Reuven Agami and Yosef Shaul

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1 The kinse ctivity of ut not v-al is potentited y direct interction with RFXI, protein tht inds the enhncers of severl viruses nd cell-cycle regulted genes Reuven Agmi nd Yosef Shul Oncogene (1998) 16, 1779 ± Stockton Press All rights reserved 0950 ± 9232/98 $ Deprtment of Moleculr Genetics, The Weizmnn Institute of Science, Rehovot 76100, Isrel, the non-receptor tyrosine kinse is ssocited with EP, DNA element found in promoters/enhncers of di erent viruses nd cell-cycle regulted genes. EP-DNA inds RFXI, memer of novel fmily of DNA-inding proteins tht is conserved through evolution nd in yest, it controls di erentition nd exit from the mitotic cycle to G 0. EP-ssocited proteins re preferentilly tyrosine phosphorylted nd the ssocited hs strong tyrosine kinse ctivity. Here we investigted the moleculr mechnism underlying this kinse ctivity. We show tht RFXI nd re in direct interction, in vitro nd in cell extrcts, through the RFXI proline rich (PxxP) motif nd the SH3 domin. Remrkly, this interction signi cntly potentites c- Al ut not v-al uto-kinse ctivity. Collectively, we descrie novel mechnism of recruitment to de ned DNA-cis element with its concomitnt kinse ctivtion. We propose tht this mechnism my ct to regulte cell-cycle control genes. Keywords: EP element; HBV enhncer; tyrosine kinse; DNA inding; ; trnscription Introduction The product of the c-l proto-oncogene is uiquitously expressed 140 kd non-receptor tyrosine kinse tht is loclized predominntly to the nucleus (Vn Etten et l., 1989; Wng, 1994). is sudivided into four regions: two N-terminl regultory domins termed Src homology (SH) 2 nd 3, tyrosine kinse domin nd unique C-terminl til (Rmkrishnn nd Rosenerg, 1989; Wng, 1993, 1994). The SH2 nd SH3 domins re known to medite protein-protein interctions with speci c tyrosine phosphorylted sequences nd proline-rich motifs, respectively (Cicchetti et l., 1992; Ren et l., 1993; Cohen et l., 1995; Pwson, 1995,). The long C-terminl til contins n ctin inding domin (McWhirter nd Wng, 1993; Vn Etten et l., 1994) nd severl PxxP motifs cple of intercting with Crk1, Ai1 nd Ai2 proteins (Ren et l., 1994; Feller et l., 1994,; Shi et l., 1995; Di nd Pendergst, 1995). Also, this region hs DNA inding ctivity (Kipreos nd Wng, 1990, 1992) with three high moility group-like domins (Mio nd Wng, 1996). Geneticlly, the C-terminus is crucil for function, s mice homozygous to C-terminlly Correspondence: Y Shul Received 18 Septemer 1997; revised 7 Novemer 1997; ccepted 7 Novemer 1997 truncted (deleted t the DNA inding domin) exhiit neontl lethlity to the sme degree s null mice do (Tyulewicz et l., 1991; Schwrtzerg et l., 1991). In cells, the kinse ctivity of is highly regulted nd hrdly detectle ut cn e detected in proliferting cells (Konopk nd Witte, 1985; Jckson nd Bltimore, 1989; Pendergst et l., 1991; Liu et l., 1996; Lewis et l., 1996). overexpression inhiits cell growth nd its kinse ctivity is required for G 1 rrest in response to tretment with DNA dmging gents (Vn Etten et l., 1989; Swyers et l., 1994; Khrnd et l., 1995). In shrp contrst, overexpression of constitutively ctive Al mutnts leds to growth stimultion nd chnge in cell shpe nd ehvior (reviewed in Wng, 1993). Deleting, mutting or swpping the SH3 domin ws su cient to render it constitutively ctive, cytoplsmic nd trnsforming (Vn Etten et l., 1989; Myer nd Bltimore, 1994). Therefore, it hs een suggested tht SH3 ssocition with cellulr fctors negtively regultes c- Al ctivity (Konopk nd Witte, 1985; Pendergst et l., 1991; Myer nd Bltimore, 1994). Indeed, two proteins Ai1 nd Ai2 were recently identi ed s modultors of trnsforming ctivity (Di nd Pendergst, 1995; Shi et l., 1995). Ech of these proteins intercts simultneously with the SH3 domin nd proline-rich motif t the C-terminl til of. Presumly, this interction might stericlly sequester c- Al kinse domin. We hve previously demonstrted the ssocition of with speci c DNA element termed EP (Dikstein et l., 1992). EP is key functionl element of the heptitis B virus (HBV) enhncer (Ostpchuk et l., 1989; Zhng et l., 1990,, 1991; Dikstein et l., 1990). A second component of the EP nucleo-protein complex is., 140 kd protein, elongs to fmily of DNA inding proteins conserved in evolution down to yest (Reith et l., 1990, 1994; Steimle et l., 1995; Wu nd McLeod, 1995; Emery et l., 1996). inds the EP site nd ctivtes the HBV enhncer (Siegrist et l., 1993). However, when exmined in isoltion, not in the context of the enhncer, ws found to hve multiple functionl domins (Ktn et l., 1997). It hs trnscriptionl repression domin which overlps with the dimeriztion domin, nd n ctivtion domin t its centrl region. These domins neutrlize ech other giving rise to mostly trnscriptionlly inctive protein. Most intriguing, contins modultor region which overlps with the proline rich region t its N- terminus tht ects its level of expression nd DNA inding ctivity. Bsed on in vivo leling nd protein puri ction ssys, it hs een found tht the components of the

