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Cell Signaling and Developmental Regulation

by: Mrs. Willis Mante

Cell Signaling and Developmental Regulation MCDB 4426

Mrs. Willis Mante

GPA 3.6

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Ding Xue
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This 67 page Class Notes was uploaded by Mrs. Willis Mante on Thursday October 29, 2015. The Class Notes belongs to MCDB 4426 at University of Colorado at Boulder taught by Ding Xue in Fall. Since its upload, it has received 33 views. For similar materials see /class/231836/mcdb-4426-university-of-colorado-at-boulder in Molecular, Cellular And Developmental Biology at University of Colorado at Boulder.

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Date Created: 10/29/15
Control of Cell Proliferation by Peptide Growth Factors Endocrine F39ararmrlnn hutmulmc Autocrine Growth Factor Production Causes Malignant Transformation Transforming Activities From Condition Media from a Tumor Cell Line Condition Media from the Moloney MuSV transformed 3T3 cell line Normal NRK Cells Tumor NRK cells Todaro and De Larco PNAS 1978 Transforming Activities From Condition Media from a Tumor Cell Line Table 2 Effect of 80 from MuSVtunslormed 3T3 cells on growth nf rat broblasts in so Agar 39 39 39 Substance and Distribution by calnnx size no concentration lt003 Z 00324964 00650130 gt0130 ugml mm mm mm mm SGF 56 i 9 so so 14 27 42 23 3 r 5 32 6 7 a E F 100 ngml 100 o o 0 None we 0 u o SGF sarcoma growth factor Tudam and De Lama PNAS 7978 Transforming Activities is Due to TGFbeta Moloney MuSV transformed 3T3 Cell Pellet l Acidethanol extraction 1 Gel filtration l HPLC TGFa TGFB EGF EG F NO colony formation Stimulate colony formation Anzano M et al 1982 Cancer Res 424776 Paradoxical Activity of TGFB Ill Ill I EF amr I39l F iiiJ11 FL I39ZnFI39ll JEEPM El monies I W2 I 1 4 ll 2i 4m it ll 3 1 III E an HI ll39 lF l j Ennimtratinn lpM llt39iFl Cnncentrati un Film Effects of TGFB on the anchorageindependent growth of Myc l cells In the presence of PDGF TGFB stimulates growth A and in the presence of EGF TGFB inhibits growth B Roberts A etal 1985 Proc Natl Acad Sci USA 82119 Purification of Bone Inducing Factors Prat Nail Acad Sci USA VOL 82 ipp 226 427 April 1955 Biochtmistry Puri cation and characterization of two cartilageinducing factors from bovine demineraliz ed bone pmwoglycanf39tyw II culligcnfd reremia onfcell cullm SAEID M SEYEDIN THOMAS C THOMAS1 ANDREA Y THOMPSON DAVID M Rowan AND KARL A P152 Conuccliv Tissue Research Laburumrics Collagen Corp 1500 Faber Place Pale Altai CA 94303 ABSTRACT Two naturally occurring was mt induce Table 1 Puri cation of CIFA and CIFB mamgenesis in culture have been puri ed in 29mm Speci c humageneity These cartilagednducing factors CIFA and Protcirh activity yidd of Punif CIF B wen isnlated mm bovine dcmimnlizcd bone by dix SW ms unitsims acliviw cation39 sedative aluminum girl ltratinm alienexchange chromn deHCl ingraphy Ind rwemdphase HPLC CIFA and ClFl a mum 6480 435 100 1 mmm om of 1 10 ngfml ml induce emqu rm mes SEPIWSW39 00 enchymal cells