Direct Interaction of p21 Cyclin-Dependent Kinase Inhibitor with the Retinoblastoma Tumor Suppressor Protein

1999 ◽  
Vol 263 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Makoto Nakanishi ◽  
Yoko Kaneko ◽  
Hitoshi Matsushime ◽  
Kyoji Ikeda
Keyword(s):  
Tumor Suppressor ◽  
Kinase Inhibitor ◽  
Direct Interaction ◽  
Tumor Suppressor Protein ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

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

2001 ◽  
Vol 21 (12) ◽  
pp. 4032-4045 ◽  
Author(s):  
Zvjezdana Sever-Chroneos ◽  
Steven P. Angus ◽  
Anne F. Fribourg ◽  
Huajing Wan ◽  
Ivan Todorov ◽  
...  
Keyword(s):  
Dna Replication ◽  
Tumor Suppressor ◽  
Cyclin A ◽  
S Phase ◽  
Tumor Suppressor Protein ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Phase Progression ◽  
Retinoblastoma Tumor

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.

scholarly journals Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo

2001 ◽  
Vol 21 (14) ◽  
pp. 4773-4784 ◽  
Author(s):  
Sergei A. Ezhevsky ◽  
Alan Ho ◽  
Michelle Becker-Hapak ◽  
Penny K. Davis ◽  
Steven F. Dowdy
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Dominant Negative ◽  
Tumor Suppressor Protein ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (∼10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.

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