Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3domain

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1 (2005) 24, & 2005 Nture Pulishing Group All rights reserved /05 $ Trnsctivtion of Al y the Crk II dpter protein requires PNAY sequence in the Crk C-terminl SH3domin Chrles Reichmn 1, Kmlendr Singh 1, Yn Liu 1, Sukhwinder Singh 1, Hong Li 1, J Edurdo Fjrdo 2, Andrs Fiser 2 nd Rymond B Birge*,1 1 Deprtment of Biochemistry nd Moleculr Biology, UMDNJ-New Jersey Medicl School, 185 South Ornge Avenue, Newrk, NJ 07103, USA; 2 Deprtment of Biochemistry, Alert Einstein College of Medicine, Bronx, NY 10461, USA To gin etter understnding of how Crk II regultes the function of the Al tyrosine kinse, we explored the function of the C-terminl linker nd SH3domin, region of Crk II tht is still poorly understood. Moleculr modeling, tryptophn fluorescence, nd covrition sequence lignment indicte tht the Crk-SH3-C hs unique inding groove nd RT loop not oserved in typicl SH3domins. Bsed on these models, we mde series of muttions in the linker nd in residues predicted to destilize the puttive inding pocket nd RT loop. In Al trnsctivtion ssys, Y222F nd P225A muttions in the linker resulted in strong trnsctivtion of Al y Crk II. However, muttions predicted to e t the surfce of the Crk SH3-C were not ctivtors of Al. Interestingly, comintions of ctivting muttions of Crk II with muttions in the highly conserved PNAY sequence in the SH3-C inctivted the ctivting muttions, suggesting tht the SH3-C is necessry for ctivtion. Our dt provide insight into the role of highly conserved residues in the Crk-SH3-C, suggesting mechnism for how the linker nd the Crk-SH3-C function in the trnsctivtion of the Al tyrosine kinse. (2005) 24, doi: /sj.onc ; pulished online 12 Septemer 2005 Keywords: Crk; Al; SH3 domin; typicl; covrition; protein modeling; kinse ctivtion Introduction The Crk II protein is n SH2 SH3 domin contining dpter protein involved in cytoskeletl reorgniztion nd events ssocited with cellulr dhesion, migrtion, phgocytosis, cellulr prolifertion, nd oncogenic trnsformtion (Feller, 2001). Crk II, the cellulr homolog of the v-crk oncoprotein, is expressed from two lterntivelyspliced mrnas to produce Crk II (p38) nd Crk I (p28). Crk II nd Crk I differ in their C- terminl regions (Mtsud et l., 1992; Reichmn et l., 1992; Feller, 2001). Crk II contins n N-terminl SH2 domin nd two SH3 domins (SH3-N nd SH3-C). *Correspondence: RB Birge; E-mil: irger@umdnj.edu Received 2 Mrch 2005; revised 23 June 2005; ccepted 24 June 2005; pulished online 12 Septemer 2005 Crk II lso contins n pproximtely45 mino-cid linker etween the two SH3 domins. In contrst, v-crk nd Crk I encode proteins with C-terminl trunctions tht do not possess regultorytyrosine residue 222 (Y222) etween the SH3 domins, or the C-terminl SH3 domin (Myer et l., 1988; Reichmn et l., 1992). Expression of v-crk or Crk I in cells roustlyincreses cellulr phosphotyrosine levels, nd trnsforms cells, despite the fct tht neither protein hs intrinsic tyrosine kinse ctivity. While severl cellulr proteins hve een identified tht ind to the SH2 nd SH3-N domin, Al nd its prlog Arg re the onlytyrosine kinses tht re known to directlyssocite with v-crk or Crk II (Feller et l., 1994; Ren et l., 1994; Wng et l., 1996). Binding of v-crk or Crk I to Al results in Al trnsctivtion, nd possilycontriutes to the trnsforming potentil of Al (Ren et l., 1994; Sttler nd Slgi, 1998; Hemmeryckx et l., 2002). In contrst, the Crk II Al interction is more trnsient, nd y signl tht is not well understood, Al-ound Crk ecomes phosphorylted on Y222 ythe Al kinse, resulting in the intrmoleculr ssocition of Y222 with the Crk SH2 domin, nd dissocition of Crk from Al (Feller et l., 1994; Rosen et l., 1995). Expression of Y222F Crk II enhnces Al trnsctivtion, increses cellulr phosphotyrosine, nd promotes cellulr trnsformtion (Shishido et l., 2001; Zvr et l., 2001). Presently, the moleculr mechnism y which Crk II ctivtes Al is not well understood, lthough previous studies suggest tht the SH2 nd SH3-C domins re necessry(shishido et l., 2001). The present studyws crried out to investigte the structurl nd functionl chrcteristics of the Crk-SH3-C. We present dt tht the linker nd the Crk-SH3-C, through conserved residues in the modeled inding pocket nd RT loop of the SH3-C, plyimportnt roles in the trnsctivtion of the Al tyrosine kinse. Results nd discussion Threding nlysis, computtionl moleculr modeling, nd iophysicl studies of the C-terminl Crk SH3 domin (Crk SH3-C) Previous efforts to identifyinding prtners of the Crk- SH3-C domin filed to detect n interction with

2 8188 cnonicl PxxPxK,R contining motifs. Feller et l. (1994) previouslyreported tht the Crk-SH3-N, ut not the Crk-SH3-C, ound Al. It hs lso een demonstrted tht the Crk-SH3-N cn interct with PXXPXK motifs from C3G, wheres Crk-SH3-C cnnot (Knudsen et l., 1994). Using 35 S peptide overlyssy(feller et l., 1995), Crk-SH3-N ws shown to ind to numer of cellulr proteins, lthough no inding prtners of Crk-SH3-C were detected. We lso filed to detect Crk- SH3-C inding to PPPALPPKK peptide ytryptophn fluorescence (dt not shown). It hs een reported, however, tht the Crk-SH3-C cn ind to the nucler export protein Crm1 (Smith et l., 2002). To define the structurl elements of the Crk-SH3-C nd scertin whether its structure conforms to tht of cnonicl SH3 domins, we utilized unised threding nlysis, sequence lignment, templte structure-sed modeling, nd tryptophn fluorescence spectroscopy on purified recominnt proteins in the present pper. We employed n unised threding nlysis to determine structures in the dtses tht most closelymtch the structure of the Crk-SH3-C ( ). Threding nlysis (using Fugue nd 3D-PSSM dtses) identified severl structures, ll SH3 domins (Figure 1), nd 10 models were uilt on ech selected structure. The est model produced, sed on the evlutions performed with Pros, ws one sed on the crystl structure of the SH3-C domin of humn Gr2 (Lowenstein et l., 1992), n SH2/SH3 domin contining dpter protein. High-qulitymodels were lso produced from the SH3 domins of Cenorhditis elegns Sem-5 (Rozkis- Adcock et l., 1992), nd of the mouse Gr2-like protein Mon/Gds (Lewitzky et l., 2004). All re typicl SH3 domins tht ind PXXP-independent motifs. Crk-SH3- C lso hd structurl similrities to p47phox, the Vv proto-oncogene SH3-N, nd Amphiphysin II (Figure 1). Bsed on the threding, the est templte we found ws Gr2-SH3-C (1gri). Therefore, we uilt the homologymodel using Gr2-SH3-C s templte. However, the homology-sed model using the N- terminl SH3 domin of Crk superimposed on the one uilt with Gr-2 SH3-C