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

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs oth er than proline-based sequences is unclear. In this study, we report SH3 do main binding to a novel proline-independent motif in immune cell adaptor SK AP55, which is comprised of two N-terminal lysine and arginine residues fol lowed 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 stu dies demonstrated a requirement for the arginine, lysine and tandem tyrosin es 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 th e charged surface of the SH3 domain, while resonance signals for other resi dues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. E xpression of the RKGDYASY peptide potently inhibited TcR zeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 d omain binding motifs to include a tyrosine-based motif and demonstrate a re gulatory role for this motif in receptor signaling.