PHD1 links cell-cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192.
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.
Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.Show MeSH
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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).
Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.