Limits...
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.

Show MeSH

Related in: MedlinePlus

Cep192 Is a Hydroxylation Target of PHD1(A) PHD1 is required for the centrosomal localization of Cep192. HeLa cells expressing GFP-centrin (green) were depleted of PHD1 or Cep192 and stained for Cep192 (red) in interphase (left panel, upper) and mitosis (left panel, lower). The scale bars represent 1 μm. The relative fluorescence intensity of Cep192 signal in interphase and mitosis was determined (right panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗∗p < 0.0001). See also Figure S2.(B) Cep192 interacts with PHD1. PHD1 was immunoprecipitated from cells and blotted for Cep192. See also Figure S3.(C) Electrospray-MS spectrum of the product of in vitro hydroxylation of the synthetic peptide WHLSSLAPPYVK showing an m/z increment of 5.333 Th (the mass increment of 15.999 Da) corresponding to proline hydroxylation of the triply charged ion at m/z 466.5894 Th and the formation of the ion 471.9224 Th (the mass of the hydroxylated peptide).(D) Absence of an m/z increment of 5.333 Th (or mass increment of 15.999 Da) of the triply charged ion, indicating that the peptide WHLSSLAAPYVK is not in vitro hydroxylated.(E) Cep192 is hydroxylated on proline 1717. Product ion spectrum of the endogenous doubly charged ion at m/z 707.3797 Th corresponding to the hydroxylated peptide WHLSSLAP(OH)PYVK showing b and y fragment ions typical for higher-energy C trap dissociation fragmentation, which allowed the identification of the peptide sequence. Asterisks denote the fragment ions incorporating the mass increment of 15.999 Da, corresponding to proline hydroxylation. See also Figure S4.(F and G) Titration of nonhydroxylated (F) and hydroxylated standards (G). Diagrams show extracted-ion chromatograms, retention times, and measured areas for the standards injected in different amounts.(H) Diagram correlating the MAs of signals for nonhydroxylated and hydroxylated standards to the amounts of material analyzed.(I) Correlation of the MA of nonhydroxylated standard and hydroxylated standard.See also Figures S3 and S4.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3757158&req=5

fig3: Cep192 Is a Hydroxylation Target of PHD1(A) PHD1 is required for the centrosomal localization of Cep192. HeLa cells expressing GFP-centrin (green) were depleted of PHD1 or Cep192 and stained for Cep192 (red) in interphase (left panel, upper) and mitosis (left panel, lower). The scale bars represent 1 μm. The relative fluorescence intensity of Cep192 signal in interphase and mitosis was determined (right panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗∗p < 0.0001). See also Figure S2.(B) Cep192 interacts with PHD1. PHD1 was immunoprecipitated from cells and blotted for Cep192. See also Figure S3.(C) Electrospray-MS spectrum of the product of in vitro hydroxylation of the synthetic peptide WHLSSLAPPYVK showing an m/z increment of 5.333 Th (the mass increment of 15.999 Da) corresponding to proline hydroxylation of the triply charged ion at m/z 466.5894 Th and the formation of the ion 471.9224 Th (the mass of the hydroxylated peptide).(D) Absence of an m/z increment of 5.333 Th (or mass increment of 15.999 Da) of the triply charged ion, indicating that the peptide WHLSSLAAPYVK is not in vitro hydroxylated.(E) Cep192 is hydroxylated on proline 1717. Product ion spectrum of the endogenous doubly charged ion at m/z 707.3797 Th corresponding to the hydroxylated peptide WHLSSLAP(OH)PYVK showing b and y fragment ions typical for higher-energy C trap dissociation fragmentation, which allowed the identification of the peptide sequence. Asterisks denote the fragment ions incorporating the mass increment of 15.999 Da, corresponding to proline hydroxylation. See also Figure S4.(F and G) Titration of nonhydroxylated (F) and hydroxylated standards (G). Diagrams show extracted-ion chromatograms, retention times, and measured areas for the standards injected in different amounts.(H) Diagram correlating the MAs of signals for nonhydroxylated and hydroxylated standards to the amounts of material analyzed.(I) Correlation of the MA of nonhydroxylated standard and hydroxylated standard.See also Figures S3 and S4.

