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PHD1 links cell-cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192.

Moser SC, Bensaddek D, Ortmann B, Maure JF, Mudie S, Blow JJ, Lamond AI, Swedlow JR, Rocha S - Dev. Cell (2013)

Bottom Line: Importantly, PHD1 is also required for primary cilia formation.Cep192 is hydroxylated by PHD1 on proline residue 1717.This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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Loss of PHD1 Inhibits Mitotic Progression(A) HeLa cells were treated with the indicated siRNAs, and the cell-cycle profile was determined by flow cytometry. Values represent averages of two different experiments. Error bars indicate ± 1 SD.(B) Transfection of GFP-PHD1 rescues the mitotic arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic index was determined. Values represent averages of three different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(C) Transfection of GFP-PHD1 partially rescues the prometaphase arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic distribution was determined. Values represent averages of two different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(D) PHD1 depletion disrupts the mitotic spindle. Immunofluorescence images of mitotic cells treated with PHD1 siRNA and stained for α-tubulin (green) and DNA (blue). The scale bar represents 5 μm.(E) Quantification of spindle morphology phenotypes observed in PHD1-depleted cells.See also Figure S1.
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fig1: Loss of PHD1 Inhibits Mitotic Progression(A) HeLa cells were treated with the indicated siRNAs, and the cell-cycle profile was determined by flow cytometry. Values represent averages of two different experiments. Error bars indicate ± 1 SD.(B) Transfection of GFP-PHD1 rescues the mitotic arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic index was determined. Values represent averages of three different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(C) Transfection of GFP-PHD1 partially rescues the prometaphase arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic distribution was determined. Values represent averages of two different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(D) PHD1 depletion disrupts the mitotic spindle. Immunofluorescence images of mitotic cells treated with PHD1 siRNA and stained for α-tubulin (green) and DNA (blue). The scale bar represents 5 μm.(E) Quantification of spindle morphology phenotypes observed in PHD1-depleted cells.See also Figure S1.

Mentions: To determine whether PHDs have a role in cell-cycle progression, we independently depleted each of the three isoforms PHD1–3 by siRNA in HeLa Kyoto cells (Figures S1A–S1D available online). Whereas depletion of either PHD2 or PHD3 caused little or no alteration of the cell-cycle profile, depletion of PHD1 increased the G2/M population of cells and decreased the G1 population (Figure 1A). In contrast, and as previously observed (Culver et al., 2011), HIF-1α depletion led to an increase of cells in G1 (Figure 1A). Further analysis revealed that PHD1 knockdown increased the mitotic index (Figure 1B). When the endogenous PHD1 protein was removed by targeting the 3′ UTR of its mRNA with siRNA, this effect could be reversed by the expression of PHD1 from a cDNA lacking the siRNA target site (Figure 1B). These data suggest that PHD1 is required for mitotic progression.


PHD1 links cell-cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192.

Moser SC, Bensaddek D, Ortmann B, Maure JF, Mudie S, Blow JJ, Lamond AI, Swedlow JR, Rocha S - Dev. Cell (2013)

Loss of PHD1 Inhibits Mitotic Progression(A) HeLa cells were treated with the indicated siRNAs, and the cell-cycle profile was determined by flow cytometry. Values represent averages of two different experiments. Error bars indicate ± 1 SD.(B) Transfection of GFP-PHD1 rescues the mitotic arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic index was determined. Values represent averages of three different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(C) Transfection of GFP-PHD1 partially rescues the prometaphase arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic distribution was determined. Values represent averages of two different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(D) PHD1 depletion disrupts the mitotic spindle. Immunofluorescence images of mitotic cells treated with PHD1 siRNA and stained for α-tubulin (green) and DNA (blue). The scale bar represents 5 μm.(E) Quantification of spindle morphology phenotypes observed in PHD1-depleted cells.See also Figure S1.
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fig1: Loss of PHD1 Inhibits Mitotic Progression(A) HeLa cells were treated with the indicated siRNAs, and the cell-cycle profile was determined by flow cytometry. Values represent averages of two different experiments. Error bars indicate ± 1 SD.(B) Transfection of GFP-PHD1 rescues the mitotic arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic index was determined. Values represent averages of three different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(C) Transfection of GFP-PHD1 partially rescues the prometaphase arrest of HeLa cells treated with PHD1 siRNA. HeLa cells were transfected with the indicated siRNAs and plasmids and the mitotic distribution was determined. Values represent averages of two different experiments (n = 100; error bars indicate ± 1 SD; p values are significant according to the Student’s t test; ∗∗p < 0.01).(D) PHD1 depletion disrupts the mitotic spindle. Immunofluorescence images of mitotic cells treated with PHD1 siRNA and stained for α-tubulin (green) and DNA (blue). The scale bar represents 5 μm.(E) Quantification of spindle morphology phenotypes observed in PHD1-depleted cells.See also Figure S1.
Mentions: To determine whether PHDs have a role in cell-cycle progression, we independently depleted each of the three isoforms PHD1–3 by siRNA in HeLa Kyoto cells (Figures S1A–S1D available online). Whereas depletion of either PHD2 or PHD3 caused little or no alteration of the cell-cycle profile, depletion of PHD1 increased the G2/M population of cells and decreased the G1 population (Figure 1A). In contrast, and as previously observed (Culver et al., 2011), HIF-1α depletion led to an increase of cells in G1 (Figure 1A). Further analysis revealed that PHD1 knockdown increased the mitotic index (Figure 1B). When the endogenous PHD1 protein was removed by targeting the 3′ UTR of its mRNA with siRNA, this effect could be reversed by the expression of PHD1 from a cDNA lacking the siRNA target site (Figure 1B). These data suggest that PHD1 is required for mitotic progression.

Bottom Line: Importantly, PHD1 is also required for primary cilia formation.Cep192 is hydroxylated by PHD1 on proline residue 1717.This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Show MeSH
Related in: MedlinePlus