MDMX Inhibits P53 DNA Binding Through Secondary Interactions With The P53 Protein

Background: The tumor suppressor protein p53 encodes a transcription factor that plays critical roles in controlling cell cycle arrest, cell growth, metablism or apoptosis in response to various types of damage and stress. P53 has two key regulars-MDM2 and MDMX. MDM2 binds to p53 and acts as an ubiquitin E3 ligase to promote its proteasomal degradation. There is an unique role for MDMX in the regulation of p53. Althought MDMX is a p53 binding protein with strong sequence homology to MDM2, lack of MDMX is able to active p53 rather than increasing the p53 protein level. Affinity purification of MDMX has identified casein kinase 1 alpha(CK1α) as major binding protein. CK1α interacts with the central region of MDMX, and enhances MDMX-p53 binding. Because of the co-localization of MDMX and p53 to regulate p53 function, we intent to investigate the possibility that MDMX regulates p53 independent of degradation mechanism and inhibits p53 DNA binding in vivo whether cooperates with CK1α.Methods: 1.DNA binding assay: FLAG-MDMX or CK1α-MDMX-p53 complexes cotransfected into H1299 cellline and purifed by M2 beads, then incubates with DNA oligos with p53 binding sites to find out weather these complexes could bind with specific DNA oligos in vitro. 2.Using PONDR to predict the flexible areas within MDMX structure, we inserted precission points and three different tags(FLAG, MYC and HA) into MDMX to create a novel plasmid MDMXc3 for further investigation. 3. MDMXc3 was first captured with GST-p53 beads and then subjected to on-bead cleavage by Pre Scission. We hypothesized that after cleavage of the complex, MDMX fragments that engage in binding to p53 will dissociate slowly compared to fragments that have no affinity for p53, thus reveal interactions that other binding assays failed to uncover due to low affinity or slow binding kinetics. We refer to such experiments as Proteolytic Fragment Release assay(PFR).The novel assay-proteolytic fragment release(PFR) was created to test the interaction between MDMX and p53. During the process, PFR was used to test whether CK1α cooperated with MDMX to enhance MDMX-p53 binding. 4. After transient knockdown of MDMX or CK1α in the A549,JEG-3,MCF-7 and U2 OS, western blot and chromatin immunoprecipitation(Ch IP) analysis were perfomed to test wether CK1α could cooperate with MDMX to inhibit p21 expression and p53 binding to the p21 promoter. To test whether MDMX inhibits p53 DNA binding in the presence of ARF, MDMX c DNA was stably transfected into a U2 OS cell line expressing IPTG-inducible ARF(NARF6).To further test the role of CK1α in MDMX inhibition of p53, we generated U2 OS cells stably expressing MDMX and MDMX-367 A mutant. We want to investigate whehter MDMX binding to p53 was reduced after knockdown of endogenous CK1α.Results: 1. When MDMX and CK1α were coexpressed with p53, the MDMX-p53-CK1α complex showed a loss of DNA binding, suggesting that MDMX has the ability to inhibit p53 DNA binding after forming a complex with CK1α. MDMX-CK1α binding and phosphorylation of MDMX S289 by CK1α is important for inhibiting p53 DNA binding. 2. A cleavable MDMXc3 was generated for further investigation in PFR assay. The PFR analysis revealed that in addition to the well-established N terminal binding, the MDMX AD and RING domain fragments also showed significant association to p53 after cleavage. Interestingly, the RING domain binding to p53 was more stable than the p53 BD fragment after cleavage of MDMXc3. 3. The results of PFR showed that the core domain of p53 contains the secondary binding sites for MDMX AD and RING domains. To test whether the MDMX W200/W201 residues are also important for AD binding to p53 core domain, PFR analysis was performed using MDMXc3-W200S/W201 G mutant. The AD containing SG mutation showed significantly reduced binding to p53, whereas the RING domain from the SG construct retained strong binding to p53. The result also suggests that MDMX AD and RING bind to p53 independently, possibly interacting with different surfaces on the core domain. 4. Using the PFR assay, we found that co-expression of MDMXc3 and CK1α stimulated the secondary MDMX AD-p53 interaction.Mutation of the CK1α phosphorylation site on MDMX(MDMXc3-289A) abrogated the stimulation by CK1α. The CK1α-136 N catalytic mutant also failed to stimulate AD-p53 binding. Furhtermore, the RING domain contributes to p53 inhibition by stabilizing the MDMX-p53 complex. The MDMX RING domain-p53 interaction also contributes to p53 inactivation. 5. In cell culture experiments, transient knockdown of MDMX in several tumor cell lines induced expression of p53 target gene p21 and p53 binding to p21 promoter, but did not cause significant increase in p53 level. Treatment of NARF6 cells with IPTG inducing ARF expression or DNA damage by IR treatment resulting in p53 stabilization and induction of p21.Wild type MDMX and MDMX-367 A mutant overexpression inhibited p21 induction and p53 binding to p21 promoter. MDMX binding to p53 was reduced after knockdown of endogenous CK1α. To test the role of CK1α in p53 regulation after DNA damage, U2 OS cells expressing MDMX-367 A were treated with CK1α si RNA and IR.CK1α knockdown also partially abrogated the ability of MDMX-367 A to inhibit p53 binding to DNA. These results further demonstrate that MDMX inhibition of p53 DNA binding requires the cooperation by CK1α.Conclusions: These are referred to as secondary interactions as 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 to inhibit p53 DNA binding. These results reveal the importance of secondary intermolecular interactions in p53 regulation by MDMX, which may be a common phenomenon in complexes containing multi-domain proteins.