Evidence for SH3 Domain Directed Binding and Phosphorylation of Sam68 by Src

Oncogene (1999) 18, 4647 ± 4653 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Evidence for SH3 domain directed binding and phosphorylation of Sam68 by Src

Zhiwei Shen1,2, Andreas Batzer3,6, Jackie A Koehler1,2, Paul Polakis4, Joseph Schlessinger3, Nicholas B Lydon5 and Michael F Moran*,1,2

1Banting and Best Department of Medical Research, University of Toronto, Canada; 2Department of Molecular and Medical Genetics, University of Toronto, Canada; 3Department of Pharmacology, New York University, New York, USA; 4ONYX Pharmaceuticals, Richmond, California, USA; 5Kinetix Pharmaceuticals Inc, Medford, Massachusetts, USA

Sam68 is a 68 kDa protein that associates with and is which aect Src subcellular localization and substrate phosphorylated by the c-Src kinase at mitosis. It interactions. Src becomes activated near mitosis contains a KH domain implicated in RNA binding and (Chackalaparampil and Shalloway, 1988; Morgan et several proline-rich motifs that resemble known SH3 al., 1989), and is required for transition from the G2 binding sites. The SH3 domains of c-Src, phosphatidy- phase to mitosis (Roche et al., 1995). linositol 3-OH kinase, phospholipase C-g and Grb2 Sam68 is a 68-kDa protein that is reported to protein (containing two SH3 domains), but not other associate with the c-Src protein-tyrosine kinase during SH3 domains tested, were capable of binding Sam68 in mitosis (Fumagalli et al., 1994; Taylor and Shalloway, vitro. Synthetic peptides corresponding to the proline 1994). Although it was ®rst cloned as a Ras-GAP motifs of Sam68 inhibited with dierent eciencies the associated phosphotyrosine-containing protein (Wong binding of SH3 domains to Sam68 suggesting that the et al., 1992), it is unrelated to the major phosphoty- proline motifs of Sam68 function as speci®c SH3 domain rosine-containing 62-kDa protein, Dok, which is binding sites. Mutation of Sam68 SH3 binding sites known to associate with Ras-GAP (Carpino et al., further indicated that the SRC SH3 domain mediates 1997; Yamonashi and Baltimore, 1997). Sam68 binding of Src to unphosphorylated Sam68. Phosphor- contains a KH domain with nucleic acid-binding ylation of Sam68 by Src kinase was inhibited when the properties, multiple potential tyrosine phosphorylation Src SH3 binding site of Sam68 was mutated or when sites and several proline-rich motifs which resemble corresponding peptides were added to in vitro kinase known src homology-3 (SH3) binding sites (Wong et reactions indicating that binding of the Src SH3 domain al., 1992). The SH2 and SH3 domains of Src can to a speci®c site near the amino-terminus of Sam68 independently interact with Sam68 and contribute to (including residues 38 ± 45: PPLPHRSR) facilitates the in vivo association of Src and Sam68 (Taylor and phosphorylation of Sam68 by the Src kinase domain. Shalloway, 1994). The KH domain of Sam68 was Sam68-based proline peptides had no eect on the found to ®nd poly(U) speci®cally (Taylor and Shallo- phosphorylation of another in vitro substrate of Src, way, 1994), but its interactions with RNA in vivo enolase. These results suggest that Src eectively mounts remains to be established. The RNA binding capability Sam68 through its SH3 domain, possibly as a mechan- of Sam68 is apparently regulated by its tyrosine ism to position the kinase domain close to substrate phosphorylation status and interaction with SH3 tyrosine residues in the carboxyl-half of the protein. domains (Wang et al., 1995; Taylor et al., 1995). Suggestions for the biological role of Sam68 include Keywords: p68; SH3; Src; kinase regulation of RNA splicing, nucleocytoplasmic trans- port, mRNA stability, translation and signal transduction (Courtneidge and Fumagalli, 1994; Richard et al., 1995), and recent evidence implicates Sam68 in Introduction poliovirus RNA replication (McBride et al., 1996). In addition, Sam68 is a substrate for Cdc2 kinase which The c-Src protein tyrosine kinase (Src) and the related further suggests it may represent an important eector Src-family kinases are normally inactive in vivo as a of cell cycle progression (Fumagalli et al., 1994; consequence of intramolecular interactions involving Resnick et al., 1997; Taylor and Shalloway, 1994). their SH3 and SH2 domains (Sicheri and Kuriyan, Tyrosine phosphorylation of Sam68 in chicken 1997). Activation of these non-receptor kinases can embryo ®broblasts transfected with activated c-Src occur by a variety of mechanisms that perturb these requires an intact Src SH3 domain suggesting that intramolecular interactions. In addition to allowing the the Src SH3 domain may function to recruit substrates catalytic domain to function, activation of Src by this for the Src kinase domain (Weng et al., 1994). The Src- mechanism may also involve the interaction of its SH2 family kinase Fyn was also found to interact with and SH3 domains with substrates or other proteins Sam68 through its SH2 and SH3 domains (Richard et al., 1995). SH3 domains consisting of about 60 amino acids are present in a wide range of proteins involved *Correspondence: MF Moran, Charles H. Best Institute, 112 College in signal transduction and associated with the Street, Toronto, Ontario, M5G 1L6, Canada cytoskeleton (Pawson, 1994). They consist of two 6 Current address: Hoechst Marion Roussel, Deutschland GmbH, DG small b-sheets and three variable loops (Musacchio et Rheumatologie, H528, Postfach 35 40, D-65174 Wiesbaden, Germany al., 1994; Noble et al., 1993) and contain an extended Received 1 June 1999; revised 14 July 1999; accepted 14 July 1999 hydrophobic patch that interacts in a sequence-speci®c SH3-mediated Sam68-Src interactions ZShenet al 4648 manner with the proline-rich core of ligands. Their To better understand the role of the c-Src SH3 binding sites typically contain proline residues that domain in its interaction with substrates, we sought to adopt a left-handed polyproline type II (PPII) helix identify SH3 binding sites on Sam68, and to address conformation. Proline-rich sequence motifs having the whether phosphorylation of Sam68 by Src is facilitated consensus sequences RXXPPXP (class I motifs) or by interaction of the Src SH3 domain with proline PPXPXR (where X corresponds to no speci®c amino motifs on Sam68. In this communication we report acid, class II motifs) bind SH3 domains with opposite results from in vitro experiments which indicate that orientations (Feng et al., 1994; Lim et al., 1994). Ionic two proline motifs in Sam68 are involved in Sam68 interactions between acidic residues on the SH3 binding to the Src SH3 domain, and the interaction of domain and the arginine residue proximal to the the Src SH3 domain with these sites is an important PPXP motif also contribute to SH3-ligand binding. eector of Sam68 tyrosine phosphorylation by Src. Human Sam68 contains ®ve proline-rich candidate SH3 binding sites (Wong et al., 1992). Results

To identify SH3 domains capable of binding Sam68, GST fusion proteins (1 mg, immobilized on glutathione agarose) containing SH3 domains from various proteins were incubated with 25 nM unphosphorylated puri®ed human Sam68-KT3. After extensive washing of the beads, bound Sam68-KT3 was detected following SDS ± PAGE and immunoblotting with the KT3 antibody. Sam68 bound to the SH3 domains of the Src tyrosine kinase, p85, PLCg, and to a fusion protein containing the entire Grb2 protein which is comprised of two SH3 domains separated by a single SH2 domain (Figure 1a). Similar experiments employ- ing various mutant versions of Grb2 indicated that the interaction with Sam68 did not involve the Grb2 SH2 domain, and was primarily a function of the amino terminal SH3 domain of Grb2 (Z Shen and A Batzer, unpublished observations). Sam68 did not bind to SH3 domains derived from the Abl tyrosine kinase, Crk, the individual SH3 domains of Grb2, or the neural isoform of Src (Src+) which contain a six residue insertion,

