CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein–protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1–CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1–P-BMAL1 loop is an integral part of the core clock oscillator.

To date, the molecular mechanism underlying rhythmic mammalian clock protein phosphorylation remains elusive. Previously, we have reported circadian BMAL1 phosphorylation at Ser90 by CK2 [12], which is generally thought to be a constitutively active kinase [20]. Interestingly, the BMAL1 protein is hyperphosphorylated in CRY1/2-deficient mice [8,14], leading us to hypothesize that CRY proteins are involved in rhythmic BMAL1 modification. Here, we investigated the universal role and oscillatory mechanism of the circadian CK2-mediated BMAL1 phosphorylation. Accordingly, a novel role of CRY as a repressor for protein phosphorylation was found. We propose a model that explains how CRY proteins produce circadian oscillations and integrate posttranslational modification events (i.e., BMAL1 phosphorylation) in the negative limb of the core transcription-translation feedback loop.

CK2-Mediated Circadian BMAL1-S90 Phosphorylation Regulates Mammalian Central and Peripheral Clocks
To investigate the critical role of CK2-mediated circadian phosphorylation of BMAL1 at Ser90 (referred to as BMAL1-S90) in regulating the central clock in the SCN and peripheral clocks in the liver, and in cultured fibroblasts, we designed a competitive inhibitor of BMAL1-S90 phosphorylation, consisting of a 14 amino acid BMAL1 peptide (BMs90p), centered around Ser90 (Fig 1Aa). As expected, and in line with S90A mutagenesis data [12], BMs90p dose-dependently (optimum at~6 μM) suppressed both the formation of BMAL1 phosphorylated at Ser90 (hereafter referred to as P-BMAL1-S90), and mPer2 promoter-driven luciferase (Per2L) bioluminescence rhythms in dexamethasone (Dex) clock-synchronized NIH-3T3 fibroblasts (Fig 1Aa, 1Ab and 1Ac). P-BMAL1-S90 was recovered only partially around 2-6h post-treatment (S1A Fig). In contrast, a control peptide with Ser90 replaced by Ala (BMa90p) did not inhibit Per2L rhythms and P-BMAL1-S90 phosphorylation, demonstrating the specificity of BMs90p (S1B Fig). Thus, BMs90p perturbs the circadian core oscillator (as evident from the suppressed Per2L rhythms) by inhibiting BMAL1-S90 phosphorylation. To test the effect on the central and peripheral clocks, we applied BMs90p to SCN and liver organotypic slices from mPER2 Luc mice [21]. BMs90p-treatment provoked an evident reduction of the amplitude (liver; 0.255, SCN; 0.322; average value pre-treatment set as 1) and peak intensity (liver; 0.420, SCN; 0.525; average value pre-treatment set as 1) of Per2L (PER2::LUC) rhythms, without any evident phase-shifting effect ( Fig 1B). Notably, this effect was only observed when BMs90p was administered at the trough of Per2L reporter gene activity (liver;~CT5, SCN;~CT2) (S2 and S3 Figs). Similar to the whole slice data, BMs90p suppressed Per2L rhythms in the SCN, as determined by imaging multiple (n = 24) small SCN cell clusters (Fig 1C, S4 Fig and S1 Movie). Taken together, these data strongly suggest a pivotal role of cyclic CK2-mediated BMAL1-S90 phosphorylation in the circadian oscillator of SCN neurons (central clock) and liver cells (peripheral clocks).

CRY Proteins Inhibit BMAL1-S90 Phosphorylation
How are circadian oscillations in P-BMAL1-S90 levels generated? A clue may be found in previous observations that BMAL1 is constitutively hyperphosphorylated in CRY1/2-deficient (CRYdKO) cells with a dysfunctional clock [8,14].
We therefore first investigated whether hyperphosphorylation of BMAL1 in CRYdKO cells includes Ser90. As shown in Fig 2A and 2B, P-BMAL1-S90 level was significantly higher in CRYdKO cells than in wild-type (WT) cells. Importantly, expression of Cry1 promoter-driven Myc-CRY1 in CRYdKO cells (S5 Fig) caused the P-BMAL1-S90 level to return to WT levels  (Fig 2A and 2B), suggesting that the CRY proteins act as suppressors of BMAL1-S90 phosphorylation.
