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Regulation of ubiquitin ligase dynamics by the nucleolus.

Mekhail K, Khacho M, Carrigan A, Hache RR, Gunaratnam L, Lee S - J. Cell Biol. (2005)

Bottom Line: Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded.The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability.Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

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Kinetics of nucleolar VHL and REV. (A and B) MCF7 cells cotransfected to express VHL-GFP and REV-BFP were incubated in hypoxia in SD or AP conditions. After localization of VHL-GFP to the nucleolus, cells were either fixed and immunostained for nucleolar B23 (A) or treated with 10 μg/ml ActD and monitored over time (B). Arrows in A indicate the same position in the cell and dashed cellular outlines are shown in B. Insets in B show a time-matched set that was not treated with ActD. (C and D) VHL-GFP-NES fusion protein is not released from nucleoli after ActD treatment. MCF7 cells were transfected to express VHL-GFP, VHL-GFP-NES, or GFP-NES alone and transferred to hypoxia in SD or AP media. Shown are images 50 min after AP media reached the pH 6.5 threshold. Where indicated, leptomycin B (LMB) was added when AP media reached pH 6.65 (usually 30–40 min) before pH 6.5 time point. Insets in C show Hoechst staining of DNA. (D) Ratiometric fluorescence measurements of hypoxic-acidotic cells initially displaying nucleolar REV-BFP, VHL-GFP, and VHL-GFP-NES after ActD treatment.
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fig2: Kinetics of nucleolar VHL and REV. (A and B) MCF7 cells cotransfected to express VHL-GFP and REV-BFP were incubated in hypoxia in SD or AP conditions. After localization of VHL-GFP to the nucleolus, cells were either fixed and immunostained for nucleolar B23 (A) or treated with 10 μg/ml ActD and monitored over time (B). Arrows in A indicate the same position in the cell and dashed cellular outlines are shown in B. Insets in B show a time-matched set that was not treated with ActD. (C and D) VHL-GFP-NES fusion protein is not released from nucleoli after ActD treatment. MCF7 cells were transfected to express VHL-GFP, VHL-GFP-NES, or GFP-NES alone and transferred to hypoxia in SD or AP media. Shown are images 50 min after AP media reached the pH 6.5 threshold. Where indicated, leptomycin B (LMB) was added when AP media reached pH 6.65 (usually 30–40 min) before pH 6.5 time point. Insets in C show Hoechst staining of DNA. (D) Ratiometric fluorescence measurements of hypoxic-acidotic cells initially displaying nucleolar REV-BFP, VHL-GFP, and VHL-GFP-NES after ActD treatment.

Mentions: Due to the role of the nucleolus in ribosomal biogenesis, perturbations to transcription, such as by treatment with low levels of actinomycin D (ActD), alter the trafficking properties of steady-state nucleolar proteins between the nucleolus and the nucleoplasm (Chen and Huang, 2001; Andersen et al., 2005). For example, the human immunodeficiency virus (HIV) mRNA exporter REV is a dynamic nucleolar protein that redistributes to the nucleoplasm and cytoplasm after transcriptional inhibition under both neutral (unpublished data; Stauber et al., 1995; Daelemans et al., 2005) and acidic (Fig. 2 A; Fig. 2 B, top) conditions. In contrast, the nucleolar localization of VHL in acidosis persisted in the absence of transcription (Fig. 2 B, bottom). Similar results were obtained in experiments using VHL-BFP and REV-GFP (unpublished data). The ability of REV to rapidly alter its steady-state distribution under these conditions is greatly enhanced by its strong nuclear export sequence (NES). We therefore tested whether fusion of this NES to VHL would enable it to release from nucleoli of acidotic cells after transcriptional inhibition. VHL-GFP-NES fusion localized almost exclusively to the cytoplasm under neutral conditions but was restricted to nucleoli at steady-state under acidosis (Fig. 2 C). ActD treatment failed to release the VHL-GFP-NES fusion protein from nucleoli (Fig. 2, C and D), suggesting that the subcellular trafficking dynamics of VHL in the nucleolus significantly differ from the dynamics of resident nucleolar proteins.


