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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 the redistribution of MDM2 from nucleoplasm to nucleoli in response to perturbations in ribosomal biogenesis alters general MDM2 dynamic state. MCF7 cells transfected to express low levels of MDM2-GFP were cultured either under standard conditions (A), or ribosomal stress (RS) (B) induced by ActD treatment (see Lohrum et al., 2003 and Materials and methods). Cells were submitted to FRAP analysis as described for VHL in Fig. 3 by bleaching the indicated nucleoplasmic regions (white squares) or specific nucleoli (arrows) within specific nuclei (dashed circles). Pseudocolored zoom of area indicated by dashed rectangle is shown in B.
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fig6: FRAP analysis reveals that the redistribution of MDM2 from nucleoplasm to nucleoli in response to perturbations in ribosomal biogenesis alters general MDM2 dynamic state. MCF7 cells transfected to express low levels of MDM2-GFP were cultured either under standard conditions (A), or ribosomal stress (RS) (B) induced by ActD treatment (see Lohrum et al., 2003 and Materials and methods). Cells were submitted to FRAP analysis as described for VHL in Fig. 3 by bleaching the indicated nucleoplasmic regions (white squares) or specific nucleoli (arrows) within specific nuclei (dashed circles). Pseudocolored zoom of area indicated by dashed rectangle is shown in B.

Mentions: MDM2 displays a diffuse nuclear localization under standard culture conditions. FRAP and FLIP experiments revealed that MDM2 is a highly dynamic protein within this setting (Fig. 6 A; Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1). MDM2 localizes to the nucleolus in response to perturbations in ribosomal biogenesis after treatment with low levels of ActD that inhibit RNA polymerase I (Fig. 6 B; Fig. S4 B; Lohrum et al., 2003). MDM2 is unable to target p53 for degradation under these conditions. We therefore assessed the dynamics of MDM2 after relocation to the nucleolus. When nucleolar, the dynamic profile of MDM2 significantly changed as GFP fluorescence did not exhibit any recovery/redistribution in FRAP experiments after bleaching of the nucleolus (Fig. 6 B) or loss in FLIP experiments after repetitive bleaching of a nucleoplasmic (Fig. S4 B) or cytoplasmic (unpublished data) region. Similar to VHL (Fig. 4, D and G), the interaction of MDM2 with the nucleolar architecture is required for modification of its trafficking dynamics as evidenced by the quick recovery of MDM2 in the nucleoplasm of transcriptionally inhibited cells expressing high levels of the protein in FRAP (Fig. 7 A). We next evaluated the dynamics of VHL and MDM2 ligases within the nucleolar space. VHL and MDM2 did not exhibit any fluorescence recovery after bleaching of an area within the nucleolus (Fig. 7, A and C). In contrast, B23 remained localized to nucleoli at steady-state within our experimental settings (SD, AP, and RS), retaining its highly mobile properties, though prolonged incubation with ActD resulted in B23 accumulating in the nucleoplasm (Fig. 7 B; unpublished data). Upon photobleaching, a border is created between the bleached area and the gradient of concentration established by the remaining fluorescent molecules (Fig. 7 C). For moving proteins, this border changes its shape as well as its position within the field of vision over time. Statically detained cellular components do not exhibit significant changes in these variables. We therefore compared the characteristics of borders of concentration gradients established by bleaching fluorescently labeled proteins when localized to different regions of the cell. Although analysis of concentration gradient borders in the nucleoplasm and cytoplasm revealed a highly dynamic profile of protein mobility, borders established within the nucleolar space neither changed in shape nor moved within the field of vision for up to 2 h after bleaching (Fig. 7, C–F; unpublished data). A similar static protein profile was observed for MDM2 in the nucleolus (unpublished data). These findings suggest that VHL and MDM2 are targeted for static detention in the nucleolus in response to physiological cues.


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 the redistribution of MDM2 from nucleoplasm to nucleoli in response to perturbations in ribosomal biogenesis alters general MDM2 dynamic state. MCF7 cells transfected to express low levels of MDM2-GFP were cultured either under standard conditions (A), or ribosomal stress (RS) (B) induced by ActD treatment (see Lohrum et al., 2003 and Materials and methods). Cells were submitted to FRAP analysis as described for VHL in Fig. 3 by bleaching the indicated nucleoplasmic regions (white squares) or specific nucleoli (arrows) within specific nuclei (dashed circles). Pseudocolored zoom of area indicated by dashed rectangle is shown in B.
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fig6: FRAP analysis reveals that the redistribution of MDM2 from nucleoplasm to nucleoli in response to perturbations in ribosomal biogenesis alters general MDM2 dynamic state. MCF7 cells transfected to express low levels of MDM2-GFP were cultured either under standard conditions (A), or ribosomal stress (RS) (B) induced by ActD treatment (see Lohrum et al., 2003 and Materials and methods). Cells were submitted to FRAP analysis as described for VHL in Fig. 3 by bleaching the indicated nucleoplasmic regions (white squares) or specific nucleoli (arrows) within specific nuclei (dashed circles). Pseudocolored zoom of area indicated by dashed rectangle is shown in B.
Mentions: MDM2 displays a diffuse nuclear localization under standard culture conditions. FRAP and FLIP experiments revealed that MDM2 is a highly dynamic protein within this setting (Fig. 6 A; Fig. S4 A, available at http://www.jcb.org/cgi/content/full/jcb.200506030/DC1). MDM2 localizes to the nucleolus in response to perturbations in ribosomal biogenesis after treatment with low levels of ActD that inhibit RNA polymerase I (Fig. 6 B; Fig. S4 B; Lohrum et al., 2003). MDM2 is unable to target p53 for degradation under these conditions. We therefore assessed the dynamics of MDM2 after relocation to the nucleolus. When nucleolar, the dynamic profile of MDM2 significantly changed as GFP fluorescence did not exhibit any recovery/redistribution in FRAP experiments after bleaching of the nucleolus (Fig. 6 B) or loss in FLIP experiments after repetitive bleaching of a nucleoplasmic (Fig. S4 B) or cytoplasmic (unpublished data) region. Similar to VHL (Fig. 4, D and G), the interaction of MDM2 with the nucleolar architecture is required for modification of its trafficking dynamics as evidenced by the quick recovery of MDM2 in the nucleoplasm of transcriptionally inhibited cells expressing high levels of the protein in FRAP (Fig. 7 A). We next evaluated the dynamics of VHL and MDM2 ligases within the nucleolar space. VHL and MDM2 did not exhibit any fluorescence recovery after bleaching of an area within the nucleolus (Fig. 7, A and C). In contrast, B23 remained localized to nucleoli at steady-state within our experimental settings (SD, AP, and RS), retaining its highly mobile properties, though prolonged incubation with ActD resulted in B23 accumulating in the nucleoplasm (Fig. 7 B; unpublished data). Upon photobleaching, a border is created between the bleached area and the gradient of concentration established by the remaining fluorescent molecules (Fig. 7 C). For moving proteins, this border changes its shape as well as its position within the field of vision over time. Statically detained cellular components do not exhibit significant changes in these variables. We therefore compared the characteristics of borders of concentration gradients established by bleaching fluorescently labeled proteins when localized to different regions of the cell. Although analysis of concentration gradient borders in the nucleoplasm and cytoplasm revealed a highly dynamic profile of protein mobility, borders established within the nucleolar space neither changed in shape nor moved within the field of vision for up to 2 h after bleaching (Fig. 7, C–F; unpublished data). A similar static protein profile was observed for MDM2 in the nucleolus (unpublished data). These findings suggest that VHL and MDM2 are targeted for static detention in the nucleolus in response to physiological cues.

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