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Casein kinase 1 alpha (CK1α) is a serine/threonine kinase with numerous functions, including regulating the Wnt/β‐catenin and p53 pathways. CK1α has a well‐established role in inhibiting the p53 tumor suppressor by binding to MDMX and stimulating MDMX‐p53 interaction. MDMX purified from cells contains near‐stoichiometric amounts of CK1α, suggesting that MDMX may in turn regulate CK1α function. We present evidence that MDMX is a potent competitive inhibitor of CK1α kinase activity (Ki = 8 nM). Depletion of MDMX increases CK1α activity and β‐catenin S45 phosphorylation, whereas ectopic MDMX expression inhibits CK1α activity and β‐catenin phosphorylation. The MDMX acidic domain and zinc finger are necessary and sufficient for binding and inhibition of CK1α. P53 binding to MDMX disrupts an intramolecular auto‐regulatory interaction and enhances its ability to inhibit CK1α. P53‐null mice expressing the MDMXW200S/W201G mutant, defective in CK1α binding, exhibit reduced Wnt/β‐catenin target gene expression and delayed tumor development. Therefore, MDMX is a physiological inhibitor of CK1α and has a role in modulating cellular response to Wnt signaling. The MDMX‐CK1α interaction may account for certain p53‐independent functions of MDMX. Synopsis: MDMX cooperates with casein kinase 1α to bind and inhibit the tumor suppressor p53. Here, the MDMX‐CK1α interaction is found to also block the ability of CK1α to phosphorylate β‐catenin and to suppress canonical Wnt signaling. MDMX is a potent competitive inhibitor of CK1α kinase activity, with a Ki of 8 nM.MDMX knockdown increases CK1α activity and β‐catenin Ser45 phosphorylation.MDMX mutation in p53‐null mice reduces Wnt signaling and delays tumor development.MDMX‐CK1α interaction is a growth‐promoting mechanism independent of p53. [ABSTRACT FROM AUTHOR]

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions. [ABSTRACT FROM AUTHOR]