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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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Hydroxylation of Cep192 Targets the SCFSkp2 Complex(A) Cep192 is ubiquitinated. HeLa cells expressing Cep192-GFP or Cep192-LAA-GFP were treated with MG132 for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-ubiquitin antibody and immunoprecipitates were blotted for Cep192 (upper panels) or ubiquitin (lower panels).(B) Cep192 stability is not dependent on pVHL. Whole-cell lysates of renal cell carcinoma (RCC) cells and RCC cells reconstituted with HA-pVHL were blotted for the indicated proteins.(C) Cep192 destabilization is dependent on Skp2. HeLa cells were treated with the indicated siRNAs and whole-cell extracts were blotted with the indicated antibodies.(D) Cep192 interaction with Skp2 depends on hydroxylation. Cells were treated with MG132 or DFX for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-Skp2 antibody and immunoprecipitates were blotted for Cep192.(E) Quantification of Cep192-Skp2 interaction. Values represent averages of four different experiments. Error bars represent SD. p values are significant according to the Student’s t test; ∗∗p < 0.01.
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fig6: Hydroxylation of Cep192 Targets the SCFSkp2 Complex(A) Cep192 is ubiquitinated. HeLa cells expressing Cep192-GFP or Cep192-LAA-GFP were treated with MG132 for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-ubiquitin antibody and immunoprecipitates were blotted for Cep192 (upper panels) or ubiquitin (lower panels).(B) Cep192 stability is not dependent on pVHL. Whole-cell lysates of renal cell carcinoma (RCC) cells and RCC cells reconstituted with HA-pVHL were blotted for the indicated proteins.(C) Cep192 destabilization is dependent on Skp2. HeLa cells were treated with the indicated siRNAs and whole-cell extracts were blotted with the indicated antibodies.(D) Cep192 interaction with Skp2 depends on hydroxylation. Cells were treated with MG132 or DFX for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-Skp2 antibody and immunoprecipitates were blotted for Cep192.(E) Quantification of Cep192-Skp2 interaction. Values represent averages of four different experiments. Error bars represent SD. p values are significant according to the Student’s t test; ∗∗p < 0.01.

Mentions: Proline hydroxylation of HIF-α proteins is necessary for their ubiquitination and subsequent proteasomal degradation. To test whether a similar mechanism is regulating Cep192 levels, we first addressed whether Cep192 is a target of the proteasome. When the proteasome was inhibited by the addition of MG132, Cep192 levels increased slightly (Figure 5D). To determine whether Cep192 was ubiquitinated and whether the hydroxylation was required for this process, we immunoprecipitated ubiquitinated proteins from cells plus or minus prior exposure to MG132, and then blotted the immunoprecipitates for Cep192. Control analysis demonstrated that ubiquitin was immunoprecipitated from both cell types (Figure 6A, lower panels). Whereas wild-type Cep192 protein isolated from cell lysates was ubiquitinated, nonhydroxylatable Cep192 mutant protein was not, as we could not detect Cep192 in the immunoprecipitates from these cells (Figure 6A, top panels).


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)

Hydroxylation of Cep192 Targets the SCFSkp2 Complex(A) Cep192 is ubiquitinated. HeLa cells expressing Cep192-GFP or Cep192-LAA-GFP were treated with MG132 for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-ubiquitin antibody and immunoprecipitates were blotted for Cep192 (upper panels) or ubiquitin (lower panels).(B) Cep192 stability is not dependent on pVHL. Whole-cell lysates of renal cell carcinoma (RCC) cells and RCC cells reconstituted with HA-pVHL were blotted for the indicated proteins.(C) Cep192 destabilization is dependent on Skp2. HeLa cells were treated with the indicated siRNAs and whole-cell extracts were blotted with the indicated antibodies.(D) Cep192 interaction with Skp2 depends on hydroxylation. Cells were treated with MG132 or DFX for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-Skp2 antibody and immunoprecipitates were blotted for Cep192.(E) Quantification of Cep192-Skp2 interaction. Values represent averages of four different experiments. Error bars represent SD. p values are significant according to the Student’s t test; ∗∗p < 0.01.
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fig6: Hydroxylation of Cep192 Targets the SCFSkp2 Complex(A) Cep192 is ubiquitinated. HeLa cells expressing Cep192-GFP or Cep192-LAA-GFP were treated with MG132 for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-ubiquitin antibody and immunoprecipitates were blotted for Cep192 (upper panels) or ubiquitin (lower panels).(B) Cep192 stability is not dependent on pVHL. Whole-cell lysates of renal cell carcinoma (RCC) cells and RCC cells reconstituted with HA-pVHL were blotted for the indicated proteins.(C) Cep192 destabilization is dependent on Skp2. HeLa cells were treated with the indicated siRNAs and whole-cell extracts were blotted with the indicated antibodies.(D) Cep192 interaction with Skp2 depends on hydroxylation. Cells were treated with MG132 or DFX for 3 hr, and then lysates were immunoprecipitated with control IgG or with anti-Skp2 antibody and immunoprecipitates were blotted for Cep192.(E) Quantification of Cep192-Skp2 interaction. Values represent averages of four different experiments. Error bars represent SD. p values are significant according to the Student’s t test; ∗∗p < 0.01.
Mentions: Proline hydroxylation of HIF-α proteins is necessary for their ubiquitination and subsequent proteasomal degradation. To test whether a similar mechanism is regulating Cep192 levels, we first addressed whether Cep192 is a target of the proteasome. When the proteasome was inhibited by the addition of MG132, Cep192 levels increased slightly (Figure 5D). To determine whether Cep192 was ubiquitinated and whether the hydroxylation was required for this process, we immunoprecipitated ubiquitinated proteins from cells plus or minus prior exposure to MG132, and then blotted the immunoprecipitates for Cep192. Control analysis demonstrated that ubiquitin was immunoprecipitated from both cell types (Figure 6A, lower panels). Whereas wild-type Cep192 protein isolated from cell lysates was ubiquitinated, nonhydroxylatable Cep192 mutant protein was not, as we could not detect Cep192 in the immunoprecipitates from these cells (Figure 6A, top panels).

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