MdmX Protein Is Essential for Mdm2 Protein-mediated p53 Polyubiquitination

Abstract

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.

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

“…10 HA-Mdm2A and HA-Mdm2-B expression plasmids were PCR-cloned with full length human Mdm2 as a template into pcDNA3.1 for mammalian expression or pFastBact vectors for recombinant protein expression in insect cells. Human Mdm2-a cDNA has a deletion of Mdm2 coding region spanning from amino acid 27 to 221 and Mdm2-b cDNA has a deletion of Mdm2 coding region spanning from amino acid 27 to 300.…”

“…The method for recombinant protein expression and purification in insect cells was described previously. 10 In vitro and in vivo ubiquitination assays for p53, Mdm2 and Mdm4 were performed as described previously. 10 …”

“…Mdm2L468A is capable of interacting with Mdm4, but it cannot bind to E2, rendering complete loss of its E3 ligase activity. 10,20 However, this E3-dead Mdm2L468A can be efficiently ubiquitinated by Mdm2-A/B in vitro, indicating trans-ubiqutination of Mdm2 full length protein by the splice isoforms (Fig. 1D).…”

“…Thus, the relative stoichiometric ratios of Mdm2, Mdm4 and p53 are determinants of the varying effects of Mdm2 on p53 and Mdm4. 10 Although Mdm2-A/B isoforms lack the p53-binding region, they both have and intact RING domain (Fig. 1A).…”

“…[7][8][9] We previously reported that the RING domain-mediated heterodimerization of Mdm2 and Mdm4 activates the polyubiquitination activity of the E3 complex that targets p53 for degradation. 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.…”