Hypoxia inducible factor 2 (HIF-2) is a transcriptional activator implicated in the cellular response to hypoxia. Regulation of its inducible subunit, HIF-2α (also known as EPAS1), involves post-translational modifications. Here, we demonstrate that casein kinase 1δ (CK1δ; also known as CSNK1D) phosphorylates HIF-2α at Ser383 and Thr528 in vitro. We found that disruption of these phosphorylation sites, and silencing or chemical inhibition of CK1δ, reduced the expression of HIF-2 target genes and the secretion of erythropoietin (EPO) in two hepatic cancer cell lines, Huh7 and HepG2, without affecting the levels of HIF-2α protein expression. Furthermore, when CK1δ-dependent phosphorylation of HIF-2α was inhibited, we observed substantial cytoplasmic mislocalization of HIF-2α, which was reversed upon the addition of the nuclear protein export inhibitor leptomycin B. Taken together, these data suggest that CK1δ enhances EPO secretion from liver cancer cells under hypoxia by modifying HIF-2α and promoting its nuclear accumulation. This modification represents a new mechanism of HIF-2 regulation that might allow HIF isoforms to undertake differing functions.
The authors declare no competing or financial interests.
E.P. performed experiments and wrote the paper, C.B. performed experiments and assisted with data analysis, I.M. assisted with the in vitro phosphorylation and subcellular fractionation assays and data processing, M.S. and G.P. performed experiments with LC-MS/MS and data analysis, G.S. supervised experiments and wrote the paper, and P.L. designed and supervised experiments and wrote the paper.
This work was partially supported by the Greek General Secretariat of Research and Technology in the context of the ARISTEIA II Action of the Operational Program Education And Lifelong Learning (grant HYPOXYTARGET, No 3129 to G.S.) co-funded by the European Social Fund (ESF) and Greek national resources.
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.191395.supplemental
- Received April 26, 2016.
- Accepted September 27, 2016.
- © 2016. Published by The Company of Biologists Ltd