2 1780 EP nucleo-protein complex re tyrosine phosphorylted (Dikstein et l., 1996). Here, we investigted the mechnism underlying kinse ctivtion. We report tht oth EP-DNA ssocited proteins; nd, re in direct interction in nucler extrcts. Moreover, in vitro, ctivtes uto-kinse ctivity. This ctivtion is medited through speci c ssocition of the -PxxP motif with the - SH3 domin, the sme domins which re required for - interction in vivo. In greement with these results we oserved tht in vivo the EP-DNA ssocited protein ut not is highly phosphorylted t tyrosine residues. As the yest homolog of regultes cell fte, our oservtions my provide moleculr mechnism for the mode of ction of in cell growth. Results kinse potentition nd DNA-inding Recominnt potentites kinse ctivity The EP nucleo-protein complex contins t lest two protein components, nd (Dikstein et l., 1992; Siegrist et l., 1993). The EP ssocited hs strong kinse ctivity (Dikstein et l., 1996). We therefore checked the possiility tht might directly regulte the kinse ctivity. This possiility ws ddressed here in vitro y employing cteril recominnt (r) nd (r) proteins. In kinse rection, r lone ws poorly phosphorylted (Figure 1, lne 1) wheres r lone ws inctive (lne 2). However, when the two proteins were mixed we oserved n increse in phosphoryltion (lne 3). Two phosphorylted nds ppered round 140 kd. Although not directly determined one could e phosphorylted nd the other phosphorylted. The e ect of r ws speci c s mixing similr mount of control recominnt protein hd no e ect on the phosphoryltion pttern when either dded lone to (lne 4) or in comintion with r (lne 5). Moreover, 2D-phosphomino cid nlysis con ned tht the proteins were phosphorylted only t tyrosine residues (Figure 1), indicting tht ws the sole kinse in the rections. In order to exmine whether the increse in phosphoryltion is the result of phosphoryltion or the result of n increse in the utokinse ctivity, we used for the following experiments truncted protein (mino cids 15 ± 667). This protein contins the SH3, SH2 nd kinse domins together with prt of the C-terminl til ut lcks the DNA nd ctin inding domins. When puri ed from cteri, through six-histidine tg t its N-terminus, in ddition to the expected 90 kd protein 65 kd truncted protein ws lso otined (see for exmple Figure 7). Using this protein we oserved n -dependent potentition of uto-phosphoryltion (Figure 2, lnes 1 nd 2) wheres r phosphoryltion ws ine cient. The potentition of c- Al kinse ctivity is r dependent nd not seen with r(15 ± 667) lone (lne 1) or in mixture with control protein (lne 3), s ws oserved with the full-length r (Figure 1). Additionlly, incresing mounts of either r or r proportionlly GST * rised the uto-phosphoryltion rte of (Figure 2), demonstrting dose response rection, chrcteristic of enzymtic ctivity. Most importnt, nti-al ntiodies immunoprecipitted the mjority of the phosphorylted proteins (Figure 2c), con rming uto-phosphoryltion. This ctivity is kinse domin dependent s kinse mutnt of Al did not support phosphoryltion. These results demonstrte the ctivtion e ect of on the uto-phosphoryltion ctivity of. Assocition of with in nucler extrcts The DNA independent potentition of kinse ctivity of rises the possiility tht these two proteins re physiclly ssocited. This possiility ws investigted y co-immunoprecipittion experiments of nucler extrcts using nti-al nd nti- ntiodies. As expected, nti-al ntiodies e ciently nd speci clly immunoprecipitted (Figure 3). Interestingly, ws co-precipitted, s reveled y incuting the sme immunolot with n nti- ntiody (Figure 3, lne 7). This co-immunoprecipittion is highly speci c, s the protein is not found in the elutes of the two control ntiodies (lnes 5 nd 6) nd in ll the wsh V frctions (lnes 2 ± 4). In reciprocl experiment, extrcts were