Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding.

摘要: We have developed a competition binding assay to quantify relative affinities of isolated Src-homology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMXM or YVXM recognition motifs bound PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, polyoma middle T antigens; matched, unphosphorylated did not bind. A scrambled corresponding GAP PLC-gamma FGF, EGF receptors 30-100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed an from PLC-gamma: site PDGF receptor Tyr1021 binds approximately 40-fold higher than YMXM-phosphopeptide. conclude essential features specific phosphoprotein/SH2 interactions can be reconstituted using truncated versions both phosphoprotein (a phosphopeptide) cognate domain-containing protein (the domain). results differences in conferred by linear sequence phosphotyrosine.