Based on these results, we next focused on CK2-mediated BMAL1-S90 phosphorylation and the role of CRY proteins therein. Whereas the majority of CK2α catalytic subunits are untreated cells were set as 1. Error bars indicate standard deviation (SD). (B) Clock performance of organotypic slices of liver and SCN from mPER2 Luc mice following treatment with 6 μM BMs90p or mock treatment with fresh medium around the PER2::LUC trough phase (liver;~CT [Circadian Time] 5, SCN; CT2). (a) Luciferase activity was monitored by real-time bioluminescence imaging. Note the recovery of BMs90P dampened rhythms by medium change. (b) Quantification of rhythm amplitude and peak bioluminescence after BMs90p treatment (n = 5), in which values in untreated slices are set as 1. Error bars indicate SD. (C) Clock performance of small cell clusters in organotypic SCN slices from PER2::LUC mice following treatment with 6 μM BMs90p around the trough phase (~CT2). (a) Luciferase activity was monitored by real-time bioluminescence imaging (n = 24). Shown are detrended (colored lines) and averaged values (dotted line). (b) Representative examples of bright field (BF) and Per2L images at CT14 (around the peak phase) pre-and post-BMs90p treatment (day 2 and 4 respectively).
doi:10.1371/journal.pbio.1002293.g001 likely recruited to a CK2β (regulatory unit) dimer to form a constitutively active CK2α 2 β 2 tetramer that can phosphorylate a wide range of substrates [20], CK2β behaves as a strong inhibitor of CK2α-mediated BMAL1 phosphorylation in a dose-dependent manner [12]. Notably, CK2β does not directly inactivate CK2α [20]. Rather, CK2α activity is thought to fluctuate by the influence of yet unidentified cellular molecules.
As reported previously [12], we demonstrated that the CK2α monomer, but not CK2α2β2, phosphorylates BMAL1 at Ser90, and that CK2 kinase activity dramatically declined at a ratio of CK2β/CK2α 1 (Fig 2C). Similarly, we confirmed that CK2β inhibits CK2α-mediated BMAL1-S90 in vitro kinase activity as a function of the CK2β/BMAL1 ratio (Fig 2C). Kinase activity dramatically declined at a ratio of CK2β/BMAL1 1, suggesting that CK2β interferes with CK2α monomer-mediated BMAL1-S90 phosphorylation by direct interaction with BMAL1. Indeed, in WT cell homogenates, CK2β is shown to co-precipitate with BMAL1 ( Fig  2A). In marked contrast, however, BMAL1-CK2β interactions were significantly reduced in hyperphosphorylated P-BMAL1-S90 containing CRYdKO cells, while the amount of CK2α bound to BMAL1-CK2β complexes was comparable to that observed in WT cells (Fig 2A and  2B). Taken together, these data suggest that the amount of CK2α interacting with BMAL1 itself does not reflect the BMAL1 phosphorylation status. Rather, it points to a model in which CK2β is recruited to BMAL1 to inhibit CK2α activity. In the absence of BMAL1-CK2β interactions in CRYdKO cells, CRY proteins are likely candidates for such recruiting function.
We therefore assessed the binding ability of CK2 subunits to mammalian CRY1/2. In vitro, recombinant GST-tagged CK2α, α 0 and β subunits could pull down CRY1 and CRY2 (S6A Fig). Thus, both CK2α and β subunits can bind to BMAL1, as well as to CRY1/2. Nonetheless, consistent with the data shown in Fig 2A, CRY proteins preferentially bind to CK2β. Notably, CRY1/2 can still interact with CK2β in BMAL1-deficient cells, demonstrating that BMAL1 is not required for CRY-CK2β interactions (S6B Fig). These results indicate that CRY proteins mediate BMAL1-CK2β binding by sequentially interacting with CK2β. Moreover, expression of Myc-CRY1 (resembling native CRY1 in that it also preferentially binds to CK2β; see Fig 2A) resets the level of BMAL1-bound CK2β to WT levels (Fig 2A and 2B). As the level of BMAL1bound CK2α remained unchanged by Myc-CRY1 expression, we propose a model in which CK2α-mediated BMAL1-S90 phosphorylation is cyclically inhibited by CRY-dependent binding of CK2β to BMAL1, resulting in rhythmic P-BMAL1-S90 levels.

CRY-Mediated BMAL1-CK2β Association Periodically Suppresses CK2α-Mediated BMAL1-S90 Phosphorylation to Produce Rhythmic Kinase
To test our hypothesis that the CRY-dependent periodic binding of CK2β to BMAL1 results in circadian P-BMAL1-S90 oscillation, we first examined the effects of CRY1/2 deficiency on the circadian pattern of P-BMAL1-S90 levels. As expected, and in contrast to the robust circadian oscillations in Dex-synchronized WT cells, P-BMAL1-S90 levels were constitutively expressed at high levels in CRYdKO cells (Fig 3 and S7 Fig). Moreover, periodic Myc-CRY1 expression (CRYdKO+CRY1) restored the circadian P-BMAL1-S90 oscillation, which peaked at a similar time (18-24 h after Dex treatment) as in WT cells (Fig 3B). The levels of BMAL1-bound CK2β in WT and Myc-CRY1 clock-rescued (CRYdKO+CRY1) cells exhibited robust circadian oscillation with peaks at 6-12 h and approximately 36 h with a phase nearly inverse to that of the P-BMAL1-S90 oscillation, whereas CRYdKO (with or without GFP) exhibited constitutively high P-BMAL1-S90 levels (Fig 3A and 3B). Reciprocally, CK2β-IP uncovered a similar temporal interaction pattern of BMAL1 with CK2β (S7 Fig). Moreover, CRY1 and CRY2 were shown to co-precipitate with BMAL1 or CK2β in a circadian manner and in phase with BMAL1-CK2β interactions (Fig 3A and S7 Fig). Notably, the circadian oscillation pattern of CK2β-CRY1/2 closely matched BMAL1-CK2β, rather than BMAL1-CRY1/2 rhythms (S7 Fig). As CK2β-CRY1/2 complexes can be formed in the absence of BMAL1 (S6B Fig), these data suggest that CRY proteins periodically facilitate BMAL1-CK2β interaction by first associating  with CK2β. Moreover, circadian changes in posttranslational modification events may affect circadian patterns of BMAL1-CRY1/2-CK2β complexes. CK2α/β levels remained constant over time (S7 Fig), indicating that CK2α/β levels do not determine circadian P-BMAL1-S90 oscillation. These data lead us to conclude that the cyclic phosphorylation of BMAL1-S90 originates from periodic suppression of CK2α-mediated BMAL-S90 phosphorylation through CRY-mediated BMAL1-CK2β association.

Live Cell Monitoring Reveals CRY-Enhanced Circadian Oscillation of BMAL1-CK2β Association
To rule out the possibility of nonspecific associations in pull-down experiments, we next used a Split Luc complementation assay [22,23] for real-time monitoring of CK2β-BMAL1 interactions in living cells. In such an assay, bioluminescence can be detected only when N-(ELucN)-and C-(McLuc1 or ELucC)-tagged proteins associate and allow Luc moieties to complement each other and form active luciferase (Fig 4A). To this end, we ectopically expressed ELucN-CK2β and McLuc1/ELucC-BMAL1 in Cos7 and U-2OS cells at a level comparable to that of the native proteins ( Fig 4A and 4C).
Dex-synchronized U-2OS cells exhibit robust circadian rhythmicity and can express high levels of ectopic proteins [24]. To investigate temporal changes in BMAL1-CK2β interactions, we monitored Split Luc activity in real-time mode in U-2OS cells and observed a robust circadian oscillation of BMAL1-CK2β binding, peaking approximately 15 h and 40 h after Dex-synchronization, and as such, inversely phased to P-BMAL1-S90 oscillations (Fig 4C and S8A and S8B Fig). These P-BMAL1-S90 patterns are consistent with those of asynchronous WT and CRY1-rescued CRYdKO MEFs (Fig 2A and 2B). As the RREx3/CMV promoter is constitutively active (see S8C Fig), the observed circadian BMAL1-CK2β Split Luc activity originates from rhythmic BMAL1-CK2β interaction rather than rhythmic BMAL1 expression. Ectopic expression of BMAL1 and CK2β in the Split Luc assay did not affect endogenous circadian phase or amplitude of the circadian core oscillator, as monitored through Bmal1-promoter driven luciferase activity (S9 Fig). Accordingly, the circadian patterns of BMAL1-CK2β association monitored by the Split Luc assay represent a nearly endogenous circadian pattern. Taken together with the result of Figs 3 and 4B, the antiphase circadian oscillations of BMAL1-CK2β interactions (as revealed by the binding Split Luc assay) and P-BMAL1-S90 rhythms strongly suggests that under physiological conditions (i.e., in the living cell) cyclic CRY-mediated BMAL1-CK2β association drives circadian BMAL1-S90 phosphorylation.

