Proteomics 2007, 7, 1775–1785 DOI 10.1002/pmic.200601006 1775 RESEARCH ARTICLE Systematic identification of SH3 domain-mediated human protein–protein interactions by peptide array target screening Chenggang Wu1*, Mike Haiting Ma1*, Kevin R. Brown2, Matt Geisler1, Lei Li1, Eve Tzeng1, Christina Y. H. Jia1, Igor Jurisica2 and Shawn S.-C. Li1 1 Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada 2 Ontario Cancer Institute, Northeast Structural Genomics Consortium, Toronto, Ontario, Canada Systematic identification of direct protein–protein interactions is often hampered by difficulties Received: December 12, 2006 in expressing and purifying the corresponding full-length proteins. By taking advantage of the Revised: February 1, 2007 modular nature of many regulatory proteins, we attempted to simplify protein–protein interac- Accepted: February 23, 2007 tions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein–protein interactions, we exam- ined a set of interactions mediated by the PLCg1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCg1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCg1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome. Keywords: HPK1 / Interactome / Peptide array / PLCg1 / SH3 domain 1 Introduction Correspondence: Dr. Shawn S.-C. Li, Department of Biochemistry and the Siebens-Drake Research Institute, Schulich School of A description of the global protein connectivity in a cell is of Medicine and Dentistry, University of Western Ontario, London, enormous value to our understanding of all essential cellular Ontario, Canada N6A 5C1 E-mail: [email protected] functions and disease mechanisms [1–3]. Until recently, ge- Fax: 11-519-661-3175 nome-wide mapping of protein interactions, or the inter- actome, has been focused on model organisms such as Sac- Abbreviations: co-IP, coimmunoprecipitation; HEK, human charomyces cerevisiae[4–6], Caenorhabditis elegans [7], and embryonic kidney; HPK1, hematopoietic progenitor kinase 1; Drosophila melanogaster [8]. By taking advantage of the high- OPHID, online predicted human interaction protein database; PATS, peptide array target screening; PIDs, peptide or protein interaction domains; SH, Src homology; Y2H, yeast two-hybrid * Both these authors contributed equally to this work. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 1776 C. Wu et al. Proteomics 2007, 7, 1775–1785 throughput ability of the affinity purification coupled with membrane array was used in the discovery of potential Cdc4 MS (AP-MS) approach and systematic yeast two-hybrid substrates in yeast [31]. However, due to significant differ- (Y2H) [9], interactome frameworks have been established for ences in genome size and protein architecture between these organisms, which in turn have provided unprece- humans and yeast, most human protein interactions, espe- dented insights into many important biological processes cially those occurring in signal transduction and cell–cell [10]. Recent work on large-scale identification of human communication, may not be directly inferred from the yeast protein interactions by Y2H has, however, changed the land- interactome. Indeed, a comparative analysis of over 70 000 scape of the human interactome drastically [11, 12]. These binary interactions identified to date for yeast, worm, fly, and studies outlined a skeleton for part of the human inter- human revealed that only 16 interactions are common to all actome and served as an impetus for more comprehensive four species [32], suggesting that networks of protein–protein mapping of the human interactome [13]. interactions differ significantly from one species to another. Although the current state of knowledge does not allow a Because most of the regulatory proteins in humans have a thorough comparison between the human interactome and modular architecture, domain-mediated interactions may that of a model organism such as S. cerevisiae, for which a constitute a significant part of the regulatory networks found plethora of genetic, mass spectrometric, and Y2H data are in a human cell. Thus, high-throughput strategies that make available [14–19], the human interactome is believed to be use of these unique features provide attractive means to more complex than that of the yeast. On one hand, the two experimentally map regulatory networks in humans. For species differ significantly in proteome size, cellular diversity, instance, MS-aided protein identification was applied to the and compartmentalization; on the other hand, protein struc- mammalian WW and 14-3-3 domain families to identify their tures vary considerably from S. cerevisiae to Homo sapiens [20– respective interaction networks [33, 34]. These studies 22]. Compared to yeast, there is a drastic expansion of modular revealed that modular domains connect multiple proteins in domains, especially the so-called peptide or protein interaction a network and are involved in regulating a wide range of cel- domains (PIDs), in the human genome. For instance, an esti- lular processes. Recently, domain macroarrays were mated 120 copies of the Src-homology (SH) 2 domains are employed to quantitatively characterize a protein interaction encoded by the human genome while no functional SH2 do- network mediated by the ErbB receptor [35]. In the present main has so far been identified in yeast [23]. Consequently, study, we applied a peptide array target screening (PATS) signaling events regulated by tyrosine phosphorylation and strategy to map interactions stemmed from a group of 12 dephosphorylation do not take place in yeast. Similarly, a hu- SH3 domains taken from eight human proteins. The result- man cell harbors approximately 300 SH3 domains while yeast ing interaction network linked 8 “bait” proteins to 284 “tar- contains only 28 copies of the same domain family [24]. get” proteins through 921 binary interactions. Using PLCg1 Protein–protein interactions occurring via the recogni- as an example, we confirmed a number of PATS-derived tion of a conserved sequence motif in one protein by a mod- interactions by in-solution peptide binding and affinity pull- ular domain in another are a common means employed by a down or coimmunoprecipitation (co-IP) assays carried out on cell to assemble complex protein networks seen in signal intact proteins. Furthermore, we demonstrate that the PLCg1 transduction and underlie specific substrate recognition by SH3 binds to the hematopoietic progenitor kinase 1 (HPK1) protein kinases and phosphatases [25–27]. Several important in vivo and thereby negatively regulates its kinase activity. features of interaction domains make them ideal targets also for large-scale human interactome mapping. First, interac- tion domains are found in thousands of human proteins. 2 Materials and methods Second, they range from 30 to 150 amino acids in size, ap- proximately one-third of a typical human protein, and gen- 2.1 Expression, purification, and fluorescein labeling erally fold into stable 3-D structures in solution. These char- of SH3 proteins acteristics render interaction domains particularly amenable to biochemical and biophysical manipulations. Third, many SH3 domains were expressed and purified as previously PIDs bind short peptide motifs as well as they do the corre- reported [36]. To facilitate specific attachment of fluorescein, sponding native proteins [28]. Therefore, by identifying spe- each SH3 domain was engineered to contain a C-terminal cific peptides to which a PID binds, one can often deduce Gly–Gly–Cys triad sequence. FPLC-purified (His)6-SH3 pro- potential interacting proteins for that domain [29]. teins were labeled with fluorescein-5-maleimide, according An SH3 domain-mediated protein interacting network to the manufacturer’s protocol (Pierce). was first identified in yeast using a strategy that combined specificity determination by phage display libraries with 2.2 Synthesis of peptide spot arrays on cellulose experimental screening for binary protein–protein interac- membranes and probing of the peptide arrays by tions by Y2H [16]. Peptides synthesized on NC membranes SH3 domains were employed recently to map the binding partners for a group of yeast SH3 domains and to scan the human pro- Peptide arrays were assembled on a functionalized cellulose teome [30]. Similarly, a proteome-wide phosphopeptide membrane following essentially the same procedure as © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics