USE OF AN ORIENTED PEPTIDE LIBRARY TO DETERMINE THE OPTIMAL SUBSTRATES OF PROTEIN-KINASES

Background: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essen tial part in the signal transduction pathways that regulate cell growt h in response to extracellular signals. A great number of protein kina ses have been discovered, and the identification of their biological t argets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are tran smitted correctly. Previous studies have demonstrated the importance o f primary sequences within substrate proteins in determining protein k inase specificity, but efficient ways of identifying these sequences a re lacking. Results: We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented libra ry of more than 2.5 billion peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing t he mixture of products generated during a brief reaction with the kina se of interest. The optimal substrate predicted for cAMP-dependent pro tein kinase (PKA) by this technique is consistent with the sequences o f known PKA substrates. The optimal sequences predicted for cyclin-dep endent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In a ddition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specific ity. We also discuss a model incorporating the optimal cyclin B-Cdc2 s ubstrate into the known crystal structure of this kinase. Conclusions: Using the new technique we have developed, the sequence specificity o f protein kinases can rapidly be predicted and, from this information potential targets of the kinases can be identified.