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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-mediated acetylation of COMMD1 is stimuli specific and is required for RelA binding in response to stress. (A–C) SW480 or AA/C1/SB10 cells were transfected with GST–COMMD1 alone (B,C) or with the siRNA species indicated (A). Cells were treated with aspirin (Asp) (10 mM) or TNF (10 ng/µl) for 2 h prior to harvest. GST–COMMD1 was immunoprecipitated (IP) as in Fig. 2, then isolated proteins were subjected to western blotting (WB) with the antibodies indicated. Input levels of the proteins of interest are shown. (D) Model for the role of acetylation in regulating the steady state levels of COMMD1 and the ubiquitylation and nucleolar translocation of RelA. See discussion for details. AC, acetylation; ub, ubiquitylation. (E) Tumour biopsies from four colorectal cancer patients were exposed to pharmacological doses of aspirin (100 µM, 1 h) ex vivo (see supplementary material Fig. S1). Western blot showing COMMD1 levels in whole-cell lysates. The asterisks indicate biopsies where COMMD1 increases in response to aspirin. (F) Western blot showing effects of aspirin (10 mM) on COMMD1 levels in the pre-tumorigenic AA/C1 cell line.
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f04: p300-mediated acetylation of COMMD1 is stimuli specific and is required for RelA binding in response to stress. (A–C) SW480 or AA/C1/SB10 cells were transfected with GST–COMMD1 alone (B,C) or with the siRNA species indicated (A). Cells were treated with aspirin (Asp) (10 mM) or TNF (10 ng/µl) for 2 h prior to harvest. GST–COMMD1 was immunoprecipitated (IP) as in Fig. 2, then isolated proteins were subjected to western blotting (WB) with the antibodies indicated. Input levels of the proteins of interest are shown. (D) Model for the role of acetylation in regulating the steady state levels of COMMD1 and the ubiquitylation and nucleolar translocation of RelA. See discussion for details. AC, acetylation; ub, ubiquitylation. (E) Tumour biopsies from four colorectal cancer patients were exposed to pharmacological doses of aspirin (100 µM, 1 h) ex vivo (see supplementary material Fig. S1). Western blot showing COMMD1 levels in whole-cell lysates. The asterisks indicate biopsies where COMMD1 increases in response to aspirin. (F) Western blot showing effects of aspirin (10 mM) on COMMD1 levels in the pre-tumorigenic AA/C1 cell line.

Mentions: Throughout the course of these studies, we observed a reduction in XIAP in response to aspirin, which leads to increased amounts of COMMD1. Therefore, it could be argued that the effects of aspirin are dependent on XIAP and independent of p300. To further address the role of p300, we utilised the fact that knocking down p300 and XIAP together results in substantial levels of COMMD1 that, if p300 is the crucial acetyltransferase, should not be acetylated (Fig. 3A). We found that, as predicted, p300 loss abrogated aspirin-mediated COMMD1 acetylation (Fig. 4A). We also found that the interaction between RelA and COMMD1, detected in cells transfected with control and XIAP siRNA, was completely lost when p300 was depleted, despite substantial COMMD1 levels (see Fig. 4A, lane 8). These data, which were confirmed in an independent cell line (Fig. 4A), reveal that p300 is required for aspirin-mediated acetylation of COMMD1 and that this is essential for nucleolar translocation of RelA not only to stabilise COMMD1, but also to enable a RelA–COMMD1 interaction. In contrast to aspirin, we observed a minimal increase in COMMD1–p300 binding or in COMMD1 acetylation in response to TNF (Fig. 4B,C).


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-mediated acetylation of COMMD1 is stimuli specific and is required for RelA binding in response to stress. (A–C) SW480 or AA/C1/SB10 cells were transfected with GST–COMMD1 alone (B,C) or with the siRNA species indicated (A). Cells were treated with aspirin (Asp) (10 mM) or TNF (10 ng/µl) for 2 h prior to harvest. GST–COMMD1 was immunoprecipitated (IP) as in Fig. 2, then isolated proteins were subjected to western blotting (WB) with the antibodies indicated. Input levels of the proteins of interest are shown. (D) Model for the role of acetylation in regulating the steady state levels of COMMD1 and the ubiquitylation and nucleolar translocation of RelA. See discussion for details. AC, acetylation; ub, ubiquitylation. (E) Tumour biopsies from four colorectal cancer patients were exposed to pharmacological doses of aspirin (100 µM, 1 h) ex vivo (see supplementary material Fig. S1). Western blot showing COMMD1 levels in whole-cell lysates. The asterisks indicate biopsies where COMMD1 increases in response to aspirin. (F) Western blot showing effects of aspirin (10 mM) on COMMD1 levels in the pre-tumorigenic AA/C1 cell line.
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f04: p300-mediated acetylation of COMMD1 is stimuli specific and is required for RelA binding in response to stress. (A–C) SW480 or AA/C1/SB10 cells were transfected with GST–COMMD1 alone (B,C) or with the siRNA species indicated (A). Cells were treated with aspirin (Asp) (10 mM) or TNF (10 ng/µl) for 2 h prior to harvest. GST–COMMD1 was immunoprecipitated (IP) as in Fig. 2, then isolated proteins were subjected to western blotting (WB) with the antibodies indicated. Input levels of the proteins of interest are shown. (D) Model for the role of acetylation in regulating the steady state levels of COMMD1 and the ubiquitylation and nucleolar translocation of RelA. See discussion for details. AC, acetylation; ub, ubiquitylation. (E) Tumour biopsies from four colorectal cancer patients were exposed to pharmacological doses of aspirin (100 µM, 1 h) ex vivo (see supplementary material Fig. S1). Western blot showing COMMD1 levels in whole-cell lysates. The asterisks indicate biopsies where COMMD1 increases in response to aspirin. (F) Western blot showing effects of aspirin (10 mM) on COMMD1 levels in the pre-tumorigenic AA/C1 cell line.
Mentions: Throughout the course of these studies, we observed a reduction in XIAP in response to aspirin, which leads to increased amounts of COMMD1. Therefore, it could be argued that the effects of aspirin are dependent on XIAP and independent of p300. To further address the role of p300, we utilised the fact that knocking down p300 and XIAP together results in substantial levels of COMMD1 that, if p300 is the crucial acetyltransferase, should not be acetylated (Fig. 3A). We found that, as predicted, p300 loss abrogated aspirin-mediated COMMD1 acetylation (Fig. 4A). We also found that the interaction between RelA and COMMD1, detected in cells transfected with control and XIAP siRNA, was completely lost when p300 was depleted, despite substantial COMMD1 levels (see Fig. 4A, lane 8). These data, which were confirmed in an independent cell line (Fig. 4A), reveal that p300 is required for aspirin-mediated acetylation of COMMD1 and that this is essential for nucleolar translocation of RelA not only to stabilise COMMD1, but also to enable a RelA–COMMD1 interaction. In contrast to aspirin, we observed a minimal increase in COMMD1–p300 binding or in COMMD1 acetylation in response to TNF (Fig. 4B,C).

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