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

Abstract

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.

ACKNOWLEDGMENTS

We thank Shelley Barton, Kenji Fukasawa, Jean Wang, and Peter Stambrook for thought provoking discussion and critical reading of the manuscript. We are grateful to Sofie Salama and Ed Harlow for the provision of Rat-16 cells. RPA antibodies were kindly provided by Thomas Melendy and Marc Wold. George Babcock and Jim Cornelius provided exquisite flow cytometric analysis. Special thanks to Gustavo Leone, who provided the recombinant adenoviruses.

This work was supported by grant CA82525 to E.S.K. from the NIH/NCI. E.S.K. is a Kimmel Scholar.

The first two authors contributed equally.