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-mediated G(1) 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.
Sever-Chroneos
Z
Z
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0521, USA.
Angus
S P
SP
Fribourg
A F
AF
Wan
H
H
Todorov
I
I
Knudsen
K E
KE
Knudsen
E S
ES
D000818:Animals; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D051716:Replication Protein A; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D016196:S Phase / Q000502:physiology*; D015398:Signal Transduction
D002843:Chromatin; D019925:Cyclin A; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D016160:Retinoblastoma Protein; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases