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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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Characteristics of the NoDSH+ sequence. (A) Shown is a schematic representation of the VBC/Cul-2 complex. The NoDSH+ of VHL is highlighted in red and its constituent amino acids (residues 100–130 of VHL, inclusively) are indicated. (B) MCF7 cells were transiently transfected to express GFP-tagged NES, NLS, NoDSH+ or GFP alone. Cells were incubated in hypoxia under AP conditions with maximal acidifications of pH 6.9 or 6.3.
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fig9: Characteristics of the NoDSH+ sequence. (A) Shown is a schematic representation of the VBC/Cul-2 complex. The NoDSH+ of VHL is highlighted in red and its constituent amino acids (residues 100–130 of VHL, inclusively) are indicated. (B) MCF7 cells were transiently transfected to express GFP-tagged NES, NLS, NoDSH+ or GFP alone. Cells were incubated in hypoxia under AP conditions with maximal acidifications of pH 6.9 or 6.3.

Mentions: Mapping analyses of MDM2 and its associated proteins have previously identified small aminopeptide sequences that can target a GFP reporter protein to the nucleolus in response to various physiological signals (Weber et al., 2000; Lohrum et al., 2003). Deletion mutant analysis of VHL was therefore conducted to identify minimal nucleolar detention sequences. Although several regions within the β-domain of VHL displayed relatively weak nucleolar localization activity in response to acidosis, a domain encoding residues 100–130 recapitulated the nucleolar targeting capability of wild-type VHL (Fig. 8, G–I). VHL(100–130) efficiently mediated the nucleolar detention of a GFP reporter in acidosis as revealed by FLIP experiments (Fig. 8 J). Neutralization of the media released VHL(100–130) into the nucleoplasm where it resumed its dynamic mobility profile (unpublished data). In addition, similar to the wild-type protein (Fig. 4, D and G), increasing the expression level of VHL(100–130) created a static nucleolar and a dynamic nucleo-cytoplasmic pool (Fig. 8, K and L). Unlike VHL(100–130), previously identified NLS and NES sequences fail to respond to increases in extracellular hydrogen ion concentrations (Fig. 9), highlighting the functional specificity of the herein identified domain. These findings identify a novel and discrete protein domain as a new type of protein localization sequence that we now refer to as [H+]-responsive nucleolar detention signal (NoDSH+).


Regulation of ubiquitin ligase dynamics by the nucleolus.

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

Characteristics of the NoDSH+ sequence. (A) Shown is a schematic representation of the VBC/Cul-2 complex. The NoDSH+ of VHL is highlighted in red and its constituent amino acids (residues 100–130 of VHL, inclusively) are indicated. (B) MCF7 cells were transiently transfected to express GFP-tagged NES, NLS, NoDSH+ or GFP alone. Cells were incubated in hypoxia under AP conditions with maximal acidifications of pH 6.9 or 6.3.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Characteristics of the NoDSH+ sequence. (A) Shown is a schematic representation of the VBC/Cul-2 complex. The NoDSH+ of VHL is highlighted in red and its constituent amino acids (residues 100–130 of VHL, inclusively) are indicated. (B) MCF7 cells were transiently transfected to express GFP-tagged NES, NLS, NoDSH+ or GFP alone. Cells were incubated in hypoxia under AP conditions with maximal acidifications of pH 6.9 or 6.3.
Mentions: Mapping analyses of MDM2 and its associated proteins have previously identified small aminopeptide sequences that can target a GFP reporter protein to the nucleolus in response to various physiological signals (Weber et al., 2000; Lohrum et al., 2003). Deletion mutant analysis of VHL was therefore conducted to identify minimal nucleolar detention sequences. Although several regions within the β-domain of VHL displayed relatively weak nucleolar localization activity in response to acidosis, a domain encoding residues 100–130 recapitulated the nucleolar targeting capability of wild-type VHL (Fig. 8, G–I). VHL(100–130) efficiently mediated the nucleolar detention of a GFP reporter in acidosis as revealed by FLIP experiments (Fig. 8 J). Neutralization of the media released VHL(100–130) into the nucleoplasm where it resumed its dynamic mobility profile (unpublished data). In addition, similar to the wild-type protein (Fig. 4, D and G), increasing the expression level of VHL(100–130) created a static nucleolar and a dynamic nucleo-cytoplasmic pool (Fig. 8, K and L). Unlike VHL(100–130), previously identified NLS and NES sequences fail to respond to increases in extracellular hydrogen ion concentrations (Fig. 9), highlighting the functional specificity of the herein identified domain. These findings identify a novel and discrete protein domain as a new type of protein localization sequence that we now refer to as [H+]-responsive nucleolar detention signal (NoDSH+).

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