hd n RMS devition of 1.73 A. This suggests tht models uilt on either templte could e used for comprison purposes to Crk SH3-C, nd for further nlysis. We wnted to explore how the SH3-C might differ in its structure nd peptide-inding ctivityfrom those of cnonicl SH3 domins. Since the inding pocket of the Crk-SH3-N is well estlished to ind conventionl PXXP peptide-contining proteins, the miniml energy moleculr modeling progrm Look (version 3.5) ws employed to model the structure of the SH3-C using the crystl structure of mouse Crk SH3-N ound to n intercting peptide s the templte. The SH3-N intercting peptide is PPPALPPKK, from the Crk intercting protein C3G (Wu et l., 1995). The predicted structure of Crk SH3-C is consistent with conventionl SH3 domins, comprising five ntiprllel sheets, nd two flexile vrile loops clled the RT loop nd 3 10 turn etween the fourth nd fifth sheets tht flnk PDB Chin Gene Orgnism Codes 1h3h A GRB2-RELATED ADAPTOR PROTEIN 2 Mus (GADS, GRBLG, GRB2L); C-TERMINAL SH3 1sem A SEM-5; (GRB2, DRK); C-TERMINAL SH3 C. Elegns 1udl A INTERSECTIN 2; (KIAA1256); SH3 DOMAIN; Humn 1gri A GROWTH FACTOR BOUND PROTEIN 2; Humn (GRB2); SH3 DOMAIN 1ng2 A NEUTROPHIL CYTOSOLIC FACTOR 1;(P47-PHOX); Humn SH3 DOMAIN; 1gcq A VAV PROTO-ONCOGENE; Humn N-TERMINAL SH3 DOMAIN 1mv3 A MYC BOX DEPENDENT INTERACTING PROTEIN 1; Humn (BIN1; AMPHIPHYSIN II) Consensus Crk(N)SH3 Crk(C)SH3 Ced-2(C)SH3 gr2(c)sh3 GKYVRALYDYEAREDDE--LSFKKGDIITVLEKSDDGWWKGRLNDTGREGLFPSNYVEEIDS PPKAVVIF FDPQNPGD--ITLRE EVLEIINREEGD YEAENLRD QR WI A FLRPVE E K GEDEE R KV LVSDDNEE LRVK S KS YV KLL VEYVRALFDFNGNDDED--LPFKKGDILKIRDKPEEQWWNAEDMD-GKRGMIPVPYVEK FYARVIQKRVPNAYDKTALALEVGELVKVTKINMSGQWEGECN--GKRGHFPFTHVRLLD AKAKVTFDRVPNAYDPTQLRVKKGQTVLVTQKMSNGMYKAELD--GQIGSVPHTYLRFT QPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACH--GQTGMFPRNYVTPVNRNV Strnd RT-Src-loop Strnd Strnd c Strnd d Strnd e Figure 1 Sequence lignment nd threding nlysis. () Structures identified ythreding nlysis s suitle templtes for modeling the Crk-SH3-C domin. The PDB code, chin, rief description of the chin, nd the source orgnism re provided. () The SH3 consensus sequence s determined ythe covrition nlysis of Lrson nd Dvidson (2000) (op cit) ws ligned with the Crk SH3-N, Crk SH3-C nd humn Gr2 SH3-C using Clustl W softwre; lue ¼ hydrophoic core residues; red ¼ inding to PXXPXK/R. Note tht in Crk-SH3C nd Ced-SH3C, the cnonicl peptide-inding mino cids re poorlyconserved. Underlined residues ¼ short, identicl region in Ced-2 nd Crk SH3-C

3 possile inding pocket (Figure 2). Superimposition of the modeled N- nd C-terminl Crk SH3 domins, while indicting sutle differences etween the RT loop nd c nd d loops, nevertheless suggests n overll conservtion of structure etween the two SH3 domins (Figure 2). Moreover, while only17% identicl to the mmmlin proteins, the model of the Ced-2 SH3-C lso hd the sic -rrel structure chrcteristic of ll SH3 domins, when modeled using the SH3-N of Crk II s the templte. It myfold in order to generte modulr domin (dt not shown). Covrition nlysis of protein sequences involves the studyof sttisticl vritions in mino-cid sequences in homologous proteins or protein domins, nd nlyzes whether chnge in n mino cid t one position correltes with chnge t nother site in the protein. If such covrying sites re found, then it is hypothesized tht these two sites likelyinterct in some mnner, for exmple ycontriuting to the overll tertirystructure, or to complex inding region. Using covrition nlysis of 266 SH3 domins, consensus sequences for c d e f L-SH3 SH3 RT-loop Y222 SH2 SH3 [N] SH3 [C] SH3[N] SH3[C] K 190 CRP F 239 YAR THVR 293 c d e f Emx 332 nm 340 nm 340 nm 340 nm 340 nm 358 nm Figure 2 Bsic SH3-C model nd tryptophn fluorescence on recominnt proteins. () Rion structure of the Crk SH3-N (gry), superimposed on the modeled structure of the Crk SH3-C (drk gry). The RT loop is seen on the upper left. () Tryptophn fluorescence nlysis of the SH3-C domin contining proteins. A liner mp of Crk II depicting the linker region ( ) nd SH3-C ( ), nd the oundries of linkersh3-c peptides, () ; () ; (c) ; (d) ; (e) (SH3-C domin), nd (f) (DSH3-C) re shown. At right in this pnel re the normlized fluorescences of peptides f; t lower left is Coomssie lue stined gel of the peptides, with size mrkers t left nd SH3-C nd Linker-SH3-C (L-SH3) t right. All proteins were nlysed y mss spectrometry to verifymss SH3 domins hve een derived. Two types of mino cids hve een identified in the consensus sequence, those conserved for folding into the SH3 hydrophoic core, nd those typiclly present t the surfce nd conserved for interction with PXXP contining peptides (Lrson nd Dvidson, 2000; Lrson et l., 2000). We compred the SH3 domins of Crk II, Ced-2, nd the Gr2 SH3-C to the SH3 consensus sequence derived ylrson et l. (2000). As illustrted in Figure 1, Crk SH3-C nd Ced-2-SH3-C re remrklyconserved in the hydrophoic core residues tht mintin the overll tertirystructure of SH3s (10/10 hydrophoic core mino cids re either identicl or conserved in Crk II nd 9/10 in Ced-2). In contrst, Crk SH3-C nd Ced-2 SH3-C hd generl lck of conservtion in the peptideinding surfce, whereyonlythree of 16 mino cids were conserved in Crk II nd five of 16 in Ced-2. Even Gr2-SH3-C, which hd the most similr structure to Crk II s evidenced ythe threding nlysis, hd more conservtion in the peptide-inding mino cids, compred to Crk-SH3-C (Figure 1). We performed tryptophn mesurements on recominnt Crk SH3-C domin proteins. While most SH3 domins contin contiguous tryptophn duet (WW) in the c strnd, the Crk SH3-C domin contins single tryptophn (W276). This fct led us to test whether this Trp is unconstrined (unfolded, E mx B nm) or constrined (E mx B nm). As shown in Figure 2, Trp fluorescence mesurements of the fulllength SH3 domin ( ) or the SH3 domin contining prts of the linker-c-terminl SH3 domin hd E mx vlues of B nm, consistent with folded polypeptide structure. Another N-terminl truncted linker-sh3c protein ( ), nd n SH3-C protein comprising , lso hd similr E mx (dt not shown). Finlly, we generted n N-terminl truncted SH3-C domin (DFYARVIQ) tht deletes the first seven mino cids in the puttive 1 SH3 strnd (Crk DSH3-C). This genertes protein with E mx of B358 nm, consistent with n unfolded, dentured structure (Figure 2). These dt do not contrdict the model of the SH3-C. The vst mjorityof SH3 domins recognize specific proline-rich sequences tht dopt the conformtion of polyproline type I or type II helices (PPII), contining the core sequence PXXP (Ky et l., 2000). In typicl SH3 domins, the PXXP core motif is stilized y stcking of the romtic residues, Trp, Phe, nd Tyr, which interct directlywith the pyrrolidine rings of the PXXP. Additionl inding specificityoccurs outside of this core interction nd confers specificitymong distinct SH3 domins. Recently, numer of SH3 domins hve een reported to ind unconventionl non-pxxp-dependent motifs in trget proteins. Such exmples include n RKXXYXXY motif in SKAP55 tht inds the Fyn nd Lck SH3 domins (Kng et l., 2000), PXXDY motif in severl proteins tht inds the Eps8 SH3 (Mongiovi et l., 1999), WXXXFXXLE motif in p67phox nd Pex5p tht inds the Pex13p SH3 domin (Brnett et l., 2000; Kmi et l., 2002), PX(V/ I)(D/N)RXXKP motif in G1 nd SLP-76 proteins 8189

