Beth Israel Hospital

Zhou Songyang

Lewis C Cantley

The invention provides a method for determining an amino acid sequence motif for a phosphorylation site of a protein kinase. In the method of the invention, a protein kinase is contacted with an oriented degenerate peptide library, peptides within the library which are substrates for the kinase are converted to phosphopeptides and the phosphopeptides are separated from non-phosphorylated peptides. The isolated phosphopeptides are sequenced and an amino acid sequence motif for the phosphorylation site is determined based upon the relative abundance of different amino acids residues at each degenerate position. The invention also provides peptide substrates for protein kinase A, cell cycle control kinases (including cyclin B/p33.sup.cdc2 and cyclin A/p33.sup.CDK2), src family kinases (including pp60.sup.c-src and pp60.sup.v-src), EGF receptor, p92.sup.c-fps/fes, lck, c-abl, PDGF receptor, FGF receptor, insulin receptor, casein kinase II, NIMA kinase, phosphorylase kinase, Cam kinase II and Erk1 based upon amino acid sequence motifs for the phosphorylation sites of these kinases.

Substrate specificity of a protein kinases

The invention provides a method for determining an amino acid sequence motif for a phosphorylation site of a protein kinase. In the method of the invention, a protein kinase is contacted with an oriented degenerate peptide library, peptides within the library which are substrates for the kinase are converted to phosphopeptides and the phosphopeptides are separated from non-phosphorylated peptides. The isolated phosphopeptides are sequenced and an amino acid sequence motif for the phosphorylation site is determined based upon the relative abundance of different amino acids residues at each degenerate position. The invention also provides peptide substrates for protein kinase A, cell cycle control kinases, src family kinases, the EGF receptor and p92.sup.c-fps/fes based upon amino acid sequence motifs for the phosphorylation sites of these kinases.

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Brookline, MA, United States of America

Zhou Songyang