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Molecular and Cellular Biology, July 2004, p. 6058-6066, Vol. 24, No. 13
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.13.6058-6066.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

Xin-Hua Zhu,1 Hoang Nguyen,2,{dagger} H. Dorota Halicka,3 Frank Traganos,3 and Andrew Koff1,2*

Memorial Sloan-Kettering Cancer Center,1 Programs in Molecular Biology and Cell Biology and Genetics, Cornell University Graduate School of Medical Sciences, New York, New York 10021,2 Brander Cancer Research Institute, New York Medical College, Hawthorne, New York 105323

Received 24 December 2003/ Returned for modification 6 February 2004/ Accepted 13 April 2004

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCFskp2 complex.

* Corresponding author. Mailing address: RRL917D, Box 207, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Phone: (212) 639-2354. Fax: (646) 422-2062. E-mail: a-koff{at}ski.mskcc.org.

{dagger} Present address: Rockefeller University, New York, NY 10021.

Molecular and Cellular Biology, July 2004, p. 6058-6066, Vol. 24, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.13.6058-6066.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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