4 8190 tht ind the Mon/Gds SH3 domin (Lewitzky et l., 2001; Hrkiolki et l., 2003; Lewitzky et l., 2004), nd PX(P/A)XXR motif tht inds to the CIN85/SETA/ Rut protein SH3 domins (Kurkin nd Bredesen, 2002; Kurkin et l., 2003). For some typicl SH3 domins, for exmple Mon/Gd, SKAP55, nd Eps8, the unconventionl peptides hve een suggested to ind to regions including the clssicl PXXP inding site (Hrkiolki et l., 2003). However, studies with Pex13p show tht it cn ind oth conventionl PXXP-contining peptides in the usul wy, s well s second, non- PXXP peptide, tht inds t novel, noncnonicl pocket on the sme SH3. Both peptides cn ind to the Pex13p SH3 simultneously(pires et l., 2003). Therefore, it would not e unprecedented to find n SH3 whose overll structure is similr to tht of most other SH3s, ut whose inding surfce is verydifferent, s we hve presented evidence here concerning the SH3-C of Crk II. The covrition nlysis suggests tht the inding residues of the SH3-C mye verydifferent from those of cnonicl SH3s. As we show elow, when the modeled SH3-C is superimposed on the templte SH3- N nd compred in detil, the potentil peptide-inding residues of the SH3-C lso look verydifferent. For these resons, we elieve it is prole tht the SH3-C of Crk II will e shown to e typicl in the peptides it cn ind, nd will likelynot ind conventionl PXXP peptides. In order to predict which residues might occupythe inding surfce of the SH3-C, nd for lter mutgenesis studies, we compred in detil the residues in the SH3-N peptide tht mde direct contct with P3, P6, nd K8 positions of the PPPALPPKK peptide to the corresponding sustituted mino cids in the SH3-C. The peptide residues t the P3, P6, nd K8 positions of the intercting peptide re the most importnt ecuse they conform to the PXXPXK consensus sequence. There re totl of 12 residues in the Crk SH3-N domin within potentil intercting distnce (3.8 A ) of the C3G peptide (Figure 3). These residues re L140, F141, Y186, P185, P183, Q168, W169, E167, E166, D147, E149, nd D150. The topologicllyequivlent residues in the modeled C-terminl Crk SH3 domin re K246, R247, H291, T290, P288, G274, Q275, S273, M272, D253, T255, nd A256. Some of these re illustrted in Figure 3. If the region in the SH3-C model nlogous to the inding groove in the SH3-N lso inds peptide, the comprison of the properties of the inding site residues in the two domins suggests tht the nture of the peptides tht ind the two proteins re proly dissimilr. Interestingly, severl of the hydrophoic residues in the N-terminl SH3 domin tht mke direct contct with the pyrrolidine rings of the PXXP motif re replced with sic or polr mino cids in the model of the SH3-C. For exmple, F141 is replced y K246 (Figure 3), Y186 is replced yh291 (Figure 3), nd W169 is replced yq275 (Figure 3). Another mjor difference is the chrcteristics of the inding site for intercting residues with K8 nd K9 of the peptide. The two lysine residues of the C3G peptide form extensive slt ridges with negtivelychrged residues of the Crk SH3-N (Figure 3c; Wu et l., 1995). K8 forms three ion pirs with D147, E149, nd D150, nd only one of the residues (equivlent to D147) is conserved in the SH3-C. The other two residues re replced y Thr (T255) nd Al (A256). Similrly, the possiility of the slt ridge etween K9 nd E167 of Crk SH3-N lso does not exist in Crk SH3-C, ecuse the equivlent position to E167 of the SH3-N is occupied y Ser residue (not shown). These dissimilrities clerlyestlish the differences in the nture of recognized nd ound peptides ythe SH3-N versus the modeled structure of the SH3-C, nd suggest tht the Crk SH3- C does not ind conventionl PXXP-contining lignds. In Figure 3d, we show spce-filling model of the SH3-C, together with peptide in the plce nlogous to the peptide inding groove of the SH3-N. Ech of the similr regions tht interct with P3, P6, nd K8 in the SH3-N re highlighted in lue, green, nd yellow, respectively. Note tht potentil inding groove still exists where the peptide is positioned, lthough s detiled ove, importnt specific interctions for PXXPXK peptides re not present in the SH3-C model. Finlly, we note tht portion of the RT loop of SH3-C hs residues tht point wyfrom the SH3-C domin (red, Figure 3d), nd ecuse of this might e more ville to interct with other molecules. These include R247 to T255. In studyof the structures of 17 SH3 domins, investigtors noted the surprising presence of stilizing interctions mong residues of the RT-Src loop tht re not directlyinvolved in peptide inding. Theyspeculted tht these conserved interctions might stilize the RT loop s whole for interctions with other peptides (Lrson nd Dvidson, 2000). The C positions of the SH3-C residues R247, V248, P249, A251, Y252, D253, nd T255, nd the corresponding residues in Ced-2 (oriented similrly) re highly conserved in wide rnge of species of Crk nd CrkL, including chicken, mmmls, Xenopus levis, Drosophil melnogster, nd C. elegns (Tle 1). Lter, we detil how we used muttionl nlysis of residues in ech of the four locks highlighted in Figure 3d to understnd the functions of the SH3-C. We did this yinvestigting how theyffect the ctivtion of the Al tyrosine kinse ycrk II. Contriutions of the linker ( ) nd the Crk- SH3-C to regulting the interction etween Al nd Crk Previous studies hve shown tht Crk inding to Al hs trnsctivting function, nd therefore we cn score the extent of Al ctivtion when coexpressed with different Crk mutnts (Shishido et l., 2001; Zvr et l., 2001). To explore the functions of the SH3 linker nd the proposed surfce of the Crk SH3-C in further detil, we generted three types of mutnts in the C-terminl region of Crk, nd coexpressed these with Al to see how theyffected Al inding nd Al trnsctivtion. These mutnts include sustitutions of (i) individul proline residues of the linker, (ii) residues predicted to e on the inding surfce of the Crk SH3-C, nd

