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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 acetylates and regulates the stability of COMMD1. (A,B,D) SW480 cells were transfected with COMMD–GST or GST alone (A,B), or together with control or p300 siRNA (D). Following aspirin (Asp) (10 mM, indicated times) treatment, GST-tagged proteins were isolated from whole-cell lysates using glutathione–agarose. Precipitated proteins were subjected to western blotting (WB) for (A) p300, (B) acetylated (Ac) lysine or (D) RelA. Gels were re-probed with GST to confirm equal GST–COMMD1 pulldown. (A,D) Input levels of indicated proteins are shown. Control, non-specific band. (C,E,F) Cells were transfected with control, p300 or p300b siRNA then (C,E) treated with aspirin (10 mM) for indicated times or (F) co-transfected with the plasmids indicated. Immunoblots show COMMD1 and p300 levels. Actin controls for loading. (G) Cells were transfected with control or p300 siRNA, treated with aspirin (5 mM, 16 h) then RNA was isolated. Real-time PCR was used to quantify COMMD1 transcripts relative to GAPDH control. The mean±s.e.m of three independent experiments is shown.
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f02: p300 acetylates and regulates the stability of COMMD1. (A,B,D) SW480 cells were transfected with COMMD–GST or GST alone (A,B), or together with control or p300 siRNA (D). Following aspirin (Asp) (10 mM, indicated times) treatment, GST-tagged proteins were isolated from whole-cell lysates using glutathione–agarose. Precipitated proteins were subjected to western blotting (WB) for (A) p300, (B) acetylated (Ac) lysine or (D) RelA. Gels were re-probed with GST to confirm equal GST–COMMD1 pulldown. (A,D) Input levels of indicated proteins are shown. Control, non-specific band. (C,E,F) Cells were transfected with control, p300 or p300b siRNA then (C,E) treated with aspirin (10 mM) for indicated times or (F) co-transfected with the plasmids indicated. Immunoblots show COMMD1 and p300 levels. Actin controls for loading. (G) Cells were transfected with control or p300 siRNA, treated with aspirin (5 mM, 16 h) then RNA was isolated. Real-time PCR was used to quantify COMMD1 transcripts relative to GAPDH control. The mean±s.e.m of three independent experiments is shown.

Mentions: Instead of RelA, we considered the possibility that p300 modulates COMMD1. Indeed, immunoprecipitation indicated that COMMD1 interacts with p300, that this interaction is greatly enhanced upon aspirin exposure (despite a decrease in total cellular levels of p300) and that this is associated with COMMD1 acetylation (Fig. 2A,B). To determine the role of this acetylation in nucleolar transport of RelA we firstly utilised a small interfering RNA (siRNA) approach. Surprisingly, we found that depletion of p300 alone caused a substantial reduction in basal and induced levels of COMMD1 (Fig. 2C) and thus, abrogated the COMMD1–RelA interaction (Fig. 2D). Use of an independent siRNA confirmed the substantial depletion of COMMD1 upon p300 knockdown (Fig. 2E). Furthermore, overexpression studies indicated this could be rescued by expression of wild-type p300, but not an acetylation defective mutant [deleted for the histone acetyltransferase domain (ΔHAT)] (Dornan et al., 2004) (Fig. 2F).


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 acetylates and regulates the stability of COMMD1. (A,B,D) SW480 cells were transfected with COMMD–GST or GST alone (A,B), or together with control or p300 siRNA (D). Following aspirin (Asp) (10 mM, indicated times) treatment, GST-tagged proteins were isolated from whole-cell lysates using glutathione–agarose. Precipitated proteins were subjected to western blotting (WB) for (A) p300, (B) acetylated (Ac) lysine or (D) RelA. Gels were re-probed with GST to confirm equal GST–COMMD1 pulldown. (A,D) Input levels of indicated proteins are shown. Control, non-specific band. (C,E,F) Cells were transfected with control, p300 or p300b siRNA then (C,E) treated with aspirin (10 mM) for indicated times or (F) co-transfected with the plasmids indicated. Immunoblots show COMMD1 and p300 levels. Actin controls for loading. (G) Cells were transfected with control or p300 siRNA, treated with aspirin (5 mM, 16 h) then RNA was isolated. Real-time PCR was used to quantify COMMD1 transcripts relative to GAPDH control. The mean±s.e.m of three independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f02: p300 acetylates and regulates the stability of COMMD1. (A,B,D) SW480 cells were transfected with COMMD–GST or GST alone (A,B), or together with control or p300 siRNA (D). Following aspirin (Asp) (10 mM, indicated times) treatment, GST-tagged proteins were isolated from whole-cell lysates using glutathione–agarose. Precipitated proteins were subjected to western blotting (WB) for (A) p300, (B) acetylated (Ac) lysine or (D) RelA. Gels were re-probed with GST to confirm equal GST–COMMD1 pulldown. (A,D) Input levels of indicated proteins are shown. Control, non-specific band. (C,E,F) Cells were transfected with control, p300 or p300b siRNA then (C,E) treated with aspirin (10 mM) for indicated times or (F) co-transfected with the plasmids indicated. Immunoblots show COMMD1 and p300 levels. Actin controls for loading. (G) Cells were transfected with control or p300 siRNA, treated with aspirin (5 mM, 16 h) then RNA was isolated. Real-time PCR was used to quantify COMMD1 transcripts relative to GAPDH control. The mean±s.e.m of three independent experiments is shown.
Mentions: Instead of RelA, we considered the possibility that p300 modulates COMMD1. Indeed, immunoprecipitation indicated that COMMD1 interacts with p300, that this interaction is greatly enhanced upon aspirin exposure (despite a decrease in total cellular levels of p300) and that this is associated with COMMD1 acetylation (Fig. 2A,B). To determine the role of this acetylation in nucleolar transport of RelA we firstly utilised a small interfering RNA (siRNA) approach. Surprisingly, we found that depletion of p300 alone caused a substantial reduction in basal and induced levels of COMMD1 (Fig. 2C) and thus, abrogated the COMMD1–RelA interaction (Fig. 2D). Use of an independent siRNA confirmed the substantial depletion of COMMD1 upon p300 knockdown (Fig. 2E). Furthermore, overexpression studies indicated this could be rescued by expression of wild-type p300, but not an acetylation defective mutant [deleted for the histone acetyltransferase domain (ΔHAT)] (Dornan et al., 2004) (Fig. 2F).

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