Identification of Functional Domains in the Neuronal Cdk5 Activator Protein

doi:10.1074/jbc.272.9.5703

Identification of Functional Domains in the Neuronal Cdk5 Activator Protein*

From the ^‡ Salk Institute for Biological Studies, La Jolla, California 92037 and
^¶Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093

^∥ American Cancer Society Research Professor. To whom correspondence should be addressed:
Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 619-453-4100 (ext. 1385); Fax: 619-457-4765.

↵^§ Fellow of the International Human Frontier Science Program. Current address: Dept. of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

Abstract

Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. In contrast to the activation of typical CDKs by cyclin subunits, p35·;Cdk5 was not further activated by the CDK-activating kinase (CAK) and was neither phosphorylated nor inhibited by the Tyr-15-specific Wee1 kinase. The previously identified proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, was found to be sufficient to bind and activate Cdk5. Other CDKs, including Cdk2, associated weakly with p25. However, their kinase activity was only activated to the low level observed for cyclin A·;Cdk2 without Thr-160 phosphorylation, and phosphorylation of Thr-160 in Cdk2 did not activate the p25·;Cdk2 complex further. We have identified distinct regions in p35 required for binding to Cdk5 or activation of Cdk5. Residues ∼150-200 of p35 were sufficient for binding to Cdk5, but residues ∼279-291 were needed in addition for activation of Cdk5 in vitro.

Footnotes

↵* This work was supported by the United States Public Health Service Grants CA14195 and CA39780 (to T. H.). 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:

CDK

cyclin-dependent kinase

CAK

CDK-activating kinase

GSH

glutathione

GST

glutathione S-transferase

PA

Staphylococcus aureus protein A

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction.
↵² L.-H. Tsai, personal communication.
↵³ R. Y. C. Poon and T. Hunt, unpublished data.
↵⁴ R. Y. C. Poon, J. Lew, and T. Hunter, unpublished data.
↵⁵ R. Y. C. Poon, unpublished observations.
↵⁶ D. Tang, A. C. S. Chun, M. Zhang, and J. H. Wang, submitted for publication.
- Received October 3, 1996.
- Revision received December 3, 1996.