Limits...
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.

Show MeSH

Related in: MedlinePlus

pH-responsive nucleolar detention signal (NoDSH+) allows VHL to target the VBC/Cul-2 ubiquitin ligase complex for static detention in the nucleolus. (A) Schematic representation of the VBC/Cul-2 complex. ΔC157 is a mutant of VHL that fails to assemble the complex. Positions of different amino acids of VHL are indicated. (B) Complex formation is not required for nucleolar localization. VHL-deficient 786-0 cells were infected to express GFP-tagged VHL, ΔC157, or Cullin-2 and transferred to hypoxia in AP media. Acidosis triggered the nucleolar relocation of VHL-GFP and ΔC157-GFP, but not of GFP-Cullin-2. (C–F) VHL targets components of its ubiquitin ligase complex to the nucleolus. (C) VHL-deficient 786-0 cells were left uninfected or infected to express flag-tagged VHL-GFP or GFP alone and incubated in hypoxia in SD or AP media. When ∼60% of the cells in AP conditions displayed ∼30% nucleolar accumulation of VHL-GFP, cells were lysed and submitted to anti-flag immunoprecipitation and silver staining. Acidosis did not cause any sudden disruption of the VBC/Cul-2 complex. (D) VHL-deficient 786-0 cells cotransfected to transiently express VHL-BFP and GFP-Cullin-2 (1:1 ratio) were transferred to hypoxia in SD or AP conditions. Cullin-2 colocalized with VHL to the nucleolus in acidosis (arrowheads). (E and F) VHL-deficient 786-0 cells were cotransfected to transiently express VHL-BFP and GFP-Cullin-2 to varying ratios (indicated on panels and graph). VHL is the limiting factor in the nucleolar colocalization (arrows) of VHL and Cullin-2. (G) VHL mapping analysis. MCF7 cells transfected to transiently express indicated GFP-tagged proteins were incubated in AP media for 20 h in hypoxia. Schematic of VHL exons and derived amino acid domains are shown. Nucleolar localization was scored according to representative images in (H). Amino acid residues 100–130 of the VHL protein were identified as the minimal domain to recapitulate the nucleolar localization potential of the wild-type VHL tumor suppressor protein in acidosis. “a.d.” denotes acidic domain. (I) VHL(100–130) represents a surface pocket on the VHL protein as revealed by molecular modeling (PyMOL). Each amino acid within that sequence is represented in a different color for better visualization. MCF7 cells were transfected to transiently express VHL(100–130)-GFP at low (J) or high (K and L) levels and transferred to hypoxia under AP conditions. Upon reaching a plateau for nucleolar targeting, cells were submitted to FLIP analysis in which a nucleoplasmic region (white square in K) was repeatedly bleached. Insets in K show a pseudocolored zoom of a nucleolus (arrow).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171338&req=5

fig8: pH-responsive nucleolar detention signal (NoDSH+) allows VHL to target the VBC/Cul-2 ubiquitin ligase complex for static detention in the nucleolus. (A) Schematic representation of the VBC/Cul-2 complex. ΔC157 is a mutant of VHL that fails to assemble the complex. Positions of different amino acids of VHL are indicated. (B) Complex formation is not required for nucleolar localization. VHL-deficient 786-0 cells were infected to express GFP-tagged VHL, ΔC157, or Cullin-2 and transferred to hypoxia in AP media. Acidosis triggered the nucleolar relocation of VHL-GFP and ΔC157-GFP, but not of GFP-Cullin-2. (C–F) VHL targets components of its ubiquitin ligase complex to the nucleolus. (C) VHL-deficient 786-0 cells were left uninfected or infected to express flag-tagged VHL-GFP or GFP alone and incubated in hypoxia in SD or AP media. When ∼60% of the cells in AP conditions displayed ∼30% nucleolar accumulation of VHL-GFP, cells were lysed and submitted to anti-flag immunoprecipitation and silver staining. Acidosis did not cause any sudden disruption of the VBC/Cul-2 complex. (D) VHL-deficient 786-0 cells cotransfected to transiently express VHL-BFP and GFP-Cullin-2 (1:1 ratio) were transferred to hypoxia in SD or AP conditions. Cullin-2 colocalized with VHL to the nucleolus in acidosis (arrowheads). (E and F) VHL-deficient 786-0 cells were cotransfected to transiently express VHL-BFP and GFP-Cullin-2 to varying ratios (indicated on panels and graph). VHL is the limiting factor in the nucleolar colocalization (arrows) of VHL and Cullin-2. (G) VHL mapping analysis. MCF7 cells transfected to transiently express indicated GFP-tagged proteins were incubated in AP media for 20 h in hypoxia. Schematic of VHL exons and derived amino acid domains are shown. Nucleolar localization was scored according to representative images in (H). Amino acid residues 100–130 of the VHL protein were identified as the minimal domain to recapitulate the nucleolar localization potential of the wild-type VHL tumor suppressor protein in acidosis. “a.d.” denotes acidic domain. (I) VHL(100–130) represents a surface pocket on the VHL protein as revealed by molecular modeling (PyMOL). Each amino acid within that sequence is represented in a different color for better visualization. MCF7 cells were transfected to transiently express VHL(100–130)-GFP at low (J) or high (K and L) levels and transferred to hypoxia under AP conditions. Upon reaching a plateau for nucleolar targeting, cells were submitted to FLIP analysis in which a nucleoplasmic region (white square in K) was repeatedly bleached. Insets in K show a pseudocolored zoom of a nucleolus (arrow).

