CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation.
Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1(Cdt2)-dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1(Cdt2) were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co-immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1(Cdt2) complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G(2) phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1(Cdt2). Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.
Nishitani
Hideo
H
Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo, 678-1297, Japan. hideon@sci.u-hyogo.ac.jp
Shiomi
Yasushi
Y
Iida
Hiroka
H
Michishita
Masato
M
Takami
Toshihiro
T
Tsurimoto
Toshiki
T
D001665:Binding Sites; D044842:Cullin Proteins / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism / Q000502:physiology*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D016196:S Phase; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays
C114234:CUL4A protein, human; D044842:Cullin Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C429530:DTL protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases