Atypical Recognition Consensus of CIN85/SETA/Ruk SH3 Domains Revealed by Target-assisted Iterative Screening

Abstract

Target-assisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, c-Cbl, BLNK, Cbl-b, AIP1/Alix, SB1, and CD2 proteins, as well as the prediction of CIN85 novel-interacting partners in protein databases. Synaptojanin 1, PAK2, ZO-2, and TAF II 70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione S-transferase pulldown experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting.One of the initial approaches to study a novel gene product is the identification of its interacting partners. As increasingly more proteins become functionally characterized it is more and more probable that the identification of interacting partners of a novel protein will suggest a possible function for this protein by associative reasoning and, thus, provide a framework for its characterization. Using this approach, a novel CIN85/CMS adaptor protein family was functionally implicated in a remarkably wide spectrum of different cellular processes such as the down-regulation of receptor tyrosine kinases and endocytosis (through the association of CIN85 with Cbl/Cbl-b and endophilins) (1, 2), apoptosis (through the association of CIN85 with p85-phosphatidylinositol 3-kinase and AIP1/Alix) (3, 4), and adhesion phenomena (through the association of CMS with p130Cas and the interactions of CD2AP/CMS with CD2 and cortactin) (5-7). However, the mapping of CIN85/CMS SH3 domains binding sites within the characterized CIN85/CMS interactors has remained an elusive goal, giving rise to discordant conclusions in the literature. The absence of information on the recognition properties of CIN85 SH3 domains hampers the further characterization of its interactions and the understanding of the biological role of this protein, which has emerged as a highly connected node linking functionally diverse protein interaction sub-networks. Intriguingly, it has been suggested that CIN85/CMS may have unique function(s) in higher eukaryotes, as CIN85 orthologues of the human and rat proteins were not found in Caenorhabditis elegans, Drosophila melanogaster, or yeast (8).At least five discrete protein interaction modules can be inferred from the analysis of the CIN85 amino acid sequence. These are three consecutive SH3 domains at the N terminus followed by a central proline-rich region and a coiled-coil domain situated at the C terminus (Fig. 1). CIN85 was shown to homodimerize through its coiled-coil domains, and a number of proteins interacting with the CIN85 proline-rich region were identified including Grb2, p130Cas , endophilins, p85-phosphatidylinositol 3-kinase, and cortactin (1,3,7,9,10). The SH3 domains of CIN85 bound c-Cbl, Cbl-b,...

Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3 domain

“…Recently, a number of SH3 domains have been reported to bind unconventional non-PXXP-dependent motifs in target proteins. Such examples include an RKXXYXXY motif in SKAP55 that binds the Fyn and Lck SH3 domains (Kang et al, 2000), a PXXDY motif in several proteins that binds the Eps8 SH3 (Mongiovi et al, 1999), a WXXXFXXLE motif in p67phox and Pex5p that binds the Pex13p SH3 domain (Barnett et al, 2000;Kami et al, 2002) that bind the Mona/Gads SH3 domain (Lewitzky et al, 2001;Harkiolaki et al, 2003;Lewitzky et al, 2004), and a PX(P/A)XXR motif that binds to the CIN85/SETA/ Rut protein SH3 domains (Kurakin and Bredesen, 2002;Kurakin et al, 2003). For some atypical SH3 domains, for example Mona/Gad, SKAP55, and Eps8, the unconventional peptides have been suggested to bind to regions including the classical PXXP binding site (Harkiolaki et al, 2003).…”

Clues to CD2-associated Protein Involvement in Cytokinesis

“…We found that the anillin N-terminal fragment (1-155), which contains a proline-rich region, was required to interact with CD2AP. The PX(P/A)XXR consensus site has been recently identified as the target of SH3 domains of CD2AP or CIN85 (for c-Cbl interacting protein of 85 kDa), which is a closely related CD2AP protein (Kurakin et al, 2003). A careful examination of the anillin sequence led us to propose its 26-PTAAPR-31 sequence as a likely candidate for this interaction.…”

The PDZ Domain as a Complex Adaptive System

“…Binding of 95 artificial phage-displayed ligands to six PDZ domains of PSD95 and SAP97. Binding histograms were obtained by individual phage ELISA performed on purified GST fusions of the indicated domains immobilized in micro-titer plate wells [40]. For accurate relative affinity evaluations and cross-domain comparison, the slopes of individual ELISA kinetics were determined and normalized by the highest slope value in each of the six sets shown.…”