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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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Reversible static detention of VHL and MDM2 by the nucleolar architecture. MCF7 cells were transfected to express GFP-tagged MDM2 (A), B23 (B), or VHL (C–G) and incubated under SD, AP, or RS conditions as indicated. A region representing about half of the nucleolar space (white squares) of a single nucleolus was bleached and cells were monitored by time-lapse fluorescence microscopy. Note the absence of any changes in fluorescence for nucleolar VHL and MDM2. Zoom of a single nucleolus is shown in C. Changes in the shape of the border (dashed white outline set by bleaching) of the protein concentration gradient from different time points as in C and D are overlayed in E. (F) Borders with changing (cytoplasm) or unaltered (nucleolus) shapes (plotted on X and Y axes) were monitored for their displacement away from their original position within the field of vision. Only borders set within the nucleolar space did not exhibit any movement. (G) After hypoxia-induced acidification of AP media and confinement of VHL-GFP to the nucleolus, NaOH was added to neutralize AP media (to pH 7.2) or cells were replenished with fresh SD media (pH 7.2) to induce the reversion of VHL-GFP to the nucleo-cytoplasm (also see Fig. 1). FRAP analyses in which small nucleoplasmic regions were bleached reveal that VHL-GFP resumes its high mobility profile after a return to neutral pH conditions.
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fig7: Reversible static detention of VHL and MDM2 by the nucleolar architecture. MCF7 cells were transfected to express GFP-tagged MDM2 (A), B23 (B), or VHL (C–G) and incubated under SD, AP, or RS conditions as indicated. A region representing about half of the nucleolar space (white squares) of a single nucleolus was bleached and cells were monitored by time-lapse fluorescence microscopy. Note the absence of any changes in fluorescence for nucleolar VHL and MDM2. Zoom of a single nucleolus is shown in C. Changes in the shape of the border (dashed white outline set by bleaching) of the protein concentration gradient from different time points as in C and D are overlayed in E. (F) Borders with changing (cytoplasm) or unaltered (nucleolus) shapes (plotted on X and Y axes) were monitored for their displacement away from their original position within the field of vision. Only borders set within the nucleolar space did not exhibit any movement. (G) After hypoxia-induced acidification of AP media and confinement of VHL-GFP to the nucleolus, NaOH was added to neutralize AP media (to pH 7.2) or cells were replenished with fresh SD media (pH 7.2) to induce the reversion of VHL-GFP to the nucleo-cytoplasm (also see Fig. 1). FRAP analyses in which small nucleoplasmic regions were bleached reveal that VHL-GFP resumes its high mobility profile after a return to neutral pH conditions.

Mentions: Cells cultured under standard neutral conditions displayed an essentially complete recovery of VHL-GFP fluorescence within seconds of bleaching nucleoplasmic (Fig. 3, A and G) or cytoplasmic (see Fig. 7 D) regions. We first assessed the capacity of the nucleolus to sustain dynamic shuttling under acidosis by monitoring resident nucleolar proteins, such as the rRNA-processing factors fibrillarin (FIB) and nucleophosmin (NPM or B23), as well as the RNA polymerase I preinitation factor upstream binding factor 1 (UBF1). Acidosis did not alter the steady-state distribution of any of the studied resident nucleolar proteins (Fig. 3, D and E) compared with neutral conditions. In addition, these proteins displayed a rapid pH-independent recovery of fluorescence after bleaching of a single nucleolus within cells with multiple nucleoli (Fig. 3, C and F), indicating dynamic protein shuttling between nucleoli of acidotic cells. In contrast, nucleolar VHL failed to display recovery of fluorescence under the same culture and bleaching parameters (Fig. 3, B and G), suggesting that acidosis alters the mobility profile of VHL. Similar to previous reports, reduction of the temperature from 37 to 22°C did not have any significant effect on the kinetics or extent of recovery of any of the tested proteins in the nucleus or cytoplasm (unpublished data; see Phair and Misteli, 2000). These data suggest that the redistribution of VHL to the nucleolus in response to increases in extracellular hydrogen ion concentrations may alter its general dynamic characteristics in the cell.


Regulation of ubiquitin ligase dynamics by the nucleolus.

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

Reversible static detention of VHL and MDM2 by the nucleolar architecture. MCF7 cells were transfected to express GFP-tagged MDM2 (A), B23 (B), or VHL (C–G) and incubated under SD, AP, or RS conditions as indicated. A region representing about half of the nucleolar space (white squares) of a single nucleolus was bleached and cells were monitored by time-lapse fluorescence microscopy. Note the absence of any changes in fluorescence for nucleolar VHL and MDM2. Zoom of a single nucleolus is shown in C. Changes in the shape of the border (dashed white outline set by bleaching) of the protein concentration gradient from different time points as in C and D are overlayed in E. (F) Borders with changing (cytoplasm) or unaltered (nucleolus) shapes (plotted on X and Y axes) were monitored for their displacement away from their original position within the field of vision. Only borders set within the nucleolar space did not exhibit any movement. (G) After hypoxia-induced acidification of AP media and confinement of VHL-GFP to the nucleolus, NaOH was added to neutralize AP media (to pH 7.2) or cells were replenished with fresh SD media (pH 7.2) to induce the reversion of VHL-GFP to the nucleo-cytoplasm (also see Fig. 1). FRAP analyses in which small nucleoplasmic regions were bleached reveal that VHL-GFP resumes its high mobility profile after a return to neutral pH conditions.
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Related In: Results  -  Collection

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fig7: Reversible static detention of VHL and MDM2 by the nucleolar architecture. MCF7 cells were transfected to express GFP-tagged MDM2 (A), B23 (B), or VHL (C–G) and incubated under SD, AP, or RS conditions as indicated. A region representing about half of the nucleolar space (white squares) of a single nucleolus was bleached and cells were monitored by time-lapse fluorescence microscopy. Note the absence of any changes in fluorescence for nucleolar VHL and MDM2. Zoom of a single nucleolus is shown in C. Changes in the shape of the border (dashed white outline set by bleaching) of the protein concentration gradient from different time points as in C and D are overlayed in E. (F) Borders with changing (cytoplasm) or unaltered (nucleolus) shapes (plotted on X and Y axes) were monitored for their displacement away from their original position within the field of vision. Only borders set within the nucleolar space did not exhibit any movement. (G) After hypoxia-induced acidification of AP media and confinement of VHL-GFP to the nucleolus, NaOH was added to neutralize AP media (to pH 7.2) or cells were replenished with fresh SD media (pH 7.2) to induce the reversion of VHL-GFP to the nucleo-cytoplasm (also see Fig. 1). FRAP analyses in which small nucleoplasmic regions were bleached reveal that VHL-GFP resumes its high mobility profile after a return to neutral pH conditions.
Mentions: Cells cultured under standard neutral conditions displayed an essentially complete recovery of VHL-GFP fluorescence within seconds of bleaching nucleoplasmic (Fig. 3, A and G) or cytoplasmic (see Fig. 7 D) regions. We first assessed the capacity of the nucleolus to sustain dynamic shuttling under acidosis by monitoring resident nucleolar proteins, such as the rRNA-processing factors fibrillarin (FIB) and nucleophosmin (NPM or B23), as well as the RNA polymerase I preinitation factor upstream binding factor 1 (UBF1). Acidosis did not alter the steady-state distribution of any of the studied resident nucleolar proteins (Fig. 3, D and E) compared with neutral conditions. In addition, these proteins displayed a rapid pH-independent recovery of fluorescence after bleaching of a single nucleolus within cells with multiple nucleoli (Fig. 3, C and F), indicating dynamic protein shuttling between nucleoli of acidotic cells. In contrast, nucleolar VHL failed to display recovery of fluorescence under the same culture and bleaching parameters (Fig. 3, B and G), suggesting that acidosis alters the mobility profile of VHL. Similar to previous reports, reduction of the temperature from 37 to 22°C did not have any significant effect on the kinetics or extent of recovery of any of the tested proteins in the nucleus or cytoplasm (unpublished data; see Phair and Misteli, 2000). These data suggest that the redistribution of VHL to the nucleolus in response to increases in extracellular hydrogen ion concentrations may alter its general dynamic characteristics in the cell.

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