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. 2016 Sep 29;11(9):e0164086.
doi: 10.1371/journal.pone.0164086. eCollection 2016.

E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation

Rui Gao  1 , Lan Wang  1   2 , Hao Cai  1 , Jingjing Zhu  3 , Long Yu  1
Affiliations

E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation

Rui Gao et al. PLoS One. .

Abstract

RNF12/RLIM is a RING domain-containing E3 ubiquitin ligase whose function has only begun to be elucidated recently. Although RLIM was reported to play important roles in some biological processes such as imprinted X-chromosome inactivation and regulation of TGF-β pathway etc., other functions of RLIM are largely unknown. Here, we identified RLIM as a novel E3 ubiquitin ligase for c-Myc, one of the most frequently deregulated oncoproteins in human cancers. RLIM associates with c-Myc in vivo and in vitro independently of the E3 ligase activity of RLIM. Moreover, RLIM promotes the polyubiquitination of c-Myc protein independently of Ser62 and Thr58 phosphorylation of c-Myc. However, RLIM-mediated ubiquitination does not affect c-Myc stability. Instead, RLIM inhibits the transcriptional activity of c-Myc through which RLIM restrains cell proliferation. Our results suggest that RLIM may function as a tumor suppressor by controlling the activity of c-Myc oncoprotein.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. RLIM interacts with c-Myc.
(A) 293T cells were transfected with HA-RLIM and myc-c-Myc expression vectors and immunoprecipitation was carried out with anti-myc or anti-HA antibodies as indicated. Immunoprecipitates were subjected to WB with anti-HA and anti-myc antibodies. (B) and (C) 293T cell lysate was subjected to immunoprecipitation with control IgG antibody and anti-RLIM (B) or anti-c-Myc (C) antibodies. Immunoprecipitates were subjected to WB with anti-c-Myc and anti-RLIM antibodies. * indicates the heavy chain of IgG. (D) Purified bacterial-expressed GST or GST-c-Myc proteins were incubated with bacterial-expressed His-RLIM protein. Interaction between RLIM and c-Myc were detected by GST pull-down and subsequent WB with anti-His antibody. (E) 293T cells were transfected with WT or catalytic dead HA-RLIM together with myc-c-Myc. Cells were subjected to immunoprecipitation with anti-myc antibody followed by WB with anti-HA and anti-myc antibodies.
Fig 2
Fig 2. RLIM promotes c-Myc ubiquitination.
(A) 293T cells were transfected with Flag-Ub, myc-c-Myc, HA-RLIM and HA-RLIMC596A in combinations as indicated. Ectopically expressed c-Myc was immunoprecipitated by anti-myc antibody followed by WB with anti-Flag antibody. (B) 293T cells were transfected as in (A) except using 6 × His-Ub instead of Flag-Ub. Ubiquitinated proteins were precipitated with nickel (Ni)-NTA beads and subjected to WB with anti-myc antibody. (C) 293T cells were transfected with His-Ub, HA-RLIM, myc-c-Myc and myc-c-MycDM (S62A and T58A) as indicated. Ubiquitinated proteins were precipitated with nickel (Ni)-NTA beads and subjected to WB with anti-myc antibody.
Fig 3
Fig 3. RLIM does not affect c-Myc protein degradation.
(A) and (B) 293T (A) and H1299 (B) cells were transfected with myc-c-Myc and increasing amount of HA-RLIM or HA-RLIMC596A plasmids as well as GFP plasmid. Ectopic c-Myc protein was detected by anti-myc antibody. GFP was used to monitor transfection efficiency. c-Myc and GFP levels were quantified using ImageJ software and the ratios of c-Myc to GFP are shown. (C) and (D) 293T (C) and H1299 (D) cells were transfected with scramble siRNAs or siRNAs against RLIM. Endogenous c-Myc protein was detected by anti-c-Myc antibody. c-Myc and actin levels were quantified using ImageJ software and the ratios of c-Myc to actin are shown. (E) and (F) 293T cells were transfected with HA-RLIM (E) plasmid or siRNA against endogenous RLIM (F). Cells were harvested at different time points after cycloheximide treatment and subjected to WB. Quantification of c-Myc protein level relative to actin are summarized from 3 independent experiments and shown in the right panels.
Fig 4
Fig 4. RLIM negatively regulates c-Myc transcriptional activity.
(A) 293T cells were transfected with an E-box-luciferase (Firefly) reporter plasmid and a Renilla luciferase plasmid (as an internal control) together with c-Myc and RLIM plasmids as indicated. Firefly luciferase activity was measured and normalized by Renilla luciferase activity. Three independent experiments were conducted with similar results and one representative result is shown. Data is presented as mean ± SD. **p<0.01, ***p<0.001. (B) and (C) U2OS cells were transfected with c-Myc and RLIM plasmids as indicated (B) or with siRNAs against RLIM (C). Real-time PCR was carried out to examine E2F2 and Nucleolin gene expression. Three independent experiments were conducted with similar results and one representative result is shown. Data is presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001. (D) RLIM overexpression and control stable U2OS cell lines were transfected with scramble or c-Myc siRNAs. 2 days after transfection, cells were plated in 96 well plates. Cell growth were monitored by cell counting kit-8. (E) U2OS cells were transfected with scramble siRNA or siRNAs against RLIM and/or c-Myc as indicated. 2 days after transfection, cells were plated in 96 well plates. Cell growth were monitored by cell counting kit-8. Three independent experiments were conducted with similar results and one representative result is shown. Data shown is relative cell number as compared to day 1 after plating. Data is presented as mean ± SD. *p<0.05, ***p<0.001. Lower WB figures show the expression of RLIM and c-Myc at day 3 after plating. (F) H1299 cells were transfected with scramble siRNA or siRNAs against RLIM and/or c-Myc as indicated. 2 days after transfection, cells were plated in 96 well plates. Cell growth were monitored by cell counting kit-8. Three independent experiments were conducted with similar results and one representative result is shown. Data shown is relative cell number as compared to day 1 after plating. Data is presented as mean ± SD. **p<0.01. Lower WB figure show the expression of RLIM and c-Myc at day 4 after plating.
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