5 8191 PPPALPPKKK Peptide Q275 PPPALPPKKK peptide H291 P6 W169 P3 Y186 F141 P3 interctions: crksh3(n) Y186 P185 F141 crksh3(c) H291 T290 K246 P6 interctions: crksh3(n) W169 P183 P185 crksh3(c) Q275 P288 T290 c PPPALPPKKK Peptide D253 D150 D147 K A E149 A256 d RT-loop K8 P A P3 K8 interctions: crksh3(n) D147 E149 D150 crksh3(c) D253 T255 A256 Figure 3 Peptide/SH3 interction detils. () Detils of the interctions of the P3 residue of the PPPALPPKK peptide with the SH3-N (gry), superimposed on the corresponding residues of the SH3-C (purple). Side chins of SH3-N (gry, red for oxygen) nd side chins of SH3-C (purple) re shown. () Detils of the interctions of the P6 residue of the PPPALPPKK peptide with the SH3-N (gry), superimposed on the corresponding residues of the SH3-C (purple). Side chins of SH3-N (gry, red for oxygen) nd side chins of SH3-C (purple) re shown. (c) Detils of the interctions of the K8 residue of the PPPALPPKK peptide with the SH3-N (gry), superimposed on the corresponding residues of the SH3-C (purple). Side chins of SH3-N (gry, red for oxygen), side chins of SH3-C (purple), nd intermoleculr distnces etween SH3-N residues nd K8 re shown. (d) Spce filling model of the SH3-C with the PPPALPPKK peptide shown in the similr loction to its position when inding to the SH3-N. The region tht intercts with P3 of the peptide (lue), P6 (green), nd K8 (yellow) re highlighted. The prt of the RT loop tht does not interct with PPPALPPKK nd projects wyfrom the SH3 is in red. This is the most conserved region in the lignment of Crk II nd Ced-2 (iii) residues tht comprise the puttive RT loop of the Crk-SH3-C. Prolines hve mnyroles in intercting with dpter molecules, nd cn thereystronglyffect the regultion of these nd other signling molecules, including kinses. The est known exmples of proline-inding ctivityre the cnonicl PXXP peptides tht interct with most SH3 domins, nd short sequences like the P 221 YAQP 225 in the Crk linker which, when phosphorylted on tyrosine, ind to SH2 domins. Proline residues in linker/connector regions hve lso een shown to e inhiitors in cis of some kinses (Mcis et l., 2002). For exmple, in Hck, the SH3 folds over nd intercts intrmoleculrlywith smll proline-contining region, locking the kinse domin (Sicheri et l., 1997). Finlly, in p47phox, n SH3 domin is inhiited from inding to intercting proteins yn intrmoleculr interction with proline-contining peptide, providing precedent for the regultion of SH3 domin inding ysuch intrmoleculr interctions (Ago et l., 1999). Shown in Figure 4A is the Clustl W lignment of the linker regions of chicken, rt, nd Xenopus Crk II proteins, s well s murine CrkL. The lignment of prolines is highlighted; mnyof these re present s PXP sequences. As the linker region of Crk II contins 10 prolines out of 49 mino cids, we decided to mutte ech one individullyto lnine, nd test the ilityof these mutnts to ctivte Al kinse ctivity. The mutnts generted were P193A, P211A, P213A, P217A, P219A, P221A, P225A, P230A, nd P238A

6 8192 Tle 1 Txonomic lignment of the Crk-SH3-C nd CrkL SH3-C domins CLUSTAL W (1.74) multiple sequence lignment mus_crkii hum rt gllus Xenopus Dnio Drosophil Ced-2 hum_crkl DnioRerio_CrkL PIYARVIQKRVPNAYDKTALALEVGELVKVTKINVSGQWEGECNGKRGHFPFTHVRLLDQ PIYARVIQKRVPNAYDKTALALEVGELVKVTKINVSGQWEGGCNGKRGHFPFTHVRLLDQ PIYARVIQKRVPNAYDKTALALEVGELVKVTKINVSGQWEGECNGKRGHFPFTHVRLLDQ PFYARVIQKRVPNAYDKTALALEVGELVKVTKINMSGQWEGECNGKRGHFPFTHVRLLDQ PIFARVIQKRVPNAYDKTALALEVGDLVKVTKINVSGQWEGECNGKYGHFPFTHVRLLEQ PVYARAIQKRVPNAYDKTALALEVGDMVKVTKINVNGQWEGECKGKHGHFPFTHVRLLDQ PAYARVKQSRVPNAYDKTALKLEIGDIIKVTKTNINGQWEGELNGKNGHFPFTHVEFVDD PAKAKVTFDRVPNAYDPTQLRVKKGQTVLVTQKMSNGMYKAELDGQIGSVPHTYLRFTAV PVFAKAIQKRVPCAYDKTALALEVGDIVKVTRMNINGQWEGEVNGRKGLFPFTHVKIFDP PVLAKAIQKRVPCAYDKTALALEVGDIVKVTRMNISGQWEGEVNNRRGLFPFTHVKILDP * *:..*** *** * * :: *: : **:.* ::...: *.*.*::.: Using the Clustl W 1.74 progrm t the EMBnet Swiss site, we ligned the SH3-C of Crk II nd CrkL from severl species, nd found tht the most conserved string of residues in Crk II ws in the RT loop. This ws lso true when the entire Crk II lignments were exmined (not shown). Shown re the SH3-C domins from the Crk II of murine (Ogw et l. 1994), humn (Mtsud et l. 1992), rt (Kizk-Kondoh et l. 1996), Gllus gllus (Reichmn et l. 1992), X. levis (Evns et l. 1997), D. rerio (IMAGE clone AAH77088; (Lennon et l. 1996), nd D. melnogster (Gllett et l. 1999); the SH3-C from ced-2 of C. elegns (Reddien nd Horvitz, 2000), nd CrkL from humn (ten Hoeve et l. 1993) nd crkl D. rerio (IMAGE clone AAH56763; (Lennon et l. 1996). The PNAY sequence studied in this pper (nd the ligned PCAY of crkl) is shown in gry. * indictes identicl or equivlent residues in ll sequences in the lignment, : indictes conserved sustitutions,. indictes semi-conserved sustitutions A Chicken Rt Xenopus MousecrkL KCRPSSASVSTLTGGNQDSSHPQPLGGPEPG-PYAQPSINTPLPNLQNGPFYARVIQ KYRPASASVSALIGGNQEGSHPQPLGGPEPG-PYAQPSVNTPLPNLQNGPIYARVIQ KYRPPSSPGSALIGGNQENSHPQPLGGPEPG-PYAQPSVNTPLPNLQNGPIFARVIQ GNRNSNSYG.. IPEPAHAYAQPQTTTPLPTVASTPGAAINPLPSTQNGPVFAKAIQ B 66 no DNA Crkwt Crk Crk Y222F Crk W170K Crk W276K Crk P193A Crk P211A Crk P213A Crk P217A Crk P219A Crk P221A Crk P225A Crk P230A Crk P238A Kinse Assy GST-Crk Densitometry (1.0) (1.7) (0.2) (1.8) (1.1) (0.7) (08) (0.6) (1.2) (1.8) (5.3) (1.9) (1.1) + Al no DNA Crkwt Crk Crk Y222F Crk W170K Crk W276K Crk P193A Crk P211A Crk P213A Crk P217A Crk P219A Crk P221A Crk P225A Crk P230A (0.1) (0.1) Crk P238A c Al --Al d Crk --Crk II Figure 4 Proline to lnine muttions in the Crk linker. (A) Alignment of the Crk II linker from three species, together with the CrkL protein from murine. This shows the strong conservtion in the linker region, including the Tyr phosphorylted y Al, nd the YXXP site which inds SH2. The Pro mutted to Al is leled ove. (B) () nd (), in vitro kinse ssyfrom 293T cells coexpressing Al nd Crk II constructs mutted t ech of the sites shown. Kinse ssys were seprted y 10% SDS PAGE nd [ 32 P] leled nds were detected y utordiogrphy. Shown t the left re sizes of protein mrkers, nd t the right the sustrte used in the kinse ssy. Quntittion ws on phosphoimger (normlized to the Crk II wild-type smple). (c) nd (d) Western lots of Al nd Crk expression. Equl mounts of protein from cell lystes were detected y Western lotting to confirm expression. Al DNA ws trnsfected in ll smples except no DNA

