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

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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 TcRζ/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.