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

Abstract

p53 is regulated by heterodimer E3 ligase Mdm2-Mdm4 via RING domain interaction. Mdm2 transcripts undergo alternative splicing, and Mdm2 splice isoforms are increased in cancer and induced by DNA damage. Although 2 major Mdm2 splice isoforms that do not bind to p53 were reported to impact the p53 pathway, the underlying biochemical mechanisms were not understood. Here, we show that these Mdm2 splice isoforms ubiquitinate Mdm2 and Mdm4 in vivo and regulate the activity of Mdm2-Mdm4 E3 complex in cells. The Mdm2 isoforms are capable of promoting p53 ubiquitination in the absence of Mdm2 or Mdm4. The 2 isoforms stimulate Mdm2 or Mdm4 activity for p53 ubiquitination in vivo and promote degradation of p53 and Mdm4 in cells. However, the Mdm2 isoforms have opposing effects on the steady-state p53 levels depending on the stoichiometric ratios of Mdm2, Mdm4 and the isoforms, causing either decreased or increased p53 levels in cells. Our data indicate that the Mdm2 splice isoforms can act as independent E3 ligases for p53 when Mdm2 and Mdm4 are absent, form potent heterodimer E3 ligases with either Mdm2 or Mdm4 for targeting p53 degradation, or act as inhibitory regulators of Mdm2-Mdm4 E3 ligase activity by downregulating Mdm4. These findings suggest that Mdm2 splice isoforms may play critical roles in the regulatory loop of p53/Mdm2-Mdm4 via a RING domain-mediated biochemical mechanism.

Keywords: Mdm2; RING domain; degradation; p53; splice isoforms; ubiquitination.

Figure 1.

Mdm2-A/B possesses intrinsic E3 ligase activity and can trans-ubiquitinate full length Mdm2 in vitro. (A) A diagram of Mdm2-A/B structure. (B) Mdm2-A/B autoubiquitination. Western blotting of polyubiquitin chain after in vitro ubiquitination reaction with indicated amounts of Mdm2-A/B. (C) Mdm2-A/B ubiquitinates full length Mdm2. (D) Trans-ubiquitination of full length enzyme-dead Mdm2L468A by Mdm2-A/B in vitro.

Figure 2.

Mdm2-A potently ubiquitinates Mdm4, p53 and strongly stimulates p53 ubiquitination by Mdm2-Mdm4 in vitro. (A) Mdm2-A is more active than Mdm2 in ubiquitination of Mdm4. (B) Mdm2-A is more active than Mdm2 in p53 ubiquitination. (C) Mdm2-A-Mdm4 is more active heterodimer E3 ligase than Mdm2-Mdm4 in promoting p53 polyubiquitination in vitro.

Figure 3.

Effect of Mdm2-A/B on steady-state levels of p53 and Mdm4 in cells. (A) Co-transfection of p53 with indicated amounts of Mdm2 or isoforms in TKO MEFs followed by WB 24h after transfection. (B) As performed in A but with addition of Mdm4. (C) As performed in B but in PC3 cells. (D) A model of RING-domain heterodimer E3 ligases in regulation of the p53/Mdm2/Mdm4 loop.