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