prepred from cells trnsfected with the HA-tgged cdna, immunoprecipitted with the nti-ha nd control monoclonl ntiodies, nd nlysed y Western lot. E cient expression of HA in the trnsfected cells ws otined (Figure 4, lne 6). ws coimmunoprecipitted y nti-ha nd not y the control rit nti-mouse (RM) ntiody (Figure 4, lnes 5 nd 6). Speci city ws lso pprent when the lot ws ph 1.9 ph 3.5 py pt ps pi Figure 1 phosphoryltion increses when oth recominnt nd proteins re incuted together. () 50ng of the following recominnt proteins:, nd the control protein ± GST, were incuted either lone (lnes 1 nd 2) or mixed (lnes 3, 4 nd 5), in kinse rection. The position of the full-length r nd r proteins is indicted y sterisk nd the moleculr weight mrkers re shown in kd. () Phosphomino cid nlysis of the phosphorylted protein(s) from the region mrked with sterisk. The gel in ws lotted to PVDF memrne (Bio-Rd), nd the indicted region ws excised from the lot, nlysed for phosphomino cids nd visulized with PhosphorImger (Boyle, 1991). The positions of the phospho-tyrosine (py), phospho-serin (ps), phospho-threonine (pt), nd the free phosphte (Pi) re mrked with circle ccording to stndrds nd the loding point is mrked with n X

3 kinse potentition nd DNA-inding 1781 wsh V elute wsh V elute long exp GST short exp ext. rαm PI αal rαm PI αal ext. rαm PI αal rαm PI αal Figure 3 is co-immunoprecipitted y n nti-al ntiody in nucler extrcts. HeL cell nucler extrct ws sujected to immunoprecipittion with either rit nti mouse (rm, lnes 2 nd 5), preimmune (PI, lnes 3 nd 6) or nti-al polyclonl 543 ntiodies (lnes 4 nd 7). Untreted extrct (lne 1), wsh 5 out of six (lnes 2 ± 4) nd elute (lnes 5 ± 7) frctions were seprted on 7.5% SDS ± PAGE nd Western lot nlysed rst y the polyclonl nti- (), then, fter dentured-rentured procedure, y the monoclonl 8E9 nti-al ntiody (). nd proteins re mrked with rrowheds ng ng IB: α-al MW IP: 190 unound wsh V elute RαM α ΗΑ RαM α ΗΑ RαM α ΗΑ c IP: Al-Km α- α-al IB: α- 190 / HA c 63 IB: α-r 125 R 5 Figure 2 Speci c ctivtion of kinse y. () Kinse rections were performed with 20 ng of r(15 ± 667) (lnes 1 ± 3) nd 20 ng of either r (lne 2) or GST (lne 3) proteins. Two di erent X-ry lm exposure times re shown. () Three concentrtions of r(15 ± 667) protein (5, 20 nd 50 ng) were tested for uto-kinse ctivity in the sence or the presence of 2 or 20 ng r. (c) Fifty ng of r(15 ± 667) ws incuted either lone (lne 1) or together with 20 ng of r (lnes 2 ± 4). The rections were sujected to immunoprecipittion with nti- (lne 3) or 8E9 nti-al ntiodies (lne 4). The positions of r nd r(15 ± 667) re indicted 5 6 Figure 4 Co-immunoprecipittion of protein with HA. SK-Hep1 cells were trnsfected with 5 mg of psg5- HA. Their nuclei were extrcted nd sujected to immunoprecipittion y rit nti-mouse (RM) or nti-ha ntiodies. The unound frction (lnes 1 nd 2), wsh #5 out of six (lnes 3 nd 4), nd elute (lnes 5 nd 6), were seprted on 7.5% SDS ± PAGE nd sujected rst to Western lot nlysis with the 8E9 monoclonl nti-al ntiody (). Following dentured-rentured procedure, the sme lot ws rected with the polyclonl nti- ntiody () nd ltter with the monoclonl 245 nti-r (c)., /HA nd R proteins re mrked

4 1782 kinse potentition nd DNA-inding rected with nti-r ntiody. Although over-exposed, no R protein ws detected in the elute of the nti-ha immunoprecipittion (Figure 4c, lnes 5 nd 6). Thus, there is speci c physicl interction etween the nd proteins in nucler extrcts. The mino terminl region of is necessry for ssocition with Inspection of the sequence reveled proline rich mino terminl region contining ve PxxP repets which re conserved etween mouse nd humn (Figure 5). PxxP motifs were shown to interct with SH3 domins (Ren et l., 1993; Cicchetti et l., 1992). To exmine the involvement of this region in the interction with, nucler extrcts from cells trnsiently trnsfected with either, HA, or two derivtive mutnts were immunoprecipitted with n nti-ha ntiody (Figure 5). Western lot nlysis with nti- demonstrted tht the di erent HA-tgged proteins were eqully expressed nd precipitted y nti-ha (Figure 5c). Anlysis of the sme immunolot with 8E9, n nti-al monoclonl ntiody, reveled tht c- Al ws co-precipitted with HA nd HAD(77 ± 588) (lnes 2 nd 4), ut not with HAD(1 ± 77) which lcked the PxxP motifs (lne 3). This is despite the fct tht the ltter ws properly expressed nd nucler loclized. As expected, ws not detected when cells were trnsfected with n plsmid tht lcked the HA-tg, ruling out cross-rection etween the nti-ha ntiody nd c- Al. Thus, the mino terminl region of is necessry for the interction in vivo. The SH3 domin speci clly intercts with the proline rich region To exmine whether the -SH3 domin directly intercts with the proline rich region of, we performed fr-western nlysis. This method previously ws employed to demonstrte SH3 interction with distinct PxxP motifs (Cicchetti et l., 1992; Ren et l., 1994). Totl extrcts of cteri tht produce either, D(1 ± 77) or control protein were seprted on SDS ± PAGE nd lotted. Anlysis with nti- ntiody con rmed similr production of oth recominnt nd D(1 ± 77) proteins (Figure 6). Also, severl degrdtion products were oserved, minly t sizes of 52, 65, 75 nd 95 kd for nd 65 nd 100 kd for D(1 ± 77). Fr-Western nlysis with 32 P-leled -SH3 domin (mino cids 15 ± 147) showed speci c interction with ut not with the control proteins (Figure 6). The rective nds corresponded exctly to the full length nd the degrdtion products of r (compre lnes 1 in Figure 6 nd ). As expected rd(1 ± 77) lcking the PxxP motif did not ind the SH3 domin (lne 2). Similr results were otined when higher c d HA HA (1-77) HA (77-588) HA HA (1-77) HA (77-588) Figure 5 The N-terminl proline-rich region of is necessry for the ssocition with in vivo. () The mino cids sequence of the N-terminl proline rich region of the humn nd mouse proteins is shown. The puttive SH3 inding sites (PxxP; where P designte proline nd x ny mino cid) re indicted. () A schemtic drw of HA with the HA epitope, proline rich region (mrked PxxP), DNA inding domin (DBD) nd dimeriztion domin (DIM), together with the two deletion mutnts, HAD(1 ± 77) nd HAD(77 ± 588). (c nd d) SK-Hep1 cells were trnsfected with 5 mg of either, HA, HAD(1 ± 77) or HAD(77 ± 588) constructs. Susequently their nuclei were extrcted nd sujected to immunoprecipittion with n nti-ha ntiody. The elutes were seprted on 7.5% SDS ± PAGE nd sujected to Western nlysis with either polyclonl nti- (c) or 8E9 monoclonl nti-al ntiody (d). The protein is mrked with n rrowhed

5 mounts nd puri ed recominnt proteins were used. The rd(1 ± 77) protein showed no SH3 inding, wheres rd(77 ± 588), contining the PxxP region e ciently ound it (Figure 6c). Together, these dt indicte tht the SH3 domin of intercts with the proline rich region of. r potentites kinse ctivity through direct interction Hving demonstrted tht nd re in direct ssocition we next sked whether this interction is importnt for tyrosine kinse ctivtion y. It is well documented tht v-al, lcking the SH3 domin, hs constitutive tyrosine kinse ctivity (Konopk nd Witte, 1985; Myer et l., 1992; Renshw et l., 1992). Interestingly, r filed to potentite v-al kinse ctivity (Figure 7, lnes 1 ± 3), despite the fct tht v-al utophosphoryltion level ws lower thn tht otined with -ctivted c- Al (compre lnes 2 to 8 in Figure 7 nd ). Thus, strongly suggesting tht the SH3 domin intercting with is required for kinse ctivtion y. rd(1 ± 77), which did not ind (Figure 5d), ws incple of potentiting kinse ctivity (lne 10) demonstrting the requirement of the PxxP motifs for kinse ctivtion. Western lot nlysis with n nti-al ntiody con rmed the presence of similr mounts of (15 ± 667) nd v-al in ll rections (Figure 7). The requirement of r ws further sustntited y the fct tht nity puri ed ntiodies essentilly reduced the kinse ctivity lmost to the sl level (lne 9). Furthermore we hve con rmed the involvement of kinse ctivity in kinse potentition nd DNA-inding the process y using either genistein or kinse mutnt. Genistein, tyrosine kinse speci c inhiitor, inhiited ll phosphoryltion (Figure 7, lne 11), nd no kinse ctivity ws otined with the kinse mutnt of v-al (Figure 7, lnes 4 ± 6). Collectively, these results suggest tht potentition of uto-kinse ctivity y r is highly speci c nd is regulted y direct interction. is phosphorylted t tyrosine residues in vivo Immunoprecipittion of in vivo leled HeL cell extrct with the 8E9 monoclonl nti-al ntiody, rought down contining low level of phosphotyrosine residues (Figure 8 nd d). This ntiody does not recognize the frction which is ssocited with (8B, lne 1). The ssocited frction is speci clly recognized y the polyclonl 543 nti-al ntiodies (Figure 3). We therefore used the 8E9 ow-through frction, fter het tretment to seperte etween nd, to immunoprecipitte either or y using 543 nd nti ntiodies, respectively. This frction, comprising