in culture in mm a cartilage morphulo y F1 395 935 m 2 15 and synthesize car lageqmcific pmmoglycam and type I wil 3 3 42 ii lagen The amino acid compositions of CIFeA and CIFB m CMWMOR quot quot 1 similar but not identical Both factors have an apparent M or NWwa 299 93 1 1 26000 as determined by Na ndsodl AGE In the pmesence CM1 879 1212 33 219 at 2mmaplmlhanol bath are converted to species all about Chi2 0333 1600 49 384 anewhsll39 that Mr1 indicating that they are dinners at identh m CM393 13934 10000 79 230 very similar chains PM CIFAi 0457 mama 274 3885 ClF Ei 0082 465000 134 10591 Purification of Bone Inducing Factors i 1 J Biol Chem 1937 Feb 152625iz1945 9 Cartilageinducing factorB is a unique protein structurally and functionally related to transforming growth factorbeta r l 39 SM Seqarini PR Rosen DM Thompson AY Bentz H Graycar J Cartilageinducing factorsA CIF A and El CIFB puri ed from bovine bone on the basis of their ability to induce the cartilage phenotype in Uitro are proteins with molecular weights of 26000 composed of two apparently identical disul deIinked chains CIFA is apparently identical to TGFbeta from human platelets Seyedin S M Thompson A Y Bentz H Rosen D M McPherson J M Conti i3 Siegel N R Galluppi G R and Piez K A 1985 J Biol Chem 261 56935695 We have now found that like CIFA and TGFbeta CIFE induces anchorageindependent proliferation of NRK 49F cells when these cells are simultaneoust treated with epidermal growth factor Furthermore CIFEl competes with CIFA for the same cell membrane receptors in NRK 49F cells Partial amino acid sequencing reveals that CIFB is a distinct molecule with extensive homology to CIFMTGFbeta These results Show that CIFB and TGFbeta are structurally and functionally similar molecules but differ more from each other than does TGFbeta from different species CIFA TGFbeta 1 CIFB TGFbeta 2 Purification of Bone Inducing Factors Novel Regulators of Bone Formation Molecular Clones and Activities JOHN M WOZNEY VICKI ROSEN ANTHONY I CELESTE LISA M MITSOCK MATTHEW I WHITI ERS RONALD W KRIZ RODNEY M HEWICK ELIZABETH A WANG Protein extracts derived from bone can ini ate the pro cess that begins with cartilage formation and ends in de novo bone formation The critical components of this extract termed bone morphogenetie protein BMP that direct cartilage and bone formation as well as the consti tutive elements supplied by the animal during this process have long remained unclear Amino acid sequence has been derived from a highly puri ed preparation of BMI 39om bovine bone Now human complementary DNA clones corresponding to three polypeptides present in this BMI preparation have been isolated and expression of the recombinant human proteins have been obtain Each of the three BMPl EMF2A and BMP3 ap pears to be independently capable of hidqu the forma tion of cartilage in viva Two of the eneogcd proteins BMPZA and BMP3 are new members of the TGFB supergene family while the third BMPl appears to be a n vel re at m l 0 gal or 0 come SCIENCES VOL 242 0 i Purification of BMPs rquot 39 quot7 BMP2A kmquvn SDVGWD NI VAPPG IHAFY I N a DHLNSTNHH