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p300-mediated acetylation of COMMD1 regulates its stability, and the ubiquitylation and nucleolar translocation of the RelA NF-κB subunit.

O'Hara A, Simpson J, Morin P, Loveridge CJ, Williams AC, Novo SM, Stark LA - J. Cell. Sci. (2014)

Bottom Line: We show that p300 is required for stress-mediated ubiquitylation and nucleolar translocation of RelA, but that this effect is indirect.In contrast, tumour necrosis factor (TNF) has no effect on COMMD1 acetylation.Finally, we demonstrate these findings have relevance in a whole tissue setting.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research Centre, IGMM, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

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p300 is required for the ubiquitylation and nucleolar translocation of RelA. (A,B) SW480-GFP-RelA cells were exposed to 0 (−) or 5 mM (+) aspirin (Asp, 16 h), RelA was immunoprecipitated (IP), then recovered proteins were analysed by immunoblot (WB) for (A) acetylated RelA (AcRelA; anti-AcRelAK310 antibody) or (B) p300. Stripped gels were re-probed for RelA. Input levels of protein are shown. Rabbit IgG acts as a control. (C–F) SW480 cells were transfected with control (scrambled) or p300 siRNA, then treated with aspirin (C,D, 10 mM, times indicated; E,F, 5 mM, 16 h). (C) Immunoblot for p300. (D) Cells were co-transfected with His6–ubiquitin. Ubiquitylated RelA was analysed in lysates using nickel (Ni) agarose bead precipitation and immunoblotting for RelA. (E,F) Immunomicrographs demonstrating the localisation of RelA. The arrow indicates a nucleolus. The number of cells showing nucleolar RelA was determined in >200 cells from more than five fields. Data shown are the mean±s.e.m. n = 3. *P<0.05 (Student's t-test). (G,H) Diagram showing p300 acetylated lysine residues that were mutated to arginine residues. Live-cell imaging demonstrates nucleolar localisation of mutants. Scale bar: 10 µm.
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f01: p300 is required for the ubiquitylation and nucleolar translocation of RelA. (A,B) SW480-GFP-RelA cells were exposed to 0 (−) or 5 mM (+) aspirin (Asp, 16 h), RelA was immunoprecipitated (IP), then recovered proteins were analysed by immunoblot (WB) for (A) acetylated RelA (AcRelA; anti-AcRelAK310 antibody) or (B) p300. Stripped gels were re-probed for RelA. Input levels of protein are shown. Rabbit IgG acts as a control. (C–F) SW480 cells were transfected with control (scrambled) or p300 siRNA, then treated with aspirin (C,D, 10 mM, times indicated; E,F, 5 mM, 16 h). (C) Immunoblot for p300. (D) Cells were co-transfected with His6–ubiquitin. Ubiquitylated RelA was analysed in lysates using nickel (Ni) agarose bead precipitation and immunoblotting for RelA. (E,F) Immunomicrographs demonstrating the localisation of RelA. The arrow indicates a nucleolus. The number of cells showing nucleolar RelA was determined in >200 cells from more than five fields. Data shown are the mean±s.e.m. n = 3. *P<0.05 (Student's t-test). (G,H) Diagram showing p300 acetylated lysine residues that were mutated to arginine residues. Live-cell imaging demonstrates nucleolar localisation of mutants. Scale bar: 10 µm.

Mentions: Previous reports have suggested that RelA deacetylation is a pre-requisite for TNF-mediated ubiquitylation (Li et al., 2012). However, here, we found that aspirin-mediated ubiquitylation of RelA (aspirin is used as a model stress stimuli) is paralleled by increased acetylation of RelA and increased binding to the acetyltransferase p300 (Fig. 1A,B). We also found that p300 depletion abrogated aspirin-mediated ubiquitylation and nucleoplasmic to nucleolar translocation of RelA (Fig. 1C–F). However, site-directed mutagenesis revealed that all the identified p300 acetylation sites (K122, K123, K218 K221 and K310) within the first 311 amino acids of RelA [the minimal required for nucleolar localisation (Stark and Dunlop, 2005)] are dispensable for nucleolar transport of the protein (Fig. 1G–H).


p300-mediated acetylation of COMMD1 regulates its stability, and the ubiquitylation and nucleolar translocation of the RelA NF-κB subunit.

