Reconstitution of Neuronal Cdc2-like Kinase from Bacteria-expressed Cdk5 and an Active Fragment of the Brain-specific Activator

摘要: Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and 25-kDa subunit which derived from brain-specific 35-kDa novel protein, p35 (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. Aebersold, R., Hunt, T., Wang, J. H. (1994) Nature 371, 423-426). Three truncated forms including the one corresponding to have been expressed in Escherichia coli shown activate bacteria-expressed with equal efficacy. The shortest form p35, p21, spanning amino acid residues 88 291, has used reconstitute active characterize activation reaction. purified displays similar specific enzyme activity phosphorylation site specificity as neuronal bovine brain. Bovine brain extract contains uncomplexed or p25 also found be activated by p21 p25. results substantiate previous suggestion that activator. Several observations suggest that, unlike other well characterized kinases whose activities depend on catalytic subunits at distinct kinase, reconstituted Cdk5/p21 does not for activity. reconstitution highly was achieved without requiring any possibility autophosphorylation putative ruled out no occurred during rate extent were significantly affected Mg2+ ATP.