC, Erk, and Akt n^utl-s in reduced eomptaii fúrtnartiůii vjllh PI-3 kinasa (JK) F«a at of, 199/?h IfflJb; U it sJ„ 1 PWJl H was also a tort n that tiypefphoaphciryialiun cd Oabl on Sea/Thr mkJwf negativny w^Ih haprtwirte orawth lactor-medtated biological respon&ea iQual at al., KK)1J. Hnwcvcr, antrihor study raparted lhat pnos-phofytatian of Gabl by MAP Kinase enhances complex Inrmatlnn With PI-3 kinasa resutung In sbrnUlatifin Ot PI-3 kinase aciiwty (Yu et &U 2QQi). Another example of a negative reflulatojy rata of MAP kinase b iloatraled by lha phosphorylation ol Smadl [□ proviant nuclear accumulation and Iranscnptioriai activity in response ■to BMP etMnulatkm Qrítetzschmar at al.r 1"9&7>. In nummary, experiments prpsrjnlcd in this report dsmcfBtrale tnet tne same moiecu*e responSble torttw KUrurtineiil of ^ihva regulators can urt*2*alsmanílEůí lha same pathway far a nagafta laedback rpachonoim resulting in signal attenuation- MAPK response x**t(*■ eHb WW 0«*Wíl PMEfi-FW WppMWllWj WVl 1 OH fMt-kHBWHlW turu ufun fOJBCCVBaL). Ouwalkm ol FRS2h ' colli. dkfuiou&-Inp 1lw cBkrarrt FRSin mutant were pmtousfy daurbbd (Hadul etal.,£00/11. FrlorIdgrowmractoritrrHJaBon, Dd:wn danwdh Eorom-rroo nod Lm (rvcrnO^l- PC-iE crHb TOprossrig j ccKllllcd FUjif-niT mutant ywpu Ireatcd wflh LKi.i.rui vVki ■_■ to iDofl ao pns-vkwuy «ť*niwu ní«idůra*i al.. íssřV ararti Fwwflt innwt&s, ^i«»dlw, »4 mawikte ECF, PDGF rjnwDgan|4 and kuubifSIgmsj vur»ituaa1 itu lUriw- Inq ooneuvtiaacra! EOF. 100 nQrtnl, PDQF, 35 ngftnL InsJn: 104 nflvm FCF1 i3p*iAif-nr«rr*ii #1 al., lust 1» nsviKi *«l twpiuin rjStVM^Ii uo-'iiih wvh 11 .iflflri tflgwwďícrsrJmifKíttW *■ FtF iWiO-to*?. Fh«mi tw» inhhtlm **ft vml m pw rrto^nownowilr*-tkHB: UEH pjOISt, ftonup, 10 mhf|i F*C+CF1ůíiKůjI, Bcnul, 3 mUf; PKA {PKI, Bunut 3 tM). and Ft-3K (Wuniw^ Slfliru, J rtů nM|i otalat ^ta^SlgmB, 3ů nl^.aiulr*rihft5iitia1fls* |Blfliru, 5 p^uimpM), and Dbiarultuncna fStgrrui, 2 ^.M). Andbudm Doolui FRUn, Cutii, fJ-Tyr, and ttas *dr« pmhHKhj duu^lbud |Koutiard«1ď., 1uS7l.AnU»dte:agMiMycLq,rMPII PikhdjioI-pau C^, fPLC-jl, and ann-nuHi£fi HRP war* nurcfiassd fewn Santa Cnit BtalBchnoiofltei. Anllbedds dliactadagalnsl tř"ri p■ Ttr ffpjt-nfeuiyblJoniltn d MAPKr-JiTFi wen DbUiudlnari 0«! eignalng. Anll-FLAG anUtnolBS (Ml) wen cblakwd rrom aigma.Jvl antibodies wera dssahBd ki IXTBEJ5K. BOA lor ImmunDMDHriq CKpadmants. Tha- vkal cnEnsshm plaxrntb wen DDnslmclsd In pBABEVpur* as desedtwd f-ukdal nl al., 20011. Al p asmldi UHd kilrarelanl ««preE-dn eaqaenmHi wan c^rctrudBd In pFKS (Kcuhaia «1 al., 1*9T|. Tno- Hyc^taq MEKpMAFK. plasmlds warn djlakwd km Mukne Cobta. Purl nod acttvalnd EHK2 tiM NaCI, 1 ipM EDTA, 1 ft THOU X-l 00, 6% ŮW&M. 10 111M pyríuívwtihůíí. 1 *?M tw/ů,. 