Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.
Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, 208066, New Haven, CT, 06520, USA.
Chad J
CJ
Miller
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, 208066, New Haven, CT, 06520, USA. ben.turk@yale.edu.
Benjamin E
BE
Turk
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D016384:Consensus Sequence; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D019151:Peptide Library; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D007202:Indicators and Reagents; D019151:Peptide Library; D011494:Protein Kinases
Enzyme specificity; Kinase assay; Peptide libraries; Protein kinases; Signal transduction; Substrate profiling