Figure 1 Anity precipitation of human Sam68 with SH3 domain-containing GST fusion proteins, and mapping of SH3 domain binding sites. (a) Puri®ed KT3 epitope-tagged Sam68 at concentration of 25 nM was recovered following incubation with 1 mg of the indicated SH3 fusion proteins, or GST alone. Bound Figure 2 Direct interaction of Sam68 and Src SH3 domain. Sam68-KT3 was identi®ed by monoclonal anti-KT3 antibody Proteins from cell lysates bound to GST-SrcSH3 beads (lanes 1 staining and chemiluminescence imaging. (b) Schematic presenta- and 2) or puri®ed GST (0.7 mg) and GST-fusions containing tion of human Sam68. The relative positions of ®ve proline-rich proline-rich motifs of Sam68, GST-I (0.4 mg), GST-II (0.6 mg) or motifs are indicated and labeled I to V. (c) Cell lysates containing GST-III (0.7 mg), were separated by SDS ± PAGE and transferred Sam68-KT3 (0.1 mM) were combined with the indicated immobi- to nitrocellulose. Sam68-KT3 in lane 1 was detected by anti-KT3 lized GST fusion proteins in the presence of no added peptide Western blotting as described above. Other lanes were probed (lane 1) or 1.0 mM of the indicated proline-rich peptides. Sam68- with biotinylated GST-Src SH3 followed by horseradish KT3 recovered on the beads was detected by anti-KT3 Western peroxidase-streptavidin. Bound GST-Src SH3 proteins were blotting as described above identi®ed by chemiluminescence SH3-mediated Sam68-Src interactions ZShenet al 4649 relative to the c-Src SH3 domain, at one end of the other three proline-rich peptides. Peptides I and III ligand binding site (Yu et al., 1992). both inhibited the binding of the PLCg SH3 domain to To determine if the proline sites of Sam68 were Sam68, but peptide I was less eective at blocking the involved in the interaction of Sam68 with SH3 interaction than peptide III. Peptides I and IV blocked domains, synthetic peptides (numbered I ± V) corre- the association of the p85 SH3 domain with Sam68, sponding to the Sam68 proline-rich motifs shown in whereas peptide III had a minor inhibitory eect Figure 1b were tested for their ability to competitively (Figure 1c). In other experiments, proteins precipitated inhibit Sam68-KT3 association with GST-SH3 pro- by GST-SrcSH3 fusion from cell lysate (Figure 2, lanes teins. Peptides were added to cell lysates containing 1 and 2) or puri®ed GST fusion proteins containing the Sam68-KT3 followed by anity precipitation with dierent proline-rich sequences of Sam68 (Figure 2, GST fusion proteins containing SH3 domains immo- lanes 3 ± 6) were separated by SDS ± PAGE, transferred bilized on glutathione-agarose beads. Figure 1c shows to nitrocellulose, and probed with an anti-KT3 that peptide I (SRQPPLPHRSRG), corresponding to antibody (Figure 2, lane 1) or soluble biotinylated Sam68 residues 35 ± 46, and peptide III GST-SrcSH3 (Figure 2, lanes 2 ± 6). Among the (PPPPPVPRGR), corresponding to Sam68 residues proteins precipitated by GST-SrcSH3 beads, Sam68 is 295 ± 304, at concentrations of 1.0 mM abolished the the major protein which showed in vitro binding with interaction between Sam68-KT3 and GST-Grb2. SrcSH3 in far-Western suggesting that the binding Peptides I and III also signi®catnly blocked the between Sam68 and GST-SrcSH3 in cell lysates was binding of Sam68 to the Src SH3 domain, while no direct (Figure 2, lane 2). Direct binding between obvious competition for binding was observed for the biotinylated Src SH3 and fusion proteins containing

Figure 3 Requirement of Sam68 proline motifs for binding to and phosphorylation by Src. (a) Schematic presentation of wild type Sam68-KT3 (WT) and mutant derivatives containing a valine substitution at position 38 in proline motif I (P38V), deletion of residues 296 ± 301 in proline motif III (D296 ± 301), and substitution of the adjacent prolines at positions 334 and 335 with alanine and glycine in proline motif IV (PP334AG). (b) The pCDNA3 vector (lane 1) or vector encoding Sam68 (WT, lane 2), or the P38V (lane 3), D296 ± 301 (lane 4) and PP334AG (lane 5) mutants were detected in lysates from transfected HEK 293 cells. Lanes 6 ± 8 of the gel contained the indicated amounts of puri®ed Sam68-KT3 as standards used to calculate the concentration of Sam68 proteins in cell lysates for subsequent experiments. (c) Sam68-KT3 (WT) and the indicated mutant proteins were recovered from HEK 293 cell lysates bound to Src SH3 domain beads and were detected