![]() These consensus sequences were shown to vary among different kinases ( Table 1). Early studies with peptides derived from the phosphorylation sites of known protein substrates established the idea of the consensus sequence, in which key residues proximal to the phosphoacceptor residue were particularly critical for efficient phosphorylation ( 3). Most kinases phosphorylate substrates in the context of specific sequence motifs that interact favorably with the kinase catalytic cleft ( 2). Kinases interact directly with their substrates through both “proximal” interactions between the kinase active site and the site of phosphorylation, as well as more “distal” non-catalytic interactions to substrate docking sites. Protein kinases target specific downstream substrates in cells through a combination of direct and indirect physical interactions ( 1). Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high throughput screening or incorporation into biosensors. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. 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. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Eukaryotic protein kinases phosphorylate substrates at serine, threonine and tyrosine residues that fall within the context of short sequence motifs. ![]()
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