7 Crk R38K (Figure 4B). Wild-type Crk or individul Pro to Al mutnts were coexpressed with Al in HEK 293T cells. Susequently, detergent lystes were prepred nd immunoprecipitted with nti-crk RF51 A ( Crk SH2-domin specific ntiody), nd ssyed for ssocited Al kinse ctivity(figure 4B, ). For positive controls, we used Y222F, which muttes the Al phosphoryltion site, nd W276K Crk, n SH3-C destilizing mutnt tht we hve previouslyshown to increse the ssocition etween Al nd Crk (Zvr et l., 2001). As shown in Figure 4B,, most of the P to A mutnts did not significntlyincrese Crkssocited Al kinse ctivity, lthough consistently, P217A nd P219A slightlyreduced the ssocition of Al nd Crk s indicted ythe kinse pull-down ssy. However, muttions in the P225 of the P 221 YAQP 225 sequence stronglyincresed the trnsctivtion of Al, s well s incresing cellulr tyrosine phosphoryltion (Figure 5; Figure 7). Since P225 nd Y222 re oth prt of the pyaqp motif tht intercts with the Crk- SH2 domin, theylikelyhve similr function in preventing SH2 docking to the Y222 when tyrosine phosphorylted. These dt re consistent with the finding tht the miniml SH2 recognition sequence of phospho-y222 includes the P225, s hs een reported for the Crk SH2 domin (Songyng et l., 1993). Coexpression of Al in the sence of Crk (lne 2, Figure 4A), or with W170K Crk (lne 5), n N-terminl SH3 mutnt tht rogtes Al inding, produced no detectle Crk-ssocited Al ctivity, or Al protein (Figure 4B, ; Figure 5d; Figure 6c). This indictes tht the ssyconditions specificllymesure Al ctivityssocited with the specified Crk mutnts. Next, we studied mutnts expected to disrupt the predicted inding groove of Crk SH3-C. Since the nturl lignd of the Crk SH3-C is not known, we 8193 no Crk Crk II Crk I Crk W170K Crk Y222F Crk P225A Crk K246A Crk R247A Crk K,R246,7 A,A Crk A256D Crk Q275W Crk W276K p-tyr Al --Crk II Al --Al c Crk --Crk II d Kinse Assy --GST-Crk e Densitometry (0.2) (1) (1.9) (1.1) (0.4) (2.2) (4.4) (1.6) (1.2) (1.2) (3.1) (4.7) (3.5) f Crk ip/al lot --Al g Crk ip/crk lot --Crk II + Al Figure 5 In vitro kinse ssynd Western lots of Al þ Crk mutnts. () 293T cells were cotrnsfected with DNAs encoding Al plus Crk mutnts s indicted. Detergent lystes were prepred 48 h lter, nd centrifuged to remove insolule mterils. Equl mounts of lystes were loded on n SDS PAGE gel nd Western lotted with nti-p-tyr ntiody. () nd (c) Western lots of lystes using the indicted ntiodies. (d) Lystes were immunoprecipitted y nti-crk ntiodies, wshed, nd used for in vitro kinse ssys in the presence of [g- 32 P]ATP nd GST-Crk ( ) s exogenous sustrte. Kinse ssys were run on SDS PAGE gels nd quntitted on phosphoimger. (e) Densitometryof kinse ssyove (normlized to the Crk II wild-type smple). (f) nd (g) Kinses ssys were Western lotted with the indicted ntiodies

8 8194 Crk W276K Crk P225A/W276K Crk Y222F/W276K Crk Q275A Crk A256K CrkP225A/ PNAY CrkY222F/ PNAY Crk PNAY Crk Y252A Crk P249A Crk P225A Crk Y222F Crk W170K Crk II Crk - Kinse -GST-Crk Densitometry (3.2) (5.3) (2.9) (2.3) (3.9) (1.3) (1.7) (0.6) (1.5) (1.6) (5.5) (5.4) (0.3) (1) (0.2) c d Crk ip/al lot Crk ip/crk lot -96 e Al py f Al py g Al Al Figure 6 In vitro kinse ssynd Western lots of Al þ Crk mutnts using phospho-specific ntiodies. () 293T cells were cotrnsfected with DNAs encoding Al plus Crk mutnts, s indicted. Detergent lystes were prepred 48 h lter nd centrifuged to remove insolule mterils. Lystes were immunoprecipitted y nti-crk ntiodies, wshed, nd used for in vitro kinse ssys in the presence of [g- 32 P]ATP nd GST-Crk ( ) s sustrte. Kinse ssys were run on SDS PAGE gels nd quntitted on phosphoimger. () Densitometryof kinse ssyove (normlized to the Crk II wild-type smple). (c) nd (d) Replicte smples were immunoprecipitted nd Western lotted with the indicted ntiodies (e), (f) nd (g) re Western lots of lystes using the indicted ntiodies mutted the corresponding mino cids tht most likely replced residues tht intercted directlywith the PPPALPPKK peptide in the SH3-N. These muttions include K246A, R247A, K246A/R247A, nd H291A (P3-equivlent position mino cids), Q275W (P6- equivlent positions), nd A256D (K8-equivlent position) (Figure 3). Susequently, we mde the dditionl muttions A256K nd Q275A in order to mutte these residues wyfrom the cnonicl SH3 residues typiclly found t these positions (in the cse of A256, mny SH3s hve D t the equivlent position, which intercts with K or R in the K/RXXPXXP or PXXPXK/R intercting peptide; in the cse of Q275, the equivlent position is usullyoccupied yw, s in SH3-N). As ove, CrkSH3-C mutnts were coexpressed with Al for coimmunoprecipittion nd trnsctivtion nlysis (Figure 5). There ws moderte increse in Al kinse ctivitywhen Crk II ws coexpressed with Al, compred to overexpression of Al lone. However, the strongest ctivtors of Al were Crk I, Y222F, nd P225A (Figure 5). Compred to expression of Al with wild-type Crk II, there ws essentillyno chnge in cellulr phosphotyrosine levels with the Crk II SH3-C mutnts K246A, R247A, K246A/R247A, A256D, Q275W, nd W276K. Thus, muttions t residues modeled to e t the SH3- C surfce did not led to upregultion of Al kinse ctivity in vivo compred to wild-type Crk II. However, when we exmined the in vitro kinse ctivityof Crk immunoprecipittes, we found incresed Al kinse ctivitynot onlyin Crk I, Y222F nd P225A, ut lso in some of the SH3-C surfce muttions, A256D, Q275W, nd W276K (Figure 5d, e); the Crk II smple in lne 2 is normlized to 1). Our results with A256K nd Q275A were similr to those with A256D nd Q275W; neither incresed in vivo cellulr phosphotyrosine (Figure 7B ), ut oth showed incresed in vitro tyrosine kinse ctivity (Figure 6, ). Interestingly, when lystes of these trnsfected cells were co-immunoprecipitted with nti-crk As, there ws correltion etween the kinse ctivitynd the extent of Al co-immunoprecipittion (Figure 6c; Figure 5f). One possile explntion is tht these mutnts ind more stronglyto Al thn the wildtype Crk II protein, lthough they do not ctivte its kinse ctivity. Thus, the incresed in vitro kinse ctivitymye due to incresed Al protein complexed

