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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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pH-dependent kinetics of VHL subcellular trafficking. (A) C2C12-differentiated myotubes were cultured in standard (SD) media and infected to express adenovirus-introduced VHL-GFP. Cells were replenished with either fresh SD media (pH 7.2) or different AP media (initial pH 7.2) that allow maximal extracellular acidification to pH 6.9, 6.6, 6.4, 6.2, or 6.0 (see Materials and methods). Cells were transferred to hypoxia (1% O2) for 18 h. VHL redistributed to nucleoli only in conditions of pH 6.4 or lower (images of pH 6.2 and 6.0 are not depicted). Extracellular pH is indicated on each panel. Insets show Hoechst staining of DNA. Bars (A, B, and E), 10 μm. (B) After localization of VHL-GFP to the nucleolus, myotubes were replenished with AP media previously maximally acidified to indicated pH levels, or with fresh SD media. VHL reverted to nucleo-cytoplasmic distribution only when the pH of the media was higher than 6.4. (C) Different cell types were treated as in A and B to identify the pH thresholds required for nucleolar targeting of VHL (n = 40 for each tested pH value). (D) Kinetics of VHL nucleolar localization in acidosis and release after neutralization in differentiated myotubes. Cells were exposed to different AP media as in A to induce VHL nucleolar redistribution, and all media were neutralized to pH 7.2 by addition of NaOH (arrow). Time zero indicates time at which the pH of AP media reached 6.4. For the 6.5 and 6.7 sets, time zero is matched to that of the pH 6.4 set. (E) Cells were incubated in AP media (initial pH 7.2) that blocks acidification beyond pH 6.0. After reaching the acidification level matching the pH threshold required to trigger nucleolar targeting of any given cell-type (time zero), the localization of VHL-GFP was monitored over time. Upon reaching the pH threshold, VHL shifts from mainly cytoplasmic to mainly nucleoplasmic before triggering nucleolar localization.
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fig1: pH-dependent kinetics of VHL subcellular trafficking. (A) C2C12-differentiated myotubes were cultured in standard (SD) media and infected to express adenovirus-introduced VHL-GFP. Cells were replenished with either fresh SD media (pH 7.2) or different AP media (initial pH 7.2) that allow maximal extracellular acidification to pH 6.9, 6.6, 6.4, 6.2, or 6.0 (see Materials and methods). Cells were transferred to hypoxia (1% O2) for 18 h. VHL redistributed to nucleoli only in conditions of pH 6.4 or lower (images of pH 6.2 and 6.0 are not depicted). Extracellular pH is indicated on each panel. Insets show Hoechst staining of DNA. Bars (A, B, and E), 10 μm. (B) After localization of VHL-GFP to the nucleolus, myotubes were replenished with AP media previously maximally acidified to indicated pH levels, or with fresh SD media. VHL reverted to nucleo-cytoplasmic distribution only when the pH of the media was higher than 6.4. (C) Different cell types were treated as in A and B to identify the pH thresholds required for nucleolar targeting of VHL (n = 40 for each tested pH value). (D) Kinetics of VHL nucleolar localization in acidosis and release after neutralization in differentiated myotubes. Cells were exposed to different AP media as in A to induce VHL nucleolar redistribution, and all media were neutralized to pH 7.2 by addition of NaOH (arrow). Time zero indicates time at which the pH of AP media reached 6.4. For the 6.5 and 6.7 sets, time zero is matched to that of the pH 6.4 set. (E) Cells were incubated in AP media (initial pH 7.2) that blocks acidification beyond pH 6.0. After reaching the acidification level matching the pH threshold required to trigger nucleolar targeting of any given cell-type (time zero), the localization of VHL-GFP was monitored over time. Upon reaching the pH threshold, VHL shifts from mainly cytoplasmic to mainly nucleoplasmic before triggering nucleolar localization.