Mentions: We next explored whether Cep192 localization depends on PHD1, and found that PHD1 depletion by siRNA resulted in an increase of Cep192 at the centrosome during interphase and a failure to accumulate Cep192 at the centrosome during mitosis (Figure 3A). To determine whether PHD1 is associated with complexes containing Cep192, we isolated PHD1 by immunoprecipitation and observed the copurification of Cep192 (Figure 3B). PHD1 also colocalized with Cep192 during mitosis, as judged by fluorescence microscopy (Figure S3). Although PHD1 is mainly observed in the nucleus (Metzen et al., 2003), biochemical analysis demonstrated that, like Cep192, a proportion of PHD1 also localizes to the cytoplasm (Figure S3B). Based on these data, we next tested whether PHD1 hydroxylates Cep192. In vitro recombinant PHD1 efficiently hydroxylated a Cep192 peptide containing the motif LXXLAP (Figure 3C) but not a peptide in which the putative modified proline was mutated to an alanine (Figure 3D). We next analyzed immunoprecipitates of Cep192-GFP isolated from HeLa Kyoto cells that stably express Cep192-GFP at endogenous levels by nano-LC (liquid chromatography) and targeted tandem MS scanning on a high-resolution mass spectrometer. In these immunoprecipitates, we identified a peptide ion of Cep192 that was hydroxylated at proline 1717 in vivo (Figure 3E; Figure S4). To determine the stoichiometry of Cep192 hydroxylation, a quantification method was developed in which the level of in-vivo-hydroxylated Cep192 was compared with known amounts of synthetic hydroxylated and nonhydroxylated standards (see Experimental Procedures). From these data, we calculate that ∼10% of Cep192 is hydroxylated at Pro1717 in cells growing in asynchronous culture (Figures 3F and 3G).


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)

Cep192 Is a Hydroxylation Target of PHD1(A) PHD1 is required for the centrosomal localization of Cep192. HeLa cells expressing GFP-centrin (green) were depleted of PHD1 or Cep192 and stained for Cep192 (red) in interphase (left panel, upper) and mitosis (left panel, lower). The scale bars represent 1 μm. The relative fluorescence intensity of Cep192 signal in interphase and mitosis was determined (right panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗∗p < 0.0001). See also Figure S2.(B) Cep192 interacts with PHD1. PHD1 was immunoprecipitated from cells and blotted for Cep192. See also Figure S3.(C) Electrospray-MS spectrum of the product of in vitro hydroxylation of the synthetic peptide WHLSSLAPPYVK showing an m/z increment of 5.333 Th (the mass increment of 15.999 Da) corresponding to proline hydroxylation of the triply charged ion at m/z 466.5894 Th and the formation of the ion 471.9224 Th (the mass of the hydroxylated peptide).(D) Absence of an m/z increment of 5.333 Th (or mass increment of 15.999 Da) of the triply charged ion, indicating that the peptide WHLSSLAAPYVK is not in vitro hydroxylated.(E) Cep192 is hydroxylated on proline 1717. Product ion spectrum of the endogenous doubly charged ion at m/z 707.3797 Th corresponding to the hydroxylated peptide WHLSSLAP(OH)PYVK showing b and y fragment ions typical for higher-energy C trap dissociation fragmentation, which allowed the identification of the peptide sequence. Asterisks denote the fragment ions incorporating the mass increment of 15.999 Da, corresponding to proline hydroxylation. See also Figure S4.(F and G) Titration of nonhydroxylated (F) and hydroxylated standards (G). Diagrams show extracted-ion chromatograms, retention times, and measured areas for the standards injected in different amounts.(H) Diagram correlating the MAs of signals for nonhydroxylated and hydroxylated standards to the amounts of material analyzed.(I) Correlation of the MA of nonhydroxylated standard and hydroxylated standard.See also Figures S3 and S4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757158&req=5