Mentions: Targeting of proteins to nucleoli is achieved by nucleolar localization sequence (NoLS) or nucleolar retention sequence (NoRS). These sequences are relatively large and differ considerably from nuclear import or export sequences, which are comprised of only a few amino acid residues. Therefore, we decided to identify the domain of the VBC/Cul-2 complex that mediates [H+]-regulated nucleolar sequestration of VHL. VHL is a component of the multi-protein ubiquitin ligase complex that targets the transcription factor HIF for proteasomal destruction. The complex is composed of at least VHL, elongin B, elongin C, Cullin-2, and Rbx1 (VBC/Cul-2) (Fig. 8 A; Kaelin, 2002). The ΔC157 deletion mutant of VHL, which is defective in E3 ligase complex formation (Fig. 8 A), retains the ability to target a GFP reporter to nucleoli in acidosis (Fig. 8 B, first four panels) (Pause et al., 1997; Cockman et al., 2000; Bonicalzi et al., 2001; Mekhail et al., 2004a). In contrast, Cullin-2 failed to target a GFP reporter to nucleoli of VHL-deficient cells under the same conditions (Fig. 8 B). Although these data suggest that complex formation is not required for nucleolar targeting of VHL, immunoprecipitation analysis of acidotic cells suggested that VHL can still assemble within the VBC/Cul-2 complex under acidic conditions (Fig. 8 C). Furthermore, when cotransfected with at least an equimolar amount of VHL, Cullin-2 colocalized with VHL in the nucleoli of acidotic cells (Fig. 8, D–F). Cullin-2 is also immobile in the nucleolus of acidic cells, suggesting that VHL responds to changes in extracellular pH and dictates the dynamic status of the assembled VBC/Cul-2 E3 ligase complex (unpublished data). Together, these data identify a novel role for the VHL tumor suppressor in regulating the subcellular trafficking dynamics of the VBC/Cul-2 ubiquitin ligase complex by targeting it to the nucleolus in response to an increase in environmental H+ 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-responsive nucleolar detention signal (NoDSH+) allows VHL to target the VBC/Cul-2 ubiquitin ligase complex for static detention in the nucleolus. (A) Schematic representation of the VBC/Cul-2 complex. ΔC157 is a mutant of VHL that fails to assemble the complex. Positions of different amino acids of VHL are indicated. (B) Complex formation is not required for nucleolar localization. VHL-deficient 786-0 cells were infected to express GFP-tagged VHL, ΔC157, or Cullin-2 and transferred to hypoxia in AP media. Acidosis triggered the nucleolar relocation of VHL-GFP and ΔC157-GFP, but not of GFP-Cullin-2. (C–F) VHL targets components of its ubiquitin ligase complex to the nucleolus. (C) VHL-deficient 786-0 cells were left uninfected or infected to express flag-tagged VHL-GFP or GFP alone and incubated in hypoxia in SD or AP media. When ∼60% of the cells in AP conditions displayed ∼30% nucleolar accumulation of VHL-GFP, cells were lysed and submitted to anti-flag immunoprecipitation and silver staining. Acidosis did not cause any sudden disruption of the VBC/Cul-2 complex. (D) VHL-deficient 786-0 cells cotransfected to transiently express VHL-BFP and GFP-Cullin-2 (1:1 ratio) were transferred to hypoxia in SD or AP conditions. Cullin-2 colocalized with VHL to the nucleolus in acidosis (arrowheads). (E and F) VHL-deficient 786-0 cells were cotransfected to transiently express VHL-BFP and GFP-Cullin-2 to varying ratios (indicated on panels and graph). VHL is the limiting factor in the nucleolar colocalization (arrows) of VHL and Cullin-2. (G) VHL mapping analysis. MCF7 cells transfected to transiently express indicated GFP-tagged proteins were incubated in AP media for 20 h in hypoxia. Schematic of VHL exons and derived amino acid domains are shown. Nucleolar localization was scored according to representative images in (H). Amino acid residues 100–130 of the VHL protein were identified as the minimal domain to recapitulate the nucleolar localization potential of the wild-type VHL tumor suppressor protein in acidosis. “a.d.” denotes acidic domain. (I) VHL(100–130) represents a surface pocket on the VHL protein as revealed by molecular modeling (PyMOL). Each amino acid within that sequence is represented in a different color for better visualization. MCF7 cells were transfected to transiently express VHL(100–130)-GFP at low (J) or high (K and L) levels and transferred to hypoxia under AP conditions. Upon reaching a plateau for nucleolar targeting, cells were submitted to FLIP analysis in which a nucleoplasmic region (white square in K) was repeatedly bleached. Insets in K show a pseudocolored zoom of a nucleolus (arrow).
© Copyright Policy
Related In: Results  -  Collection

