A synthetic peptide corresponding in sequence to residues 6-20 of p34, cdc2(6-20), and a substitution analogue, cdc2(6-20)F15K19, which contains Thr-14 as the only phosphorylation target were used as substrates to identify a novel protein kinase in bovine thymus cytosol. The kinase catalyzed the phosphorylation of Thr-14 in both peptides and was purified extensively on the basis of its peptide phosphorylation activity. Upon SDS-polyacrylamide gel electrophoresis analyses, the purified samples consistently displayed a prominent 43-kDa protein band which could undergo in gel autophosphorylation, thus suggesting that this band represented the kinase protein. The suggestion was supported further by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhibited by millimolar concentrations of cAMP. The kinase was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide analysis of the phosphorylated Nclk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this thymus kinase is a novel Cdk inhibitory protein kinase, distinct from the recently cloned dual functional and membrane-associated Cdc2 inhibitory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G.(1995) Science 270, 86-90).

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