fig3: Cep192 Is a Hydroxylation Target of PHD1(A) PHD1 is required for the centrosomal localization of Cep192. HeLa cells expressing GFP-centrin (green) were depleted of PHD1 or Cep192 and stained for Cep192 (red) in interphase (left panel, upper) and mitosis (left panel, lower). The scale bars represent 1 μm. The relative fluorescence intensity of Cep192 signal in interphase and mitosis was determined (right panel) (n = 20; error bars indicate ± 1 SEM; p values are significant according to the Student’s t test; ∗∗∗p < 0.0001). See also Figure S2.(B) Cep192 interacts with PHD1. PHD1 was immunoprecipitated from cells and blotted for Cep192. See also Figure S3.(C) Electrospray-MS spectrum of the product of in vitro hydroxylation of the synthetic peptide WHLSSLAPPYVK showing an m/z increment of 5.333 Th (the mass increment of 15.999 Da) corresponding to proline hydroxylation of the triply charged ion at m/z 466.5894 Th and the formation of the ion 471.9224 Th (the mass of the hydroxylated peptide).(D) Absence of an m/z increment of 5.333 Th (or mass increment of 15.999 Da) of the triply charged ion, indicating that the peptide WHLSSLAAPYVK is not in vitro hydroxylated.(E) Cep192 is hydroxylated on proline 1717. Product ion spectrum of the endogenous doubly charged ion at m/z 707.3797 Th corresponding to the hydroxylated peptide WHLSSLAP(OH)PYVK showing b and y fragment ions typical for higher-energy C trap dissociation fragmentation, which allowed the identification of the peptide sequence. Asterisks denote the fragment ions incorporating the mass increment of 15.999 Da, corresponding to proline hydroxylation. See also Figure S4.(F and G) Titration of nonhydroxylated (F) and hydroxylated standards (G). Diagrams show extracted-ion chromatograms, retention times, and measured areas for the standards injected in different amounts.(H) Diagram correlating the MAs of signals for nonhydroxylated and hydroxylated standards to the amounts of material analyzed.(I) Correlation of the MA of nonhydroxylated standard and hydroxylated standard.See also Figures S3 and S4.
Mentions: We next explored whether Cep192 localization depends on PHD1, and found that PHD1 depletion by siRNA resulted in an increase of Cep192 at the centrosome during interphase and a failure to accumulate Cep192 at the centrosome during mitosis (Figure 3A). To determine whether PHD1 is associated with complexes containing Cep192, we isolated PHD1 by immunoprecipitation and observed the copurification of Cep192 (Figure 3B). PHD1 also colocalized with Cep192 during mitosis, as judged by fluorescence microscopy (Figure S3). Although PHD1 is mainly observed in the nucleus (Metzen et al., 2003), biochemical analysis demonstrated that, like Cep192, a proportion of PHD1 also localizes to the cytoplasm (Figure S3B). Based on these data, we next tested whether PHD1 hydroxylates Cep192. In vitro recombinant PHD1 efficiently hydroxylated a Cep192 peptide containing the motif LXXLAP (Figure 3C) but not a peptide in which the putative modified proline was mutated to an alanine (Figure 3D). We next analyzed immunoprecipitates of Cep192-GFP isolated from HeLa Kyoto cells that stably express Cep192-GFP at endogenous levels by nano-LC (liquid chromatography) and targeted tandem MS scanning on a high-resolution mass spectrometer. In these immunoprecipitates, we identified a peptide ion of Cep192 that was hydroxylated at proline 1717 in vivo (Figure 3E; Figure S4). To determine the stoichiometry of Cep192 hydroxylation, a quantification method was developed in which the level of in-vivo-hydroxylated Cep192 was compared with known amounts of synthetic hydroxylated and nonhydroxylated standards (see Experimental Procedures). From these data, we calculate that ∼10% of Cep192 is hydroxylated at Pro1717 in cells growing in asynchronous culture (Figures 3F and 3G).

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