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

fig8: pH-responsive nucleolar detention signal (NoDSH+) allows VHL to target the VBC/Cul-2 ubiquitin ligase complex for static detention in the nucleolus. (A) Schematic representation of the VBC/Cul-2 complex. ΔC157 is a mutant of VHL that fails to assemble the complex. Positions of different amino acids of VHL are indicated. (B) Complex formation is not required for nucleolar localization. VHL-deficient 786-0 cells were infected to express GFP-tagged VHL, ΔC157, or Cullin-2 and transferred to hypoxia in AP media. Acidosis triggered the nucleolar relocation of VHL-GFP and ΔC157-GFP, but not of GFP-Cullin-2. (C–F) VHL targets components of its ubiquitin ligase complex to the nucleolus. (C) VHL-deficient 786-0 cells were left uninfected or infected to express flag-tagged VHL-GFP or GFP alone and incubated in hypoxia in SD or AP media. When ∼60% of the cells in AP conditions displayed ∼30% nucleolar accumulation of VHL-GFP, cells were lysed and submitted to anti-flag immunoprecipitation and silver staining. Acidosis did not cause any sudden disruption of the VBC/Cul-2 complex. (D) VHL-deficient 786-0 cells cotransfected to transiently express VHL-BFP and GFP-Cullin-2 (1:1 ratio) were transferred to hypoxia in SD or AP conditions. Cullin-2 colocalized with VHL to the nucleolus in acidosis (arrowheads). (E and F) VHL-deficient 786-0 cells were cotransfected to transiently express VHL-BFP and GFP-Cullin-2 to varying ratios (indicated on panels and graph). VHL is the limiting factor in the nucleolar colocalization (arrows) of VHL and Cullin-2. (G) VHL mapping analysis. MCF7 cells transfected to transiently express indicated GFP-tagged proteins were incubated in AP media for 20 h in hypoxia. Schematic of VHL exons and derived amino acid domains are shown. Nucleolar localization was scored according to representative images in (H). Amino acid residues 100–130 of the VHL protein were identified as the minimal domain to recapitulate the nucleolar localization potential of the wild-type VHL tumor suppressor protein in acidosis. “a.d.” denotes acidic domain. (I) VHL(100–130) represents a surface pocket on the VHL protein as revealed by molecular modeling (PyMOL). Each amino acid within that sequence is represented in a different color for better visualization. MCF7 cells were transfected to transiently express VHL(100–130)-GFP at low (J) or high (K and L) levels and transferred to hypoxia under AP conditions. Upon reaching a plateau for nucleolar targeting, cells were submitted to FLIP analysis in which a nucleoplasmic region (white square in K) was repeatedly bleached. Insets in K show a pseudocolored zoom of a nucleolus (arrow).
Mentions: Targeting of proteins to nucleoli is achieved by nucleolar localization sequence (NoLS) or nucleolar retention sequence (NoRS). These sequences are relatively large and differ considerably from nuclear import or export sequences, which are comprised of only a few amino acid residues. Therefore, we decided to identify the domain of the VBC/Cul-2 complex that mediates [H+]-regulated nucleolar sequestration of VHL. VHL is a component of the multi-protein ubiquitin ligase complex that targets the transcription factor HIF for proteasomal destruction. The complex is composed of at least VHL, elongin B, elongin C, Cullin-2, and Rbx1 (VBC/Cul-2) (Fig. 8 A; Kaelin, 2002). The ΔC157 deletion mutant of VHL, which is defective in E3 ligase complex formation (Fig. 8 A), retains the ability to target a GFP reporter to nucleoli in acidosis (Fig. 8 B, first four panels) (Pause et al., 1997; Cockman et al., 2000; Bonicalzi et al., 2001; Mekhail et al., 2004a). In contrast, Cullin-2 failed to target a GFP reporter to nucleoli of VHL-deficient cells under the same conditions (Fig. 8 B). Although these data suggest that complex formation is not required for nucleolar targeting of VHL, immunoprecipitation analysis of acidotic cells suggested that VHL can still assemble within the VBC/Cul-2 complex under acidic conditions (Fig. 8 C). Furthermore, when cotransfected with at least an equimolar amount of VHL, Cullin-2 colocalized with VHL in the nucleoli of acidotic cells (Fig. 8, D–F). Cullin-2 is also immobile in the nucleolus of acidic cells, suggesting that VHL responds to changes in extracellular pH and dictates the dynamic status of the assembled VBC/Cul-2 E3 ligase complex (unpublished data). Together, these data identify a novel role for the VHL tumor suppressor in regulating the subcellular trafficking dynamics of the VBC/Cul-2 ubiquitin ligase complex by targeting it to the nucleolus in response to an increase in environmental H+ 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