We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the ؉1 and ؊3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.The essential role of protein kinases in regulating signal transduction was established with the discovery of cyclic AMPdependent protein kinase (PKA) (12). To respond to different extracellular stimuli, distinct groups of protein kinases have evolved. Each protein kinase is thought to phosphorylate a unique set of targets in the cell. The substrate specificities of protein kinases are therefore crucial for the fidelity of signaling events.The classical approach for studying the specificity of a protein kinase is to compare the phosphorylation kinetics of synthetic peptides on the basis of known sequences phosphorylated by the kinase. This procedure is helpful in identifying the amino acids critical for efficient phosphorylation. However, it is not practical to synthesize and study each of the billions of possible variations of sequences that must be considered. Moreover, it is extremely difficult to apply this approach to study the specificity of a protein kinase with no known substrates. To overcome these problems, we developed a method for determining the primary sequence specificities of protein kinases by using an oriented degenerate peptide library (21). Optimal peptide substrates of a given protein kinase are identified by phosphorylation of a pool of degenerate peptides containing billions of different species. The specificities determined for PKA, CDC2, and CDK2 by using this technique were consistent with known substrates of these kinases. The results also allowed the prediction of in vivo kinase substrates. Synthetic peptides based on predicted optimal motifs were shown to act as low-K m substrates for the kinases studied. Therefore, this method is a useful tool for studying substrate specificities of protein kinases.We present here the specificities of eight additional protein Ser/Thr kinases: CDK5, casein kinase I (CKI) ␦ and ␥, casein kinase II (CKII), NIMA, calmodulin-dependent (Cam) kinase II, Erk1, and phosphorylase kinase. Our findings demonstrate that each of these protein kinases has a distinct optimal peptide substrate. Critical determinants for recognition by the protein kinas...