Interaction of Cyclin-dependent Kinase 5 (Cdk5) and Neuronal Cdk5 Activator in Bovine Brain

doi:10.1074/jbc.271.3.1538

Interaction of Cyclin-dependent Kinase 5 (Cdk5) and Neuronal Cdk5 Activator in Bovine Brain (*)

From the (1)Medical Research Council Group in Signal Transduction, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada and the
(2)Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

^¶Alberta Heritage Foundation for Medical Research Scientist. To whom correspondence should be addressed. Tel.: 852-358-8272; Fax: 852-2358-1552.

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/p35. 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-p21, a heterodimer of Cdk5 and p25 that displays high kinase activity, and a Cdk5•p35 complex that is inactive and refractory to GST-p21 activation. Analysis of the Cdk5•p35 complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of 670 kDa. When the macromolecular complex was subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35 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.

Footnotes

↵^§ Alberta Heritage Foundation for Medical Research Postdoctoral Fellow.
↵* This work was supported by operating grants from the Medical Research Council, National Cancer Institute, and Alzheimer Society of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Cdk

cyclin-dependent kinase

DTT

dithiothreitol

FPLC

fast protein liquid chromatography

GST

glutathione S-transferase

Nck5a

neuronal Cdk5a activator

Nclk

neuronal cdc2-like kinase.
↵²K. T. Shetty, S. Kaech, W. T. Link, H. Jaffe, C. M. Flores, S. Wray, H. C. Pant, and S. Beushausen, submitted for publication.
- Received May 4, 1995.
- Revision received November 6, 1995.