1 inM Pr|iijlkHi IniiimiiOtirH^rHPcri vkI hdniuiwuMiTia wmi ipr- antrwn aillowHí ■pwt pM4Miy op-wlm O&stůť« ůi . iSW: MenvmjKii^i $1,. i-ýfín Cell MHfrswo. Cell P':it in 1. anuColony fo*mii1k>tt In ivrt Aoer Cel m(nf»isň wo- portermw wig ft^w tmwiwig LC«ii«t m twf, cnsrnnw pr^wnlofl *1th 1 v "flflni rcr 1 hf al arů. Chcrnaattuclonni n 1.5 ml PBS or control ±alidl«i mm idrtftíi n tf» bemom chamber. MEFi inch lumd in»nilgti1, haiMAHMl ■tlh uumr canaWng 25 irU htfEPEs indBmU EPTA ipH 7.S],pdki-UMJ,dwiI'AUiSfHHUkidlijSc««srtnlhF«a. Col ucpdnskNi ip.i mi} wa& pbcod ui ttw np mamtvan«, and chamtMe: můru Inoilutad Tor A hr at 37 C -Ofils vura 1lMn ramev&d rram 1ha 1up pan ot the mambrsna and Itu eels attached 1u 11m bottDm marntHanú Mra Ikw] ultti methaml, sdarud wlti lumatoi-yln, jrd counted fl-udal at al., ÍU011. Cell prulderallHi asu^r. wdud pofliKmecl acrananoj 1d publtshod promdura raptvak-Hriilzrnanien al. 15JM|. Fcr soil anar colonr amayv cats- wen plalad onabyer □f i>.75fi scfl o^anm at tfflemnt Ddl donsattos (If, 1 , l'j"l In □.3Tvcnrtanarp:c.Archoraq^ InncccnckTiorewmcrnucrcecopIc cnkHikB wn semd alter 14 dtiyiL A C kfl DWtHl n marni A.W. Is a rKipiLiil ol a p?ťld«lDral InPtmntplrcm Iho-Canadian Inslttulm cf i-teiiui arJ RpvHiuh t3MFJ. JLHL war unnrtod bv a prpdwlorai lralnrig uran* ri Druit Canmr: moarch MMDI 1-W51 *mi»1Iiu USAWWC HowivoiB*aia a not*ior Ftf aflftjUoo m pr*9wnto?wn mBiruiuuan noscenprwM. Pnět hjW AíW M UW SS. SOÍř-SMř. BoiE*. a,s . Bonn, 0. Února, j.M., Wom*, E.v .ínfl 3ihh»«ifgw, J. ilfOlh Hluorchy of bnsrq £ttu QoS aim Sht on Aplddrnul flra^mlsciiir lacopuv. Prta ůůll. Btol H.£l*J-£SOt. EUHtdngtun, H.S, ^>d Hobafeon, EJ. flMq. Aids dMhipnwit^Hl Duty atymmany h manurub. Col €t, 136-SO> Gvuna, &a,SDJtmrtz,L, Harpal, K., v^maoucM, T.P., andficusanl, J. (10071. CHmfldc ^naysls ol Ibroblaal growth lar.lcr nboeptnr-1 ffiSil MwJkTl^oi NViKllh^-firtiai* i^fl^-lvni t1í IT-Qfrlj ViCflW; * W* njr FSfRI In ntfBriiJIPIPr HWW CudmL C, Oil. ŮH. MHtntndOW. J., Qoopoft JA, *nd HAW, T, CIW^O. PwiíMi píxpw-c;: piKKpwyqffra « (poenitůi growth fWjrůr «WBťaiM nriUĎK t1S*(li«fí|íňlfr(Wiri lúCICf-SUrfiulSlM ryiwlra prolM k"»$ UCH*!, J. fltol Ciww, £5Í. 2WJ-25W Ctůíůtrj, P.H., and Warlh. ůJl iiů*6(. "nw můuů* Fa*) aww «*■ tOOrt a rampy. J irtKpeplkhři ¥4 « aflwwwa " n^ukun dmi iwnt iiih4*M4i, Dn- Fm. K.. ami Kolli, RlA. (1 Hflřa) Moduiatler ur inwiPn rjoopLrr suUrlroJto-l lyirane uT uiiJfiL>yU)uu ami rumflon bj mrtnoon-acP-Yiflwl pr^n um™-. J. 6lul Cli*ni. Z7?. SUflO-JIdBj. IhRn, K., and Hoffi. HA (liwrb|. ProWn hhawi C PTKluBKoFurr InsJIn rocepkw subslraln-i lynKrephnsptHHylallcm nqJmin-Irw E12. rfcct^mrrlry jij, 1EWB-1 SMÍ. □ong, COL, WynshM-Borti, A., Strm, M.M., Dougherty. C, OfT*r, D.M., and Lador. F. <1*W|. Murine FGFR.1 Is naqirirad