CRY1 and BMAL1 Regions Critical for BMAL1-CK2β Association
Next, we generated a panel of ELucC-mBMAL1 deletion constructs ( Fig 5A) to determine the region of BMAL1 [25] critical for BMAL1-CK2β interaction. BMAL1-CK2β binding was detected at comparable levels in Cos7 cells expressing ELucN-CK2β with either ELucC-B-MAL1-WT (full length) or deletion mutants in the N-terminal half of BMAL1 (Bd1-3), and was stimulated by co-expression of CRY1 or CRY2 ( Fig 5B). However, irrespective of the   [26,27] resulted in significantly lower levels of bioluminescence (approximately 30%; p < 0.001) as compared to BMAL1-WT (Fig 5B), indicating that the C-terminal region of BMAL1 is critical for BMAL1-CK2β binding. Yet, BMAL1-Bd5:CK2β Split-Luc activities were significantly enhanced by co-expression of CRY1 (p < 0.001) or CRY2 (p < 0.001), suggesting that CRY binding to CK2β can also enhance CK2β binding to BMAL1 without direct physical interaction between CRY and BMAL1. Moreover, independent of the presence of CRY1/2, expression of the Bd4 mutant lacking the BMAL1 PAC (PAS-associated C-terminal) domain resulted in significantly higher levels of bioluminescence (approximately 2-fold; p < 0.001) as compared to BMAL1-WT ( Fig 5B), implicating this region as a potential regulatory site for BMAL1-CK2β binding. CK2β binding with WT BMAL1, Bd4, and Bd5 was largely enhanced by ectopic CRY1/2 expression. These findings identified critical regions in BMAL1 for CRYenhanced binding to CKβ.
To identify CRY1 protein regions critical for facilitating BMAL1-CK2β interaction, we generated a panel of mCRY1 deletion constructs ( Fig 5C) for co-expression with ELucN-CK2β and ELucC-BMAL1 in Cos7 cells. CRY-facilitated BMAL1-CK2β binding, as detected by the Split-Luc assay, was not significantly altered by deletion of either the N-terminal DNA photolyase domain (Cd1) or the C-terminal region (Cd4) of CRY1 ( Fig 5D). However, co-expression of a CRY1 mutant protein lacking the FAD-binding domain (Cd2) [28] resulted in significantly lower levels of bioluminescence (approximately 20%; p < 0.001) as compared to CRY1-WT (Fig 5D), indicating that the FAD-binding domain of CRY1 is critical in enhancing BMAL1-CK2β binding. Notably, co-expression of a mutant CRY1 protein (Cd3), lacking the pivotal region for inhibition of BMAL1-CLOCK-mediated transcription in the FAD-binding domain [28,29] also resulted in significantly lower levels of bioluminescence (approximately 40%; p < 0.001) as compared to CRY1-WT ( Fig 5D). This finding indicates that the FAD-binding region is not only critical for suppression of CLOCK-BMAL1 -mediated transcription activation but also for CK2α-mediated BMAL1-S90 phosphorylation by facilitating BMAL1-CK2β binding.
Next, we performed a Split-Luc live cell assay experiment using an ElucC-mBMAL1-K537R mutant protein. In the absence of CRY1/2, the BMAL1-K537R-CK2β binding was not significantly different from BMAL1-CK2β binding. However, in the presence of CRY proteins BMAL1-CK2β interactions increased by approximately 4-fold, while BMAL1-K537R-CK2β binding did not significantly increase (p < 0.001 in comparison with WT) (Fig 6A). Thus, CLOCK-mediated acetylation of K537 in the C-terminal region of BMAL1 appears critical for CRY-enhanced BMAL1-CK2β binding.
Taken together, these data demonstrate that acetylation of BMAL1 at Lys537 facilitates BMAL1-CK2β association, and as such represses BMAL1-S90 phosphorylation.

