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.
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit which is derived from a brain-specific 35-kDa novel protein, p35 (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426). 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 to 291, has been used to reconstitute active Cdk5 kinase and to characterize the activation reaction. The purified kinase displays similar specific enzyme activity and similar phosphorylation site specificity as the neuronal Cdc2-like kinase purified from bovine brain. Bovine brain extract contains Cdk5 uncomplexed with p35 or p25 which has also been found to be activated by p21 or p25. The results substantiate the previous suggestion that p35 is a specific Cdk5 activator. Several observations suggest that, unlike other well characterized Cdc2-like kinases whose activities depend on the phosphorylation of the catalytic subunits at a specific site by a distinct kinase, the reconstituted Cdk5/p21 does not depend on the phosphorylation of Cdk5 for activity. The reconstitution of the highly active Cdk5 kinase was achieved without requiring any other kinase in the reconstitution reaction. The possibility of autophosphorylation of Cdk5 on the putative activation site has been ruled out as no phosphorylation occurred on Cdk5 during the enzyme reaction. The rate and extent of the kinase reconstitution were not significantly affected by Mg2+ ATP.
Qi
Z
Z
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Huang
Q Q
QQ
Lee
K Y
KY
Lew
J
J
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins
D017931:DNA Primers; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C089495:neuronal Cdk5 activator (p25-p35); D000255:Adenosine Triphosphate; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases