Degradation of p21(Cip1) through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/C-Cdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Laboratory of Genome Structure and Regulation, School of Biomedical Science, Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2011; 286(51):44057-66. DOI: 10.1074/jbc.M111.236711
Source: PubMed


Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully
understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of
ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability
of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled
cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

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    • "Moreover, we found that p21 accumulated rapidly in Bat3-KD cells released from a nocodazole arrest, whereas the phosphorylation of p21 was reduced. Degradation of p21 by APC/CCdc20 during G2/M progression has also been reported to occur during prometaphase and the interaction between Cdk2 and p21 inhibits this process [17], [46]. These findings suggest that the decreased p21 phosphorylation in the Bat3-KD cells results in continuous p21 synthesis. "
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    ABSTRACT: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.
    PLoS ONE 06/2012; 7(6):e38085. DOI:10.1371/journal.pone.0038085 · 3.23 Impact Factor
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    ABSTRACT: p21(Cip1) is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/cyclin B1, and is important for a fine-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will briefly summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.Oncogene advance online publication, 26 May 2014; doi:10.1038/onc.2014.133.
    Oncogene 05/2014; DOI:10.1038/onc.2014.133 · 8.46 Impact Factor
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    ABSTRACT: Our study aimed to investigate the gene expression at different myocardial infarction (MI) phases and to understand the development mechanisms of congestive heart failure (CHF) after MI. Dataset GSE1957 including 24 samples of rat left ventricles at 1-day post MI or sham operation and 7-day post MI or sham operation was downloaded from Gene Expression Ominibus. The data were normalized with an affyPLM package and differentially expressed genes (DEGs) were identified with a Linear Models for Microarray Data package. Heat maps of the DEGs were constructed using Cluster 3.0. GO (Gene Ontology) enrichment analysis of the DEGs was performed in Database for Annotation, Visualization, and Integrated Discovery. A protein-protein interaction (PPI) network was constructed by Biomolecular Interaction Network Database and visualized by Cytoscape, and a subnetwork was analyzed using plugin ClusterONE in Cytoscape. A total of 5 DEGs at 1-day post-MI, 5 DEGs at 7-day post-MI, and 7 DEGs between the MI and sham groups at 1-day and 7-day post-MI were identified. For the GO category analysis, DEGs at 1-day post-MI were enriched in response to cytokine stimulus. DEGs at 7-day post-MI were enriched in response to inorganic substance and chemical homeostasis. DEGs between 1-day and 7-day post-MI including CDK2 and CDC20 were significantly enriched in mitosis. CDK2, ANXA1, CDC20, and AQP2 were included in the PPI network, and CDK2 was the only DEG included in the subnetwork. In conclusion, the induction of DEGs at 7-day post-MI might participate in the response to a hormone and endogenous stimulus to regulate the development of CHF after MI.
    International Heart Journal 06/2015; 56(4). DOI:10.1536/ihj.14-422 · 1.07 Impact Factor


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