doi: 10.1038/nsmb.1578. Epub 2009 Mar 29.
CK2alpha phosphorylates BMAL1 to regulate the mammalian clock
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- PMID:19330005
- PMCID: PMC6501789
- DOI: 10.1038/nsmb.1578
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CK2alpha phosphorylates BMAL1 to regulate the mammalian clock
Teruya Tamaru et al. Nat Struct Mol Biol.2009 Apr.
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Abstract
Clock proteins govern circadian physiology and their function is regulated by various mechanisms. Here we demonstrate that Casein kinase (CK)-2alpha phosphorylates the core circadian regulator BMAL1. Gene silencing of CK2alpha or mutation of the highly conserved CK2-phosphorylation site in BMAL1, Ser90, result in impaired nuclear BMAL1 accumulation and disruption of clock function. Notably, phosphorylation at Ser90 follows a rhythmic pattern. These findings reveal that CK2 is an essential regulator of the mammalian circadian system.
Figures
A) Mixtures of CK2α (50ng; visible in CBB staining) and CK2β (10ng; visible in autoradiography after phosphorylation) subunits were used inin vitro kinase assay with or without ATP using GST (lane 1) or GST-BMAL1 (lane 2) as substrates.B) CK2α (4 pmol) and CK2β (0-8 pmol) were used in kinase assays with GST-BMAL1 as substrate (500ng; optimal ratio to CK2α) kinase assay. GST-BMAL1 kinase activities (average values) is plotted at each CK2β/CK2α ratio, and normalized against activity in the absence of CK2β with error bars (±SD values). Photographs are representative of duplicate experiments.
(A-C)) CK2α silencing/depletion affects BMAL1 nuclear localization and clock function. NIH-3T3 cells were transfected with microRNAi (miR) targeting vectors for mCK2α and the validated negative-control (control) vector, then Dex-synchronized.A) Immunoblot analysis for CK2α and Actin in cell lysates, with one representative experiment shown. Normalized average CK2α levels from triplicate experiments are shown below with error bars (±SD values).B) CircadianmPer2 expression was monitored by a real-time bioluminescence assay. Normalized average values from multiple experiments (n=5) are plotted with error bars (±SD values of 1st/2nd peaks).C) Localization of BMAL1, visualized by immunofluorescence (red) at 24h post Dex treatment. Nuclei were stained with DAPI (blue) and miRNAi-transfected GFP-positive cells (green). Photographs are representative of triplicate experiments. Percentages of cells with BMAL1 predominantly nuclear among the GFP-positive cells from triplicate experiments are plotted as means ± SEM. (***; P < 0.001). (D-F) Mutation of BMAL1 Ser90 affects nuclear localization and clock function.D) Lysates fromBmal1−/− MEFs stably expressing Myc-BMAL1-WT, Myc-BMAL1-S90A and Myc-GFP were analyzed with anti-Myc antibody.E) CircadianmPer2 expression was not restored by BMAL1 S90A, as monitored as was monitored by a real-time bioluminescence assay. Normalized average values from multiple experiments (n=5) are plotted with error bars (±SD values of 1st/2nd peaks).F) MEFs at 24h post Dex treatment were stained with anti-BMAL1 (Green) antibody. Nuclei were stained with DAPI (Blue). Photographs are representative of mutiple (n=4) experiments. Values of nuclear BMAL1-dominant cells (%) in MEFs from multiple (n=4) experiments are plotted as means ± SEM (***; P < 0.001).
A) NIH-3T3 cells expressing miR-CK2α and control vector at 24h post Dex treatment were subjected to BMAL1-immunoprecipitation (IP) followed by immunoblot analysis (WB) for BMAL1, P-BMAL1-S90, CK2α and Actin (Lysate). Arrows and bars designate BMAL1 and P-BMAL1-S90.B) BMAL1-immunoprecipitates and lysates of NIH-3T3 cells at each time point post Dex treatment were used in immunoblot analysis for P-BMAL1-S90; BMAL1, PER1, CK2 subunits and Actin. Arrows and bars designate BMAL1 and P-BMAL1-S90. Normalized average values for P-BMAL1-S90 and PER1 from triplicate experiments are plotted in the graph with error bars (±SD values). All the photographs are representative of triplicate experiments.
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