lor eariy pnitlrT)F+anlilHjf» jiHvtti ami anMotnanlznlaQix (itrKfl Cw. J, 3057 DhaUn, Cl, ran, HI, Plcdnlto¥a, O., Lna, KJW., Zeng. L, HUH. M MulLiba, El,. Qoldtarbj, M.F., and ZhcuL H.M. 13000|. Structural base or EHT FTB daman rfloracUuns wttfi asttnd mu not reptile receptors. Mot. Cafl 6, «214». FWdman, El, Frjuayrwfeou. MM., PaflaJaanr™. V.E., DeCNara, T.M., and Gdtffjrt, U. (1B05J. Haqulromsntar FQF-4 lar posllmplanlallnn mouse development. Bobnce 25T, 345-24P. OcUtarb, UL fl DOB). hftcJVofts or Ibniola^l growth ractcfshuorte-brain devalopmarri. Cytokine Oman Faalor FUw. 7.311-32EL Oudl, P., Ctonuuw, Ei., Anguluola. i, Purtaf, PJ., and Conugio, P.M.p0ud).Qan1 pntBfďůrylawtri a raid mazhdrtsm Itť oagadid ragLijidon cd HCf net aptof signal ng. Oncogene TO. 1-E£-1ifi H#Wft V.R„ KQUMre. H„ Lem. I.. fUHl ^hlMOTSW. J. |1*flflf SlWlnfl a" tyrosine phbtfrialau lo FriES,, Is Aftunddl tot Ibtcttuň orMt,1act<*-\MĚ*&fPGW caectanarantlatton. Uoi Cull. Bid IB, aoflu-wa huiwft V"";. Mi, x. Kwifiw* H„ uv, l„ 4ir4 ew™*ífjrHj*r. -J, l»ď-íiwdl)»eí íUjnal tf*H«hl«lW p4PrHťt, P^W, K» ^4Kl 44 USA OR, SE-Tft-aEflS. KflOflfifft, H„ HHtarl, Y.P,, SKWHiUinWrnůn. T„ J . Baf- SiflL &., Lav 1.1 «*1 SetKiwho*! J. (iftWl. A tpd-anclHwd pJnodHJ pf^^PlWinriFííFjisr^wia^KffllíltteF^ Kur^ara. K„ iPiífKi. T.r mu4kmdi, H„ HoyůífK. H., K*pal. R., arm T*m(wil M, eaooi). hKf*icůih)n «1 SMTtfiRSz «nlnii slla «i RET reupKf lywiliw »Jn«t* bihj 116 rote ivmpai irar«du«ll4ri, CrtcoSnWiC'. iWw-ii+M, KfHrttrrtit, Ml, be«ty. JU and Masa**, J, UK?) OwHkig BMPfttdetiP Éifj^hti^li**íůůli»*1^*i1li*Tíř-u4tSiraiiiPy in^Jlulur ^adi. HaHinr 344, LI, J., DuFm. K .and Ruth, FLA. H TOTfl. MwUilhm c* hnuthmopfor suUslraJto-l tyrorlm pticnpncyUhn Uy an AMrproiprFJPdirlFKTilLjol a-Wna» palnwavr. J. Bi<*. Cnrnn. ^ 'i, B»i-B9»e. Lhnon, E..M, T.J., Btmrri, EL,Prync, FL, Urtch, A.,aid-Echlnv srQor, J. |1"3H^ □ ptntrf wLrKlndiKDd rrttnotrt; btrii bv mutalkm at Thr at epidermal urmvlh lartur. Met. Ced IM. U, TJ0i!-z™. McrJcuaaJI, K., Kubu, CL, VordL JUM, and Moahln, a.O-. C2M1]. Dov* oprnortaioi! prow lo»i pal tom sol Iho signaling adaphrs FRS-a and FH5-3 during mty nmbrrocja-Kety Hpctl Oct. 1UJ, I flD-l 4S. MdllD, P.M., Eanteve, M., Org a.H., Eltaud, M., Fusoc^A., Hadarl, T, EcNiKshgar, J., and Lai; L rZDSIL DodUnq proldn FR92 links the ordain tvmslna Uusa rsl and Itx oncoganlc 1nmns «riti lha mllDjjon.aLlhrjtod prccdn kausa slgnalnq casEadkt. McJ. Odl. Btd. 21, aiTMiar. MDCOdta, m.V., Mathaos, Cl, Faua, Mb, and Emma. DjE. fSOHI. Ftagulallcin Dl UAPKIuncllcKi nydkucl Inlar^bori *ttti IturTutlna. spacric G-i In yaasL SclorKH iEĚ, HB3-I4SÍ. Mrtfiňmnwťi, M„ DhhS.i .6orL*|n,A, tHirfl*w,W,H„4WS,W„JPd iotifewjiiger, J, (iiJWjJ, Id^ííttfltJPW fld tlx a^opho^nhe/yiA-rkfl 9t(W OH flhrr^KftH grsMfli Ifttitof *9flsn(iůf n wtiipm™ íi1 ttwlr mp&naíKů ň nacap»racil.flrkin,a«1 slgiui haftsj*^iksn. Mot. Cat, BKH. 1$,1K77-uMi. Nashl,UC.aMlCimrhr,D.U |1USf FGFr^YilhvgInsJtwcdd*bSl-norrunt Frcn. BKacl. a. TB1-TDJ. Nlswandn', Land klamn, Q.P. (1 Mři FgM bipnulon dudng qai-Iruatlcn, myoganails. lar* and loath dktulopmard h tha meosa. rjaidnomant 114,7SE-7&S. una, 3.H., Guy, CJt, Hadarl, V.H., Laks, 3,, Oototi, N., Schkushgar, JL. and Lam, I. {20001. FR8S pralatu nacml hkacolutar dgmlhg pathMUfs by binding la dlv«rH 1arn«ts onlbn^lasl gjnwlh racier and rK-Tiú onuvth lactor rooccdns. Ul Dall. ElloL 20, S7tt-SS* inc. 3.H., Hadarl, rjt, QalDh, NL, Quy, OR, Sctitednggr, JL and Lai;L^1^SdmulmlonarrjrKnpUfeclrtho4tol 3-Una» b> nbrn Lla:: oTv^ti J^c:^r rcccL:^nj l; -cd Jicd bt l^LrJm:řJ ■l'i.'LlI monl or mulhpb ncckJng pn>tvln=. F'roc. Nail. Acad. Gel. LFBA SB, 6074^07». Echlnnlnoor, J. IBCCHf. OHI slgnalnq bt roccplnr lymetio klnaios. C«* H1-B2E. Efmak'Krebman. 7., Lcttftkhi, m a., CHilc. I., Landbuiy, J.E, Finch-ad, □., Huang, J., Jave-, ML, Cnirnhn", d.. 6^tikTD*Tflt", J., and Lax, I. [1 Ptifi Hopurln-Huiwd olla^rnwIďPDn or FGF nHrtocutus tim-řpOnřibH iw PGr" r*rtt*tf ^ I rirf-1 á iRt*, jiltrj tHW *nJ «HI |j*r-allOú. Cti 7%1*1IHaM, y.. vii^ih-.. i n. i i'w.". i"iki.jii. m. .».i wiirii-,. riH ,;imiHi Taigtud (teftidhM cd FaB tsw«. felM-s cHi rulgrseart |M dufltuiullin] litCw** WliCtVO GM« D**. U, íaífl-iňíij, nnMBrf**- prnpwfti Of (>r««» r«cgnn}ori code, (VrWttfrfrt i T, 14*3-1*74, Tť^w. Í„ airt A*TWn. J, CJWfl), 14-3-5 PWW, Wlhy f or*ew*? In cdlklar hagdUBcn bj unnbmwiacMwu p^u^riiLvykinon. J. Elot. > XAt-XĚA Titww. t, 3twn. ¥H.. and 2hu. J. >|KM>11. Eklnglng hh da1W*rkms to icatlcMngL Dnaogona SO, eu£l-4u2uL H£(>g, A., Unudw, El, Lm, a., Schlbtshgw, J., and Lax. L (20021. FRUtt nUkruahu. FuF-nacaptof slgnakig by Grb2-mddla1ad rvi-cfLlImant ní 1tw iMquHn agaia CbL F*dc. HaLAoaoL BdL Si,OB£4-snm Xul HL, L«l KJMM., and Oi«dtarb.H. |1«6f HcmdI rccugWOiin medfl on IbrnUasI graMth laclor rocepdnr madlatasdhacl assoxaaDonand acthabon ol ahTT □dQf*arprotdni. J. Bid. Cíkítl 27\ 17387-171100. Varta. H.El, and EU, A.F.ÍS0O1I. FliospliciHrriarlhreorJna blidiiq dcmaln. Curr.Cpti.Cal BJoL 1i, 131-13?. Yamaguchl. F.P., Harpal, ML, HanlwmeyBf, H.. and RnosanL J.(PD041. Fgh ' l^reqLir^^rivnbrvcnlcojav/lh jndrrcsoderrnal nattomng dunngnwusoaatinjla1lo*i. Gene? Dev. ^KfSr-VMA. ran. K.El,KuU, H., ran, S. Mipato. a., FaroDq.A. Qotdtarb, M.P., and Zitou, P4_M_ |3CKC>. FRGZ PTB domati «mtnrrna1lon rogJaloe tMaracuonc w*H dtwonaert nouralrcphlc mccptore. J. Blot. Chom. iřř, 1 row-i iwa. ruLC.r., Roehan, C, Uu. 2L,and Carriky, LjG. pWl 1. EFK regutatee 114 hCirnTTjLjM MvaťlhM of Gaul arid tne Pi J-btů«. j. ftuJ. Cl>Ml. 2^.325i7-j?3S«.