AJVQI LVNS WSKIPKA v39Z39TEZunZ SHL ILDENEK WIJCREQ i EMF23 GRRHSLYVDP SDVGWDMI VAPPGYQRFY CHGDCPF PLA mims39mu AIVQTLVNS W 551 PTELSE SHLYLDE ID WLKNYQ u EMF 3 FARRYLKVDP ADIGHSENI ISFKSPDMIV r KSLKPSHH AJ IQSIVRA VGWPGIPEP SILFFDENL CH WWYP H n 11 W39 Pauuluunvun SDVGWDENI VAFLG IDAY I DHFNSTH H AWQTLVNN HNFGKVFKA EmLDSV MLYLNDQST WWYQ s n Vgl cKIGHLYVET KDVGHQMN 1 PQGY39MANY EILNGSNH AJDQTLVHS IEPEDIPLP Hm SHLFYDHNDN WLRHEE Inh Ba FCRQ FFIDF RLIGWND NI IAPTGYYGN TAWNQYM GLHPGWJS VLFLKL l m IVICRDVP NHAVLL a Inh 13A nnurrva KDIGWD NI IAPSGYHAN39I H NIA GTSGSSLSFH SWINHYRHR u WE LRP SHLYVDDGQN IIKKDIQH HMH LL Inh Cl W 3 39439 39 QELGHERWI VYPPSFIPHY H AHIV PNLELPVPGA PPTPAQPYSL LPGAQP quot quot quotquotquotquot PWTTSDG quotquotQquot TGF B1 in n RXDWVKJPI HEPKGYEANF My 15H LDTQ ISK VLALYNQ HHPGASM PIV YWGRRP MOI S n TGFEZ LRPLYIDF mommy HEPKG INANF AGAEPY LWSSDTQH SRVLSLYNT IHJEASASP SQDLEPL TILYYIGKTP K120i s dMLV b a MIS CAL RE LSVDL MERE V LIPETYQMN G gt WPQS DRNPRYGNH HWLLLKHQ quot HHBE P P I AYAGK quotT T 7 quot SAHHV P L quotD DGWNI PYAY 6 P H v39 T E quot 7 quot quot B 9 Q 5 9 Q Q g g s EMF2A 29 33 36 23 45 u 5 11 49 92 EMF23 29 36 35 22 43 M 56 76 48 EMF3 32 34 3 30 37 3 9 I3 app 21 37 37 13 I u 48 vol 32 36 33 23 7 39 lnh BB 2139 3B 38 25 6 Inn 3 25 46 1 27 SCIENCES VOL 242 Inna 21 28 26 TGFBZ 24 7 TGF 8 i TGFi Structure and Function Leader Prodomain Mature domain 1525 an so375 ny T u mm up furln While balls cysline residues and disul de bonds cystine knot motif Daopm 7992 Science 257 369373 TGFp Maturation mam Mmrooes Wear 7999 1mm 1 Namc Homu ngu 541112 sly unib BMPZ DppD BM P4 8MP wiguntib39 P an AD an mum in GDFS DMPl GDF DMP2 rig subfzuuilr c DF Vg 1 X x 312 mm mbfumily BMPLmIcogcmn F x o 100 71 4 TC Frm 39 Rowmum 0 30m mos Remcum ncurogcncsm chnndmgcnm intcnhgiml Gamumion 1 mmdcrm pancming m y domumm popm in ccswinggt 1 Along uh BM P5 2 mm mm subfamil panxcalum m the dcvclopmcm m can all mgans many mm in ncumgcn 5 112 c mndmgcmqs m duclnping mm 114 Vgl Mm mcmdcrm mmeth m Flag and sh 4 O cogcmc Mmmnmmn cndmhondml bnnc furmmmn monocle chcmomm 39 Maxqu Am Rev Blathem 1993 litrurmwliuru mmbm N 2 1X comm 1 n 42 Axialmcandcrminductionlc rnghtmymmcn39y116 mm 44 Regumnmmfcc di 39crcnuannnwithinhcncumhubo17D con 41 Inhibmon mmm mmch youth 8 GDFO 34 mm xnlgfmnl39ly Acm39u 42 Pim39nm 39follxclcrillmumunglmrmoncFSHmeduuinn mm 243 42 CrythrnidccHdlffncn duc in flog mmdcrm Acth m M 3 induction 1m 1m Acmin ME 40 mm mlg um39h TG Fr l 35 cu qdc uncst m cpimchul ma hcmumymcnc com control Dr TC r422 34 mownchymzd can mom an and di bzcmmnon mum TG r733 so howling cxtmrc ular mzm39n produc on1mmmozupprcssinn H 17 4 0mm nulmbw Ml AMH 27 Mi39IHcrmndudchcasium15 Inlubmzx 22 Inhib onofFSHproducnununinthclamonsnfucm39in910 N 23 Dopamincxglc neuron m ML mm dccopmcm rm m u 2 v onwmm y m MW 9 quotu pmccm m mm m Munmx uh human bmw mumhugcww wumn M m M39H 43IgtNrfgmccm MY dew m wmolmphw mm Massague Ann Rev Blathem 1998 TGFbeta Binds to Cell Surface Receptors A o xquot xe 1 an TGFbeta was labeled 1GF l5nM with radioactive 1125 30 Type III huTBRUx kaBRII Type H Type I TBRI 39 TGFbeta Inhibits