FAK integrates growth-factor and integrin signals to promote cell migration

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1 integrtes growth-ftor nd integrin signls to promote ell migrtion rtiles Dvid J. Sieg*, Christof R. Huk*, Dusko Ili, Cndie K. Klingeil*, Erik Shefer, Croline H. Dmsky nd Dvid D. Shlepfer* *Deprtment of Immunology, IMM 26, The Sripps Reserh Institute, 155 N. Torrey Pines Rod, L Joll, Cliforni 9237, USA Deprtment of Stomtology, University of Cliforni Sn Frniso, Sn Frniso, Cliforni 94143, USA QCB-A Division of BioSoure Interntionl, Hopkinton, Msshusetts 1748, USA e-mil: dshlep@sripps.edu These uthors ontriuted eqully to this work Here we show tht ells lking fol dhesion kinse () re refrtory to motility signls from pltelet-derived nd epiderml growth ftors (PDGF nd EGF respetively), nd tht stle re-expression of resues these defets. ssoites with tivted PDGF- nd EGF-reeptor (PDGFR nd EGFR) signlling omplexes, nd expression of the nd-4.1-like domin t the mino terminus is suffiient to medite n intertion with tivted EGFR. However, effiient EGF-stimulted ell migrtion lso requires to e trgeted, y its roxy-terminl domin, to sites of integrin-reeptor lustering. Although the kinse tivity of is not needed to promote PDGF- or EGF-stimulted ell motility, kinse-intive is trnsphosphorylted t the indispensle Sr-kinse-inding site, Y397, fter EGF stimultion of ells. Our results estlish tht is n importnt reeptor-proximl link etween growth-ftorreeptor nd integrin signlling pthwys. T rnsmemrne integrins ind to extrellulr mtrix proteins nd generte importnt signls tht regulte ell prolifertion nd migrtion events stimulted y reeptors for solule growth ftors. Integrin nd growth-ftor signlling pthwys n intert through severl mehnisms, from memrne-proximl lustering of the two reeptor types 1,2 to the tivtion of ommon downstrem signlling pthwys 3 5. Although there is welth of knowledge regrding the signlling pthwys tivted y oth integrin nd growth-ftor reeptors, little is known out how these signls re integrted y ells nd whether there re ommon reeptor-proximl ontrol points tht synhronize the exeution of iologil funtions suh s ell motility. is non-reeptor protein-tyrosine kinse (PTK) tht indiretly lolizes to sites of integrin-reeptor lustering through C- terminl-domin-medited intertions 6 with integrin-ssoited proteins suh s pxillin 7,8 nd tlin 9. eomes phosphorylted t seven to eight different tyrosine residues in vivo fter enggement of integrin with mtrix proteins 1. Severl studies hve shown tht funtions s prt of ytoskeleton-ssoited network of signlling proteins, lso inluding the Sr-fmily PTKs, p13 Cs, Sh nd Gr2, whih t in omintion to trnsdue integrin-generted signls to the ERK/JNK mitogen-tivted protein (MAP) kinse sdes (reviewed in ref. 11). Evidene indites tht tyrosine phosphoryltion of my e importnt for ell migrtion, s expression of the protein-tyrosine phosphtse PTEN leds to dephosphoryltion of nd inhiition of ell motility 12,13. Experiments using -null ells 14 16, overexpression 17 nd dominnt-negtive onstruts 18 hve estlished tht is essentil for integrin-stimulted ell migrtion. Null muttions of the murine fironetin 19 or fk 2 genes result in similr lethl phenotype where emryos fil to develop pst dy 8.5 of emryoni development euse of defetive gstrultion events. Cultured -defiient ( ) ells exhiit rounded morphology, enhned formtion of fol ontts with mtrix proteins, nd migrtion defets 21. Expression of the -relted PTK Pyk2 is elevted in firolsts, nd the omined phosphoryltion tivities of Pyk2 nd Sr-fmily PTKs filitte integrin-stimulted ERK2 tivtion in the sene of 14. Interestingly, Pyk2 does not strongly lolize to fol ontt sites nd its overexpression does not resue the ell-migrtion defets used y the muttion 14. When stly re-expressed in ells, epitope-tgged lolizes to fol-ontt sites, promotes morphology hnges nd reverses the integrin-stimulted migrtion defets 15. Here we show tht ts s reeptor-proximl ridging protein tht links growth-ftor-reeptor nd integrin signlling pthwys. By diretly ompring +/+, nd -reonstituted firolsts, we find tht ssoites with tivted growth-ftor reeptors through its N-terminl domin nd tht hs n importnt funtion in promoting PDGF- nd EGFstimulted ell migrtion. Results funtion is required for PDGF-stimulted ell migrtion. To test the importne of in promoting growth-ftor-stimulted ell migrtion, we rried out hemotxis ssys in modified Boyden hmer with +/+ nd firolsts, nd with lonl firolsts, whih re-express 15 (Fig. 1). At low onentrtions of PDGF (2.5 1 ng ml 1 ), +/+ nd ells, ut not ells, redily migrted to PDGF-BB in dose-dependent mnner (Fig. 1). The lk of migrtion to PDGF-BB oserved in ells ws not due to the refrtory responsiveness of the PDGFR, s norml levels of PDGF-stimulted tyrosine phosphoryltion (1 ng ml 1, 5 min) were deteted in lystes from ells (Fig. 1). Previous studies hve shown tht phosphoryltion of tyrosine residues inreses upon ell stimultion involving growth ftors, hemokines or G proteins (reviewed in ref. 11). Surprisingly, stimultion with PDGF used only smll inreses in totl phosphotyrosine levels in nd Pyk2. However, PDGF tretment indued the ssoition of phosphotyrosine-ontining protein of reltive moleulr mss 19, (M r ~19K) with in +/+ nd ells, ut not with Pyk2 in ells (Fig. 1). Reiprol o-immunopreipittion experiments using ntiodies to the PDGFR nd lystes from strved or PDGF-stimulted ells NATURE CELL BIOLOGY VOL 2 MAY Mmilln Mgzines Ltd 249

