Neuronal Cdc2-like Kinase (Nclk) Binds and Phosphorylates the Retinoblastoma Protein

doi:10.1074/jbc.272.9.5622

Neuronal Cdc2-like Kinase (Nclk) Binds and Phosphorylates the Retinoblastoma Protein*

From the Departments of ^‡ Anatomy and
^¶Medical Biochemistry, The University of Calgary, Calgary, Alberta, Canada and the
^∥Department of Biochemistry, The Hong Kong University of Science and Technology, Hong Kong

^§ To whom correspondence should be addressed:
Dept. of Anatomy, The University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada T2N 4N1.
Tel.: 403-220-8723; Fax: 403-283-8727; E-mail: kylee{at}acs.ucalgary.ca

Abstract

The tumor suppressor retinoblastoma protein (RB) plays a central role in cellular growth regulation, differentiation, and apoptosis. Phosphorylation of RB results in a consequent loss of its ability to inhibit cell cycle progression. However, how RB phosphorylation might be regulated in apoptotic or postmitotic cells, such as neurons, remains unclear. Here we report that neuronal Cdc2-like kinase (Nclk), composed of Cdk5 and a neuronal Cdk5 activator (p25^nck5a), can bind and phosphorylate RB. Since RB has been shown recently to associate with D-type G₁ cyclins and viral oncoproteins through a common peptide sequence motif of LXCXE, Nclk binding may be mediated by a related sequence motif (LXCXXE) found in p25^nck5a. We demonstrate (i) in vitro binding of bacterially expressed p25^nck5a to a GST-RB fusion protein, (ii) coprecipitation of GST-RB and reconstituted Cdk5·;p25^nck5a, and (iii) phosphorylation of GST-RB by bacterially expressed Cdk5·;p25^nck5a kinase and by Cdk5·;p25^nck5a kinase purified from bovine brain. Finally, we show that immunoprecipitation of RB from embryonic mouse brain homogenate results in the coprecipitation of Cdk5 and that Cdk5 kinase activity is maximal during late embryonic development, a period when programmed cell death of developing neurons is greatest. Taken together, these results suggest that Nclk can bind to and phosphorylate RB in vitro and in vivo. We infer that Nclk may play an important role in regulating the activity of RB in the brain, including perhaps in apoptosing neurons.

Footnotes

↵* This work was supported by postdoctoral fellowships from the Alberta Cancer Board and the Leukemia Research Fund (to C. H.); by research grants from The University of Calgary, the Arthur Henry & Alice Elizabeth Zoe Fitzgerald Endowment, as well as an Alberta Heritage Foundation for Medical Research Fellowship and the Alzheimer's Association (to K.-Y. Lee); by an operating grant from the Leukemia Research Fund of Canada (to R. N. J.); and by operating grants from the Medical Research Council and National Cancer Institute of Canada (to J. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

RB

retinoblastoma protein

Nclk

neuronal Cdc2-like kinase

MOPS

4-morpholinepropanesulfonic acid

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase

DTT

dithiothreitol.
↵² K.-Y. Lee, W. Durham, S. Bou, A. W. Clark, and R. N. Johnston, manuscript in preparation.
- Received December 2, 1996.
- Revision received December 24, 1996.