9 A B c p-tyr py245al Crk N145 CrkW276K CrkP225A/W276K Y252 N146 P249 CrkY222F/W276K CrkQ275A A251 N250 F144 CrkA256K CrkP225A/ PNAY CrkY222F/ PNAY Crk PNAY + Al Figure 7 Predicted RT loop structure nd phosphotyrosine Western lots of Al þ Crk mutnts. (A) Superimposed rion digrms of SH3-N (light gry) nd SH3-C (drk gry). Residues of the SH3-N tht do not interct with the PPPALPPKK peptide (green), nd corresponding residues of the SH3-C (purple) re lso shown. These residues re predicted to point wyfrom the SH3-C. (B) () Equl mounts of lystes from 293T cells trnsfected with Al plus Crk mutnts were loded on 8/11% step-grdient SDS PAGE gels nd Western lotted with nti-p-tyr ntiody. () nd (c) Western lots of lystes with the indicted ntiodies CrkY252A CrkP249A CrkP225A CrkY222F CrkW170K Crk II Crk with Crk in the immunoprecipittion, rther thn n increse in Al kinse specific ctivity. Importntly, however, while Q275A, Q275W, A256K, A256D, nd W276K Crk II myhve incresed the ssocition etween Crk nd Al, their mode of Al ctivtion ws distinct from tht of the Y222F nd P225A muttions in the linker. In this respect, the C- terminl SH3 domin mutnts did not trnsctivte Al to result in incresed cellulr tyrosine phosphoryltion (Figure 5, compre, d; lso compre Figure 6 with Figure 7B ). As result, some linker mutnts, ut not SH3-C mutnts tested, increse Al kinse ctivity in vivo, it suggests tht the linker region nd the SH3-C perform different functions. For exmple, the wild-type linker mye directlyinhiiting trnsctivtion, perhps yinding to the kinse domin in mnner similr to the Al kinse domin ctivtion loop, which is lso sustrte of the Al kinse. The SH3-C my ind elsewhere on Al (see elow), or myserve to modulte the ffinityof Crk II for Al. Al trnsctivtion y CrkY222F nd P225A is medited y PNAY sequence in the RT loop of the Crk-SH3-C In ddition to the residues of the RT loop of SH3-N tht interct with K8 of the PPPALPPKK peptide, other residues point wyfrom the inding groove nd do not interct with PPPALPPKK, notlyd148. Similrly, there re residues in our model of the SH3-C tht lso point wyfrom the min odyof the SH3-C. Interestingly, these comprise the most conserved stretch of residues etween the evolutionrilydistnt Crk II of vertertes nd the ortholog Ced-2 protein of C. elegns (Tle 1 nd dt not shown). Shown in the rion digrms in Figure 7A re numer of those residues of the SH3-C (P249, N250, A251 nd Y252), together with similr superimposed residues in the structure of the SH3-N (F144, N146). We sustituted numer of these residues in order to test their effect on Al ctivtion. When co-expressed in cells, the individul mutnts P249A, Y252A nd DPNAY hd little effect on cellulr phosphotyrosine compred to the wild-type Crk II protein (Figure 7B ). In ddition, their effect on the in vitro Al kinse ssy ws not drmtic (Figure 6). This suggests tht the RT loop residues re not sufficient for the direct ctivtion of Al ycrk II. However, we lso mde numer of mutnts in which we comined ctivting mutnts of Crk II with either DPNAY or the SH3-C structuredisrupting mutnt W276K (W276 is predicted to e uried in the hydrophoic core). Interestingly, these p-tyr Western lots show tht the W276K nd DPNAY muttions were dominnt over the ctivting muttions; the enhnced co-immunoprecipittion nd ctivtion ws either rogted (Y222F/DPNAY, P225A/DPNAY, nd Y222F/W276K) or reduced (P225A/W276K) (Figure 7B ). In ddition, the enhnced co-immunoprecipittion nd ctivtion ws lso rogted in the cse of the Y222F/DPNAY nd P225A/DPNAY muttions, nd reduced for the Y222F/W276K mutnt (Figure 6 c). This suggests tht the RT loop is necessryfor ctivtion of Crk II, lthough it is not sufficient. Detection of Crk mutnts nd Al proteins with nti-phospho-al ntiodies The kinse ctivityof Al cn e uto-inhiited through internl interctions in which its myristoylted N-terminus, nd SH2 nd SH3 domins ind to the two loes of the kinse domin, termed the N- nd C-terminl loes (Ngr et l., 2003). The crystl structure of the N-terminl cp region of Al ound to its own kinse domin hs shown tht the utoinhiited structure requires P 242 TIY 245 sequence, which is just N-terminl to the kinse domin. The side chin 8195

10 8196 of the unphosphorylted Y245 projects into the kinse domin, stilizing the structure nd inhiiting the kinse domin. This structure is further stilized ythe SH3 of Al, which intercts with the unphosphorylted Y245 s it rests in the kinse domin (Figure 8). Auto phosphoryltion of Y245, or muttions tht disrupt this region, ctivte Al (Brsher nd Vn Etten, 2000). In ddition, the kinse domin hs n ctivtion loop including Y412, which sticks into the kinse domin nd inhiits it. Upon ctivtion, this tyrosine is lso utophosphorylted, nd the ctivtion loop ecomes plced outside the kinse domin (Ngr et l., 2003; reviewed in Hntschel nd Superti-Furg, 2004; Wng, 2004). We utilized ntiodies mde ginst Al phosphopeptides contining either py245 or py412 to explore whether Al ws ctivted when coexpressed with Crk II. We found tht oth ntiodies detected Al in trnsfections in which cellulr phosphotyrosine ws elevted: Y222F, P225A nd P225A/W276K (Figure 6e, f; Figure 7B ). For py245, the mount of detection correlted with the levels of incresed phosphotyrosine in cells, with P225A/W276K less thn the other two. In ddition, there ws some detection on the other two W276K lystes, W276K lone nd Y222F/ W276K. The py412 ntiodydetected Al coexpressed with the three Crk II mutnts most ctive in incresing cellulr phosphotyrosine. As Al ctivtion in the presence of Crk mutnts correltes with the tyrosine phosphoryltion on Al seen in other modes of Al ctivtion, it suggests tht the trnsctivtion of Al y Crk occurs y mechnism similr to other kinds of ctivtion of Al. Most interestingly, we lso found tht the Al py245 ntiodycrossrected with Crk in severl lystes, notlyp225a, Q275A, W276K/P225A, nd W276K Figure 8 Model for the trnsient trnsctivtion of Al ycrk II. We propose multi-step process tht involves (1) the initil interction of Crk nd Al vi the SH3-N domin of Crk II nd the proline-rich domin of Al, (2) displcement of the P 242 TVY 245 motif in Al ythe P 249 NAY 252 motif of Crk, nd (3) interction of residues surrounding the Y 222 AQP 225 in the Crk II linker with the Al ctivtion loop, nd possile displcement of tht loop. Susequently, Crk II is phosphorylted t Y222, intercts with its own SH2, nd is relesed from Al. The SH3 domins of Crk re shown in ornge. N nd C refer to the N-terminus nd C-terminus of Al. The PRD is the proline-rich domin of Al (Figure 7B ). While the mening of this is not cler t present, there re instnces where n ntiodyprepred ginst one ctivtion-specific epitope cn cross-rect with nother epitope, which selects the sme inding prtner s the first epitope. For exmple, the STAT5 SH2 domin inds oth py694 of STAT5 to form STAT5 homodimer, nd py343 nd/or py401 of the erythropoietin receptor (Epo R). Two seprte ntiodies rised ginst the py694 of STAT5 crossrected with the tyrosine phosphorylted Epo R; this crossrection ws loclized to epitopes t py343 nd/or py401 (Brer et l., 2001). Bynlogy, there myexist one or severl PXXY motifs in Crk tht re structurllysimilr to the negtive regultoryp 242 TVY 245 motif in humn Al (PTIY in mouse Al). There re three PXXY motifs in the C- terminus of Crk (PVPY 186 in the C-terminus of the SH3- N; PGPY 222 t the conventionl Al phosphoryltion site, nd P 249 NAY 252 in the RT loop of the SH3-C). The P 249 NAY 252 sequence is prticulrlyinteresting not only ecuse it mypoint wyfrom the predicted SH3 domin core, ut ecuse it is identicl in