SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55

Abstract

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.

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).…”

The Three-dimensional Structure of the ZAP-70 Kinase Domain in Complex with Staurosporine

“…Cloning of ZAP-70 Truncated Kinase Domains-Human ZAP-70 cDNA fragments (GenBank TM accession number L05148) were synthesized by PCR and cloned using the NcoI (5Ј) and XhoI (3Ј) sites of pTriEx (Novagen), the BamHI (5Ј) and NotI (3Ј) sites of pFastBacHTb (Invitrogen), the BamHI (5Ј) and EcoRI (3Ј) sites of pVL1393 (Invitrogen), or the BamHI (5Ј) and the NotI (3Ј) sites of pEBB (16). The pFastBacHTb plasmids were designed to express N-terminal His-tagged proteins containing the sequence MSYYHHHHHHDYDIPTTENLYFQGAMGSHM prior to the first ZAP-70 residue.…”

Structure and regulation of Src family kinases

“…For example, the SH3 domain of the Gads T-cell adapter protein binds an RxxK motif rather than the characteristic PxxP motif (Berry et al, 2002;Liu et al, 2003). Within the Src family, the Fyn SH3 can bind the proline-independent motif RKxxYxxY found in the immune cell adaptor SKAP55 (Kang et al, 2000). Additionally, the Fyn kinase is regulated by a nonproline 'surface-surface' interaction of its SH3 domain with the SH2 protein SAP (Chan et al, 2003;Latour et al, 2003).…”