BMAL1-S90 Phosphorylation Is Prerequisite for BMAL1-K537 Acetylation and Subsequent Recruitment of CRY to BMAL1
For a variety of proteins phosphorylation has been shown to trigger subsequent acetylation events [31,32]. Accordingly, S90 phosphorylation is potentially involved in the regulation of BMAL1 acetylation. To assess the link between BMAL1-S90 phosphorylation and BMAL1-K537 acetylation and to further establish the integral role of the CK2-mediated BMAL1-S90 phosphorylation in the circadian core oscillator, we examined the effect of BMAL1-S90 mutation on BMAL1-K537 acetylation, which, as shown above, is, critical for CRY-mediated BMAL1-CK2β binding and P-BMAL1-S90 rhythms.

Discussion
Circadian BMAL1-S90 phosphorylation has been shown to be an important regulatory step in the mammalian core clock oscillator [12]. In the present study, we addressed the underlying mechanism and uncovered a vital interplay between CRY proteins and circadian BMAL1 phosphorylation. First, by applying a novel clock-perturbing peptide (BMs90p) to SCN and liver organotypic slices from mPER2 Luc mice and subsequent live monitoring of circadian clock performance, we further highlighted a universal critical role of BMAL1-S90 phosphorylation in central and peripheral clocks. BMs90p (a small 14 amino acid peptide containing the BMAL1-Ser90 phosphorylation site targeted by CK2) behaves as a competitive inhibitor of BMAL1-S90 phosphorylation and was shown to reversibly blunt Per2L bioluminescence rhythms in a doseand circadian time-dependent manner Next, triggered by the observation that a CRY1/2-deficiency causes hyper-phosphorylation of BMAL1 [8,14], we focused on the molecular mechanism underlying circadian BMAL1-S90 phosphorylation and showed that in wild-type cells, circadian phosphorylation of BMAL1-S90 is accompanied by inverse phase cyclic association of BMAL1 with CK2β, a known inhibitor of CK2α-mediated BMAL1 phosphorylation [12]. Notably, a CRY1/2-deficiency abolishes BMAL1-CK2β interactions, and as such prevents cyclic inhibition of BMAL1-S90 phosphorylation, resulting in constitutively hyperphosphorylated BMAL1. P-BMAL1-S90 in CRY1/2 deficient cells could be rescued by rhythmic Cry1 expression, which points to a model in which CRY proteins cyclically recruit CK2β to BMAL1 to inhibit CK2α activity.
To provide further evidence for this model, we developed a Split-Luc-based assay system for real-time monitoring of clock protein-protein interactions in living cells. Using this assay, we have shown that BMAL1 cyclically binds to CK2β and that circadian BMAL1-CK2β binding is enhanced by CRY proteins. Moreover, using the same Split-Luc approach in combination with mutant versions of the BMAL1 protein, we have shown that the PAC and CRY-binding domains in the C-terminal region of BMAL1, as well as BMAL1-K537 acetylation (known to enhance CRY-recruitment to BMAL1 [4]) are important in regulating BMAL1-CK2βbinding. Indeed, using SIRT1KO cells, we demonstrated that BMAL1-K537 hyper-acetylation reduces BMAL1-S90 phosphorylation through enhanced CRY-driven BMAL1-CK2β association. As BMAL1-S90 phosphorylation is prerequisite for BMAL1-K537 acetylation (see below), the low but significant P-BMAL1-S90 level in SIRT1KO MEFs is apparently sufficient to trigger BMAL1-K537 acetylation ( Fig 6B). Reciprocally, BMAL1-S90A expressing MEFs, lacking BMAL1-S90 phosphorylation, cannot trigger significant BMAL1-K537 acetylation (Fig 7A  and 7B).
By BMAL1-S90A mutagenesis, we showed that BMAL1-S90 phosphorylation is prerequisite for BMAL1-K537 acetylation. The S90A mutation significantly reduces the nuclear BMAL1-CLOCK levels [12,33] and S90-phosphorylated BMAL1 is mostly detected in the nuclear fraction (S11 Fig), strongly suggesting BMAL1 enters the nucleus promptly after S90 phosphorylation. Taken together with the lower K537 acetylation and CLOCK binding capacity of BMAL1-S90A, as compared to BMAL1-WT, it is assumed that K537 acetylation mainly occurs after BMAL1-CLOCK nuclear entry. The enhanced K537 acetylated/S90-phosphorylated BMAL1 level in Sirt1 knockout cells suggests a mutual regulatory loop between K537 acetylation and S90 phosphorylation and supports the notion that S90 phosphorylation is prerequisite for K537 phosphorylation, while K537 acetylation represses S90 phosphorylation.