Normal Cell Proliferation Isolation of TGFbeta Resistance Mutant Cells Mink lung epithelial Cells 16X107cells treated with EMS ethyl methansulfonate for 24 hr 40 killing rate 25 pM TGFbeta TGF beta Resistance Clonies Cell Proliferation Assay in the Presence of TGFB 125 Hall Incorporation oi quota Inhibillwa ion 7 so o Mvm n 334 I 371 A 971 50 A Eli Ac 20 v 2771 v 254 20 1 0 352 0 n i 3 z i 1 I o 00 1 000 TGFB1 pM Laiho M 1990 JBC Some of the Mutant Cell Lines ShowAbnormaI TGFb Receptor Expression Phenotype LFt DRa as cm Wm H gt Type 1 gt Larho M 7990 180 Genetic Screening of the Cell lines Lacking of TGFbeta Receptors Three types of cell clones R cells Loss of Type I receptor binding DR Cells loss of both Type I and Type II binding S Cells Receptor binding normal but no signaling 114315 1 T0151 receptor phenotypes in TGFiwesisumt MUlLu cell mutants Na olclomx Isolated menmm Reteptur Type 1 Type n TGN 151 2 tlones usld m phenotype recepbur racepmr 5mm mam 1mm may 5 Normal Normal 20 15 R s m Normal 3 7 LR Low level Normal 1 3 DRa A sem Absent 4 DH Low level Low level 10 4 DR Low level Low 2 Anomalous size Total no nfr lm res39 35 35 Why there are no mutant cell clones in Which the type I receptors are normal but lacking the type 11 receptors 7 Laiho M 1990 JBC TGFbeta Receptor Signaling Mechanism TGF B Jig TBR ll Somatic cell l hybrids No signal h m 339 quot I l TBR ll l TBFH N0 bindingm g I TBFFI w No signal 1 44 2 l TBR H l TBFlml No signal Laiho M 1990 JBC Expression Cloning of TGFbeta Receptors lb Initial screening al cDNA pools I cDNA coding for dealred receptor kj Irrelevant protein Transient transfection of COS cells R A t 1 KW L1gandb1nd1ng 059399 KO l39gtll siz Isolating positive cells Plasmids replicate in nucleus V V Recovenng plasmld DNA encoding gene as red L Y r v of 1nterest ece1039 X m al Plasmid expression vector Add radinlabeled ligand l Doublestranded cDNA from cell normally synthesizing xi xi receptor protein 5 gtoooooooooooolt i K 4 42 SV4D origin of Signals for termination DNA replication and polyadenylation 01 Autoradicgraphy and promoter NA x Signals for DNA Plasmid DNA replication in bacterial cells A Lin and Lodish 1991 Cell 755785 Cloning of TGFbeta Receptor Serinethreonine kinase Carboxyterminal Membrane domain I I 39 tail receptor Typel receptor GS domain Kinase insert Cysteinerich region Franze etal Cell 75 681 92 TGFbeta Receptor Signaling Modeling a TQM b c a l 1 gt if gt l 9 3 e I 5 1 I 1 w 1 w 1 3 39 J l 7 9 K 4 a 13 1 5 3 TBR II T R l T R l 51 y ASignali1 Type and II TGFbeta receptor families Type I receptor family Type II receptor family L gand AcR B mm a xTrR IX AclRIIB ALm MD 39 R4 T SR39quot BMPRIA BMPR TTSGSGSGLP Emmm AMHR 8 LMJJ 8 Lm c m Acuvanng 5 Da g phusphovyla uns g AcR X x axD FKBWZ 1ka bmdmg sue Dal1C quotguru 3 Type I and II Tow rcccpml ix pmchcd b he as donmm QSr Th ilmmclcmuc 63 maximum the phosphorylarion sites rum he FKBP cltcbmrca unh ss orrrerrmc indicated DIH7m71f T V V 7 NH Vn39h qD mmrlm In type I lcrcylmk rhc pmrcln mm Jummn scqucn monf of w Rel ls shown mm mcmbch are from Aurarm 2 L39rrerrarrrrrrrrm ILgr quot 39 M rRllBanmxhch ldCHUl V Maxxague Am Rev Blathem 1993 Functional Reconstitution of TGFbeta Signaling A 9 5 g Q1 2 mmnsnm o V t r 39 km 39 39 39 39 gt209 humE 1 A94 quot awquotj TEN 0 ac 45 WIS75 am Massague 7992 Ce Reporter Gene Assay for Studying TGFbeta Signaling A THE PAI1 UJ Mv1 Lu DR26 Rd S Luciferase Activity x103 0 hTBRJI TGF B1 lt Wrana and Massague 1992 Cell TBTRI Kinase Complexed with Inhibitor FKBP12 FKBP12 ac Helix Em x Activation Caialylic 39w quot Segment 1 39 x WR I Kinase Decapentaplegic dpp Wild type Class I Class 11 Class III Figure 1 Comparative Scanning Electron Micrographs of Adult Via ble Decapentaplegic Mutants at Lateral view of a wildtype adult fly showing normal morphology Antenna an eye e genitalia and analia gal halter h tarsal claws la and wing w are indicated Eb Dorsal view of a class adult fly showing the heldcrut wing phenotype In Lateral View at a class II adult showing reduced wing and halter Iid Lateral view 0 a class It adult showing reduced eye wing and halter Bar represents 1000 In Identification and Cloning of dpp Downstream Targets Wild Type dppMad l ill in Iiil imaginal lixk Imlliml Illu39nulipl39 iliill wiin mm L i ildihpc Ill INM 397 39IHquot illi lr JH 2 3520 Iml39 Tlll39 tIIJwipI quot Wng i ii i AliplmimLIMlx I uIhi ll norm v0 Lind hm lt39lk39fllVL39 inuliun ermul ml lilll39 mp ul Hml it rmulis in 1 lullm l39ll inn ll lpln39mimalrli mil lIil RI l llm 1s nl Mull Immin l lip1quotquot I I lll ll2ll hall normal lL llllll39l39l39 l illn lllllllill 01 in t ll l mpy nl ill111 D lmlllLIHl nnclimd L N Ilu plwnnirpv Itsulling in Whip vin l usionx wingx 11 Ihl sumc Inngnillt39ulimi nl llnl39lllill izu illul hm l39l NlIlllil Loss of function mutations of Mad are dominant maternal effect enhancers ofdpp during early embryogenesis and dominant Newfeld et al 1995 zygotic enhancers of dpp in imaginal discs PhosphorylatiOn of Smad3 by TGFbeta RI at the Cterminus 3 of 3 39 9 SSXS Motif Liu et al PNAS 1997 TGFBdependent ampIndependent SmadZ Phosphorylation Mv1 Lu wt R1B TBRI Defective TGFB Nocodazole TGFB Nocodazole 12 15 20 12 15 20 T Rll 39 g r Phosphorylation of RSmads by TGFbeta Receptor Kinase Smadl SmadS BNIP Signaling TGFB and ActiVin Signaling R Smads Smad6 Smad7 MM Domain Cancer mum m missense nmm TGFBActivinBMP Signaling CoSmad TGFBActivinBIVIP gt InhibitorySmad MHZ Domain Linker El DNA bInjlng halrpln SIS Hecemur kinasa Ewe RSmada necapmr Il39lKOrill t39JGf l L2 Imp Farmaria I SAHEI ccnIaL39tE Ema2 SmacSrrac Imamcznn snaa I MAP kinewv Hil fj LSrnad131v IE Dmvbinding mlailrzr sila I39Flas 39l ts I CaCM kxnase Ell E Snadzl 5 39 39 I Smurr uEIquilln Iiga e Ella 1 MOE da mm Mb SImuquot Smads Are DNA Binding Proteins A C Mock Smad4eiemed Smadsselemed 7 brainme CID egmvmmumm 39 Clonwrwmvmmhmm g i l i Shiftedb probe Probe Wm B D clnuexGTCTAGAC 75 clunett GTCTAGAC 75mm 32 49 1 43 7ct4l 9g g 33 14 t 39 13 31 23 i i 7 4ci39 14quotv gata 31 6 gg3o 27777977L29 18 r r r t 3o 7 g ct29 1777gc7c729 5gtv g t26 9 g25 6gtq25 16gt g c 21 10 catt23 13 r r Vc 19 12 g g 23 lor rgc cvlg 3 gCt 22 12 c g 18 8 ct vg 11 15 quot t 16 4 cCtC34 11 cccgt8 11 31 Reporter