Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity.
To search for peptides which serve as substrates for protein kinases, an approach based on peptide libraries has been developed. These peptide libraries are chemically synthesized by a modified "divide-couple-recombine" strategy. After reaction with the kinase of interest, the most highly phosphorylated substrate (selected from the library) is identified using on-line liquid chromatography-electrospray mass spectrometry (LC-ESMS). Negative ion LC-ESMS with stepped collision energy is used to identify phosphorylated peptides in the enzyme reactions. As predicted, the cAMP-dependent protein kinase is shown to preferentially phosphorylate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in a library consisting of 19 variants of Kemptide substituted at position 2. Additional experiments have been carried out on the nonreceptor tyrosine kinase v-Abl using a peptide library based on the v-Src autophosphorylation site (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly). These results indicate that Ile is the optimal residue at the position N-terminal to tyrosine. Individual peptides containing the Glu-Asp-Ala-Ile-Tyr motif have Vmax/Km values 6-fold higher than the peptide based on the autophosphorylation site itself, confirming the results of the library experiments. This motif has been identified in several tyrosine kinases at a position in the sequence not previously reported to serve as a phosphorylation or autophosphorylation site.
Till
J H
JH
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794.
Annan
R S
RS
Carr
S A
SA
Miller
W T
WT
D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis; D010748:Phosphopeptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; D010455:Peptides; D010748:Phosphopeptides; C037642:kemptide; D011494:Protein Kinases