Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-m ediated G(1) inhibition has been extensively studied, the mechanism utilize d for S-phase inhibition is unknown. To delineate the mechanism through whi ch RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-p hase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or dis rupt early initiation events. However, activation of RB in S-phase cells di srupts the chromatin tethering of PCNA, a requisite component of the DNA re plication machinery. The action of RB was S phase specific and did not inhi bit the DNA damage-mediated association of PCNA with chromatin. We also sho w that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Import antly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PC NA activity partially restored S-phase progression in the presence of activ e 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.