DEMONSTRATION OF CYCLIN-DEPENDENT KINASE INHIBITORY SERINE THREONINE KINASE IN BOVINE THYMUS/

A synthetic peptide corresponding in sequence to residues 6-20 of p34( cdc2), cdc2(6-20), and a substitution analogue, cdc2(6-20)F15K19, whic h contains Thr-14 as the only phosphorylation target were used as subs trates to identify a novel protein kinase in bovine thymus cytosol. Th e kinase catalyzed the phosphorylation of Thr-14 in both peptides and was purified extensively on the basis of its peptide phosphorylation a ctivity. Upon SDS-polyacrylamide gel electrophoresis analyses, the pur ified samples consistently displayed a prominent 43-kDa protein band w hich could undergo in gel autophosphorylation, thus suggesting that th is band represented the kinase protein. The suggestion was supported f urther by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhib ited by millimolar concentrations of cAMP. The kinase was found to ina ctivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (N cIk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of NcIk by the purifi ed thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide an alysis of the phosphorylated NcIk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this t hymus kinase is a novel Cdk inhibitory protein kinase, distinct from t he recently cloned dual functional and membrane-associated Cdc2 inhibi tory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Du rphy, W. G. (1995) Science 270, 86-90).