[10] Design and use of peptide substrates for protein kinases

Publisher Summary This chapter focuses on the design and use of peptide substrates for protein kinases. There are three approaches to the design of protein kinase peptide substrates. The most widely used approach is to synthesize analogs of known phosphorylation site sequences. These may be from natural substrates, autophosphorylation sites, or phosphorylation sites in exogenous substrates such as histone, myelin basic protein, or caseins. The second approach is to use random polymers of amino acids such as Tyr and Glu for tyrosine kinases. The third approach is to prepare substrate analogs of the pseudosubstrate autoregulatory regions that have been found in a number of protein kinases. Synthetic peptide substrates have proved extremely useful reagents in the study of protein kinases across a wide spectrum of studies, extending from those carried out with crude extracts to structural studies with NMR and X-ray crystallography. By using peptides containing a single phosphorylatable residue, it is possible to detect protein kinases in cell extracts containing multiple protein kinase activities. The sensitivity and specificity of synthetic peptide substrates has made them the substrate of choice in the study of hormonal regulation of protein kinases. Stability and chemical purity are also major benefits of using synthetic peptide substrates. Since quantities are not usually limiting, they can often be used at saturating concentrations making it possible to obtain maximum rates of phosphorylation.