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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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FLIP analysis reveals that nucleolar VHL does not traffic between the nucleolus and nucleoplasm in acidosis. MCF7 cells transiently transfected to express low (A–C, and F) or high (D, E, and G) levels of VHL-GFP or B23-GFP were incubated in hypoxia under SD or AP conditions. At time points matching the relocation of VHL-GFP (low levels set) to nucleoli, all cells were submitted to a FLIP analysis where nucleoplasmic regions (white squares) within specific nuclei (dashed circles) were repeatedly bleached. Cells were imaged between pulses. (F and G) Corresponding kinetics of loss of fluorescence.
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fig4: FLIP analysis reveals that nucleolar VHL does not traffic between the nucleolus and nucleoplasm in acidosis. MCF7 cells transiently transfected to express low (A–C, and F) or high (D, E, and G) levels of VHL-GFP or B23-GFP were incubated in hypoxia under SD or AP conditions. At time points matching the relocation of VHL-GFP (low levels set) to nucleoli, all cells were submitted to a FLIP analysis where nucleoplasmic regions (white squares) within specific nuclei (dashed circles) were repeatedly bleached. Cells were imaged between pulses. (F and G) Corresponding kinetics of loss of fluorescence.

Mentions: The dynamics of VHL were next analyzed by fluorescence loss in photobleaching (FLIP) experiments (Lippincott-Schwartz et al., 2003). In FLIP, a living cell is repeatedly hit with a laser beam in the same region. Loss of fluorescence in an area outside the bleached spot is reflective of protein mobility between that area and the bleached spot. A rapid loss of VHL-GFP fluorescence was observed in essentially the whole nucleus after repetitive bleaching of a small nucleoplasmic region in cells incubated under neutral conditions (Fig. 4 A). Studies presented further in this report (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1) will study the nuclear-cytoplasmic trafficking properties of VHL. These observations indicate that VHL participates in dynamic molecular networks.


Regulation of ubiquitin ligase dynamics by the nucleolus.

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

FLIP analysis reveals that nucleolar VHL does not traffic between the nucleolus and nucleoplasm in acidosis. MCF7 cells transiently transfected to express low (A–C, and F) or high (D, E, and G) levels of VHL-GFP or B23-GFP were incubated in hypoxia under SD or AP conditions. At time points matching the relocation of VHL-GFP (low levels set) to nucleoli, all cells were submitted to a FLIP analysis where nucleoplasmic regions (white squares) within specific nuclei (dashed circles) were repeatedly bleached. Cells were imaged between pulses. (F and G) Corresponding kinetics of loss of fluorescence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171338&req=5

fig4: FLIP analysis reveals that nucleolar VHL does not traffic between the nucleolus and nucleoplasm in acidosis. MCF7 cells transiently transfected to express low (A–C, and F) or high (D, E, and G) levels of VHL-GFP or B23-GFP were incubated in hypoxia under SD or AP conditions. At time points matching the relocation of VHL-GFP (low levels set) to nucleoli, all cells were submitted to a FLIP analysis where nucleoplasmic regions (white squares) within specific nuclei (dashed circles) were repeatedly bleached. Cells were imaged between pulses. (F and G) Corresponding kinetics of loss of fluorescence.
Mentions: The dynamics of VHL were next analyzed by fluorescence loss in photobleaching (FLIP) experiments (Lippincott-Schwartz et al., 2003). In FLIP, a living cell is repeatedly hit with a laser beam in the same region. Loss of fluorescence in an area outside the bleached spot is reflective of protein mobility between that area and the bleached spot. A rapid loss of VHL-GFP fluorescence was observed in essentially the whole nucleus after repetitive bleaching of a small nucleoplasmic region in cells incubated under neutral conditions (Fig. 4 A). Studies presented further in this report (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1) will study the nuclear-cytoplasmic trafficking properties of VHL. These observations indicate that VHL participates in dynamic molecular networks.

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