CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.
Bottom Line: We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT.This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation.These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.
Affiliation: Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Larissa, Greece.Show MeSH
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Mentions: To investigate the effect of CK1-mediated phosphorylation on HIF-1 in living cells, we applied fluorescence recovery after photobleaching (FRAP) for the determination of HIF-1α intranuclear mobility and kinetics that reflect its ability to form heterodimers that bind stably to chromatin. To this end, we used HeLa cells ectopically expressing GFP-HIF-1α or its mutant forms that abolish or mimic its CK1δ-dependent phosphorylation (Supplementary Fig. 1b). Mutation of Ser247 to alanine (S247A) has been shown to increase the affinity of HIF-1α for ARNT in in vitro binding assays, whereas, the phosphomimetic mutation of the same site to aspartate (S247D) has been shown to exhibit the opposite effect . As negative control, we used HeLa cells expressing a GFP-tagged fragment of HIF-1α that lacks the heterodimerization domain (HIF-1α-ΔΝ) and has, therefore, no ability to form heterodimers. FRAP was performed 24 h post-transfection by bleaching a circular area within the nucleus and then monitoring the recovery of fluorescence in the bleached region over time (Fig. 3a, circles). A visual, qualitative examination of the resulting FRAP recovery curves (Fig. 3b and Supplementary Fig. 2) revealed distinct dynamics for the GFP-HIF-1α constructs under normoxia. GFP-HIF-1α-ΔΝ is characterized by rapid and full recovery of fluorescence, indicating a purely diffusive behavior. Wild-type HIF-1α and its phosphomimetic mutant (S247D) are characterized by similar dynamics, whereas S247A, which is unable to be phosphorylated by CK1δ, exhibits decreased recovery. Finally, recovery was further decreased when cells expressing wild type GFP-HIF-1α were treated with the CK1δ inhibitor D4476.
Affiliation: Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Larissa, Greece.