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 transduc- tion (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).