Abstract
Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J., and Wang, J. H. (1995) Trends Biochem. Sci. 20, 33-37). The regulatory subunit is an N-terminal truncated derivative of a 35-kDa protein expressed specifically in brain, hence the name neuronal Cdk5 activator, p25/p35nck5a. In this study, we probe the relationship between the two different forms of Nck5a and their interaction with and activation of Cdk5 in bovine brain extract. Using protein fractionation procedures in combination with Western blot analysis and protein kinase assay, three forms of Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be activated by bacterially expressed GST-p21nck5a, a heterodimer of Cdk5 and p25nck5a that displays high kinase activity, and a Cdk5.p35nck5a complex that is inactive and refractory to GST-p21nck5a activation. Analysis of the Cdk5.p35nck5a complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of approximately 670 kDa. When the macromolecular complex was subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35nck5a and Cdk5, although eluting at the same position as control, displayed high kinase activity. The result is compatible with the suggestion that the macromolecular complex contained a kinase inhibitory factor that dissociated from the complex in 10% ethylene glycol.
Highlights
The animal cell division cycle is controlled by the coordinated actions of a family of protein Ser/Thr kinases, called cdc2-like kinases, which are activated and deactivated at discrete phases of the cell cycle
We have examined the states of Cdk5 in crude bovine brain extract using protein fractionation procedures in combination with kinase activity and immunological analysis
Initial characterization using specific antibodies showed that both Cdk5 and Nck5a were quantitatively absorbed on a hydroxylapatite column or a cation exchanger such as an FPLC Mono S column but negligibly bound to anion exchangers such as DEAE-Sepharose and FPLC Mono Q columns
Summary
DEAE-Sepharose CL-6B, FPLC Mono-S, and Superose-12 columns were obtained from Pharmacia Biotech Inc.(Baie d’Urfe). Hydroxylapatite and molecular weight markers were purchased from Bio-Rad. [␥-32P]ATP (4500 Ci/mmol) was obtained from ICN. All other chemicals were purchased from Sigma
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