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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 TcR
zeta/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.