Gene Assays for Studying TGFbeta Signaling 31 0 I mama PAl1 50 MV I Lu DH25 FI1B 532 6 39s 300 7 w 040 39 a rig g 330 eAZDD D 3 lt mg 3 EE sm sa 2 100 4 a lo 53 r 535 at OJ TGFBa quot39 TBR39 quotH quotH quotH quotH TGFB1 TGF b39etaSmad Signaling Dynamics Cytoplasmic retention factor W 00000000000000000 000000000000 00000000000 0000000 0000000000 00000 00000000000000000 000000000000 00000000000 0000000 0000000000 00000 N u cl ear retention factor Transcription Transcription Activation and Repression by TGFbeta Sequencespecific transcription factors AP 1bZlP AMLRunx bHLH Homeodomain Sp1 Winged helix Intracellular receptors Coactivators CBPp300 SMIF MSG1 ARC105 Coactivator repressor TBPTAFs Pol II TATA Co repressors cSkiSnoN cMyc Evi1 ATF3 TGIF SNP1 SIP1 Tob Derynck and Zhang Nature 2002 Mutations of TGFbeta Signaling Components in Human CancerCell s Transcription Massague et al Cell 2000 Nucleocytoplasmic Shuttling of Smads 2 3 and 4 Permits Sensing of TGF Receptor Activity Gareth J Inman Francisco J Nicola39s and Caroline S Hill A Tarrs Timemil lhn 1 5 m 15 2030451h2h3h lhsh h n Eh no cycloheximide What is pSmad2 How to detect it Why is cycloheximide used HaCaT iCAGAu Lucifernse 15 Fold ac va an Time of 53431542 addi nn minM57 To a a u 1 s In znsngsmzhlhmmah we H HaCzT DELucifeme 5 Fold activation n a a a a a I sanlszuanasmmmnsnsn Mixer What was the inhibitor added at different time points TGFbeta receptors remain active for at least 3 4 hr in HaCaT cells n 1 z 5 mlsznaouswmnm ECF Time min LeerPrLucifcmsu Fnld acLivaLion a Time M31478 addi an min a wh v r r Nch lkC A TGF D Time Imin z 5 m m 3 5 uSMIWS 3 Treatment Targs Time min so 1 10 no m m asmdza I I nSmadl IIIIWZ What does this experiment tell us about Smad2 and Smad4 in relationship to receptor activity C Treatment Timeltg i 0 60190 120 180 240 PSmadz M Smadz Smad3 P Smadz I SIB431542 Salads What happened to PSmad2 upon treatment with SB431542 69 5 TCFB Timumin n so go 130 an m SmadZIP Imuupmipimes 5m El Smad4 wa Inputs 23 SmadZS ws 1 6 2 3 4 5 Why there is less Smad4 in SmadZ IP samples treated with SB 431542 A Treatment Tarp Time min 0 6quot 1 9a 120 180 240 M3 l uSma What accounts for low nuclear staining of Smad23 in the presence of 53431542 Trtatmem TUE3 Timelmin n so 90 12a Ian 24m 4 31 Smade 39 ii 5mad3 g g PSmadz 5343154 Email I r Eli431542 Treatment Eli431542 LMB LMB TGFa i 130 180 130 18 Time min 393 60 l I What is LMB What does it do What does this experiment tell us 2 NaN3 ZdG TGFl l 53431542 Endyuinl Time min 0 10 40 60 120 180 0 w 4 5393 xx 4 9 W e Treatment 9 9Q Qx 399 TGF b Time min 0 60 60 180 180 130 150 ulsmadya I I wsmad4 Opposing BMP and EGF signalling pathways converge on the TGIFB family mediator Smad 1 Marcus Kretzeehmar Jacqueline Dandy 311mm Massagu 465 ailing mm GumTits PragI39rI Ia ri39miH 39isn3113fHirgfh S quotL riquot Isrsrrirmz quotnimm1 rimi Simw39Kiz rnarmg Cairn1 iquot Cmrw va EMS New 311 IF 09317 USA Domain Organization of Smad Homaxrimerizauon hemm arlgomsrvzauan psxps mom Em phnspnaryrlllnn m in signaling Homalrimerln an haremengamerizmon Receptor Interaction L3 loop 2 bindlng PY mam