DNA damage-induced phosphorylation of MdmX at serine 367 activates p53 by targeting MdmX for Mdm2-dependent degradation.
Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.
Okamoto
Koji
K
Radiobiology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan. kojokamo@gan2.res.ncc.go.jp
Kashima
Kenji
K
Pereg
Yaron
Y
Ishida
Michiko
M
Yamazaki
Satomi
S
Nota
Ayumi
A
Teunisse
Amina
A
Migliorini
Domenico
D
Kitabayashi
Issay
I
Marine
Jean-Christophe
JC
Prives
Carol
C
Shiloh
Yosef
Y
Jochemsen
Aart G
AG
Taya
Yoichi
Y
D048948:14-3-3 Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D004249:DNA Damage; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D012694:Serine; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex