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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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FRAP analysis reveals that VHL does not shuttle between nucleoli in acidosis. MCF7 cells transiently transfected to express low levels of VHL-GFP, B23-GFP, FIB-GFP, or UBF1-GFP were incubated in hypoxia under SD or AP conditions. (A–C) Cells were imaged before and after bleaching of indicated nucleoplasmic regions (dashed square) or specific nucleoli (arrows) within nuclei (dashed circles). Post-bleach time is indicated in seconds. Areas of interest are shown as pseudocolored panels to better illustrate minimal changes in fluorescence. (D and E) Steady-state localization of GFP-tagged proteins after incubation in SD or AP media under hypoxia. (F and G) Quantitation of recovery kinetics. Fluorescence intensity in the bleached region was measured and expressed as relative recovery (see Materials and methods). For quantitation, at least 10 cells were analyzed for each result.
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fig3: FRAP analysis reveals that VHL does not shuttle between nucleoli in acidosis. MCF7 cells transiently transfected to express low levels of VHL-GFP, B23-GFP, FIB-GFP, or UBF1-GFP were incubated in hypoxia under SD or AP conditions. (A–C) Cells were imaged before and after bleaching of indicated nucleoplasmic regions (dashed square) or specific nucleoli (arrows) within nuclei (dashed circles). Post-bleach time is indicated in seconds. Areas of interest are shown as pseudocolored panels to better illustrate minimal changes in fluorescence. (D and E) Steady-state localization of GFP-tagged proteins after incubation in SD or AP media under hypoxia. (F and G) Quantitation of recovery kinetics. Fluorescence intensity in the bleached region was measured and expressed as relative recovery (see Materials and methods). For quantitation, at least 10 cells were analyzed for each result.

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)

FRAP analysis reveals that VHL does not shuttle between nucleoli in acidosis. MCF7 cells transiently transfected to express low levels of VHL-GFP, B23-GFP, FIB-GFP, or UBF1-GFP were incubated in hypoxia under SD or AP conditions. (A–C) Cells were imaged before and after bleaching of indicated nucleoplasmic regions (dashed square) or specific nucleoli (arrows) within nuclei (dashed circles). Post-bleach time is indicated in seconds. Areas of interest are shown as pseudocolored panels to better illustrate minimal changes in fluorescence. (D and E) Steady-state localization of GFP-tagged proteins after incubation in SD or AP media under hypoxia. (F and G) Quantitation of recovery kinetics. Fluorescence intensity in the bleached region was measured and expressed as relative recovery (see Materials and methods). For quantitation, at least 10 cells were analyzed for each result.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171338&req=5

fig3: FRAP analysis reveals that VHL does not shuttle between nucleoli in acidosis. MCF7 cells transiently transfected to express low levels of VHL-GFP, B23-GFP, FIB-GFP, or UBF1-GFP were incubated in hypoxia under SD or AP conditions. (A–C) Cells were imaged before and after bleaching of indicated nucleoplasmic regions (dashed square) or specific nucleoli (arrows) within nuclei (dashed circles). Post-bleach time is indicated in seconds. Areas of interest are shown as pseudocolored panels to better illustrate minimal changes in fluorescence. (D and E) Steady-state localization of GFP-tagged proteins after incubation in SD or AP media under hypoxia. (F and G) Quantitation of recovery kinetics. Fluorescence intensity in the bleached region was measured and expressed as relative recovery (see Materials and methods). For quantitation, at least 10 cells were analyzed for each result.
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