2 Migrtion (A 6 ) Chemotxis ssy +/ PDGF (ng ml 1) - -Pyk2 - Blot: Pyk2 HA ell lystes PDGFR Sh- PDGF + PDGF 2- +/+ -Pyk2 - - PDGF IPs Pyk2 IPs HA IPs P Y -PDGFR Plsmidenoded proteins PDGFR- - PDGFr- PDGFR- PDGFR PDGFr + PDGF (1 ng ml 1) Cell lystes Cell lystes PDGFR HA IPs PDGFR Blot - HA - PDGFr - HA IPs HA PDGFr IPs Figure 1 forms omplex with the tivted PDGFR nd is required for PDGF-stimulted ell motility., In hemotxis ssy using modified Boyden hmer, +/+ firolsts nd ells migrte towrds PDGF-BB wheres ells do not. Vlues re mens ± s.d. of t lest three seprte experiments. Strved firolsts respond to PDGF-BB stimultion with enhned tyrosine phosphoryltion, s deteted y ting of ell lystes ginst phosphorylted tyrosine (P Y). Numers in the right pnel represent M r in thousnds., After stimultion with PDGF-BB, phosphotyrosine-ontining protein of M r ~19K oimmunopreipittes with ntiodies to in +/+ ells nd ntiodies to HAtgged (HA ) in ells, ut not with ntiodies to Pyk2 in ells. HAtgged ssoites with PDGFR immunopreipittes (IPs) in firolsts fter PDGF-BB stimultion., Trnsient o-trnsfetion of PDGFR-null 293T ells shows the speifi ssoition of HA nd PDGFR immunopreipittes fter PDGF-BB stimultion. HA PDGF-stimulted migrtion (ftor of indution reltive to pdna) ell hemotxis ssy pdna (F397) (R454) (A712/713) (F925) -HA Poly-Glu Tyr (4:1) phosphoryltion (1 3 Cerenkov pm) ells -ssoited kinse tivity PDGF + Strved PDGF In vitro kinse ssys Sr- 32 P IVK ssy HA IPs PDGF Csk p5 HA Sr PTK PDGF-stimulted migrtion (ells per field) ell hemotxis ssy Control Control p5 Csk p5 Csk Csk p5 (K222M) Csk p5 (K222M) Figure 2 The SH2-inding site nd Sr-fmily protein-tyrosine-kinse tivity re neessry for PDGF-stimulted ell migrtion., The Y397 site is neessry to enhne PDGF-stimulted migrtion of ells, wheres kinse-intive (R454) or mutnts tht do not ind p13 Cs ((A712/713)) or Gr2 ((F925)) funtion to promote PDGF-stimulted motility. Vlues re mens ± s.d. of t lest three seprte experiments., Effet of PDGF stimultion on the kinse tivity of, s mesured y in vitro poly-glu Tyr phosphoryltion nd oserved using n in vitro utophosphoryltion ( 32 P IVK) ssy. The oserved differene my reflet the reruitment of tive Sr-fmily PTKs to signlling omplex. IPs, immunopreipittes. Numers in the entrl pnel represent M r in thousnds., Inhiition of Sr-fmily PTK tivity y overexpression of p5 Csk ut not kinse-intive p5 Csk (K222M) prevents PDGF-stimulted motility of ells. Vlues re mens ± s.d. of two seprte experiments. showed tht HA-tgged ws tyrosine-phosphorylted nd ssoited with PDGFR immunopreipittes only fter ells were stimulted with PDGF (Fig. 1). forms omplex with tivted PDGFR. hs een shown to o-immunopreipitte with n unknown phosphotyrosine-ontining protein of M r ~2K fter ell stimultion with PDGF 22. To onfirm the ility of nd the PDGFR to ssoite, we trnsiently trnsfeted humn 293T ells, whih do not express PDGFR, with expression vetors for the PDGFR -suunit or HA-tgged (Fig. 1) Mmilln Mgzines Ltd NATURE CELL BIOLOGY VOL 2 MAY 2

3 EGF-stimulted 293T ells Migrtion (A 6 ) Chemotxis ssy +/ EGF (ng ml 1) ell lystes EGF (1 ng ml 1) + -EGFr Cell lystes -Sh + EGF +/+ ells EGFR IPs -EGFr pdna (F397) (R454) (A712/713) FRNK - EGFR- - - FRNK EGFR HA-tg HA-tg - HA-tg IPs - Ig - FRNK HA-tg IPs Figure 3 forms omplex with the tivted EGFR nd is required for EGF-stimulted ell motility., In hemotxis ssy using modified Boyden hmer, +/+ firolsts nd ells migrte towrds EGF, wheres ells do not migrte. Vlues re mens ± s.d. of t lest three seprte experiments. Strved firolsts respond to EGF stimultion with enhned tyrosine phosphoryltion, s deteted y nti-phosphotyrosine (P Y) ting of ell lystes. Numers in the right pnel represent M r in thousnds., Assoition of EGFR nd endogenous upon stimultion with EGF of +/+ ells, s deteted y oimmunopreipittion (IP) nlyses with ntiodies to., Trnsfetion of onstruts into 293T ells shows tht the kinse tivity of, tyrosine phosphoryltion, nd the integrity of the Y397 site re not required to medite EGFR ssoition in EGF-stimulted 293T ells. No EGFR ssoition with the C- terminl domin (FRNK) ws deteted. Ig, immunogloulin. Expression of the PDGFR in 293T ells led to growth-ftorindependent phosphoryltion of the PDGFR (Fig. 1, lnes 1, 5). In the sene of HA expression, ntiodies to the HA tg did not o-immunopreipitte PDGFR fter PDGF-BB stimultion (Fig. 1, lnes 1, 2). In ells not trnsfeted with PDGFR (Fig. 1, lnes 3, 4), HA-tg ntiodies did not o-immunopreipitte with either phosphotyrosine-ontining protein of M r ~2K (dt not shown) or PDGFR-immunoretive nd. However, upon oexpression of PDGFR nd HA, ntiodies to the HA tg wekly pulled down the PDGFR in the sene of growth ftor, wheres strong PDGFR ssoition with HA ws deteted fter PDGF-BB stimultion of the 293T ells (Fig. 1, lnes 5, 6). These results indite tht PDGF-BB stimultion my promote the reruitment of into n tive PDGFR signlling omplex. Y397 phosphoryltion is required for oth integrin- nd PDGF-stimulted ell motility. mutnts lking the utophosphoryltion/sh2-inding site ((F397)), kinse tivity ((R454)), or the primry p13 Cs SH3-inding site ((A712/ 713)) re ineffetive in promoting fironetin-stimulted ell migrtion when trnsiently expressed in ells 15. These dt indite tht severl protein protein intertions involving my e neessry to promote integrin-initited ell motility (hptotxis). To determine whether similr intertions with re required to support PDGF-stimulted ell motility (hemotxis), we trnsiently trnsfeted ells with vrious onstruts nd tested them for migrtion in response to PDGF-BB (Fig. 2). Expression of HA-tgged, (R454), (A712/713) or Gr2-inding-site mutnt ((F925)) promoted roughly eightfold inrese in PDGF-stimulted ell migrtion, wheres equivlent expression of (F397) did not effetively restore PDGFstimulted ell motility (Fig. 2). All of the mutnts exept (F397) were tyrosine-phosphorylted in PDGF-stimulted ells (dt not shown). PDGF stimultes the ssoition of Sr-fmily PTKs with. The one ommon funtionl site in needed to promote oth fironetin- nd PDGF-BB-stimulted ell migrtion is the utophosphoryltion/sh2-inding site, Y397. As the kinse tivity of is not required for PDGF-stimulted ell migrtion, it is possile tht other PTKs my trnsphosphorylte t the Y397 site fter PDGF stimultion of ells. To evlute the potentil ontriution of other -ssoited kinses, we nlysed HA immunopreipittes from strved or PDGF-BB-stimulted ells for in vitro kinse tivity using poly-glu Tyr s sustrte (Fig. 2). In PDGF-BB-stimulted (1 ng ml 1, 5 min) ells, poly-glu Tyr trnsphosphoryltion ws inresed sevenfold reltive to serum-strved ells (Fig. 2). Surprisingly, in prllel utophosphoryltion ssys, this inrese in the PDGF-stimulted kinse tivity of ws not refleted y strong PDGFR or utophosphoryltion tivity in the HA immunopreipittes, ut ws orrelted with the reruitment of tive Sr-fmily PTKs to the tivted omplex (Fig. 2). A similr Sr-PTK omplex ourred fter the ddition of low onentrtions of PDGF (1 1 ng ml 1 ) to Swiss 3T3 ells 23. SH2-medited inding of Sr-fmily proteins to the Y397 site is ompeted y severl other SH2-ontining signlling proteins, suh s Sh 24, the p85 suunit of phosphtidylinositol-3-oh kinse 25, SHP-2 (ref. 26), phospholipse C-1 (ref. 27) nd Gr7 (ref. 28). To determine the importne of Sr-fmily PTK tivity in PDGF-stimulted migrtion of ells, we speifilly inhiited Sr kinse tivity y trnsiently trnsfeting ells with n expression vetor for C-terminl Sr kinse (p5 Csk ). Overexpression of p5 Csk mrkedly inhiited PDGF-stimulted (5 ng ml 1 ) motility, wheres overexpression of the kinse-intive mutnt p5 Csk (K222M) did not (Fig. 2). These results indite tht tive inding of Sr-fmily PTKs to the Y397 site my e the first of severl importnt signlling events neessry to promote PDGFstimulted ell migrtion. funtion is required for EGF-stimulted ell migrtion. To test whether the role of in promoting ell migrtion is growth-ftor-speifi, we rried out hemotxis ssys of +/+, nd ells in modified Boyden hmer using low onentrtions of EGF (Fig. 3). Wheres nd ells expressed equivlent levels of EGFR (see Supplementry Informtion), ells were not ple of inititing migrtory response, even though enhned tyrosine phosphoryltion of EGFR nd its downstrem trgets, suh s Sh, redily ourred in EGF-stimulted (1 ng ml 1, 5 min) ells (Fig. 3). Similr to the stimulted formtion of omplex ontining tivted PDGFR nd, the ssoition of endogenous with tivted EGFR ws deteted y o-immunopreipittion with nti- ntiody only fter ells were stimu- NATURE CELL BIOLOGY VOL 2 MAY Mmilln Mgzines Ltd 251

4 EGF-stimulted migrtion (ftor of indution reltive to pdna) EGF-stimulted 293T ells HA IPs ( 1 1) ( 2 1) ( 3 1) pdna (R454) ( 1 1, R454) EGFR -EGFR (R454) ( 1 1) (R454) HA IPs ell hemotxis ssy (R454) ( 1 1, R454) - ( 1 2) (R454) ( 1 3) (R454) EGFR P Y397 HA EGF EGF (1 ng ml 1) (1 ng ml 1) - ( 1 1) - EGFr Trnsfeted ells ( 1 1, R454) ( 1 1, R454) HA IPs HA P Y P Y397 P Y47 P Y576 P Y577 ( 1 1, R454) ( 1 1, R454) HA IPs + + HA IPs 32P In vitro kinse ssy P Y861 P Y925 P Y95 EGF (1 ng ml 1) ( 1 1, R454) ( 1 1, R454) - - ( 1 1) Figure 4 The N-terminl domin is required for ssoition with EGFR nd EGF-stimulted ell motility., Wild-type nd kinse-intive (R454), ut not (1 1, R454), ssoite with tivted EGFR in 293T ells. (R454) is tyrosine phosphorylted t the Y397 site fter EGF stimultion, wheres (1 1, R454) is only minimlly tyrosine phosphorylted t this site. IPs, immunopreipittes. Numer etween the two pnels represents M r in thousnds., Upper pnel, ting for HA-tgged trunted versions of. Lower pnel, wild-type nd (R454) promote EGF-stimulted migrtion of ells eqully, wheres (1 1, R454), (1 2, R454) nd (1 3, R454) do not funtion to promote EGFstimulted migrtion. Vlues re mens ± s.d. of t lest three seprte experiments., is phosphorylted t severl sites nd exhiits high level of in vitro kinse tivity when trnsiently expressed in ells. Wild-type, ut not (1 1, R454), ssoites with tivted EGFR in ells. Phosphoryltion of (1 1, R454) t the Y47 nd Y95 sites is enhned y EGF stimultion. lted with EGF (Fig. 3). In further nlogy with the PDGF results, o-immunopreipittion nlyses showed no detetle ssoition of Pyk2 with n tivted EGFR omplex fter EGF stimultion of ells (dt not shown). forms omplex with tivted EGFR. To investigte further the ssoition of nd EGFR, we trnsiently trnsfeted 293T ells with vrious mutnt onstruts nd evluted their ssoition with endogenous EGFR using o-immunopreipittion nlyses fter EGF stimultion (1 ng ml 1, 5 min; Fig. 3). All the mutnts used, exept the isolted C-terminl domin (FRNK), formed omplexes with tivted EGFR (Fig. 3). The ft tht (F397) redily formed omplex with tivted EGFR, nd yet showed only miniml levels of tyrosine phosphoryltion, indites tht SH2-medited inding of n dptor protein to the Y397 site my not e ruil to the EGFR intertion. By nlogy with the results otined from PDGF-stimulted ells (Fig. 2), trnsient expression of (F397) did not signifintly enhne EGF-stimulted ell motility (dt not shown). It hs lso een shown tht induile expression of (F397) in ells inhiits serum-stimulted migrtion 16. Together, these results support the hypothesis tht (F397) fils to promote oth integrin- 15 nd growth-ftor-stimulted ell motility euse it fils to form produtive signlling omplex with downstrem trgets suh s the Sr-fmily PTKs. The N-terminl domin is required for EGF-stimulted motility. As the isolted C-terminl domin did not detetly ssoite with tivted EGFR in 293T ells (Fig. 3), we investigted the possiility tht the EGFR ssoition is medited y the N-terminl domin. Previous studies hve shown tht truntions of the N-terminl domin led to gretly rised level of tyrosine phosphoryltion 1. We therefore introdued kinse-intive muttion (R454) into trunted version of to form (1 1, R454). We expressed wild-type, (R454) nd (1 1, R454) in 293T ells nd exmined their ility to o-immunopreipitte with tivted EGFR fter EGF stimultion (1 ng ml 1, 5 min; Fig. 4). Wheres (R454) ws strongly ssoited with EGFR nd lso wekly tyrosine phosphorylted, (1 1, R454), lthough tyrosine phosphorylted, did not detetly ssoite with tivted EGFR (Fig. 4). Wild-type nd (R454) were phosphorylted t the Y397 site, s deteted y ntiodies speifi to phosphorylted Y397, wheres (1 1, R454) ws only minimlly phosphorylted t this site (Fig. 4). These results indite tht the integrity of the N-terminl domin my e importnt for mediting intertions with tivted EGFR. To test the funtionl orreltion etween the EGFR ssoition nd EGF-stimulted ell migrtion, we trnsiently expressed mutnt or trunted onstruts in ells nd ssessed their ility to promote migrtion fter EGF stimultion (5 ng ml 1 ; Fig. 4). Expression of either wild-type or (R454) promoted migrtion of ells eqully well, wheres (1 1, R454), (1 2, R454) nd (1 3, R454) did not signifintly promote ell migrtion ove ontrol levels (Fig. 4). Trnsfetion of kinse-tive (1 1) lso filed to promote EGFstimulted migrtion of ells (dt not shown), ut its trnsient expression signifintly redued ell dhesion, whih Mmilln Mgzines Ltd NATURE CELL BIOLOGY VOL 2 MAY 2

