Degradation of p21Cip1 through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/CCdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Ikuko Goto,Mimi Tamamori-Adachi,Kazuhiko Yamada,Shigetaka Kitajima,Tomoki Okazaki,Teijiro Aso,Takashi Yasukawa,Masayoshi Iizuka

doi:10.1074/jbc.m111.236711

Ikuko Goto, Mimi Tamamori-Adachi + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.236711

Copy DOI

Abstract

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.

Highlights

Cell cycle inhibitor p21Cip1 (p21) plays a crucial role in several biological aspects, including cell cycle, differentiation, senescence, and apoptosis [1]
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
We have previously shown that cyclin D1 is induced by mitogenic stimuli [29, 30], but its nuclear import is impaired in terminally differentiated cardiomyocytes

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Reagents—Wild type rat p21 was PCR-amplified using pCRW0.8 that was provided by Dr Vogelstein. In Vitro Ubiquitylation Assay—Whole cell extracts (40 ␮g of protein) were obtained from cells infected with D1NLS/CDK4 for 48 h and mixed with 15 ␮l of ExactaCruz C (Santa Cruz Biotechnology) that had been incubated with anti-Cdc antibody (3 ␮g) and further incubated for 2 h, followed by washing four times in buffer (50 mM HEPES (pH 7.6), 100 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10 mM glycerophosphate, 1 mM NaF, 0.1 mM Na2VO4, 10% glycerol, 0.1% Tween 20, 1 mM DTT, and a mixture of protease inhibitors), and two times in reaction buffer (40 mM Tris (pH 7.5), 60 mM NaCl, 1 mM DTT, and a mixture of protease inhibitors). Statistical analysis was performed with the Student’s t test

RESULTS

DISCUSSION