Analysis of protein kinase specificity by peptide libraries and prediction of in vivo substrates

Abstract

This chapter discusses the analysis of protein kinase specificity by peptide libraries and prediction of in vivo substrates. Protein kinases are generally divided into three categories based on their abilities to phosphorylate serine/threonine, tyrosine, or both residues. Protein kinases are most commonly obtained through overexpression as recombinant proteins in either bacteria or eukaryotic cells. The specificity of a protein kinase is governed by several factors including its intracellular localization and interaction with its substrates. The most important factor is the specificity of the kinase domain. The chapter outlines peptide library design strategies, detail the experimental techniques for peptide library synthesis and selection, and discuss how the data obtained are analyzed and used to predict in vivo substrates for protein kinases. The chapter presents a three-step peptide library screening process. Proper orientation of the library is easily achieved for protein kinases because the phosphoryl table amino acids can be used to orient the library. The chapter concludes with a discussion on predicting kinase substrates and developing inhibitors of protein kinases.