only 5 ± 10% of totl (Figure 8c, compre lnes 1 nd 4) is highly phosphorylted (Figure 8, lne 4) with high level of phospho-tyrosine (Figure 8d). This is in full greement with the in vitro dt tht suggest tht ssocited is preferentilly phosphorylted t tyrosine. Under these conditions ws poorly phosphorylted, minly t serine/threonine nd very low level t tyrosine residues (Figure 8 nd lne 2d). Speci city of the immunoprecipittion ws exmined y the non relevnt nti-gst ntiody (Figure 8, lne 3). Altogether these results demonstrte tht in vivo is poorly tyrosine phosphorylted while the ssocited 1783 c (1-77) NI GST (1-77) NI GST (1-77) (77-588) r r (1-77) (77-588) Figure 6 The SH3 domin inds speci clly to the N-terminl proline rich region of., D(1 ± 77) nd D(77 ± 588) proteins were expressed in cteri nd nlysed. Totl inclusion odies of (lne 1), D(1 ± 77) (lne 2) nd control non induced (lne 3), s well s whole cell lyste of the GST control protein (lne 4), were used for oth Western nlysis with n nti- ntiody () nd lter inding ssy with the leled SH3 domin of s proe (). (c) Proteins puri ed y NiNTA resin were lso nlysed. The speci c rective nds re mrked with rrowheds

6 1784 genistein α- (1-77) Al Km v-al v-al kinse potentition nd DNA-inding In vivo phosphoryltion IP: α-al 8E E9 FT α- α-gst α-al 543 IB: nti- v-al Figure 7 The interction - is required for potentition of uto-kinse. () 20 ng of the following solule nd puri ed recominnt proteins: v-al(119 ± 472) (lnes 1 ± 3) v-al kinse mutnt (Km, lnes 4 ± 6) nd r(15 ± 667) (lnes 7 ± 11) were incuted either lone (lnes 1, 4 nd 7) or with 20 ng of either r (lnes 2, 5, 8, 9 nd 11) or rd(1 ± 77) (lnes 3, 6 nd 10). In lne 9, 0.2 mg of nity puri ed nti- ntiody ws dded to the rection. In lne 11, the kinse rection ws performed in the presence of 20 mg/ml of the speci c tyrosine kinse inhiitor, genistein. Bnds corresponding to utophosphorylted v-al nd r(15 ± 667) re indicted. () The sme extrcts s in were lotted nd Western nlysed with the 8E9 monoclonl nti-al ntiody c d 190 Pi 125 α-al 8E9 IB: nti- PAAA 8E9 FT α- α-al frction is preferentilly phosphorylted t tyrosine residues. Discussion Here we report the ssocition of with tht results in remrkle ctivtion of the tyrosine kinse. These oservtions provide moleculr explntion to the previous nding tht the EP- DNA ssocited protein hs strong kinse ctivity (Dikstein et l., 1996). In cells, the kinse ctivity of is tightly regulted. A mjor inhiitory component in is the SH3 domin, since deleting, mutting, or swpping it results in Al kinse ctivtion (Vn Etten et l., 1989; Myer nd Bltimore, 1994). This SH3-medited inhiition might e due either to interction with inhiitory proteins or to intrmoleculr folding. Since we hve used recominnt the involvement of inhiitory proteins in our system is unlikely. We show tht in vitro nd in nucler extrcts is physiclly ssocited with. The - interction ws loclized to the N-terminl PxxP motif of nd the SH3 domin of. These Figure 8 ssocited protein is preferentilly phosphorylted t tyrosine residues in vivo. () HeL cells were leled with 32 P-orthophosphte nd the nucler extrct ws sujected to immunoprecipittion with the 8E9 monoclonl nti- Al ntiody (lne 1). The superntnt of this immunoprecipittion ws het treted to seprte etween nd nd ws sujected to second immunoprecipittion with either polyclonl nti- ntiodies (lne 2), or polyclonl 543 nti-al ntiody (lne 4) nd the control non-relevnt polyclonl nti-gst ntiody (lne 3). The sme extrcts were nlysed y Western lotting to either nti- () or nti-c- Al (c) ntiodies. The indicted 140 kd phospho-proteins were excised from the gel in nd sujected to phosphomino cid nlysis nd visulized y PhosphorImger (d). Free phosphte is mrked Pi, phospho-serine, phospho-threonine nd phosphotyrosine re mrked ps, pt nd py respectively domins re lso required for kinse ctivtion y, suggesting tht - interction is prerequisite step for this process. However, s this interction leds minly to uto-phosphoryltion with low level of phosphoryltion, oth in vitro nd in vivo, we do not think tht we re evidencing here simple enzyme-sustrte interction. The ctivtion of the kinse domin y might e explined, t lest in prt, y the