Regulation of ubiquitin ligase dynamics by the nucleolus.

Mekhail K, Khacho M, Carrigan A, Hache RR, Gunaratnam L, Lee S - J. Cell Biol. (2005)

Kinetics of nucleolar VHL and REV. (A and B) MCF7 cells cotransfected to express VHL-GFP and REV-BFP were incubated in hypoxia in SD or AP conditions. After localization of VHL-GFP to the nucleolus, cells were either fixed and immunostained for nucleolar B23 (A) or treated with 10 μg/ml ActD and monitored over time (B). Arrows in A indicate the same position in the cell and dashed cellular outlines are shown in B. Insets in B show a time-matched set that was not treated with ActD. (C and D) VHL-GFP-NES fusion protein is not released from nucleoli after ActD treatment. MCF7 cells were transfected to express VHL-GFP, VHL-GFP-NES, or GFP-NES alone and transferred to hypoxia in SD or AP media. Shown are images 50 min after AP media reached the pH 6.5 threshold. Where indicated, leptomycin B (LMB) was added when AP media reached pH 6.65 (usually 30–40 min) before pH 6.5 time point. Insets in C show Hoechst staining of DNA. (D) Ratiometric fluorescence measurements of hypoxic-acidotic cells initially displaying nucleolar REV-BFP, VHL-GFP, and VHL-GFP-NES after ActD treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171338&req=5

fig2: Kinetics of nucleolar VHL and REV. (A and B) MCF7 cells cotransfected to express VHL-GFP and REV-BFP were incubated in hypoxia in SD or AP conditions. After localization of VHL-GFP to the nucleolus, cells were either fixed and immunostained for nucleolar B23 (A) or treated with 10 μg/ml ActD and monitored over time (B). Arrows in A indicate the same position in the cell and dashed cellular outlines are shown in B. Insets in B show a time-matched set that was not treated with ActD. (C and D) VHL-GFP-NES fusion protein is not released from nucleoli after ActD treatment. MCF7 cells were transfected to express VHL-GFP, VHL-GFP-NES, or GFP-NES alone and transferred to hypoxia in SD or AP media. Shown are images 50 min after AP media reached the pH 6.5 threshold. Where indicated, leptomycin B (LMB) was added when AP media reached pH 6.65 (usually 30–40 min) before pH 6.5 time point. Insets in C show Hoechst staining of DNA. (D) Ratiometric fluorescence measurements of hypoxic-acidotic cells initially displaying nucleolar REV-BFP, VHL-GFP, and VHL-GFP-NES after ActD treatment.
Mentions: Due to the role of the nucleolus in ribosomal biogenesis, perturbations to transcription, such as by treatment with low levels of actinomycin D (ActD), alter the trafficking properties of steady-state nucleolar proteins between the nucleolus and the nucleoplasm (Chen and Huang, 2001; Andersen et al., 2005). For example, the human immunodeficiency virus (HIV) mRNA exporter REV is a dynamic nucleolar protein that redistributes to the nucleoplasm and cytoplasm after transcriptional inhibition under both neutral (unpublished data; Stauber et al., 1995; Daelemans et al., 2005) and acidic (Fig. 2 A; Fig. 2 B, top) conditions. In contrast, the nucleolar localization of VHL in acidosis persisted in the absence of transcription (Fig. 2 B, bottom). Similar results were obtained in experiments using VHL-BFP and REV-GFP (unpublished data). The ability of REV to rapidly alter its steady-state distribution under these conditions is greatly enhanced by its strong nuclear export sequence (NES). We therefore tested whether fusion of this NES to VHL would enable it to release from nucleoli of acidotic cells after transcriptional inhibition. VHL-GFP-NES fusion localized almost exclusively to the cytoplasm under neutral conditions but was restricted to nucleoli at steady-state under acidosis (Fig. 2 C). ActD treatment failed to release the VHL-GFP-NES fusion protein from nucleoli (Fig. 2, C and D), suggesting that the subcellular trafficking dynamics of VHL in the nucleolus significantly differ from the dynamics of resident nucleolar proteins.

Bottom Line: Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded.The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability.Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

Show MeSH
Related in: MedlinePlus