CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock
Fig 4
CRY-enhanced circadian oscillation of BMAL1–CK2β association in living cells.
(A) Graphic representation of ELucN-CK2β, McLuc1-BMAL1, and ELucC-BMAL1 vectors for real-time bioluminescence imaging of CK2β-BMAL1 interactions as monitored by the Split Luc complementation system. IB analysis with anti-BMAL1 and CK2β antibodies reveals ectopic expression of His-tagged ELucC-BMAL1 (~100 kDa) and ELucN-CK2β ELucN-CK2β (~70 kDa) in Cos7 cells. (B) Cos7 cells were transfected with ELucN-CK2β and ELucC-BMAL1 (2.5 μg), and either Myc-HA-CRY1, Myc-CRY2, or (control) pcDNA (−) (5 μg). The presence of ectopically expressed CRY1/2 and native CK2α was confirmed by IB analysis (lower panel). BMAL1–CK2β binding was monitored by real-time bioluminescence imaging and plotted as the average value of peak bioluminescence levels (n = 5 experiments; upper panel). Error bars indicate SD. Note that BMAL1–CK2β binding was significantly facilitated by CRY1 and CRY/2. (C) Circadian rhythms in BMAL1–CK2β binding in Dex-synchronized U-2OS cells were monitored by real-time bioluminescence imaging of Split Luc activity (upper panel). The circadian profiles (Period = 25.3 h, Acrophase = 15.1 h) (n = 5; blue line) are shown with normalized values to the maximum value that was set as 1. Expression of McLuc1-BMAL1, native BMAL1, ELucN-CK2β, and native CK2β was confirmed by IB analysis (upper panel, insets). The circadian profile of P-BMAL1-S90 levels (representative examples of n = 3 experiments) was determined by IB analysis (lower panel) with anti-P-BMAL1-S90), quantified and normalized to actin content. Maximum values were set as 1 (upper panel: dotted red line). Error bars indicate SD.