DN-CKIδ or ε disrupt activities of both kinases since the kinases bind PER through their conserved catalytic domain. Blots representative of three experiments are shown. (A) In vitro kinase assay resolved by Western blot: high levels of PER2 phosphorylation cause slower mobility in SDS/PAGE. PER2 was highly phosphorylated by CKIε, but not by DN-CKIε. The reactions were stopped at the indicated times (in hours) by adding 2× sample buffer. Lane 1 represents PER2 alone. (B) Cell culture kinase assay resolved by immunoblot using anti-PER2 and anti-CKIδ/ε antibodies. In NIH 3T3 cells, the DN-CKIδ/ε do not phosphorylate PER, while wt CKIδ/ε and tau CKIε do. Both wt and DN-CKIε have a C-terminal MYC tag, while tau CKIε has no tag. Both wt- and DN-CKIδ have a FLAG tag. (C) PER2 was coexpressed with a full-length CKIδ/ε, or N-terminal (Catalytic domain; amino acid 1–277) + C-terminal (aa278–416 for CKIε and aa278–415 for CKIδ) peptides in HEK293 cells. Cell extracts were subjected to immunoprecipitation (IP) for PER2. Note that only catalytic domains were copurified with PER2. Both full-length and truncated mutant CKIδ/ε have an N-terminal FLAG tag, which was detected on the immunoblots. Note that in the rightmost panel, the full length CKIδ-FLAG comigrates with the IgG heavy chain. (D) The DN-CKIδ(ε) inhibits wt-CKIδ(ε)-dependent phosphorylation of PER2. NIH 3T3 cells were transfected with PER-V5 and pcDNA3.1, wt-CKI, or a 1:10 ratio of wt and DN-CKI. The cell lysates were subjected to immunoblotting using anti-PER2 and anti-CKIδ or ε antibodies. Both wt and DN-CKIδ have an N-terminal FLAG tag. wt- and DN-CKIε have a MYC and a FLAG tag, respectively. (E) DN-CKIδ(ε) can inhibit wt-CKIε(δ)-mediated PER2 phosphorylation. wt-CKIδ and DN-CKIε or wt-CKIε and DN-CKIδ were transfected into NIH 3T3 cells in 1:10 ratio as above. The kinases are the same as in (D). To detect both CKIδ and ε on the same blot, two antibodies were used at the same time. (F) MEF extracts were subjected to IP for CKIδ or ε, and assayed by immunoblot for PER1 and 2.

Circadian period is lengthened in ckΙδ−/− MEFs. (A) Brain extracts at embryonic day 12 and MEF extracts from the embryos were immunoblotted for CKIδ. (B) Bioluminescence (PER2:Luc) rhythms in MEFs isolated from homozygote and heterozygote ckΙδ mutant littermate embryos. (C) Periods are shown as mean ± SEM of triplicate samples. These data are representative of at least three experiments. *, P < 0.05

Disruption of PER interaction with CKIδ/ε by CKBD-P2 abolishes circadian rhythms and destabilizes PER in MEFs. (A and B) Casein kinase binding domain from PER2 (CKBD-P2) (A) and the corresponding domain from PER3 (CKBD-P3) (B) were overexpressed in wt MEFs and bioluminescence rhythms were measured as above. The FLAG tag added to the N-termini of both CKBDs was used to ensure similar expression levels. Note that the basal line for CKBD-P2 is extremely low. This was consistently observed in several experiments. (C) MEFs from (A) and (B) were immunoblotted for FLAG. (D) GFP and CKBD-P2 MEFs at indicated times were harvested and the extracts were immunoblotted. Note that endogenous CKIδ/ε levels were increased in CKBD-P2 MEFs. A dark exposure for PER2 blot in the left panels is shown in Fig. S7A to demonstrate that low levels in CKBD-P2 MEFs are not due to smearing of PER2 band. Blots are representative of three experiments. (E) PER2, CKIε and/or CKBD-P2 were overexpressed in wt MEFs, the cells were harvested 24 h after the infection and the extracts were subjected to IP for CKIε. The resulting immunocomplexes were immunoblotted for PER2, CKIε, and FLAG. Top and bottom arrow indicate exogenous and endogenous CKIε, respectively. Blots are representative of four experiments.

The DN-CKIδ/ε lengthened the period of bioluminescence (PER2:Luc) rhythms in MEFs. (A–C) MEFs were infected with adenovirus expressing DN-CKIδ (A), DN-CKIε (B,) or wt CKIε (C) for 2 h, serum-shocked for 2 h and placed into the real-time luminometer. The 3XFLAG tag added to the N-termini of wt and DN kinases was used to ensure similar expression among different adenoviruses. ½× represents half titer of 1×. The numbers are shown as mean ± SEM of triplicate samples. The results are representative of several experiments. *, P < 0.05; **, P < 0.01.

Overexpression of DN-CKIε completely disrupts circadian rhythms and compromises the molecular clock in ckΙδ−/− MEFs. (A) Bioluminescence rhythms were measured in GFP (green) and DN-CKIε (blue) MEFs as above. Rhythms could not be detected after a very weak second peak in DN-CKIε cells when analyzed using the Clocklab software. The results are representative of three experiments. (B) PER1 and 2 rhythms were measured in GFP and DN-CKIε MEFs as above. Bottom graphs are quantification of PER abundance indicated as mean ± SEM of three experiments. (C) CHX was added to MEFs and the cells were harvested as in Fig. S4A. (D) The results from (C) were quantified as above by densitometric scanning. The numbers are shown as mean ± SEM of three experiments. (E) wt MEFs were treated with DMSO or protease inhibitors, MG132+PSI, at T24 and harvested at indicated times. * indicates non-specific bands, the red bar indicates extra slow-migrating PER2, and the blue bar indicates multiubiquitinated β-Catenin. Note that the extra slow-migrating PER2 isoforms are not observed in control MEFs. Data are representative of at least three experiments. (F) PER1 and 2 were stained in GFP and DN-CKIε ckIδ−/− MEFs fixed at T24 after serum shock. Representative cells are shown from three independent experiments. PERs were predominantly (>90%) nuclear in Adv-GFP, CKIδ-deficient cells whereas they were observed in both compartments in most (>90%) Adv-DN-CKIε, CKIδ-deficient cells; more cells are shown in Fig. S6.