p35, the Neuronal-specific Activator of Cyclin-dependent Kinase 5 (Cdk5) Is Degraded by the Ubiquitin-Proteasome Pathway*
- From the ‡Howard Hughes Medical Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Abstract
Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. We have investigated the regulation of the p35/Cdk5 kinase. Here we show that p35, the activator of Cdk5, is a short-lived protein with a half-life (t 1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants RO1-CA 64888-3 (to P. M. H.) and GM53049 to (L-H. T.) and National Science Foundation Grant MCB-9507109 (to L-H. T.).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.
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↵§ To whom correspondence should be addressed: Howard Hughes Medical Institute, Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0320; Fax: 617-432-3975; E-mail:gpatrick{at}fas.harvard.edu.
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↵¶ A Special Fellow of the Leukemia Society of America (3196-97).
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↵‖ An assistant investigator of the Howard Hughes Medical Institute, a Rita Allen Foundation Scholar, and a recipient of an Esther A. and Joseph Klingenstein Fund.
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↵2 L. H. Tsai and K. S. Kosik, unpublished data.
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↵3 E. Gilmore, personal communication.
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↵4 Y. Ramos and L. H. Tsai, unpublished data.
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↵5 G. Patrick and L. H. Tsai, unpublished data.
- Abbreviations:
- Cdk5
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cyclin-dependent kinase 5
- LLnL
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N-acetyl-l-leucyl-l-leucyl-l-norleucinal
- Z-L3V5
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carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone
- CMV
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cytomegalovirus
- GST
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glutathione S- transferase.
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- Received April 6, 1998.
- Revision received June 10, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
