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 determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further 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-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.
Chan
P M
PM
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook 11794, USA.
Keller
P R
PR
Connors
R W
RW
Leopold
W R
WR
Miller
W T
WT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D019151:Peptide Library; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor; D017421:Sequence Analysis; D013379:Substrate Specificity
D019151:Peptide Library; D010748:Phosphopeptides; D000255:Adenosine Triphosphate; C099852:PDGF receptor tyrosine kinase; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor