The use of synthetic peptides for defining the specificity of typrosine protein kinases

Abstract

The tyrosine protein kinases are a large family of enzymes that may be involved in regulating cell growth and differentiation. An important property of these enzymes is their substrate specificity. Defining the specificity of these enzymes will contribute to a greater understanding of their biological functions. Synthetic peptides provide a useful means for studying the specificities of protein kinases. The utility of synthetic peptides for specificity studies is exemplified by the results obtained with the cyclic nucleotide-dependent protein kinases. A large number of synthetic peptides have been tested as substrates for these enzymes. There are three important conclusions from this work. First, in terms of primary sequence, the two cyclic nucleotide-dependent protein kinases both require the presence of a pair of basic residues on the N-terminal side of the phosphorylatable residue. Second, recognition of a specific secondary structure of these enzymes. Third, the specificities of the two cyclic nucleotide-dependent protein kinases are remarkably similar in terms of both primary and secondary structure recognition. Sequences at the sites of tyrosine phosphorylation often show the presence of numerous acidic residues on the N-terminal side of the tyrosine. This result led to the suggestion that these acidic residues might be important for the recognition of these sites by the tyrosine protein kinases. Specificity studies using synthetic peptidese have provided some experimental verification of this concept. In the case of four tyrosine protein kinases, LSTRA cell tyrosine protein kinase, epidermal growth factor receptor kinase, insulin receptor kinase and pp60 gag-yes tyrosine protein kinase, the presence of acidic residues on the N-terminal side of the tyrosine in a synthetic peptide was a favorable determinant. In the case of a fifth member of the tyrosine protein kinase family, namely pp60 src kinase, the data are less clear that the presence of acidic residues is involved in substrate recognition. This enzyme readily phosphorylated several peptides that did not have acidic residues on the N-terminal side of the tyrosine. Although there is some indication that the presence of acidic residues on the N-terminal side of the tyrosine is a factor in substrate recognition by several of the tyrosine protein kinases, the changes in kinetic parameters with the various peptide substrates were rather small. In addition, some sites phosphorylated in proteins do not have any acidic residues on the N-terminal side of the phosphorylated tyrosine residue. The presence of acidic residues on the primary sequence near the tyrosine is not an absolute determinant for the recognition of sites of phosphorylation by the tyrosine protein kinases. As in the case of the cyclic nucleotide-dependent protein kinases, secondary structure is probably also important for the recognition of sites of phosphorylation by the tyrosine protein kinases.