7 sequestrtion of the SH3 inhiitory role. In the Alfmily, regultion of the tyrosine kinse ctivity y cis-sh3 domin seems to e generl theme. Both c- Src nd Hck protein tyrosine kinses were shown to e intr-moleculrly inhiited minly y the SH3 domin (Xu et l., 1997; Sicheri et l., 1997). Moreover, inding of the Nef PxxP motif to the SH3 domin, in vitro, relieves its inhiition nd fully ctivtes the kinse (More et l., 1997). The intr-moleculr interction nd inhiition of the tyrosine kinse y the SH3 domin is indirect nd medited through 14 mino-cids linker region etween the SH2 nd kinse domins (Xu et l., 1997; Sicheri et l., 1997). Most intriguingly, similr linker region is found in with out 40% identity to c-src. This conservtion my indicte tht the medited ctivtion cts y sequestering the inhiitory e ect of the SH3 domin on the kinse. However, the potency of kinse ctivtion y is extreme nd exceeds y fr tht of v-al (Figure 7 nd, compre intct nds in lnes 2 nd 8). The ltter, lcking the SH3 domin, is considered constitutive nd very e cient protein tyrosine kinse. Thus, kinse super-ctivtion y cnnot e ttriuted only to sequestrtion of the SH3 domin, ut lso to some dditionl c- Al llosteric chnges. Recently, two proteins of the sme fmily, Al Interctor protein 1 nd 2 (Ai1 nd 2), were found to modulte ctivity y inding to oth the SH3 domin nd the proline rich region t the N-nd the C- terminl regions of, respectively (Di nd Pendergst, 1995; Shi et l., 1995). Notly, the proline rich sequence is highly similr to tht of Ai1. Interestingly, Ai1 nd 2, in ddition to inding, lso hve puttive DNA inding domin, suggesting tht, like, these proteins my ttrct to DNA. Therefore, it is possile tht is ssocited with di erent DNA cis-elements through proteinprotein interctions with the cognte DNA-inding protein. However, nd Ai disply opposite e ects on kinse ctivity, in tht the former ctivtes, while the ltter inhiits it. Thus, the SH3 domin my exert oth positive nd negtive e ects on its ctlytic domin. Moreover in Drosophil positive regultion of Al protein, nmely recognition of its sustrte En, ws medited y its SH3 domin (Gertler et l., 1995). This dul role of the SH3 domin my o er mechnism tht is highly sensitive to signls nd extremely selective for sustrtes. Our results lso uncover mechnism for recruitment of tyrosine kinse domin to DNA-cis element with its concomitnt ctivtion. Oviously, the most immedite im is to de ne the role of the kinse domin in the trnscription mchinery. In contrst to the positive role of EP in virl enhncers, the EP-DNA element, s multimer nd n isoltion, does not ctivte trnscription (Ostpchuk et l., 1989; Dikstein et l., 1990; Reinhold et l., 1995; Lrie et l., 1995; Sfrny nd Perry, 1995; Ktn et l., 1997). itself contins severl independent functionl domins. An ctivtion nd repression domins which re mutully neutrlized, producing nerly inctive trnscription fctor (Ktn et l., 1997). Curiously, lso contins modultor region t its extreme N-terminus which overlps with the inding site (mino cids 1 ± 77). This region is importnt for the kinse potentition nd DNA-inding level of expression nd DNA inding ctivity. Whether is responsile for this modultor ctivity is yet n open question. This context-dependent ctivity of EP-site rises the possiility tht the ssocited my modify either other enhncer inding proteins or components of the generl trnscription mchinery. Two enhncer elements, GB nd E, were shown to cooperte with EP (Ghzl et l., 1988; Dikstein et l., 1990; Grci et l., 1993). Extremely intriguing is the E element, which inds the ATF2 nd Cyclic-AMP responsive element inding protein (CREB) fmilies (Fktor et l., 1990; Mguire et l., 1991). One memer of this fmily, CREB, ws found to e trnscriptionlly potentited y through physicl interction (Birchenll et l., 1995). Modultion of the HBV enhncer inding ctivtors y the nery ssocited is n interesting possiility to e investigted, however, s the ville - 7/7 cells re not of liver origin, t the moment this study is not trivil. Alterntively, it is possile tht recruited modultes trnscription y phosphoryltion of the POLII C-terminl domin (CTD, Bskrn et l., 1993). Among severl cellulr inding sites of the EP nucleo-protein complex, one is locted in the rst intron of the c-myc gene (MIE) nd nother in the promoter of the PCNA gene (Zjc et l., 1988; Lrie et l., 1995) oth of which re expressed in cell cycle dependent mnner. The role of EP in regulting these genes is not fully understood. A hint for possile role of MIE in myc gene expression hs een noticed from the study of the myc gene sequence in di erent Burkitt's lymphoms. It hs een found tht in these tumor cells, whose myc gene expression is deregulted, MIE is frequently