Crk II from wide rnge of species (humn, rt, mouse, chicken, zerfish, Drosophil, nd X. levis). It is lso the most conserved contiguous sequence etween the entire mmmlin Crk II nd Ced-2 proteins (Tle 1 nd dt not shown). It is lso interesting tht the Y222F nd P2225A Crk proteins were tyrosine phosphorylted when expressed with Al, suggesting tht sites distinct from the negtive regultoryy222 re phosphorylted yal. Although we hve yet to mp these phosphoryltion sites, it is noteworthy tht Crk strongly crossrected with nti-al py245, ut not nti-al py412, gin suggesting tht one or more of the PXXY sequences of Crk re phosphorylted. Consistent with this ide, expression of P225A/DPNAY or Y222F/ DPNAY rogted Crk II phosphoryltion, suggesting tht the puttive RT loop of the SH3-C is necessryfor Al ctivtion ycrk II. Tken together, we posit tht the Crk SH3-C, in prticulr the RT loop residues, is required for the trnsient ctivtion of Al ycrk II, possilyymimicking nd destilizing the negtive regultoryptiy motif in Al. As shown in Figures 6 nd 7, phosphoryltion on Y245 of Al ws not sufficient to ctivte Al in the cse of coexpression of Crk mutnts contining W276K, suggesting tht this muttion cn lock Al ctivtion, perhps yremining ound to Al under conditions in which wild-type Crk II would normlly dissocite from Al. This suggests tht W276K trps the Crk II/Al interction in ound, prtilly ctivted stte. If W276K inds more stronglyto Al thn wild-type Crk II, it would ring down more Al in the Crk immunoprecipittes. This myexplin why the W276K mutnts show incresed Al kinse ctivityin the in vitro kinse ssynd incresed co-immunoprecipittion with Al, lthough theydo not increse the levels of cellulr phosphotyrosine ( in vivo kinse ssy ). If the W276K mutnt does trp Al in ound, nonproductive stte, this would leve the kinse domin

11 inctive, with Crk II rther thn the Al N-terminl domins controlling/inhiiting Al kinse ctivity. This lso suggests tht the wild-type Crk II/Al heterodimer wits some signl to Crk or Al itself tht results in ctivtion of Al. It would e interesting to test whether DPNAY or W276K Crk-SH3-C mutnts ct s dominnt negtive mutnts for the physiologicl ctivtion of the Al or Bcr-Al kinses. Since it hs een shown tht phosphoryltion of Al Y245 relieves the intrmoleculr inding of the Al P 242 TVY 245 region to the Al kinse domin, it is conceivle tht phosphoryltion of one or more of the PXXY sequences in Crk II could result in Al ctivtion. If W276K mutnts of Crk II remin ound to Al in stte in which the heterodimer complex is prtillyctivted, theymyecome trgets for dditionl phosphoryltion on PXXY regions of Crk nd/or Al, without the susequent ctivtion of Al. This might explin whyw276k mutnts hd incresed phosphoryltion on Al of Y412 compred to Crk II lone, on Al nd Crk of Y245 epitopes, nd incresed Al in vitro kinse ctivity(i.e., possilyincresed inding of Crk to Al). The inding of Crk to Al could chnge the ilityof Al to interct with other proteins. The Al P 242 TVY kinse intrmoleculr interction is stilized ythe Al SH3 domin; should the P 242 TVY 245 of Al e displced ycrk II, then the SH3 of Al might ecome ville to ind to other prtners. Susequently, this could lso potentillymke the SH2 of Al ville s well, since it is lso prt of the Al intrmoleculr N-terminl-tokinse domin interction (Ngr et l., 2003). It is lredyknown tht Al phosphoryltes Crk Y222, nd utophosphoryltes on Al Y245 nd Al Y412. It would e interesting to explore whether other kinses cn phosphorylte these sites, nd lso whether Crk cn e phosphorylted t PXXY sites y Al or nother kinse. Phosphoryltion of Al t Y245 nd Y412 myoccur yutophosphoryltion, or perhps trnsphosphoryltion y nother Al molecule; it hs een shown tht Al cn e ctivted yhomodimeriztion (Smith nd Vn Etten, 2001). Since Src nd Hck hve een shown to phosphorylte nd ctivte Al, it is lso possile tht Src fmilymemer, or nother tyrosine kinse, could e involved in phosphorylting Al or Crk when the two re in complex (Tnis et l., 2003). Overll, these nd other oservtions suggest tht while Crk I cn constitutivelyctivte Al kinse ctivity in vivo, Crk II ctivtion of Al (towrds the phosphoryltion of exogenous sustrtes) is more tightlyregulted nd requires multistep process. Furthermore, it ppers tht nyctivtion of Al y Crk II tht does occur requires n inititing signl in which, t the minimum, n inhiitoryeffect of the Crk II linker (centering on Y222) is relieved, nd nother ctivting step involving the SH3-C occurs s well. As shown in Figure 8, we propose multistep process in the interction nd trnsient ctivtion of Al ycrk II. These include (1) the initil interction of Crk nd Al vi the SH3-N domin of Crk nd the proline-rich domin of Al, (2) displcement of the P 242 TVY 245 motif in Al ythe P 249 NAY 252 motif of Crk, nd (3) interction of residues surrounding the Y 222 AQP 225 in the Crk II linker with the Al kinse domin, replcing the Al ctivtion loop (the Al kinse ctivtion loop mintins Al in the inctive configurtion). Susequently, phosphoryltion y Al of Crk t Y222, nd intrmoleculr inding of Crk SH2 to this phosphotyrosine, leds to dissocition of Crk from Al nd signl downmodultion. In summry, we exmined the structurl nd functionl properties of the Crk II linker nd SH3-C domin to understnd how theyregulte the iologicl properties of Crk II. Using severl independent criteri including tryptophn fluorescence, homology-sed moleculr modeling, nd informtic nlysis, we provide model where the miniml functionl unit of the Crk SH3-C ( ) is modulr undle contining ntiprllel -sheets pcked ginst ech other with W276 t the hydrophoic core. However, in contrst to cnonicl SH3 domins, Crk SH3-C nd Ced-2 SH3-C re unlikelyto ind conventionl PXXP-contining peptides, ecuse the residues tht re required for these interctions re mostlyunconserved. Our dt suggest tht the SH3-C domins of Crk nd Ced-2 re stlefolded entities chrcteristic of ll known SH3 domins, ut contin inding residues unique mong SH3 domins. We lso present evidence tht the Crk-SH3- C, through conserved PNAY sequence in the puttive RT loop, is necessryfor the trnsient ctivtion of Al following Crk II inding. Consistent with this study, studies ykizk-kondoh et l. (1996) hve lso shown tht this region of Crk is importnt for EGFinduced signling to rs in rodent firolsts, lthough these investigtors did not explore the role of Al in this pthwy. SH3 nd SH2 domins not only ind to py peptides nd PXXP peptides intermoleculrly, ut they ct intrmoleculrlyin some kinses s inhiitors (Al, Src, Hck). We hypothesize tht Crk, s protein tht hs een demonstrted to regulte Al kinse ctivity, is using the sme themes in its regultion of Al. Thus, the method of regultion of Al lredyseen intrmoleculrlymye conserved when Crk inds Al, except tht in the heterodimer the domins of Crk tke over the regultoryroles from those of Al. This studysuggests nother degree of modulrityin the function of the Crk dpter protein, nd possile explntion for whythe C-terminl SH3 domin is so conserved. Mterils nd methods Threding nlysis, homology-derived moleculr modeling, nd covrition sequence nlysis A homologymodel of the Crk SH3-C ws generted using the progrm Look (version 3.5; Moleculr Appliction Group, Plo Alto, CA, USA). The templte for homologymodeling (Gr-2 SH3-C) ws identified ythreding nlysis using FUGUE (Shi et l., 2001) nd 3D-PSSM (Kelley et l., 2000). The qulityof models produced yfugue nd 3D-PSSM ws evluted with PROSA (Sippl, 1993). 8197