5 Trnsfeted onstruts EGFr β 1 integrin My P Y397 IPs EGF-stimulted 293T ells My tg My tg pdna My (1 42) HA FRNK HA My (1 42) HA tg Trnsfeted GST-fusion onstruts (1 42) HA tg EGFR -2 -EGFr -HA EGFr -β1-66 -My (1 42) -66 -My (1 42) Figure 5 Exogenously expressed N-terminl domin n form omplex with tivted EGFR., 293T ells were trnsfeted with the indited onstruts nd stimulted with EGF. Sequentil ting of Mytg, HA-tg, or EGFR immunopreipittes (IPs) shows tht the My-tgged N-terminl domin (My (1 42); M r ~6K) ssoites with EGFR. Wild-type nd the C-terminl domin (FRNK), ut not (1 42), form omplex with 1 integrins. (1 42) is trnsphosphorylted t the Y397 site in EGF-stimulted ells. Numers represent M r in thousnds., (1 42) promotes EGF-stimulted migrtion of ells hlf s well s untrunted. Vlues re mens ± s.d. of two seprte experiments., Expression of GST (1 42), ut not GST Pyk2(1 47), leds to the formtion of omplex with tivted EGFR in vivo upon EGF stimultion of 293T ells. Diret inding of GST Gr2 to EGFR is muh stronger thn GST (1 42) ssoition. Both GST (1 42) nd GST Pyk2(1 47) re tyrosine phosphorylted. GST (1 42) phosphoryltion t the Y397 site is enhned fter EGF stimultion. Stining of the memrne with Coomssie lue shows the mounts of GST onstruts ssoited with glutthione eds. EGF-stimulted migrtion (ells per field) Strved EGF (1 ng ml 1) (1 42) Pyk2(1 47) Gr2 GST only (1 42) Pyk2(1 47) Gr2 GST only ells - 2 pdna (1 42) EGFR p52 Sh P Y397 Coomssie lue stin GST hmpered onlusions from motility ssys. Nevertheless, the results otined from -truntion mutnts show tht formtion of the EGFR omplex is orrelted to the promotion of EGF-stimulted ell motility. By nlogy with PDGF-stimulted motility (Fig. 2), the kinse tivity of ws not required for EGF-stimulted ell migrtion. This n e explined mehnistilly y the ft tht my e sustrte for other PTKs upon EGF stimultion of ells. This ide is supported y our finding tht untrunted (R454) ssoited with tivted EGFR nd ws phosphorylted t the Y397/SH2-inding site when expressed in 293T ells (Fig. 4). To investigte further the trnsphosphoryltion of fter EGF stimultion nd to determine whether (1 1, R454) is phosphorylted t the Y397 site when trnsiently expressed in ells, we sequentilly nlysed immunopreipittes of HA-tgged nd (1 1, R454), using phosphospeifi ntiodies tht reognize different phosphoryltion sites (Fig. 4). Under oth strved nd EGF-stimulted onditions, HA ws highly tyrosine phosphorylted t the Y397, Y47, Y576, Y577 nd Y861 sites, nd wekly phosphorylted t the Y925 nd Y95 sites. HA trnsiently expressed in ells ssoited with EGFR fter EGF stimultion, yet it exhiited high level of in vitro kinse tivity under oth strved nd EGF-stimulted onditions (Fig. 4). Assoition of (1 1, R454) with EGFR ws not deteted in ells, lthough the totl level of tyrosine phosphoryltion of (1 1, R454) mrkedly inresed fter EGF stimultion (Fig. 4). Using the phosphospeifi ntiodies, we deteted (1 1, R454) phosphoryltion t the Y47 nd Y95 sites, ut no signifint phosphoryltion t the Y397 site or in the kinse domin t Y576 nd Y577 (Fig. 4). The mehnism of tyrosine phopshoryltion of (1 1, R454) is unknown. However, the ft tht (1 1, R454) does not detetly ssoite with tivted EGFR nd is not phosphorylted t the Y397 site supports our previous onlusions tht these two events re importnt for funtion in promoting EGF-stimulted motility. The isolted N-terminl domin n ssoite with tivted EGFR. To determine whether the EGFR intertion n our in the sene of the fol-dhesion-trgeting signls loted in the C-terminl domin, we expressed residues 1 42 ((1 42)) s My-epitope-tgged protein in 293T ells. Immunopreipittion nd ting ginst the My tg deteted protein of M r ~6K in ells trnsfeted with this onstrut (Fig. 5, lne 2). Co-immunopreipittion nlyses showed tht My-tgged (1 42) nd HA-tgged ssoited with tivted EGFR (Fig. 5, lnes 2, 4), wheres HA-tgged FRNK did not show strong or reproduile ssoition (Fig. 5, lne 3 nd Fig. 3). Anlysis of ssoition of 1 integrin with the onstruts used showed tht it ws present in oth the FRNK nd the immunopreipittes ut not in those of My-tgged (1 42) (Fig. 5, lnes 2 4). This result indites tht the ssoition of the N- terminl domin with EGFR my not involve linkges through 1 integrins; in this respet our results re not onsistent with previous report of in vitro inding of 1 integrin peptides to the N- terminl domin 29. To onfirm the ssoition of the N-terminl domin with EGFR, we rried out reiprol o-immunopreipittion experiments using nti-egfr ntiodies, nd ted the immunopreipittes for ssoited My-tgged proteins (Fig. 5, lne 5). Mytgged (1 42) ws phosphorylted t the Y397/SH2-inding site fter EGF stimultion of 293T ells, whih is inditive of trnsphosphoryltion event (Fig. 5, lnes 2, 5). A low level of 1 - integrin-suunit signl ws lso deteted in the EGFR immunopreipitte (Fig. 5, lne 5), whih is onsistent with previous reports of EGFR nd integrin o-lustering 2. (1 42) expressed in ells promoted ell migrtion in response to EGF t hlf the level stimulted y wild-type (Fig. 5). These results support the ide tht lthough (1 42) forms omplex with tivted EGFR nd is phosphorylted t the Y397 site, NATURE CELL BIOLOGY VOL 2 MAY Mmilln Mgzines Ltd 253