SmurfDinding B hairpin Binding to DNAbinding omer moraine Hvurupnumc comdur Receptor inlerac on a phosphnryla an Homo and nexgrouligomerizaxiun DNA Nuclear I hon Binding m DNA n my colacluls Nuclear Impnrllexporl Bindlnn m DNAbinding calaclnrs Elndlng m noaulvamrs and corepreasurs Phosphorylation of Smad1 E WT AAAAV TAA 4SPr AP 1 u a RumpturaEMP2 4 i s I a x y v I 33F labelling Emac vhrrmmunnhl at i 5m l nim Protease Digestion of Labeled Smad1 a 9 W 144r 32 A 5 f Protease Digestion of Labeled Smad1 dst F WT Maxim MUA HEFIMP Smad1 p REEEPf fkEMP 39 i v w v 3392P labelling Smad1 Ir A Imrnu mth 7 7 Smad1 I Phosphorylation of Smad1 Amino acid 1 loo 2 300 400 Proleas Ndoma om Trypsm fan 9 lragmems l A i6 llagmenls uChymol ypsin mm Isagmemslr Ira mansquot 450 VLTQMGSPHNPImE 465 BMP recep or pilosphorylation sites I A l w my w u 227 Erk MAPkinase phosohorylalion sites Why the predicted digestion pattern of Smad1 allowed us to identify the phosphorylated region as part of the linker region of Smad1 U ptimal pH Average MW Chymotrypsin Cleaves peptides at the carboxyl side of tyrosine tryptophan and phenylalanine Time Ilium Em 31111 33F labelling Sunam r lmmunahhat Smutquot EGF induced Smad1 Phosphorylation ES F TN Fux T 31 If 3 h EGF min Smad1 WI Smad1 ASWAP MEK 1ihibi1or Worlrnannm Serum 31F labelling Smad1 F lmmumblnt Smad1 II 4 4 w w M PM if k 7 4 3 I39 H EGF induced Smad1 Phosphorylation at the Linker Region 1 a Y Q quot Marm Mil1 Wanker 52 l 325 L a 39 8 C45 HGF induced Smad1 Phosphorylation d HGF HGF Time min 139 an 7 a Smad1 WT Smad14SPmP A 4 L 33F labelling Smad1 F lmm unublut Smad1 gt e Time min 3 g 3 D Smad1 I 1 MEK1inhibllor 33F labelling Smad1 gt lmmunoblol Smad1 tv In vitro Phosphorylation of Smad1 by Erk2 and BMPR1 Smad1 I WT 45PIAP 7 g m 1 BMPHI B QZGED W39I39 I15 F39J39AP Smad1 39Ir c Smam linker Smad1 linker has potential MAPK sites MAPK E MIP R a 6mm MAPK amen Jr t aSm Serines were mutated to alanine to prevent MAPKinduced phosphorylation EGFinduced phosphorylation of the Smad1 linker region does not prevent Smad1Smad4 complex formation BMP EGF Smaml quotIr Smad1 l I d 39Wpe d l fAP EmadI Ernatill Mterwllgmmr AnilaFiagi lri39lmunohim RTK block in BMP signaling is downstream of Smad1Smad4 complex formation Phosphorylation of Linker Region Represses Smad1 alal n a ac39liualinn GEM Gal4 Galrh Smad1 Smad1 WT 4EFAF39 BMP induces nuclear accumulation of Smad1 39 No treatment BMP BMP EGF BMPEGF uo126 U0126 Smadlinker mutant EGFinduced phosphorylation of Smad inhibits nuclear accumulation induced by BMP Phosphorylation of Linker Region Represses Smad1 ME K1 serum EM FoEGF in h lbitor starved c EMF EGF Wildly I dSPIA P MAPK phosphorylation of Smad1 prevents translocation of Smad1Smad4 complexes EGF BMP RTK RST K Nucleus Smad1Smad4 Gene responses BMP target gene expression is blocked due to a block in Smad1Smad4 nuclear translocation BMP signaling is regulated at multiple steps in the pathway Churdin Nuggin Ext race uar Brirp l l a a 77 a w Receptors l BAMBl T 3 5 Smacl I a madB l Ema15 l Erk Target genes Intracellular


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