RECONSTITUTION OF NEURONAL CDC2-LIKE KINASE FROM BACTERIA-EXPRESSED CDK5 AND AN ACTIVE FRAGMENT OF THE BRAIN-SPECIFIC ACTIVATOR KINASE ACTIVATION IN THE ABSENCE OF CDK5 PHOSPHORYLATION

Three truncated forms of p35 including the one corresponding to the 25 -kDa subunit of the kinase have been expressed in Escherichia coli and shown to activate a bacteria-expressed Cdk5 with equal efficacy. The shortest truncated form of p35, p21, spanning amino acid residues 88 t o 291, has been used to reconstitute active Cdk5 kinase and to charact erize the activation reaction. The purified kinase displays similar sp ecific enzyme activity and similar phosphorylation site specificity as the neuronal Cdc2-like kinase purified from bovine brain, Bovine brai n extract contains Cdk5 uncomplexed with p35 or p25 which has also bee n found to be activated by p21 or p25. The results substantiate the pr evious suggestion that p35 is a specific Cdk5 activator, Several obser vations suggest that, unlike other well characterized Cdc2-like kinase s whose activities depend on the phosphorylation of the catalytic subu nits at a specific site by a distinct kinase, the reconstituted Cdk5/p 21 does not depend on the phosphorylation of Cdk5 for activity. The re constitution of the highly active Cdk5 kinase was achieved without req uiring any other kinase in the reconstitution reaction. The possibilit y of autophosphorylation of Cdk5 on the putative activation site has b een ruled out as no phosphorylation occurred on Cdk5 during the enzyme reaction. The rate and extent of the kinase reconstitution were not s ignificantly affected by Mg2+ ATP.