Child, F. | Search bioRxiv

bioRxiv2025

PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression

Prolyl-hydroxylases (PHDs) are oxygen sensing enzymes that mediate the hydroxylation of proline residues. In mammals, three PHD isoforms (PHD1-3) are responsible for proline hydroxylation of Hypoxia Inducible Factor (HIF) alpha, a key regulator of the hypoxia response. In the accompanying paper (Jiang et. al., 2025) we report development of a mass spectrometry-based method to reliably identify proline hydroxylation (OH-Pro) sites on proteins and use this to identify a PHD-dependent OH-Pro modification at Pro604 on the protein RepoMan (CDCA2), a regulatory subunit for protein phosphatase PP1{gamma}, with important roles in mitotic progression and cell viability. Here, we investigate the functional significance of hydroxylation of RepoMan at P604. During M phase, the PP1-RepoMan complex dephosphorylates Thr3 of Histone H3 (H3T3) on chromosomes arms to ensure the correct localisation of the chromosomal passenger complex (CPC) at centromeres. We show that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in prometaphase-arrested cells. In cells depleted of endogenous RepoMan, exogenous expression of wild type RepoMan, but not a RepoMan P604A mutant, restored normal H3T3 phosphorylation localisation in prometaphase arrested cells. RepoMan P604 is located proximal to the Short Linear Motifs (SLiMs) that function as binding sites for the serine/threonine Protein Phosphatase 2A (PP2A). The interaction of RepoMan and PP2A-B56{gamma} is reduced in cells expressing RepoMan P604A. Moreover, analyses in both fixed and live cells released from a prometaphase arrest, show that expression of the RepoMan P604A mutant delays completion of mitosis, results in defects in chromosome alignment and segregation and increases levels of cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis, acting, at least in part, via hydroxylation of RepoMan at P604 regulating the interaction of RepoMan with PP2A during chromosome alignment and thereby controlling the levels of Histone H3 phosphorylation at Thr3.

cell biology↗

bioRxiv2022

Disruption of HIF1A translational control attenuates the HIF-dependent hypoxic response and solid tumour formation in vivo

Adaptation to reduced oxygen availability is mediated by the hypoxia-inducible factor (HIF) family of transcription factors. The activity and availability of HIF proteins is primarily driven by the stability of the HIF alpha subunits. However, it is becoming increasingly apparent that preferential translation of HIF1 mRNA is also necessary for full activation of the HIF1-dependent hypoxic response. Consequently, the mechanisms controlling HIF1 translation are of equivalent importance to the proline hydroxylase-dependent degradation pathways. Here we investigate the role of the 5UTR of the HIF1 mRNA in controlling preferential translation of endogenous HIF1 in hypoxic cells. CRISPR/Cas9-mediated genetic deletion of the 5 UTR of HIF1 results in reduced HIF1 levels following hypoxia, without alteration in mRNA or protein stability. HIF1 mRNA lacking the 5UTR was efficiently translated in adequately oxygenated cells but this was inhibited during hypoxia, consistent with the global block on protein synthesis. The HIF1 translational defect observed in cells missing the 5UTR led to reduced viability in hypoxic conditions in vitro and an impaired ability to form solid tumours in murine xenografts. Prevention of preferential HIF1 translation limits the duration and intensity of the HIF-dependent hypoxic response and disrupts the formation of solid tumours. Together these results demonstrate the importance of translation control over HIF1 and suggest that strategies to inhibit preferential HIF1 protein translation in hypoxic cancer cells will be an effective strategy to limit the growth of solid hypoxic tumours.

cancer biology↗