6 EGFr- Plsmidenoded proteins 116- EGF-stimulted ells EGFr + FRNK EGFr IPs + FRNK + FRNK HA-tg -2 -HA Plsmidenoded proteins EGF-stimulted ells + FRNK HA-tg + FRNK Anti--N-terminus + FRNK - py397 HA-tg EGF-stimulted migrtion (ells per field) hemotxis ssy pdna FRNK FRNK (S134) -HA FRNK Figure 6 Expression of the C-terminl domin inhiits the EGFR ssoition nd EGF-stimulted ell motility., Co-expression of the C- terminl domin (FRNK) with untrunted in EGF-stimulted ells inhiits ssoition with tivted EGFR omplexes. IPs, immunopreipittes. Numers represent M r in thousnds., Co-expression of FRNK with untrunted in EGF-stimulted ells suppresses phosphoryltion t the Y397 site., Expression of FRNK, ut not the point mutnt FRNK(S134) tht does not lolize to fol ontt sites, inhiits EGF-stimulted ell motility, s mesured y modified Boyden-hmer ssy. Vlues re mens ± s.d. of t lest three seprte experiments. full funtion in promoting EGF-stimulted ell motility my lso require trgeting of the C-terminl domin to sites of integrin-reeptor lustering. EGFR ssoites with the N-terminl domin of ut not of Pyk2. Reent sequene omprisons hve reveled tht the N-terminl domins of oth nd Pyk2 ontin divergent domin with homology to nd 4.1 tht hs een implited in mediting intertions with trnsmemrne reeptors 3,31. To evlute the speifiity of the ssoition of the N-terminl domin with EGFR, nd to show tht this ssoition ours in n ntiodyindependent fshion, we mplified sequenes enoding (1 42), (1 42, 1 1), the Pyk2 N-terminl domin (Pyk2(1 47)) nd the Gr2 SH2/SH3 dptor protein nd loned them into the pebg mmmlin glutthione-s-trnsferse (GST)- fusion expression vetor. GST (1 42), GST Pyk2(1 47) nd GST Gr2 were suessfully expressed nd ound to glutthione grose eds (Fig. 5), ut GST (1 42, 1 1) ws proteolytilly degrded nd not stly expressed (dt not shown). Both the GST (1 42) nd the GST Pyk2(1 47) fusion proteins were tyrosine phosphorylted in strved 293T ells (Fig. 5, lnes 1, 2), nd GST Pyk2(1 47) nd GST Gr2 ound to n unknown phosphotyrosine-ontining protein of M r ~19K (Fig. 5, lnes 2, 3). As expeted, EGF stimultion of 293T ells gretly enhned the ssoition of vrious phosphotyrosine-ontining proteins suh s Sh nd EGFR with GST Gr2 (Fig. 5, lne 7). Signifintly, EGF stimultion lso promoted ler, lthough weker, ssoition of phosphotyrosine-ontining protein of M r ~2K with GST (1 42); this protein ws positively identified s EGFR y immunoting (Fig. 5, lne 5). EGF stimultion lso inresed the tyrosine phosphoryltion of GST (1 42) t the Y397 site, ut did not promote the ssoition of GST Pyk2(1 47) or GST lone with EGFR (Fig. 5, lnes 6, 8). Frwestern protein protein nlyses showed tht 32 P-lelled GST Gr2 ound diretly to immunopreipitted nd tyrosine-phosphorylted EGFR, wheres 32 P-lelled GST (1 42) did not (dt not shown). Tretment of ells with ytohlsin D (1 M, 2 min) efore EGF stimultion disrupted the ility of to ssoite with the tivted EGFR omplex (dt not shown). These dt support the ide tht this EGFR ssoition my reflet, similrly to integrin ssoitions, n indiret intertion. Expression of the C-terminl domin inhiits EGF-stimulted ell motility. Intertions, medited y the C-terminl domin, of with proteins suh s pxillin 7,8 nd tlin 9 funtion to promote the trgeting to, nd the indiret ssoition of, with integrins t fol-ontt sites. As expression nd tyrosine phosphoryltion of (1 42) did not promote EGF-stimulted migrtion of ells to the sme extent s with wild-type (Fig. 5), full tivity my require C-terminl-domin-medited trgeting of to fol ontt sites. As C-terminl truntions of Growth-ftor reeptor PTKs (e.g. EGFR, PDGFR...) GF? N Bnd-4.1 domin Kinse domin Atin stress fires Y397 Signls MOBILITY Fironetin Vitronetin Pxillin Figure 7 Model of funtion. Indiret loliztion of to sites of integrin nd growth-ftor-reeptor lustering ples in position to t s reeptorproximl regultory protein. Phosphoryltion of t the Y397 site nd the integrity of the tin ytoskeleton re oth required for funtion in promoting oth growth-ftor (GF)- nd integrin-medited ell motility. Tlin ECM Integrin βα Atin stress fires C Mmilln Mgzines Ltd NATURE CELL BIOLOGY VOL 2 MAY 2

7 were sujet to proteolyti degrdtion when expressed in ells (dt not shown), we exogenously expressed the C- terminl domin (FRNK) s ompetitive inhiitor of loliztion to sites of integrin-reeptor lustering 32. Although FRNK did not strongly ssoite with the tivted EGFR omplex when expressed in 293T ells (Fig. 3d), o-expression of FRNK with in ells disrupted the ssoition of with the tivted EGFR omplex (Fig. 6) nd suppressed EGF-stimulted phosphoryltion t the Y397 site (Fig. 6). Signifintly, FRNK expression potently inhiited EGF-stimulted migrtion of ells (Fig. 6), wheres equivlent expression of FRNK point mutnt (FRNK(S134)) tht does not strongly lolize to fol-ontt sites 15 did not inhiit motility (Fig. 6) or disrupt the stimulted formtion of EGFR omplex (dt not shown). These results support the onlusion tht full funtion in promoting EGF-stimulted motility involves omintion of distint intertions medited y oth the N nd the C termini (Fig. 7). In ddition, the results otined from FRNK overexpression indite tht link etween the N terminus nd EGFR my e filitted nd/or stilized y intertions etween the C terminus nd integrins. Disussion It is well doumented tht hs importnt funtions in integrininitited signlling events. Chnges in the tyrosine-phosphoryltion sttus of lso our fter stimultion with solule growth ftor in severl different ell types. We hve shown here tht is importnt in linking PDGFR nd EGFR tivtion to the ellulr mhinery tht promotes direted ell migrtion. Our results support new funtion for the unique N-terminl domin whih, upon exogenous expression, ssoited with n tivted EGFR omplex. The inding of the N-terminl domin to EGFR ws not s strong s diret Gr2 SH2-domin-medited inding to the phosphorylted EGFR. In ddition, the ssoition of with tivted omplexes of oth EGFR nd PDGFR ould e disrupted y pretretment of ells with ytohlsin D, whih prevents tin polymeriztion ut does not lok tyrosine phosphoryltion of PDGFR or EGFR. As we ould not detet diret ssoition etween the N-terminl domin nd EGFR in frwestern nlyses, this intertion, like the o-loliztion of with integrin reeptors, my e medited y one or more intermediry ridging proteins. The overll signifine of our iohemil findings is highlighted y the ft tht is reruited to sites of growth-ftorreeptor nd integrin lustering in intt ells (see Supplementry Informtion) nd tht expression is required for oth PDGFnd EGF-stimulted ell motility. This ples in ritil position s reeptor-proximl omponent of oth integrin- nd growth-ftor-reeptor PTK signlling pthwys (Fig. 7). We found tht deletions of the N-terminl domin disrupted the ssoition of with n tivted EGFR omplex. onstruts lking n intt N-terminl domin did not promote EGF-stimulted motility when nlysed in gin-of-funtion hemotxis ssys using ells. However, tyrosine phosphoryltion of nd exogenous expression of its N-terminl domin only prtilly funtioned to promote EGF-stimulted ell motility. These results indite tht loliztion of to sites of integrin-reeptor lustering is lso importnt for ell motility. Indeed, overexpression of the C-terminl domin ted to disrupt the ssoition of with tivted EGFR omplexes, promote dephosphoryltion t the Y397 site nd inhiit EGF-stimulted ell motility. Thus, onnetions to oth growth-ftor reeptors nd integrins re required to oordinte ell migrtion. The ssoition of with n tivted CCR5 reeptor omplex hs reently een demonstrted using o-immunopreipittion nlyses 33. In ddition, hs een shown to ssoite with the intive EphA2 PTK 34. Although these reports did not eluidte the moleulr mehnisms mediting these reeptor intertions, we speulte tht the involvement of with other trnsmemrne-reeptor signlling pthwys my e refleted y the ft tht tyrosine phosphoryltion of n e modulted y severl different extrellulr stimuli (reviewed in ref. 11). Speifiity etween - nd Pyk2-medited signlling events my e determined y differenes in the intertions undertken y their respetive N-terminl domins. With respet to the mehnism of funtion in ell motility, we found, surprisingly, tht the kinse tivity of ws not required to promote either PDGF- or EGF-stimulted ell migrtion in ells, even though the sme ells require s kinse tivity to promote fironetin-stimulted ell motility 15. Diret inding of p13 Cs to ws lso not required for PDGF-stimulted ell migrtion, lthough previous nlyses hve shown tht p13 Cs intertions re importnt for fironetin- nd integrin-stimulted hptotxis 15,17. These results indite tht the moleulr mehnisms of -medited motility differ depending upon whether the primry ell stimulus is initited y the tivtion of integrin or growth-ftor reeptors. Mehnistilly, the requirement for s kinse tivity in promoting integrin-stimulted motility n e explined y the ft tht integrins lk intrinsi tlyti tivity. For growth-ftor-stimulted motility, phosphoryltion my e initited y reeptor-ptk tivtion. We found tht untrunted kinse-intive nd the N-terminl domin were trnsphosphorylted t the Y397 site fter EGF stimultion, n indition tht other PTKs prtiipte in -medited signlling events. A ommon nd importnt mehnisti onnetion needed to promote oth integrin- nd growth-ftor-stimulted ell migrtion ws the Y397 site. Phosphoryltion t this site retes n SH2-inding site for numer of different signlling nd dptor proteins. Our results show tht PDGF stimultion promotes the reruitment of tive Sr-fmily PTKs to, nd tht inhiition of Sr-fmily PTK tivity y overexpression of p5 Csk prevents PDGF- nd -medited migrtion. As ells exhiit n overundne of fol ontts nd deresed ell motility, our results lso support working model for funtion in promoting fol-ontt turnover. The tlyti tivity of Sr-fmily PTKs hs een shown to promote the turnover of fol-ontt strutures during ell motility 35, nd Sr intertions re importnt for EGF-stimulted ell migrtion 36. Therefore, -medited loliztion of tive Sr-fmily PTKs to sites of growth-ftor- nd integrin-reeptor lustering my initite fol-ontt remodelling nd migrtory signls. In summry, our results show tht n funtion s n importnt reeptor-proximl omponent to promote ell migrtion. As is overexpressed in vriety of invsive humn tumours 37,38 nd the gene is mplified in vriety of humn ner ells 39, strtegies to redue levels of protein or phosphoryltion t the Y397/SH2-inding site my provide wy to interfere with enhned growth-ftor- nd integrin-stimulted ell motility in oth vsulr nd neoplsti diseses. Methods Cell ulture. +/+, nd firolsts were ultured, serum strved nd trnsfeted s desried 15. +/+, nd ells hve the sme p53 geneti kground. Humn 293T ells were ultured nd trnsfeted s desried 4. DNA onstruts. HA-tgged onstruts for wild-type, (F397), (R454), (A712/713), (F925), FRNK nd FRNK(S134) were used s desried 15. Expression vetors for p5 Csk nd p5 Csk (K222M) were used s desried 14. (1 1) (ref. 4) ws loned into (R454) s KpnI/ClI frgment. My (1 42) ws reted y ligting BmHI/ClI wild-type frgment into the BglII/XI sites of pcs My 14 using the primers 5-CGATGGTGGTTGAT nd 5-CTAGATCAACCACCAT s linkers. BmHI/ClI-ended frgments ontining the nuleotide sequene enoding (1 2) nd (1 3) were reted using the polymerse hin retion (PCR) nd loned into the sme sites in pdna enoding (R454). PCR ws used to rete BmHI/XhoI-ended frgment ontining the nuleotide sequene enoding (1 42), (1 42, 1 1), Pyk 2(1 47) nd humn Gr2. The 3-flnking PCR primer ontined the nuleotide sequene enoding protein kinse A onsensus NATURE CELL BIOLOGY VOL 2 MAY Mmilln Mgzines Ltd 255

8 phosphoryltion site (mino-id sequene RRASVG) upstrem of the XhoI site in ll onstruts. Construts were loned into the BmHI/XhoI sites of pgex-4t nd suloned s BmHI/NotI frgments into pebg mmmlin GST-fusion expression vetor. All DNA onstruts reted y PCR were verified y sequening. Chemotxis. For modified Boyden-hmer (Millipore, Bedford, MA) motility ssys, oth sides of memrnes were oted with rt-til ollgen (5 g ml 1 in PBS; Boehringer) for 2 h t 37C nd wshed with PBS; hmers were pled in 24-well dishes ontining migrtion medi (.4 ml DMEM ontining.5% BSA) with or without humn reominnt PDGF-BB or EGF (Cliohem) t the indited onentrtions. For trnsient-trnsfetion ell-migrtion ssys, 2.5 g pdna3 lz were omined with 2.5 g expression plsmids to identify trnsfeted ells. Serum-strved ells (1 1 5 ells in.3 ml migrtion medi) were dded to the upper omprtment nd, fter 3 h t 37 C, ells on the memrne upper surfe were removed with otton-tip pplitor; migrtory ells on the lower memrne surfe were fixed y tretment with methnol nd id nd then either stined (.1% rystl violet,.1 M orte ph 9. nd 2% ethnol) or nlysed for trnsfeted -gltosidse tivity using X-gl s sustrte. Cell-migrtion vlues were determined either y dye elution nd sorene mesurements t 6 nm or y ounting -gl-stined ells (mesured s ells per field using 4 ojetive). Mens were tken from three individul hmers. Bkground levels of ell migrtion (less thn 5% of totl) in the sene of hemotxis stimuli (.5% BSA only) were sutrted from ll points. Immunopreipittion. Cells on 1-m pltes were lysed with.5 ml modified RIPA lysis uffer 15 ; lystes were immeditely diluted with.5 ml HNTG uffer (5 mm HEPES ph 7.4, 15 mm NCl,.1% Triton X-1 nd 1% glyerol), inuted with grose eds nd lered y entrifugtion. Immunopreipittes with the indited ntiodies were rried out for 4 h t 4 C nd olleted on grose eds with protein A (Repligen, Cmridge, MA) or protein G-plus (Cliohem); the preipitted protein omplexes were wshed t 4 C in Triton-only lysis uffer (modified RIPA without sodium deoxyholte nd SDS) nd then in HNTG uffer efore diret nlysis y SDS PAGE. For GST-fusion proteins, lystes of 293T ells were prelered nd inuted with 2 l glutthione grose-ed slurry (Sigm) for 2 h t 4 C. Beds were pelleted y entrifugtion nd wshed three times in 1% Triton uffer efore diret nlysis y SDS PAGE. Assoited GST-fusion proteins were oserved y stining with Coomssie lue nd immoiliztion on polyvinylidene fluoride memrnes (PVDF; Millipore). In vitro kinse (IVK) ssy. IVK ssys of immunopreipittes in the presene or sene of poly-glu Tyr (4:1; Sigm) were rried out s desried 14. Immunoting. Proteins nlysed y SDS PAGE were trnsferred to PVDF memrnes, loked in 2% BSA nd Trisuffered sline with.5% Tween-2 for 2 h t room temperture nd then inuted with either 1 g ml 1 monolonl ntiody or 1:1, dilution of polylonl ntiodies for 2 h t room temperture. Bound primry ntiody ws oserved y enhned hemiluminesene detetion nd susequent reproing of memrnes ws rried out s desried 15. Anti-phosphotyrosine nd nti-pdgfr ntiodies were from Upstte Biotehnology (Lke Plid, NY); nti-my- nd nti-ha-tg ntiodies were from Covne Reserh (Berkeley, CA); nti-egfr nd nti-sr ntiodies were from Snt Cruz. Negtively pre-dsored nd ffinity-purified polylonl ntiodies to peptides orresponding to the sequenes surrounding tyrosine-phosphoryltion sites Y397, Y47, Y576, Y577, Y861, Y925 nd Y95 were from BioSoure Interntionl (Cmrillo, Cliforni, USA). Affinity-purified polylonl ntiodies to, Pyk2 nd Csk were used s desried 14. RECEIVED 7 OCTOBER 1999; REVISED 26 JANUARY 2; ACCEPTED 1 MARCH 2; PUBLISHED 27 MARCH Shneller, M., Vuori, K. & Ruoslhti, E. v3 integrin ssoites with tivted insulin nd PDGF reeptors nd potentites the iologil tivity of PDGF. EMBO J. 16, (1997). 2. Miymoto, S., Teremoto, H., Gutkind, J. S. & Ymd, K. M. Integrins n ollorte with growth ftors for phosphoryltion of reeptor tyrosine kinses nd MAP kinse tivtion: roles of integrin ggregtion nd oupny of reeptors. J. Cell Biol. 135, (1996). 3. Howe, A., Aplin, A. E., Alhri, S. K. & Julino, R. L. Integrin signling nd ell growth ontrol. Curr. Opin. Cell Biol. 1, (1998). 4. Ginotti, F. G. & Ruoslhti, E. Integrin signling. Siene 285, (1999). 5. Shwrtz, M. A. & Bron, V. Intertions etween mitogeni stimuli, or, thousnd nd one onnetions. Curr. Opin. Cell Biol. 11, (1999). 6. Hildernd, J. D., Shller, M. D. & Prsons, J. T. Identifition of sequenes required for the effiient loliztion of the fol dhesion kinse, pp125, to ellulr fol dhesions. J. Cell Biol. 123, (1993). 7. Thin, K., Sto, T., D Avirro, N. & Morimoto, C. Diret ssoition of pp125 with pxillin, the fol dhesion-trgeting mehnism of pp125. J. Exp. Med. 182, (1995). 8. Liu, S. et l. Binding of pxillin to lph 4 integrins modifies integrin-dependent iologil responses. Nture 42, (1999). 9. Chen, H. C. et l. Intertion of fol dhesion kinse with ytoskeletl protein tlin. J. Biol. Chem. 27, (1995). 1. Shlepfer, D. D. & Hunter, T. Evidene for in vivo phosphoryltion of the Gr 2 SH 2-domin inding site on fol dhesion kinse y Sr-fmily protein-tyrosine kinses. Mol. Cell. Biol. 16, (1996). 11. Shlepfer, D. D., Huk, C. R. & Sieg, D. J. Signling through fol dhesion kinse. Prog. Biophys. Mol. Biol. 71, (1999). 12. Tmur, M. et l. Inhiition of ell migrtion, spreding, nd fol dhesions y tumor suppressor PTEN. Siene 28, (1998). 13. Gu, J. et l. Sh nd differentilly regulte ell motility nd diretionlity modulted y PTEN. J Cell Biol. 146, (1999). 14. Sieg, D. J. et l. Pyk2 nd Sr-fmily protein-tyrosine kinses ompenste for the loss of in fironetin-stimulted signling events ut Pyk2 does not fully funtion to enhne - ell migrtion. EMBO J. 17, (1998). 15. Sieg, D. J., Huk, C. R. & Shlepfer, D. D. Required role of fol dhesion kinse () for integrinstimulted ell migrtion. J. Cell Si. 112, (1999). 16. Owen, J. D., Ruest, P. J., Fry, D. W. & Hnks, S. K. Indued fol dhesion kinse () expression in -null ells enhnes ell spreding nd migrtion requiring oth uto- nd tivtion loop phosphoryltion sites nd inhiits dhesion-dependent tyrosine phosphoryltion of Pyk 2. Mol. Cell Biol. 19, (1999). 17. Cry, L. A., Hn, D. C., Polte, T. R., Hnks, S. K. & Gun, J.-L. Identifition of p13 Cs s meditor of fol dhesion kinse-promoted ell migrtion. J. Cell Biol. 14, (1998). 18. Gilmore, A. P. & Romer, L. H. Inhiition of fol dhesion kinse () signling in fol dhesions dereses ell motility nd prolifertion. Mol. Biol. Cell 7, (1996). 19. George, E. L., Georges-Louesse, E. N., Ptel-King, R. S., Ryurn, H. & Hynes, R. O. Defets in mesoderm, neurl tue nd vsulr development in mouse emryos lking fironetin. Development 119, (1993). 2. Furut, Y. et l. Mesoderml defet in lte phse of gstrultion y trgeted muttion of fol dhesion kinse,. Onogene 11, (1995). 21. Ili, D. et l. Redued ell motility nd enhned fol dhesion ontt formtion in ells from defiient mie. Nture 377, (1995). 22. Chen, H. C. & Gun, J. L. The ssoition of fol dhesion kinse with 2-kD protein tht is tyrosine phosphorylted in response to pltelet-derived growth ftor. Eur. J. Biohem. 235, (1996). 23. Slzr, E. P. & Rozengurt, E. Bomesin nd pltelet-derived growth ftor indue ssoition of endogenous fol dhesion kinse with Sr in intt Swiss 3T3 ells. J. Biol. Chem. 274, (1999). 24. Shlepfer, D. D., Jones, K. C. & Hunter, T. Multiple Gr2-medited integrin-stimulted signling pthwys to ERK 2/mitogen-tivted protein kinse: Summtion of oth -Sr nd -initited tyrosine phosphoryltion events. Mol. Cell Biol. 18, (1998). 