The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo

Abstract

There are currently two distinct models proposed to explain why both MDM2 and MDMX are required in p53 control, with a key difference centered on whether these two p53 inhibitors work together or independently. To test these two competing models, we generated knockin mice expressing a point mutation MDMX mutant (C462A) that is defective in MDM2 binding. This approach allowed a targeted disassociation of the MDM2/MDMX heterocomplex without affecting the ability of MDMX to bind to p53, and while leaving the MDM2 protein itself completely untouched. Significantly, Mdmx C462A/C462A homozygous mice died at approximately day 9.5 of embryonic development, as the result of a combination of apoptosis and decreased cell proliferation, as shown by TUNEL and BrdU incorporation assays, respectively. Interestingly, even though the MDMX mutant protein abundance was found slightly elevated in the Mdmx C462A/C462A homozygous embryos, both the abundance and activity of p53 were markedly increased. A p53-dependent death was demonstrated by the finding that concomitant deletion of p53 completely rescued the embryonic lethality in Mdmx C462A/C462A homozygous mice. Our data demonstrate that MDM2 and MDMX function as an integral complex in p53 control, providing insights into the nonredundant nature of the function of MDM2 and MDMX.knockin mouse model | p53 regulation U nder normal physiological conditions, wild-type p53 protein levels must be kept low owing to its growth-inhibitory activities, and this control is mainly modulated via regulation of p53 protein stability. Although a number of different regulators have been reported to be involved in this protein regulation, MDM2 has been shown to be the principal player in control of p53 turnover (1). MDM2 primarily functions as an E3 ubiquitin ligase targeting p53 for ubiquitination and subsequent degradation. At the same time, p53 induces the expression of the Mdm2 gene, forming a negative feedback loop (1). The importance of MDM2 in p53 control is highlighted by the finding that Mdm2 knockout results in p53-dependent embryonic lethality in mice (2, 3).MDMX (also known as MDM4), which was originally isolated as a novel p53-interacting protein, shares substantial structural homology with MDM2 (4, 5). The highest sequence similarity between MDM2 and MDMX lies at the N terminus and contains a p53-binding domain, and the two also share high sequence homology in a RING-finger domain, a region that mediates the association between MDMX and MDM2 (6,7). Genetic studies have demonstrated that like MDM2, MDMX is another essential negative regulator of p53 (8-10). Although it remains unclear why both MDM2 and MDMX are required for p53 control, a model has been proposed that these two proteins function independently. On the basis of the fact that unlike MDM2, MDMX lacks an intrinsic ubiquitin E3 ligase activity, it has been proposed that MDMX inhibits p53 chiefly by binding to the p53 transactivation domain and antagonizing p53 transcription activity, whereas MDM2 inactivates p53 primarily by wo...

p53 regulation: Teamwork between RING domains of Mdm2 and MdmX

“…Consistent with this prediction, two recent reports from Yuan's and Lozano's groups demonstrated that knock-in of MdmX RING domain mutants either as point mutant MdmxC462A or as RING domain deletion in mice caused a p53-dependent embryonic lethal phenotype. 81,82 In both cases, the time point of embryonic lethality was embryonic day 9.5, which is similar to knockout of the whole gene of MdmX. 57 These results suggest that the RING domain of MdmX carries the whole function of MdmX protein in p53 inhibition during development.…”

“…Knockout of Mdm2 and knock-in of Mdm2 RING mutant kill the embryos at the earlier developmental stage of Day 5.5-7.5, 61,83,84 while knockout of MdmX or knock-in of MdmX RING mutant kill the embryos at the later stage of day 9.5. 57,81,82 These observations imply that Mdm2-mediated p53 inhibition at an earlier developmental stage depends on its RING domain but does not need MdmX. The possible mechanisms underlying this stage of p53 inhibition might be (1) Mdm2-mediated monoubiquitination drives p53 nuclear export which is sufficient for inactivation of p53 nuclear activity or (2) on top of nuclear exporting of p53 by Mdm2-catalyzed monoubiquitination, other E3 ligases or E4 factors can complete the ubiquitin-dependent degradation process in the cytoplasm.…”

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity

“…Mutation of the conserved cysteine residue in the MdmX RING domain, C462A, disrupts dimerization with Mdm2 and allows for p53 activation, leading to embryonic lethality by day 9.5 (17). Notably, in the …”

Mdm2 Splice isoforms regulate the p53/Mdm2/Mdm4 regulatory circuit via RING domain-mediated ubiquitination of p53 and Mdm4

“…The rescue of Mdm4 knockout and Mdm4 RING domain mutant mice by p53 deficiency concluded that Mdm4 has a non-overlapping function of Mdm4 with Mdm2. 12,13,19 However, this conclusion is difficult to reconcile with the finding that an Mdm2 transgene containing Mdm2 gene and its native promotor can completely rescue Mdm4-deficiency-induced p53-dependent lethality, leading to a conclusion that Mdm2 and Mdm4 are functionally overlapping in development. 28 Based on our biochemical model, it is higly likely that the Mdm2 splice isoforms expressed from the Mdm2 transgene, rather than the overexpression of full-length Mdm2 per se, may have compensated for the loss of Mdm4 as a RING-domain stimulater of full length Mdm2 activity to fully suppress p53 during development.…”

“…10 Mouse genetics studies indicated that RING-RING interaction of Mdm2-Mdm4 domain is essential for restricting p53 activity during embryonic development, since mutation of either Mdm2 or Mdm4 RING domain causes p53-dependent embryonic lethality. [11][12][13] Mdm2 alternative splice isoforms were reported years ago. 14 Two major splice isoforms Mdm2-A and Mdm2-B (referred thereafter as Mdm2-A/B) do not bind to p53 and induce p53-independent cell growth in vivo and tumorigenesis in vivo.…”