Mentions: Ischemic tissues or hypoxic cells normally acidify their extracellular milieu as a physiological consequence of anaerobic glycolysis. This is best exemplified by muscle fatigue, in which myotubes produce lactic acid after exposure to hypoxia. Study of the ubiquitin ligase component VHL within this setting revealed its functional regulation by changes in environmental H+ concentrations (Mekhail et al., 2004a). VHL engages in nuclear/cytoplasmic trafficking in neutral conditions but accumulates in the nucleolus upon a decrease in extracellular pH, a process that results in stabilization of its substrate HIF. Differentiated myotubes can be incubated in standard (SD) media, which prevents fluctuations in pH, or in acidification-permissive (AP) media, which is prepared to enable hypoxic cells to acidify their extracellular milieu to varying degrees (see Materials and methods) (Mekhail et al., 2004a). VHL-GFP is observed in its typical diffuse nuclear cytoplasmic distribution under neutral pH conditions, independent of oxygen tension (Fig. 1 A, left; Mekhail et al., 2004a). A rapid redistribution of VHL-GFP to nucleoli was observed only when hypoxic myotubes were incubated in AP media that allow the myotubes to acidify their environment to pH 6.40 or lower (Fig. 1, A and D; see Fig. S1, A and B, for cell viability controls, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1), indicating the existence of a pH threshold required for triggering nucleolar localization of VHL. The threshold displayed cell type–specific differences within the 6.60–5.80 pH range (Fig. 1 C; Fig. S2). VHL efficiently reverted to a diffuse nuclear-cytoplasmic localization under hypoxic conditions only when cells were replenished with media at values higher than the pH threshold required to trigger nucleolar localization (Fig. 1, B–D). Closer examination of this system reveals that the relocation of VHL to the nucleolus is a two-step process, where the protein displays an initial shift from mainly cytoplasmic to mainly nucleoplasmic localization before initiating any detectable targeting to the nucleolus, in all tested cell lines (Fig. 1 E; Fig. S1, C and D). These data suggest that the subcellular trafficking dynamics of VHL are regulated by a multilayered cellular mechanism that gauges environmental hydrogen ion concentrations.


Regulation of ubiquitin ligase dynamics by the nucleolus.

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

pH-dependent kinetics of VHL subcellular trafficking. (A) C2C12-differentiated myotubes were cultured in standard (SD) media and infected to express adenovirus-introduced VHL-GFP. Cells were replenished with either fresh SD media (pH 7.2) or different AP media (initial pH 7.2) that allow maximal extracellular acidification to pH 6.9, 6.6, 6.4, 6.2, or 6.0 (see Materials and methods). Cells were transferred to hypoxia (1% O2) for 18 h. VHL redistributed to nucleoli only in conditions of pH 6.4 or lower (images of pH 6.2 and 6.0 are not depicted). Extracellular pH is indicated on each panel. Insets show Hoechst staining of DNA. Bars (A, B, and E), 10 μm. (B) After localization of VHL-GFP to the nucleolus, myotubes were replenished with AP media previously maximally acidified to indicated pH levels, or with fresh SD media. VHL reverted to nucleo-cytoplasmic distribution only when the pH of the media was higher than 6.4. (C) Different cell types were treated as in A and B to identify the pH thresholds required for nucleolar targeting of VHL (n = 40 for each tested pH value). (D) Kinetics of VHL nucleolar localization in acidosis and release after neutralization in differentiated myotubes. Cells were exposed to different AP media as in A to induce VHL nucleolar redistribution, and all media were neutralized to pH 7.2 by addition of NaOH (arrow). Time zero indicates time at which the pH of AP media reached 6.4. For the 6.5 and 6.7 sets, time zero is matched to that of the pH 6.4 set. (E) Cells were incubated in AP media (initial pH 7.2) that blocks acidification beyond pH 6.0. After reaching the acidification level matching the pH threshold required to trigger nucleolar targeting of any given cell-type (time zero), the localization of VHL-GFP was monitored over time. Upon reaching the pH threshold, VHL shifts from mainly cytoplasmic to mainly nucleoplasmic before triggering nucleolar localization.
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Related In: Results  -  Collection

