Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice
Cho-Rok Jung, … , Seung-Moo Noh, Dong-Soo Im
Cho-Rok Jung, … , Seung-Moo Noh, Dong-Soo Im
Published November 8, 2010
Citation Information: J Clin Invest. 2010;120(12):4493-4506. https://doi.org/10.1172/JCI42674.
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Enigma negatively regulates p53 through MDM2 and promotes tumor cell survival in mice

Abstract

The human E3 ubiquitin ligase murine double minute 2 (MDM2) targets the tumor suppressor p53 for ubiquitination and degradation but also promotes its own ubiquitination and subsequent degradation. As the balance between MDM2 and p53 levels plays a crucial role in regulating cell proliferation and apoptosis, we sought to identify factors selectively inhibiting MDM2 self-ubiquitination. Here we have shown that the LIM domain protein Enigma directly interacts with MDM2 to form a ternary complex with p53 in vitro and in human hepatoma and colon carcinoma cell lines and mouse embryonic fibroblasts. We found that Enigma elicited p53 degradation by inhibiting MDM2 self-ubiquitination and increasing its ubiquitin ligase activity toward p53 in cells. Moreover, mitogenic stimuli such as serum, FGF, and HGF increased Enigma transcription via induction of serum response factor (SRF), leading to MDM2 stabilization and subsequent p53 degradation. We observed similar results in the livers of mice treated with HGF. In humans, we found SRF and Enigma coexpressed with MDM2 but not p53 in several liver and stomach tumors. Finally, we showed that Enigma promoted cell survival and chemoresistance by suppressing p53-mediated apoptosis in both cell lines and a mouse xenograft model. Our findings suggest a role for Enigma in tumorigenesis and uncover a mechanism whereby mitogens attenuate p53 antiproliferative activity through an SRF/Enigma/MDM2 pathway.

Authors

Cho-Rok Jung, Jung Hwa Lim, Yoonjung Choi, Dae-Ghon Kim, Koo Jeong Kang, Seung-Moo Noh, Dong-Soo Im

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Figure 1

Enigma regulates p53 and p21 protein levels through MDM2.

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Enigma regulates p53 and p21 protein levels through MDM2. (A) Enigma sta...
(A) Enigma stabilizes MDM2. HLK3 cells were transfected with F-Enigma vector (0, 2, 5 μg [designated –, +, ++]), incubated for 12 hours in the presence or absence of 10 μM MG132, and immunoblotted as indicated. (B) p53 levels are reflected in p53 activity. We transfected cells with the indicated reporter constructs (0.5 μg each) and with or without F-Enigma vector, and incubated them with or without MG132 for 12 hours. A luciferase assay was performed. Rel. luc. unit, relative luciferase units. (C) The effects of Enigma on mRNA levels of MDM2, p53, and p21. We transfected HLK3 cells with or without F-Enigma vector, prepared cell lysates or extracted total RNAs after 48 hours, and performed Northern blotting (NB) or IB as indicated. We stained 28S RNA with ethidium bromide as a control to indicate equivalent loading. (D) Enigma regulates p53 levels Mdm2-dependently. We transduced MEFs with Ad-p53 at an MOI of 100. After 16 hours, we transduced them with Ad–F-Enigma or Ad-LacZ at an MOI of 50, or Ad-siEnigma or Ad-siControl at an MOI of 100, and incubated them for 32 hours before IB as indicated. (E) Enigma regulates p53 and p21 levels MDM2-dependently. We transfected cells with F-Enigma (5 μg), F-EniΔLIM3 (5 μg), siEnigma (10 μg), or siControl (10 μg) vectors, and incubated them for 48 hours before IB as indicated.