mutted (Zjc et l., 1988). It is therefore ssumed tht MIE is involved in the silencing of c-myc trnscription, which is correlted with cellulr differentition nd cell growth rrest. Interestingly, oth c- Al nd re implicted in these processes, ws shown to inhiit cell growth (Swyers et l., 1994) nd Sk1, yest homolog of, ws shown to e involved in sex di erentition nd exit from the mitotic cell cycle to G 0 (Wu nd McLeod, 1995). Thus, the EP protein complex my represent novel trnscription component tht regultes cell growth through the tyrosine kinse ctivity of. Mterils nd methods Plsmid constructions nd cell trnsfection psg5-ha, in which the expression of HA-tgged is driven y SV2, ws constructed y ltering the ses t position 73 to 3, reltive to the initition strt site of open reding frme, to form n NdeI site. Then, n EcoRI ± NdeI HA epitope ws ligted to EcoRI digested psg5 together with n frgment (NdeI± EcoRI). PSG5-HAD(1 ± 77) ws generted y NdeI± ScI doule digest nd ligtion together with n in-frme NdeI±ScI linker. psg5-had(77 ± 588) ws generted y ScI digest. For cteril expression of, D(1 ± 77) nd D(77 ± 588) DNA frgments were excised from their corresponding psg5 plsmids y NheI± EcoRIndinsertedinfrmeintopRSET-B(Qigen). For preprtion of recominnt -SH3 domin, for fr western lter inding ssy, humn 1 cdna 1785

8 1786 kinse potentition nd DNA-inding (Shtivelmn et l., 1986) ws digested with HinDIII ± HinCII (mino cids 15 ± 147 nd inserted in-frme to prset-c. A protein kinse phosphoryltion site (PKS) ws creted y nneling of the two oligoes 5'-CTAGCCTCCGGA- GAGCTTCTCTTG-3' nd 5'-CTAGCAAGAGAAGCTCT- CCGGAGAGG-3' nd insertion into the NheI site, forming prset-pks--sh3 to produce rpks-l-sh3. Vectors for production of recominnt were constructed y inserting either n intct or truncted (mino cids 15 ± 667) humn 1 cdna into the HinDIII nd PVuII sites of prset-c, respectively. In ech cse the structure of the constrcts ws veri ed y enzymtic digestion nd sequencing. For trnsfection, SK-Hepl cells were cultured in Duleco modi ed Egle miniml essentil medium contining 100 U of penicillin per ml nd 100 mg strepomycin per ml supplemented with 10% fetl clf serum. Sixteen to 20 h efore trnsfection, cells were plted per 60 mm dish. Cells were trnsfected s descried (Honigwchs et l., 1989). Preprtion of recominnt proteins To generte solule recominnt proteins, the plsmids prset-, prset-d(1 ± 77), prset nd prset-(15 ± 667) were trnsformed into BL21-Lys S cteri cells, plted on LB-Amp pltes nd single colony ws chosen for n overnight strter culture. M9ZB0.4% glucose, hlf liter, ws seeded with 1/30 of the strter nd expression ws induced t n optiml density OD595=0.5 y dding IPTG to nl concentrtion of 0.5 mm. After 60 min, cells were hrvested, wshed with cold PBS, resuspended in lysis u er (25 mm HEPES (7.5), 5 mm MgCl 2,10% glycerol, 10 mm -mercptoethnol nd 200 mm NCl) nd lysed y ve times 30 s-sonictions. The lysis u er contined 1 mm freshly-mde phenylmethylsulfonyl uoride, 10 mm enzmidine hydrocloride, nd 10 mg/ml ech of 1,10 phenntroline, protenin nd leupeptin protese inhiitors. After removl of insolule inclusion odies y centrifugtion t r.p.m. for 20 min t 48C, solule histidine-tgged or Glutthion-Strnsferse (GST)-tgged proteins were puri ed from lystes using either NiNTA resin (Qigen) or glutthion grose eds (Sigm) nd eluted with lysis u er contining either 200 mm Imidzole or 5 mm of glutthion, respectively. For puri ction of GST-tgged proteins, 1 mm DTT nd 1 mm EDTA were dded to the solutions. Puri ed proteins were dilyzed ginst u er D (20 mm HEPES-KOH (ph 7.9), 50 mm KCl, 1 mm DTT, 1mM EDTA, 10% glycerol nd protese inhiitors). Proteins were puri ed to homogeneity nd ech step of the extrction nd puri ction ws followed y Coomssie stining, ws puri ed s 140 kd with 80 kd degrduted polypeptide, (15 ± 667) ws puri ed s 90 kd with degrdution product of 65 kd polypeptide. As ll were His-tgged t the N-terminus they must e truncted t the C-terminus. For lter inding ssys, totl cteril proteins contining either (r) or rd(1 ± 77) were used. These proteins were expressed s descried ove, only they were induced with 0.5 mm IPTG for 3 h. The resulting insolule frctions were collected nd wshed three times with PBS nd resuspended in 1 ml sodium dodecyl sulftepolycrylmide gel electrophoresis (SDS ± PAGE) smple u er. Typiclly, 20 ± 60 ml ws used for lter inding ssy to chieve *1 mg of the induced full length proteins on the