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Molecular and Cellular Biology, June 2001, p. 4032-4045, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.4032-4045.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Retinoblastoma Tumor Suppressor Protein Signals through Inhibition of Cyclin-Dependent Kinase 2 Activity To Disrupt PCNA Function in S Phase

Zvjezdana Sever-Chroneos,1,dagger Steven P. Angus,1 Anne F. Fribourg,1 Huajing Wan,1 Ivan Todorov,2 Karen E. Knudsen,1 and Erik S. Knudsen1,*

Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0521,1 and Dumont Transplant Center, University of California---Los Angeles School of Medicine, Los Angeles, California 90095-70542

Received 5 December 2000/Returned for modification 16 January 2001/Accepted 13 March 2001

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G1 and S-phase progression. While RB-mediated G1 inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.

* Corresponding author. Mailing address: Dept. of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521. Phone: (513) 558-8885. Fax: (513) 558-4454. E-mail: erik.knudsen{at}uc.edu.

dagger Present address: University of Texas Health Center at Tyler, Tyler, TX 35708.

Molecular and Cellular Biology, June 2001, p. 4032-4045, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.4032-4045.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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