RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage.
In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.
Fu
Xiaoyong
X
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Yucer
Nur
N
Liu
Shangfeng
S
Li
Muyang
M
Yi
Ping
P
Mu
Jung-Jung
JJ
Yang
Tao
T
Chu
Jessica
J
Jung
Sung Yun
SY
O'Malley
Bert W
BW
Gu
Wei
W
Qin
Jun
J
Wang
Yi
Y
D000595:Amino Acid Sequence; D002453:Cell Cycle / Q000528:radiation effects; D002460:Cell Line; D045744:Cell Line, Tumor; D004249:DNA Damage; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011839:Radiation, Ionizing; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000528:radiation effects
D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C549038:RFWD3 protein, human; D044767:Ubiquitin-Protein Ligases