by anti-KT3 immunoblotting followed by chemiluminescence imaging on ®lm and quanti®cation by chemiluminescence-imager analysis (histogram). (d) KT3 immunoprecipitates containing equivalent amounts of the indicated Sam68 proteins were phosphorylated by Src in vitro in the presence of Mg2+ and [g-32P]ATP. The control reaction contained wild type p68 and no Src. Phosphorylation of Sam68 was quanti®ed by phosphorimager analysis and the amount of precipitated Sam68 proteins were quanti®ed by chemiluminescence-imager analysis following Western blotting. The speci®c activity of Sam68 phosphorylation is plotted relative to wilde type Sam68 which was set at 100% (histogram). Error bars in (c) show the standard deviation from two experiments and in (d) from four experiments SH3-mediated Sam68-Src interactions ZShenet al 4650 proline-rich motif I or III was also con®rmed, while proline-rich motifs II and GST alone did not bind to Src SH3 under the same condition (Figure 2, lanes 3 ± 6). Similar experiments performed with 35S-labeled GST-Grb2 also indicated that Grb2 binds directly to Sam68 sequences corresponding to peptides I and III (data not shown). Having established that the binding of Src and Sam68 was direct and since Src and Sam68 are reported to interact in vivo, their interaction was further investigated. To examine the role of the polyproline motifs of Sam68 in the context of the native protein, mutations in three dierent proline-rich motifs of Sam68 were introduced as either deletions or amino acid replacements, as depicted in Figure 3a, and tested for their interaction with the Src SH3 domain. Figure 4 Proline-rich peptides that bind the Src SH3 domain inhibit the phosphorylation of Sam68, but not denatured enolase, Wild type (WT) and mutant KT3-tagged proteins were by Src. Histogram showing 32P incorporated into puri®ed Sam68 expressed to similar levels in HEK 293 cells after or denatured enolase following their addition as substrates to Src transient transfection and their concentrations were kinase reactions containing no added peptides (open bars) or determined by comparison with puri®ed Sam68-KT3 proline peptides, I, II and III at concentrations of 0.5 mM (shaded bars) and 2 mM (black bars). Radiolabeled Sam68 (left panel) and standards (Figure 3b). Lysates containing equivalent enolase (right panel) were visualized by autoradiography and amounts of WT (56 ng) and the mutant Sam68-KT3 quanti®ed by phosphorimager analysis. The error bars show the proteins: P38V (49 ng), D296 ± 301 (54 ng) and standard deviation from three experiments PP334AG (53 ng) were then combined with immobilized GST-Src SH3. WT Sam68-KT3 bound eciently to GST-Src-SH3, but substitution of proline with implicated in the Src SH3-Sam68 interaction (de- valine at position 38 in proline motif I of Sam68 scribed above), resulted in an approximately 15% greatly diminished the ability of the mutant protein to reduction in Sam68 phosphorylation, and this eect bind the Src SH3 domain (Figure 3c). Binding of the was not dose-dependent (Figure 4). Denatured enolase P38V mutant or the site III mutant (D296 ± 301) to the is a convenient in vitro substrate for Src and other non- Src SH3 domain was reduced by 84 and 54%, receptor tyrosine kinases (Stone et al., 1991). Unlike respectively (Figure 3c). Peptide IV did not compete Sam68 however, phosphorylation of enolase by Src was signi®cantly with Sam68 for Src SH3 (see Figure 1c), not aected by the proline-containing peptides; however, substitution of two adjacent prolines in the peptides I, II and III each caused less than 20% corresponding motif in Sam68 diminished the binding inhibition of enolase phosphorylation, compared to its of the mutant protein (PP334AG) to the Src SH3 phosphorylation without the addition of the peptides, domain by approximately 21% (Figure 3c). and these minor eects were not dose-dependent To determine whether the Src SH3 binding sites are (Figure 4). Sam68 was a much more ecient substrate important for the phosphorylation of Sam68 by Src in than enolase for the Src kinase activity; approximately vitro, WT and mutant Sam68 proteins in the form of 40-fold more label was incorporated per mole of anti-KT3 immunoprecipitates, containing equivalent Sam68 compared to denatured enolase (Figure 4). amounts of Sam68 protein, were used as substrates in Src in vitro kinase reactions. WT Sam68 was eciently phosphorylated by Src in vitro, whereas no signal was Discussion detected with a KT3 immune complex which had been incubated with lysate from cells transfected with the Sam68 bound in vitro to the SH3 domains of Src, p85, control vector which does not encode Sam68 (Figure PLCg and Grb2. SH3-mediated interactions with p85 3d). Phosphorylation of the P38V mutant protein was and PLCg have been observed previously, and Grb2 greatly diminished compared to wild type Sam68, has been demonstrated to bind in an SH2 domain- containing only approximatley 18% the amount of dependent manner to Sam68 (Richard et al., 1995; 32P incorporated into an equivalent amount of wild Taylor et al., 1995). Our results indicate that Sam68 type Sam68. The D296 ± 301 and PP334AG mutants has the potential to interact with Grb2 as a function of were phosphorylated to an extent equal to approxi- the Grb2 SH3 domains, and the amino-terminal SH3 mately one-half and two thirds, respectively, of WT domain in particular (data not shown). We detected Sam68 (Figure 3d). ectopically expressed Sam68 in Grb2 immunoprecipi- The ability of the Sam68-based peptides to aect the tates suggesting these proteins can interact in vivo, phosphorylation of Sam68 by Src in vitro was however, our results suggested this was only a very examined. Addition of either peptide I or peptide III minor complex and therefore does not constitute caused a dose-dependent decrease in the phosphoryla- compelling evidence that Grb2 and Sam68 interact in tion of Sam68 by Src (Figure 4). Compared to cells (data not shown). reactions not containing added peptide, peptide I, at The demonstration that Sam68 binds only a subset a concentration of 2 mM typically resulted in an of SH3 domain containing proteins in vitro, and that approximately 62% reduction in the phosphorylation these interactions can be blocked by competing proline- of Sam68. Similarly, 2 mM peptide III resulted in an containing peptides in a sequence-dependent manner approximately 72% reduction in the phosphorylation reveals the potential of Sam68 to interact with dierent of Sam68. Peptide II, whose sequence was not SH3 containing proteins in vivo. Based on the results of SH3-mediated Sam68-Src interactions ZShenet al 4651 the in vitro binding and competition experiments, we The peptide corresponding site I in p68 employed in can conclude that at least proline motifs I, III and IV Taylor et al. (1995) contained only residues 32 ± 42 are bona ®de SH3 binding sites. However, while the (QTPSRQPPLPH) and was therefore lacking Arg-43 peptide inhibition results shown in Figure 1 suggest the which is required to satisfy a consensus sequence for various SH3 domains bound to the corresponding sites SH3 interactions. Our data that site III binds the Src in Sam68, these measurements by themselves do not SH3 domain, and that sites III and IV bind to the SH3 prove the binding is direct or whether the inhibition of domain of p85 are consistent with the ®ndings of binding caused by the peptides was competitive in Taylor et al. (1995). Richard et al. (1995) used a nature. The inability of the neuronal Src SH3 domain, version of p68 lacking amino acids 1 ± 67, and hence which contains an altered ligand binding domain missing site I, in their experiments to map the binding relative to non-neuronal c-Src (Yu et al., 1992), to site for the SH3 domain of Fyn. bind Sam68 is further evidence that Sam68 interaction The fact that the addition of the Src SH3 binding with c-Src is a conventional SH3-proline motif ligand peptides had no eect on the phosphorylation of interaction. enolase supports our model that engagement of the Src Since Sam68 has been found to interact with Src SH3 domain does not modulate the kinase domain of during mitosis and to be a substrate of Src, the nature c-Src that is not phosphorylated at Tyr 527, but rather and