Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding

Significance

MDMX is a critical regulator of p53 and a potential drug target. The mechanisms by which MDMX inhibit p53 are not fully understood. Results in this report suggest that MDMX inhibits p53 DNA-binding function. Using a protein fragment release assay, MDMX and p53 were found to engage in multiple strong secondary interactions following initial binding through the canonical binding domains. These secondary interactions are involved in blocking p53 DNA binding and stabilizing the MDMX–p53 complex. The results suggest that secondary interactions play important roles in regulating the function of multidomain protein complexes.

Abstract

The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX–p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins.