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MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9608–9620

DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation. MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9608–9620. Mdmx interacts with 14-3-3 proteins while Mdm2 does not. 14-3-3 binding is abolished by S367A substitution.

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MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9608–9620

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  1. DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9608–9620

  2. Mdmx interacts with 14-3-3 proteins while Mdm2 does not

  3. 14-3-3 binding is abolished by S367A substitution

  4. Mdmx S367 is phosphorylated in vivo - + l-phosphatase a - Flag IP: Flag a - P-S367 Supplementary figure indicating specificity of the antibody for phosphorylated S367 sites According to the author, this data also indicates that 14-3-3 proteins require a phosphorylated serine site in order to bind Mdmx consistent with previous findings related to 14-3-3 binding consensus sequences.

  5. Increased repression of p53-dependent transcriptional activation with S367A P53 deficient cells H1299 cells P53-/- Mdmx-/- MEF cells HA-p53 AIP: p53 responsive promoter Bax: Bax p53 responsive promoter (14-3-3 protein) Measured by luciferase activity assay

  6. Levels of p53 and Mdm2 are unaffected by S367A The C463A mutation of the RING domain, required for Mdmx2 association, serves nicely as a control. The authors claim that the increased levels of S367S are likely the cause of the reduced AIP activity in the previous experiment

  7. S367A blocks Mdm2/proteasome dependent degradation Little dose dependence experiment from the supplemental figures shows a nice example of the mutant resistance Flag-Mdmx S367A WT Myc-Mdm2 a - Flag a - LacZ a - Myc 1 2 3 4 5 6

  8. Proteasome dependent Mdmx degradation due to reduced ubiquitination His-Ub 2.0 p53 - - + + + + Myc-Mdm2 + + + + + + Flag-MdmX D361A S367A S367A C463A WT WT a-Flag 9 10 11 12 13 14 Co-IP of the Flag Mdmx reveals a reduced proportion of pS367 by antibody probing. This indicates preferential degradation of the phosphorylated protein.

  9. DNA damage induces Mdmx degradation in a proteasome dependent manner DNA damage induces phosphorylation of S367 and subsequent degradation according to the authors

  10. Mutant S367A Mdmx cells exhibit increased growth rate Increased growth rate cannot be attributed to p53 levels in S367A mutants as there is no appreciable change in the protein expression. However, p21 which is regulated by p53 levels are markedly reduced indicating suppression of p53 activity

  11. Authors Theoretical Model

  12. Conclusions • Mdmx associates with 14-3-3 proteins in a phosphorylation dependent manner • S367A decreases AIP activity due to degradation resistance (increased levels) • Phosphorylation leads to reduced levels of 14-3-3 in presence of Mdm2 in a proteasome dependent manner • Ubiquitination by Mdm2 requires binding and phospho S367 • Mdmx and Mdm2 work in tandem to regulate p53 activity

  13. Questions • What kinases regulate the S367 site on Mdmx? (potentially ATM/ATR kinases) • Do 14-3-3 proteins play a role in the degradation of Mdmx or is the role more functionally related? • Do S367A and C463A mutations alter E3 ligase activity of Mdmx? Mdm2? • How do anti-apoptotic promoters (Bcl-2, Bcl-xl) respond to this method of p53 deactivation? • Does phosphorylation affect activity on p53 responsive promoters?

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