possible function of their interaction was investi- facilitates the substrate-kinase interaction. It is likely gated. The Src SH3 domain bound to puri®ed Sam68 that Sam68 is a superior substrate of Src compared suggesting their interaction was direct, and this was with denatured enolase, which is itself rich in tyrosine further supported by data shown in which recombinant residues near acidic residues preferred by Src, because Src SH3 proteins bound directly to ®lters containing its SH3 binding sites provide a means to bind and either whole Sam68 molecules or GST fusion proteins possibly properly position Src to facilitate its bearing either proline motif I or III of Sam68 (Figure phosphorylation. One consequence of the Src SH3 2). The peptide competition experiments further domain interaction with Sam68 would be the creation indicated that sites I and III mediate Src binding to of Src SH2 binding sites in Sam68. Therefore, the Sam68. In the context of full length Sam68, wherein the binding and phosphorylation of Sam68 by Src may accessibility of any given site might be aected, the occur in two waves, wherein the initial SH3-mediated most amino-terminal proline motif was most important interactions are followed by the formation of for Src interaction (Figure 3d). secondary SH2 domain-mediated complexes. In vitro kinase reactions indicated that Sam68 is an Phosphorylation of amino-terminal sites in c-Src by ecient substrate for the Src kinase activity, and that cyclin-dependent and other kinases is accompanied by mutation of the site I proline motif dramatically the release of Src from the auto-inhibitory eects of the reduced the phosphorylation of Sam68 by Src in SH2 domain-phosphotyrosine 527 interaction, and may vitro. Since the SH3 domain of Src can participate in coincide with breakdown of the nuclear membrane an auto-inhibitory interaction with a motif located when Src may gain access to Sam68, which is otherwise between the SH2 and kinase domains (Xu et al., 1997), nuclear (Chackalaparampil and Shalloway, 1988; it is conceivable that SH3 interactions may serve to Wong et al., 1992). The intramolecular interactions of activate the Src kinase domain. However, our results the adjacent SH3 domain may also be broken as a suggest that this is not the case with Sam68, since consequence of Src phosphorylation at mitosis, which addition of proline peptide I to Src in vitro kinase would allow for SH3-mediated interactions with Sam68 reactions containing Sam68 as substrate did not leading to its phosphorylation. The resultant interac- stimulate, but rather inhibited Sam68 phosphoryla- tions and modi®cations of Sam68 may aect its tion. This result suggests that proline site I located near interactions with speci®c RNA molecules that may be the amino-terminus of Sam68 may function to provide important for cell cycle progression. a docking site for the Src SH3 domain which may thereby position the kinase domain to phosphorylate Sam68 at sites known to be located near the carboxyl Materials and methods terminus (Wong et al., 1992). We note that the puri®ed c-Src kinase used in this study is not tyrosine Antibody and peptides phosphorylated at position 527 (Lydon et al., 1992), and hence resembles mitotic Src in this regard Polyclonal antibody against Sam68 (S331) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), (Bagrodia et al., 1994). monoclonal antibody against Grb2 was from Transduction While the results shown in Figures 1 and 2 indicate Laboratory (Lexington, KY, USA) and monoclonal anti- that site I in Sam68 is the major binding site for Src, KT3 antibody was produced from hybridoma culture mutation of site III decreased to a lesser extent Sam68 supernatant. Five peptides were synthesized (Vetrogen Ltd., binding to Src and phosphorylation by Src. Whether London, ON, Canada) which correspond to the proline rich site III becomes a more dominant determinant of motif sequences of human Sam68: I, SRQPPLPHRSRG Sam68 binding and phosphorylation when the pre- (residues 35 ± 46); II, TQPPPLLPPSAT (residues 51 ± 72); III, ferred site is absent, or whether site III provides an APPPPPVPRGRG (residues 294 ± 305); IV, independent docking site that directs the phosphoryla- TRGVPPPPTVRG (residues 330 ± 341); V, QRIPLPPP- tion of dierent sites in Sam68 from those eected by PAPE (residues 353 ± 364). The peptides were veri®ed by mass spectroscopy analysis. site I remains to be determined and is under investigation. Two previous reports did not implicate site I as Plasmids important in p68 interactions with the SH3 domains of Human Sam68 cDNA (Wong et al., 1992) containing the Src (Taylor et al., 1995) and Fyn (Richard et al., 1995). DNA sequence for the C-terminal amino acid extension SH3-mediated Sam68-Src interactions ZShenet al 4652 TPPPEPET (KT3 epitope from the simian virus 40 large-T 958C for 5 min, and the eluted proteins were then subjected antigen (MacArthur and Walter, 1984)) was subcloned from to SDS ± PAGE and anti-KT3 Western blotting. the SF9 cell transfer vector p62R2 as an EcoRI/BamHI For anity precipitation of Sam68 from cell lysates, lysate fragment into similarly-cut pCDNA3 (Invitrogen) and containing 5 mg Sam68 (for experiments depicted in Figure 1) pUC119 to generate pCDNA3-Sam68 and pUCSam68, or lysate containing 100 ng Sam68-KT3 (for Figure 2) was respectively. Mutations were introduced by polymerase chain incubated with 25 mg GST or GST fusion proteins reaction-mediated site-directed mutagenesis by using proof- immobilized on beads at 48C. After 90 min the beads were reading thermostabile DNA polymerase (Stratagene and New washed three times with lysis buer, dissociated in 30 mlof England Biolabs) and pUCSam68 as template. The speci®c 26 Laemmli buer (see below) and heated at 908C for 4 min nucleotide changes introduced were: nucleotides 112 ± 114 prior to SDS ± PAGE. were changed from CCG to GTA; nucleotides 922 ± 926 were Immunoprecipitations were performed by incubating cell changed from CCACC to GCCGG; nucleotides 886 ± 903 lysates (0.6 mg total protein) and 1 mg antibody at 48C for (CCTCCACCACCTGTTCCC) were deleted. DNA restric- 60 min followed by the addition of 5 ml protein A agarose tion fragments containing these mutations were subcloned beads of anti-mouse IgG agarose (Sigma, St. Louis, MO, into pCDNA3-Sam68. USA). After incubation for 60 min the beads were washed GST-Grb2 fusion proteins were prepared as previously and the protein was subjected to SDS ± PAGE as described described (Lowenstein et al., 1992) with the exception that above. pGEX-2T (Pharmacia) was used. The following fusion proteins of Grb2 were prepared: full length (amino acids 1 ± 217), N-SH3 (4 ± 55) and C-SH3 (161 ± 216). GST fusion Far-Western and Western blotting proteins containing Sam68 residues 18 ± 45 (GST-I) and residues 50 ± 77 (GST-II) were constructed by using cDNAs Electroblotted proteins on nitrocellulose ®lters were blocked ampli®ed by PCR from the human Sam68 cDNA (Wong et in PBST (10 mM Na2HPO4,1mM KH2PO4, pH 7.4, 137 mM al., 1992) and the appropriate oligonucleotide primers. The NaCl, 2.7 mM KCl, 0.2% Tween-20) supplemented with 3% insert used to make GST-III, containing Sam68 codons 277 ± BSA and then incubated with PBST containing KT3 303, was made by annealing synthetic oligonucleotides. The antibody (0.5 mg/ml). Bound antibody was detected follow- accuracy of all constructs was con®rmed by sequencing. ing incubation of washed ®lters with horseradish peroxidase- conjugated anti-mouse Ig (Bio Rad, 1 : 20 000 dilution in PBST) for 60 min followed by chemiluminescence develop- Cell cultures ment imaging and quanti®cation. Alternatively, ®lters were blocked in PBST supplemented with 3% BSA and then Rat-1 ®broblasts expressing Sam68-KT3 were maintained in incubated with PBST containing GST-SH3 protein (10 mg/ Dulbecco's modi®ed Eagle's medium (DMEM) supplemented ml) or biotinylated GST-SH3 proteins (1 mg/ml) for 1 h. The with 10% fetal bovine serum (FBS) and 200 mg/ml G418 membranes were then incubated with horseradish peroxidase (Gibco-BRL) and kept in a 5% CO atmosphere. Human 2 conjugated streptavidin in PBST containing 1% BSA embryonic kidney (HEK 293) cells were maintained in followed by enhanced chemiluminescence detection as DMEM supplemented with 4 m -glutamine and 10% ML described above. FBS. For transient expression, HEK 293 cells were plated in 10-cm tissue culture dishes 18 ± 22 h before transfection with calcium-phosphate precipitates containing 20 mg plasmid DNA (Fam et al., 1997). Two days after transfection cells Tyrosine kinase assays were harvested by scraping with a rubber policeman and Puri®ed Sam68-KT3 was phosphorylated by incubating 0.1 ± collected by low speed centrifugation, washed once with ice- 0.15 mg of the puri®ed protein in 20 ml of buer containing cold phosphate-buered saline (PBS) and stored at 7708Cor 20 mM TrisCl, pH 8.0, 5 mM MgCl2,5mM ATP, 1 mM DTT, lysed immediately. For lysis, cell pellets from approximately 1% glycerol, 0.1% ovalbumin, 0.8 mCi [g-32P]ATP (3000 Ci/ 56106 cells were resuspended in 2 ml of lysis buer (50 mM mmole, Du Pont, Boston, MA, USA) and 20 ng non- TrisCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5 mM 4- myristylated c-Src kinase, which was puri®ed as described (2-aminoethyl)-benzenesulfonyl¯ouride (AEBSF and 20 mg previously (Lydon et al., 1992). After 15 min incubation at aprotinin per ml) and incubated on ice for 10 ± 30 min, 258C the reaction was stopped by adding 10 mlof36 followed by clari®cation by centrifugation at 20 000 g for Laemmli buer (6% SDS, 30% glycerol, 182 mM Tris-Cl, 20 min. Protein concentrations were determined by using a pH 6.8, 0.015% bromophenol blue), heated at 908C for 4 min Coomassie Blue assay (Pierce Inc., Pockford, IL, USA) and followed by SDS ± PAGE (8% polyacrylamide). The gel was using BSA as a protein standard. The concentration of soaked in 10% acetic acid and 10% isopropranol overnight, Sam68-KT3 in cell lysates was determined by comparative dried, and then radiolabeled Sam68 was detected with Kodak anti-KT3 immunoblotting of (SDS ± PAGE-resolved) lysates XAR ®lm and quanti®ed by a phosphorimager analysis. and puri®ed Sam68-KT3 produced in insect cells (for To analyse the phosphorylation of immunoprecipitated example, see Figure 3B, lanes 6 ± 8) (Wong et al., 1992); Sam68, immune complexes were washed three times with lysis chemiluminescence of peroxidase-stained proteins were buer and then resuspended in 20 ml of buer containing quanti®ed with a Bio Rad phosphorimager (model GS-250). 20 mM Tris-Cl, pH 8.0, 3 mM MgCl2,6mM ATP, 1 mM DTT, 1% glycerol, 0.1% ovalbumin, 4 mCi [g-32P]ATP (3000 Ci/ mmole, Du Pont, Boston, MA, USA) and 90 ng of puri®ed Anity precipitation, immunoprecipitation Src kinase. After incubation at room temperature for 20 min Sam68-KT3 was puri®ed by immunoanity chromatography the reaction was stopped by adding 10 mlof36 Laemmli as described previously (Wong et al., 1992). GST fusion buer. Rabbit muscle enolase (Worthington) was dissolved at proteins were puri®ed on glutathione-agarose according to 4 mg/ml in a buer containing 30 mM HEPES, pH 7.0,

Smith and Johnson (1988). GST SH3 proteins (1 mg protein) 1.2 mM DTT, 1.2 mM MgCl2 and 30% glycerol and immobilized on glutathione beads were incubated with gentle denatured by addition of 2 vol 50 mM acetic acid. After agitation for 60 min at 48C in binding buer (phosphate 10 min at 378C an equal vol 100 mM TrisCl, pH 8.0, 20 mM

buered saline, 10% glycerol, 2 mM dithiothreitol, 0.05% MgCl2 was added. The kinase reaction mixture was similar to Triton X-100) containing 25 nM Sam68-KT3. Beads were that used with Sam68, except that Sam68 was replaced by collected by centrifugation and washed three times with 0.8 mg denatured enolase and the concentrations of TrisCl,

binding buer and then resuspended in Laemmli sample glycerol, and MgCl2 were 25 mM, 1.5%, and 6 mM, buer. Bound proteins were released by heating samples at respectively. SH3-mediated Sam68-Src interactions ZShenet al 4653 Acknowledgements National Cancer Institute of Canada with funds provided We thank Li-Jia Zhang and Hui Chen for technical by The Canadian Cancer Society. MF Moran is a Medical assistance. This work was supported by an operating Research Council of Canada Scientist. grant and Scientist Award to MF Moran from The

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