25. Chen, H. C., Appeddu, P. A., Isod, H. & Gun, J. L. Phosphoryltion of tyrosine 397 in fol dhesion kinse is required for inding phosphtidylinositol 3-kinse. J. Biol. Chem 271, (1996). 26. Mnes, S. et l. Conerted tivity of tyrosine phosphtse SHP-2 nd fol dhesion kinse in regultion of ell motility. Mol. Cell Biol. 19, (1999). 27. Zhng, X. et l. Fol dhesion kinse promotes phospholipse C-gmm 1 tivity. Pro. Ntl Ad. Si. USA 96, (1999). 28. Hn, D. C. & Gun, J. L. Assoition of fol dhesion kinse with gr7 nd its role in ell migrtion. J. Biol. Chem. 274, (1999). 29. Shller, M. D., Otey, C. A., Hildernd, J. D. & Prsons, J. T. Fol dhesion kinse nd pxillin ind peptides mimiking integrin ytoplsmi domins. J. Cell Biol. 13, (1995). 3. Girult, J. A., Lesse, G., Mornon, J. P. & Clleut, I. The N-termini of nd JAKs ontin divergent nd 4.1 domins. Trends Biohem. Si. 24, (1999). 31. Lev, S. et l. Identifition of novel fmily of trgets of PYK2 relted to Drosophil retinl degenertion B (rdgb) protein. Mol. Cell. Biol. 19, (1999). 32. Rihrdson, A. & Prsons, J. T. A mehnism for regultion of the dhesion-ssoited protein tyrosine kinse pp125. Nture 38, (1996). 33. Cil, C. et l. Indution of phosphoryltion nd intrellulr ssoition of CC hemokine reeptor 5 nd fol dhesion kinse in primry humn CD4+ T ells y mrophge-tropi HIV envelope. J. Immunol. 163, (1999). 34. Mio, H., Burnett, E., Kinh, M., Simon, E. & Wng, B. Ativtion of EphA2 kinse suppresses integrin funtion nd uses fol dhesion kinse dephosphoryltion. Nture Cell Biol. 2, (2). 35. Finhm, V. J. & Frme, M. C. The tlyti tivity of Sr is dispensle for trnslotion to fol dhesions ut ontrols the turnover of these strutures during ell motility. EMBO J. 17, (1998). 36. Brunton, V. G., Oznne, B. W., Prskev, C. & Frme, M. C. A role for epiderml growth ftor reeptor, -Sr nd fol dhesion kinse in n in vitro model for the progression of olon ner. Onogene 14, (1997). 37. Owens, L. V. et l. Overexpression of the fol dhesion kinse (p125) in invsive humn tumors. Cner Res. 55, (1995). 38. Kornerg, L. J. Fol dhesion kinse nd its potentil involvement in tumor invsion nd metstsis. Hed Nek 2, (1998). 39. Agohiy, M. et l. Inresed dosge nd mplifition of the fol dhesion kinse gene in humn ner ells. Onogene 18, (1999). 4. Shlepfer, D. D., Broome, M. A. & Hunter, T. Fironetin-stimulted signling from fol dhesion kinse- -Sr omplex: involvement of the Gr 2, p13 Cs, nd Nk dptor proteins. Mol. Cell Biol. 17, (1997). ACKNOWLEDGEMENTS We thnk A. Moore nd S. Reider for ssistne, M. Shwrtz for polylonl ntiserum direted to 1 integrins, T. Hunter for the PDGFR--expression vetor nd polylonl ntiodies to the PDGFR, B. Myer for the pebg mmmlin GST-fusion expression vetor nd J.-L. Gun for the HA-tgged (C14) expression vetor. This work ws supported y Ntionl Cner Institute, Amerin Cner Soiety nd Amerin Hert Assoition grnts to D.D.S. D.J.S ws supported y n NIH postdotorl trining grnt; C.R.H y the Deutshe Forshungsgemeinshft (HA-2856/1-1); D.I. y the UCSF Ademi Sente; nd C.H.D y the Amerin Hert Assoition. Correspondene nd requests for mterils should e ddressed to D.D.S Mmilln Mgzines Ltd NATURE CELL BIOLOGY VOL 2 MAY 2

9 supplementry informtion +/+ +/+ Beds HA Beds EGFR BSA FN +/+ HA PDGFR EGF FN+EGF Pyk2 p13 Cs +/+ +/+ HA Pyk2 Supplementry Figure 1 Comprisons of the expression levels of, Pyk2, EGF reeptor (EGFR), PDGF reeptor (PDGFR) nd p13 Cs in +/+, nd ells. All ells re p53. FN+EGF Equl expression of EGFR nd PDGFR in nd ells. In ells, the level of expression of hemgglutinin (HA-tgged) ws lower thn in ells, wheres expression of Pyk2 ws higher in ells reltive to nd ells (Fig. S1). Equl expression levels of the PDGFR or EGFR were deteted in lystes of nd ells (Fig. S1). Equl mounts of p13cs were deteted in +/+, nd ells. ut not Pyk2 is reruited to sites of integrin nd EGFR lustering. To determine whether the speifi reruitment of to sites of integrin or EGFR lustering ould e deteted in intt ells, we used miroeds oted with BSA, fironetin, or omintion of fironetin nd EGF s stimuli (Fig. S2). We used indiret immunofluoresene to oserve the intrellulr distriution of in +/+ nd ells, nd Pyk2 in ells. In +/+ nd ells, loliztion is normlly restrited to fol-ontt regions t the sl surfe, nd this distriution did not hnge upon inding of ontrol BSA-oted eds to ells (Fig. S2). Diffuse stining ppered round fironetin-oted eds tthed to the pil surfe of +/+ ells, wheres streks of stining were oserved round the tthment sites of EGF-oted eds (Fig. S2). Stining with ntiodies to nd HA, in +/+ nd ells respetively, showed redistriution of round eds oted with fironetin nd EGF (Fig. S2). In ontrst, inding of eds oted with fironetin nd EGF to ells did not promote detetle redistriution of Pyk2 from its norml perinuler distriution (Fig. S2). These results show tht in intt ells, n e reruited to sites of integrin nd EGFR tivtion, nd tht it my hve funtion s reeptor-proximl omponent of oth integrin nd EGFR signlling pthwys. Methods Bed oting. M-5 Dyneds (Dynl In., Oslo, Norwy) were oted in.2 M orte uffer, ph 8.5, ontining 1.25 g BSA, fironetin or EGF (Peproteh, Roky Hill, NJ), or 1.25 g EGF nd.625 g fironetin. Beds were oted in rotting silionized tues for 24 h t room temperture. Remining tosyl-groups were loked y rotting for 4 h t 37 C with.1% BSA nd.2 M Tris, ph 8.5. Immunofluoresene. Coverslips were fixed 2 min fter the ddition of eds nd treted s desried 15. ws oserved using oktil of rit polylonl nti- ntiodies, N-17 nd C-2 (Snt Cruz). Pyk2 ws oserved using oktil of rit polylonl ntiodies, no. 596 ffinity-purified 14 nd Pyk2 N-19 (Snt Cruz). HA-tgged proteins were oserved using rit polylonl ntiody Y-11 (Snt Cruz). FN+EGF Supplementry Figure 2 In vivo reruitment of to pil tthment sites of eds oted with fironetin (FN), EGF or fironetin nd EGF (FN+EGF) in nd +/+ firolsts. Pyk2 does not o-lolize to sites of tthment to FN+EGFoted eds in firolsts. Bed size is 5 µm; ntiody stining (upper left), nd ells used (lower right) re indited in eh pnel. 2 Mmilln Mgzines Ltd