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Interplay between MDM2, MDMX, Pirh2 and COP1: the negative regulators of p53

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Abstract

MDM2, Pirh2 and COP1 are important E3 ubiquitin ligases, which directly interact with p53 and target p53 for proteasome-mediated degradation. MDMX, the MDM2 homologous protein, inhibits p53-mediated transcription activity. The interplay between MDM2, MDMX, Pirh2 and COP1 has not been reported, except the interaction between MDM2 and MDMX. Here, we reported that there were interactions between these four proteins independently of p53. The protein levels of MDM2, MDMX, Pirh2 and COP1 changed when any two of them were co-transfected. Our data also showed that the integrity of MDM2 RING finger domain was crucial for its ability to elevate the protein levels of COP1 and Pirh2. Any two of these four proteins could inhibit p53-mediated transcriptional activity synergistically. Furthermore, COP1 inhibited MDM2 self-ubiquitination and interfered with MDMX ubiquitination by MDM2. Our results suggest that MDM2, MDMX, Pirh2 and COP1 might inhibit p53 activity synergistically in vivo.

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Acknowledgements

We thank Chenji Wang for supply of COP1 expression vector, Qian Wang for discussions and Zhen Zhang from University of Wisconsin for proofreading.

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, People’s Republic of China

    Lan Wang, Guifen He, Xiang Wang, Mei Jiang & Long Yu

  2. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, People’s Republic of China

    Lan Wang, Pingzhao Zhang & Long Yu

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  2. Guifen He
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Correspondence to Long Yu.

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11033_2010_99_MOESM1_ESM.tif

Supplemental Fig. 1 (A) 4 × 105 H1299 cells in six-well plate were transfected with myc-MDM2 (300ng), myc-MDM2-C464A (300 ng), myc-Pirh2 (100 ng), HA-MDMX (500 ng) and FLAG-Ub (1 μg) expression vectors as indicated. (B) 4 × 105 H1299 cells in six-well plate were transfected with myc-MDM2 (1 μg), myc-MDM2-C464A (1μg), myc-MDMX (300 ng), HA-Pirh2 (200 ng) and FLAG-Ub (1 μg) expression vectors as indicated. (C) 4 × 105 H1299 cells in six-well plate were transfected with myc-Pirh2 (400 ng), FLAG-COP1 (600 ng) and HA-Ub (1 μg) expression vectors as indicated. (D) 4 × 105 H1299 cells in six-well plate were transfected with myc-MDM2 (300 ng), myc-MDM2-C464A (300 ng), myc-MDMX (300 ng), myc-Pirh2 (100 ng), FLAG-COP1 (1 μg) and HA-Ub (1 μg) expression vectors as indicated. (E) 4 × 105 H1299 cells in six-well plate were transfected with myc-MDM2 (1 μg), myc-MDM2-C464A (1 μg), HA-MDMX (300 ng), HA-Pirh2 (200 ng), and FLAG-Ub (1μg) expression vectors as indicated. Cells were treated with 20 μM MG132 for 8h before harvested. Whole cell lysates (WCL) were analyzed by direct immunoblotting (IB) and detected using indicated antibodies in each figure. In figure (A) and (B), immunoprecipitations (IP) were performed using anti-HA antibody. The precipitated proteins were analyzed by immunoblotting and detected using anti-FLAG antibody. In figure (C) and (D), immunoprecipitations were performed using anti-myc and anti-FLAG antibody, respectively. The precipitated proteins were analyzed by immunoblotting and detected using anti-HA antibody. In figure (E), immunoprecipitations were performed using anti-myc antibody. The precipitated proteins were analyzed by immunoblotting and detected using anti-FLAG antibody. (TIFF 33404 kb)

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Wang, L., He, G., Zhang, P. et al. Interplay between MDM2, MDMX, Pirh2 and COP1: the negative regulators of p53. Mol Biol Rep 38, 229–236 (2011). https://doi.org/10.1007/s11033-010-0099-x

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  • DOI: https://doi.org/10.1007/s11033-010-0099-x

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