DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation

Abstract

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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org.

ACKNOWLEDGMENTS

The original human Mdm2 and Mdmx clones are kind gifts from Donna George and Steven Berberich, respectively. We are indebted to Jiangdong Chen, Christian Gaiddon, and Ari Elson for providing us with the wild-type Flag-tagged Mdm2 expression vector, the HA-tagged p53 expression vector, and the GST-14-3-3 expression vectors, respectively. The His-ubiquitin expression plasmid is a kind gift from Dirk Bohmann. We thank Gigi Lozano for providing us p53/Mdm2-deficient fibroblasts. We also thank Tomomi Shinozaki for her experimental assistance at the initial part of the project.

This work is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.T and K.O.), a Grant-in-Aid for Third Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare, Japan (Y.T.), a Grant-in-Aid from the Tokyo Biochemical Research Foundation (Y.T.), Research Grants from the Princess Takamatsu Cancer Research Fund and Takeda Science Foundation (Y.T.), and the Program for Promotion of Fundamental Studies in Health Sciences of Organization for Pharmaceutical Safety and Research of Japan (Y.T.). The work was also supported by NCI grant 87497 (to C.P). Work in the laboratory of Y.S. is supported by the A-T Children's Project, the A-T Medical Research Foundation, and the National Institute of Neurological Disorders and Stroke (NS31763).