AMINO-TERMINAL SEQUENCE DETERMINANTS FOR SUBSTRATE RECOGNITION BY PLATELET-DERIVED GROWTH-FACTOR RECEPTOR TYROSINE KINASE

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determin ants for platelet-derived growth factor receptor (PDGFR) tyrosine kina se specificity, a peptide library with three degenerate positions N-te rminal to tyrosine was constructed, After reaction with PDGFR, the mos t abundant phosphopeptides were isolated by immunoaffinity chromatogra phy on a column containing monoclonal anti-phosphotyrosine antibody, F urther separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala- Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-A rg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where unde rlined residues are in the degenerate positions of the peptide library . Kinetic analyses of the three individual peptides (synthesized separ ately) showed these peptides to be among the best reported substrates for PDGFR, Our results expand the range of amino acid residues that ha ve been shown to serve as recognition elements for receptor tyrosine k inases.