Show All Figures
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fig1: pH-dependent kinetics of VHL subcellular trafficking. (A) C2C12-differentiated myotubes were cultured in standard (SD) media and infected to express adenovirus-introduced VHL-GFP. Cells were replenished with either fresh SD media (pH 7.2) or different AP media (initial pH 7.2) that allow maximal extracellular acidification to pH 6.9, 6.6, 6.4, 6.2, or 6.0 (see Materials and methods). Cells were transferred to hypoxia (1% O2) for 18 h. VHL redistributed to nucleoli only in conditions of pH 6.4 or lower (images of pH 6.2 and 6.0 are not depicted). Extracellular pH is indicated on each panel. Insets show Hoechst staining of DNA. Bars (A, B, and E), 10 μm. (B) After localization of VHL-GFP to the nucleolus, myotubes were replenished with AP media previously maximally acidified to indicated pH levels, or with fresh SD media. VHL reverted to nucleo-cytoplasmic distribution only when the pH of the media was higher than 6.4. (C) Different cell types were treated as in A and B to identify the pH thresholds required for nucleolar targeting of VHL (n = 40 for each tested pH value). (D) Kinetics of VHL nucleolar localization in acidosis and release after neutralization in differentiated myotubes. Cells were exposed to different AP media as in A to induce VHL nucleolar redistribution, and all media were neutralized to pH 7.2 by addition of NaOH (arrow). Time zero indicates time at which the pH of AP media reached 6.4. For the 6.5 and 6.7 sets, time zero is matched to that of the pH 6.4 set. (E) Cells were incubated in AP media (initial pH 7.2) that blocks acidification beyond pH 6.0. After reaching the acidification level matching the pH threshold required to trigger nucleolar targeting of any given cell-type (time zero), the localization of VHL-GFP was monitored over time. Upon reaching the pH threshold, VHL shifts from mainly cytoplasmic to mainly nucleoplasmic before triggering nucleolar localization.
Mentions: Ischemic tissues or hypoxic cells normally acidify their extracellular milieu as a physiological consequence of anaerobic glycolysis. This is best exemplified by muscle fatigue, in which myotubes produce lactic acid after exposure to hypoxia. Study of the ubiquitin ligase component VHL within this setting revealed its functional regulation by changes in environmental H+ concentrations (Mekhail et al., 2004a). VHL engages in nuclear/cytoplasmic trafficking in neutral conditions but accumulates in the nucleolus upon a decrease in extracellular pH, a process that results in stabilization of its substrate HIF. Differentiated myotubes can be incubated in standard (SD) media, which prevents fluctuations in pH, or in acidification-permissive (AP) media, which is prepared to enable hypoxic cells to acidify their extracellular milieu to varying degrees (see Materials and methods) (Mekhail et al., 2004a). VHL-GFP is observed in its typical diffuse nuclear cytoplasmic distribution under neutral pH conditions, independent of oxygen tension (Fig. 1 A, left; Mekhail et al., 2004a). A rapid redistribution of VHL-GFP to nucleoli was observed only when hypoxic myotubes were incubated in AP media that allow the myotubes to acidify their environment to pH 6.40 or lower (Fig. 1, A and D; see Fig. S1, A and B, for cell viability controls, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1), indicating the existence of a pH threshold required for triggering nucleolar localization of VHL. The threshold displayed cell type–specific differences within the 6.60–5.80 pH range (Fig. 1 C; Fig. S2). VHL efficiently reverted to a diffuse nuclear-cytoplasmic localization under hypoxic conditions only when cells were replenished with media at values higher than the pH threshold required to trigger nucleolar localization (Fig. 1, B–D). Closer examination of this system reveals that the relocation of VHL to the nucleolus is a two-step process, where the protein displays an initial shift from mainly cytoplasmic to mainly nucleoplasmic localization before initiating any detectable targeting to the nucleolus, in all tested cell lines (Fig. 1 E; Fig. S1, C and D). These data suggest that the subcellular trafficking dynamics of VHL are regulated by a multilayered cellular mechanism that gauges environmental hydrogen ion concentrations.

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