O'Hara A, Simpson J, Morin P, Loveridge CJ, Williams AC, Novo SM, Stark LA - J. Cell. Sci. (2014)

p300 is required for the ubiquitylation and nucleolar translocation of RelA. (A,B) SW480-GFP-RelA cells were exposed to 0 (−) or 5 mM (+) aspirin (Asp, 16 h), RelA was immunoprecipitated (IP), then recovered proteins were analysed by immunoblot (WB) for (A) acetylated RelA (AcRelA; anti-AcRelAK310 antibody) or (B) p300. Stripped gels were re-probed for RelA. Input levels of protein are shown. Rabbit IgG acts as a control. (C–F) SW480 cells were transfected with control (scrambled) or p300 siRNA, then treated with aspirin (C,D, 10 mM, times indicated; E,F, 5 mM, 16 h). (C) Immunoblot for p300. (D) Cells were co-transfected with His6–ubiquitin. Ubiquitylated RelA was analysed in lysates using nickel (Ni) agarose bead precipitation and immunoblotting for RelA. (E,F) Immunomicrographs demonstrating the localisation of RelA. The arrow indicates a nucleolus. The number of cells showing nucleolar RelA was determined in >200 cells from more than five fields. Data shown are the mean±s.e.m. n = 3. *P<0.05 (Student's t-test). (G,H) Diagram showing p300 acetylated lysine residues that were mutated to arginine residues. Live-cell imaging demonstrates nucleolar localisation of mutants. Scale bar: 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f01: p300 is required for the ubiquitylation and nucleolar translocation of RelA. (A,B) SW480-GFP-RelA cells were exposed to 0 (−) or 5 mM (+) aspirin (Asp, 16 h), RelA was immunoprecipitated (IP), then recovered proteins were analysed by immunoblot (WB) for (A) acetylated RelA (AcRelA; anti-AcRelAK310 antibody) or (B) p300. Stripped gels were re-probed for RelA. Input levels of protein are shown. Rabbit IgG acts as a control. (C–F) SW480 cells were transfected with control (scrambled) or p300 siRNA, then treated with aspirin (C,D, 10 mM, times indicated; E,F, 5 mM, 16 h). (C) Immunoblot for p300. (D) Cells were co-transfected with His6–ubiquitin. Ubiquitylated RelA was analysed in lysates using nickel (Ni) agarose bead precipitation and immunoblotting for RelA. (E,F) Immunomicrographs demonstrating the localisation of RelA. The arrow indicates a nucleolus. The number of cells showing nucleolar RelA was determined in >200 cells from more than five fields. Data shown are the mean±s.e.m. n = 3. *P<0.05 (Student's t-test). (G,H) Diagram showing p300 acetylated lysine residues that were mutated to arginine residues. Live-cell imaging demonstrates nucleolar localisation of mutants. Scale bar: 10 µm.
Mentions: Previous reports have suggested that RelA deacetylation is a pre-requisite for TNF-mediated ubiquitylation (Li et al., 2012). However, here, we found that aspirin-mediated ubiquitylation of RelA (aspirin is used as a model stress stimuli) is paralleled by increased acetylation of RelA and increased binding to the acetyltransferase p300 (Fig. 1A,B). We also found that p300 depletion abrogated aspirin-mediated ubiquitylation and nucleoplasmic to nucleolar translocation of RelA (Fig. 1C–F). However, site-directed mutagenesis revealed that all the identified p300 acetylation sites (K122, K123, K218 K221 and K310) within the first 311 amino acids of RelA [the minimal required for nucleolar localisation (Stark and Dunlop, 2005)] are dispensable for nucleolar transport of the protein (Fig. 1G–H).

Bottom Line: We show that p300 is required for stress-mediated ubiquitylation and nucleolar translocation of RelA, but that this effect is indirect.In contrast, tumour necrosis factor (TNF) has no effect on COMMD1 acetylation.Finally, we demonstrate these findings have relevance in a whole tissue setting.

View Article: PubMed Central - PubMed

Affiliation: Edinburgh Cancer Research Centre, IGMM, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

Show MeSH
Related in: MedlinePlus