gel. For the Western nlysis, 100 ng of recominnt proteins ws used. Immunoprecipittion nd immunolotting For coimmunoprecipittion of with, HeL cells nucler extrcts were generted s descried (Dignm et l., 1983, nd ). Immunoprecipittion ws done with polyclonl nti-al 543 ntiodies tht were rised ginst mino cids of humn fused to Gl (An et l., 1992; Dikstein et l., 1992) nd oosted y the sme 350 mino cids tgged with histidine epitope. Immunolotting ws performed y stndrd procedure (Towin et l., 1979) nd lters were rected with the secondry ntiodies, either sheep ntimouse or protein A, conjugted to horserdish peroxidse (Phrmingen). Immunocomplexes were detected using the ECL detection system (Amershm). For immunolot detection of nd, we used the polyclonl nti-, generous gift of B Mch (Reith et l., 1990), the speci c monoclonl 8E9 ntiody (Phrmingen, Sn Diego) nd s control, the monoclonl 245 nti-r ntiody (Phrmingen, Sn Diego). For coimmunoprecipittion of with, three 100 mm dishes of SK- Hep1 cells tht were trnsfected with HA-tgged expression plsmids, were lysed on ice for 10 min in 200 ml of 50 mm Tris : HCl (8.0), 1 mm EDTA, 250 mm NCl, 1% Triton X-100 nd 1 mm DTT. All solutions lso contined protese nd phosphtse inhiitors. Lystes were clered y centrifugtion t g for 10 min t 48C. Immunoprecipittion ws done in NET u er (50 mm Tris (8.0), 1 mm EDTA nd 150 mm NCl nd 10 mg/ml BSA) on ice for 2 h using 10 mg of monoclonl nti-ha 12CA5 (Phrmingen, Sn Diego) or 20 mg ofritntimouse(rm) ntiody (Phrmingen, Sn Diego). Immune complexes were rought down y protein A/G Sephrose eds (Snt-Cruz) nd wshed six times with ice cold NET u er. Filter inding ssy For preprtion of 32 P leled l-sh3, to e used s proe for lter inding ssys, puri ed solule rpks-l- SH3 (0.5 mg) ws phosphorylted with 8 mg/ml protein kinse (PK, Sigm) in 50 ml, contining 20 mm Tris : HCl (7.4) 1 mm DTT, 100 mm NCl, 12 mm MgCl 2 nd 50 mci [g- 32 P]ATP (5000 mci/mmol, Amershm) for 15 min t 378C. Kinse rection ws stopped with 1 ml stop solution (10 mm NPi, 10 mm NPPi nd 1 mg/ml BSA). Free [g- 32 P]ATP ws removed y employing the Ni-NTA nity resin (Qigen). Bound proteins were eluted with 100 ml of stop solution contining 200 mm Imidzole (Sigm) nd were used s proe. Nitrocellulose lters, contining totl cteril proteins together with the recominnt protein of interest, were locked in TBST (50 mm Tris (ph 8.0), 150 mm NCl 0.1% Tween-20, 0.1% Geltin, 2% non-ft milk powder) contining lso 0.5 mg, non-leled, 10 min oiled rpks-l-sh3 protein. 32 After 2 h incution t 48C with constnt shking, p rpks-l-sh3 ws dded t speci c rdioctivity of 10 6 c.p.m. per ml, nd incution ws continued t 48C for dditionl 2 h. Susequently, the lters were wshed ve times with cold TBST, ech for 5 min, nd exposed to utordiogrm for 10 h. In vitro nd in vivo phosphoryltion ssys For in vitro rections puri ed solule recominnt proteins were incuted in kinse u er contining 20 mm HEPES- KOH (ph 7.5), 20 mm MnCl 2 nd 1 mm DTT, t 48C for 30 min in 15 ± 18 ml rection. Phosphoryltion ws initited y ddition of 20 ± 50 mci [g- 32 P]ATP (5000 mci/mmol, Amershm) nd incution t 308C for 30 min (Khrnd et l., 1995). Phosphoryltion rte, under these conditions, ws liner for more thn 1 h. Rections were stopped y ddition of 1 ml stop solution (see lter inding ssy) nd free [g- 32 P]ATP ws removed y either employing the NI- NTA (Qigen) column for the His-tgged proteins or the glutthion grose eds (Sigm) for GST-fused proteins.

9 For the kinse-immunoprecipittion ssy, rection mixtures were oiled for 2 min fter the kinse rection nd then sujected to immunoprecipittion with either the polyclonl nti- or the monoclonl 8E9 nti-al ntiodies, s descried ove. In vivo phosphoryltion nd extrct preprtion ws crried out s descried (Dikstein et l., 1996). Phosphomino cid nlysis Proteins fter seprtion on SDS ± PAGE were lotted to PVDF memrne (Bio-Rd). Then, the leled nds were excised from the memrne nd incuted for 2 h in 6 M HCl t 1108C. Susequently, phosphominocid nlysis ws crried out s descried (Boyle, 1991). Phosphomino kinse potentition nd DNA-inding cid stndrds were visulized y 0.25% Ninhydrin stining in cetone. Acknowledgements We wish to thnk Dr B Mch nd Dr W Reith for the plsmids nd ntiodies, Prof E Cnnni nd Dr E Feinstein for the humn l cdna. We lso thnk Y Ktn, H Greif nd O Brk for criticlly reding the mnuscript nd S Budilovsky for her excellent technicl help. 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