12 8198 Lrson nd Dvidson (2000) used 266 SH3 domin sequences for covrition nlysis, nd derived set of consensus sequences for SH3 domins. We used the first, most highlyconserved consensus sequence in our lignments. A multiple sequence lignment of four sequences comprising the SH3 consensus sequence, Crk SH3-N, Crk-SH3-C, nd Ced-2 SH3-C, ws generted with Clustl W version The lignment ws scored for conservtion of structurl (hydrophoic core) nd protein-inding residues. Tryptophn fluorescence mesurements Fluorescence of constrined versus unconstrined tryptophn ws mesured s n indictor of protein folding. Fluorescence ws mesured using Photon TechnologyInc. (Lwrenceville, NJ, USA) fluorometer with model 840 photomultiplier. Excittion ws t 295 nm. Emission dt were collected etween 320 nd 400 nm, with step size of 0.5 nm nd integrtion time of 0.25 s. Slit width for oth excittion nd emission ws set t 5 nm. Mesurements were mde t room temperture in uffer contining 50 mm Tris ph 7.4, 150 mm NCl, 10% glycerol, nd 1 mm DTT. Plsmids nd mutgenesis pebb plsmids contining Crk mutnt DNAs were generted using the PCR-sed Quikchnget mutgenesis system (Strtgene, L Joll, CA, USA). The following mutnts were generted: K246A, R247A, K246A/R247A, A256D, A256K, Q275W, Q275A, H291A, P193A, P211A, P213A, P217A, P219A, P221A, P225A, P230A, P238A, Y252A, nd P249A. The DP 249 NAY 252 deletion mutnt ws generted using modified Quikchnge PCR procedure, using primers contining the desired deletion (Wng nd Mlcolm, 1999). The Crk II mutnts, R38K, W170K, Y222F, nd W276K, hve een descried erlier (Zvr et l., 2001). For ll studies, the murine Al IV gene ws used (Vn Etten et l., 1989). Construction of GST-fusion proteins cdna mplicons encoding the linker, SH3-C nd deletion mutnts were generted ypcr, with primers llowing for directionl cloning into the pgex 6P-1 vector (Amershm Biosciences Corp, Pisctwy, NJ, USA), for expressing GSTfusion proteins. Primer pirs were s follows: GST-linker-SH3-C; ( );5 0 AAGAATTCAAGTG TAGACCTTCCTCTGCTTCA 3 0 nd 5 0 ATAGATATCT CAGCGGACATGTGTGAATGGAAAGTG 3 0 GST D1Linker-SH3-C ( ); 5 0 ATGAATTCACT CTGACTGGAGGTAACCAG 3 0 nd 5 0 ATAGATATCT CAGCGGACATGTGTGAATGGAAAGTG 3 0 ; GST D2-Linker-SH3-C ( ); 5 0 ATGAATTCCAC CCACAACCACTGGGTGG 3 0 nd 5 0 ATAGATATCTCA GCGGACATGTGTGAATGGAAAGTG 3 0 ; GST D3-Linker-SH3-C ( );5 0 ATGAATTCGGG CCCTATGCCCAGCCC 3 0 nd 5 0 ATAGATATCTCAGC GGACATGTGTGAATGGAAAGTG 3 0 ; GST D4-Linker-SH3-C ( ); 5 0 ATGAATTCCCG CTCCCTAACCTTCAGAA 3 0 nd 5 0 ATAGATATCTCA GCGGACATGTGTGAATGGAAAGTG 3 0. GST-SH3-C ( ); 5 0 ATGAATTCTTTTATGCC CGGGTTATCCAG 3 0 nd 5 0 ATAGATATCTCAGCGG ACATGTGTGAATGGAAAGTG 3 0 ; D5 0 GST-SH3-C ( ); 5 0 AAGAATTCAAGCGA GTCCCTAATGCCTACG 3 0 nd 5 0 ATAGATATCTCAG CGGACATGTGTGAATGGAAAGTG 3 0 ; GST-SH3-C ( ); 5 0 ATGAATTCTTTTATGCC CGGGTTATCCAG 3 0 nd 5 0 ATAGATATCTCATTGA TCCAGCAGGCGGACATG 3 0 All inserts were cloned into the EcoRI nd SmI sites of pgex6p-1 nd sequenced to verifythe integrityof the plsmid DNA. Expression nd purifiction of GST-fusion proteins GST-fusion proteins were produced ystndrd methods from cteri expressing inserts cloned into the pgex 6P-1 vector (Amershm Biosciences Corp, Pisctwy, NJ, USA). Briefly, plsmid constructs were trnsformed into competent Escherichi coli. Approximtely500 ml of fresh Luri-Bertni (LB) medi contining mpicillin (100 mg/ml) were inoculted with smll liquot of n overnight cteril culture grown t 371C. The culture ws susequentlyinduced with 0.15 mm isopropyl-d-thioglctopyrnoside (IPTG) for 2 h t 371C. The purifiction nd elution of GST-fusion proteins ws ystndrd protocols. The fusion proteins were lierted from GST with 2.5 U of PreScission Protese (Amershm Biosciences Corp, Pisctwy, NJ, USA) t 41C overnight. The identitynd integrityof cleved proteins ws further verified using MALDI-TOF mss spectrometer nd Voyger softwre (Applied Biosystems, Foster City, CA, USA). Mmmlin cell trnsfection, electrophoresis, nd kinse ssys Humn emryonic kidney (HEK) 293T cells were mintined in DMEM with 10% FBS (het inctivted) nd penicillin/ streptomycin. Cells were trnsfected with 0.5 mg of ech DNA plsmid using the Lipofectminet trnsfection regent (Invitrogen Corp., Crlsd, CA, USA), nd collected 2 dys post-trnsfection. Cells were lysed in HNTG 1% uffer (Hepes 20 mm, ph 7.5; NCl 150 mm; glycerol 10% nd Triton X-100 1%; plus protese nd phosphtse inhiitors). Lystes were exmined ywestern lotting nd/or immunoprecipittion. Western lots were nlyzed on electrophoreticlly resolved proteins on PVDF memrnes (Millipore Corp., Billeric, MA, USA), using ntiphosphotyrosine ntiody PY99 (Snt Cruz BiotechnologyInc., Snt Cruz, CA, USA), nti-al A- 3 (Cliochem rnd, EMD Biosciences, Inc., Sn Diego, CA, USA), nti-py245, nd nti-py412 Al IB (Cell Signling TechnologyInc., Beverly, MA, USA). The py245 nd py412 Al ntiodies crossrect with mouse Al. For Crk immunolots, we used nti-crk RF-51, polyclonl ntiody rised ginst the SH2 domin of Crk. Crk immunoprecipittes were performed with nti-crk RF-51 A, followed yprotein A sephrose eds (Amershm Biosciences Corp., Pisctwy, NJ, USA). Kinse ssys were performed on Crk immunoprecipittes in kinse uffer (HNTG uffer contining 0.1% Triton X-100, 10 mm MnCl 2, 2mg/rection of GST-Crk mino-cid , 100 mm ATP, nd 5 mci [g 32 P] ATP (3000 Ci/mmol). After 30 min mixing t room temperture, rections were terminted ythe ddition of SDS PAGE smple uffer. Rections were exmined yseprting proteins ysds PAGE nd exposing the gels directlyto film or to phosphoimger plte, nd yquntifiction using Typhoon Storm Phosphoimger (Amershm Biosciences Corp., Pisctwy, NJ, USA). Acknowledgements We thnk Drs Cecile Bougeret, Brin Ky, Tom Muir, Stephn Feller, SllyKornluth, nd Alln Dvidson for insightful discussions nd shring unpulished dt. This work ws supported y Pulic Helth Service Awrd (NIH-GMS55760) to RBB nd n NJCCC fellowship to Sukhwinder Singh.