In conclusion, we established a circadian clock-controlling role of CK2 kinase, formerly thought to be a constitutively active kinase [20] in BMAL1 phosphorylation and uncovered a novel role of CRY as a regulator of cyclic CK2-mediated BMAL1 phosphorylation. Fig 8 illustrates our model for the molecular mechanism of the CK2-mediated posttranslational loop and its role in regulating the intracellular circadian core oscillator. In this model, cyclic CK2αmediated BMAL1-S90 phosphorylation serves as the periodic gateway that controls BMAL1-CLOCK heterodimerization (step I) and time-delayed nuclear accumulation of BMAL1-CLOCK (step II) [12].
Step I and II may play a critical role in the events described below and serve as a time-delay factor that fine-tunes the circadian periodicity [14]. Therefore, we refer to CK2-mediated BMAL1-S90 phosphorylation as the first gate, probably located at the boundary between the cytoplasm and nucleus. Consistently, constitutive nuclear predominant BMAL1 localization in CRYdKO MEFs through the circadian cycle might be largely due to constitutive active BMAL1-S90 phosphorylation [14]. After BMAL1-CLOCK accumulates in the nucleus, E-box promoter containing clock genes, including CRY1/2, are temporally transactivated (step III). This is followed by negative feedback suppression of BMAL1-CLOCK transcription of Ebox genes by the recruitment of CRY1/2 to BMAL1 (step IV), which is regulated by CLOCKmediated BMAL1-K537 acetylation [4] and requires phosphorylated BMAL1-S90. In the next step (step V), because of the delayed surge in CRY1/2-CK2β binding, the BMAL1-CLOCK-CRY complex is released from the E-box. Thereafter, we hypothesize that CRY proteins are released from the complex to make way for newly incoming CRY1/2-CK2β complexes that bind to BMAL1-CLOCK via direct CRY-BMAL1 interaction. Deletion of the CRY-binding In short, upon phosphorylation of BMAL1-S90 by CK2α (step I), BMAL1 binds to CLOCK (step II) to form a transcriptional activator complex for transcription of E-box promoter containing clock genes (i.e., Cry and Per genes) and clock-controlled genes (step III). CK2α remains bound to BMAL1 in a catalytically active state (as indicated by the red color). After a delay, CRY proteins will bind to the CLOCK-BMAL1-CK2α complex to inhibit E-box gene transcription (step IV). Upon dissociation of the BMAL1-CLOCK-CRY-CK2α complex from the DNA, CRY is released from the complex to allow CRY-CK2β binding, and subsequent BMAL1-CK2β binding, resulting in the formation of BMAL1-CLOCK-CRY-CK2β-CK2α complex (step V). This step, triggered by acetylation of BMAL-K537, renders CK2α inactive (as indicated by the grey color). After dissociation of the BMAL1-CLOCK-CRY-CK2β-CK2α complex, BMAL1 is degraded and/or dephosphorylated and deacetylated (step VI) to start a new cycle. For a detailed description of the model, see the Discussion section. For simplicity, PER proteins have not been included in the model and CRY1 and CRY2 proteins are collectively shown as CRY.
doi:10.1371/journal.pbio.1002293.g008 domain in BMAL1 does not completely abolish CRY-mediated enhancement of BMAL1-CK2β binding in the Split Luc assay (Fig 5), suggesting direct BMAL1-CRY interaction is not absolutely necessary for the enhancement of BMAL1-CK2β binding. Given that CRY has also been shown to bind to CLOCK [29], docking of CRY-CK2β to BMAL1-CLOCK may also involve CRY-CLOCK interactions. Through formation of CRY1/2-CK2β intermediates, CRY1/2 facilitates BMAL1-CK2β association. Notably, the release of CRY from and re-entry of CRY-CK2β in the BMAL1-CLOCK complex (instigated by the observation that CRY can bind CK2β in the absence of BMAL1) is the most speculative step in the model. In the absence of experimental/mechanistic evidence we cannot fully exclude that CRY proteins enhance BMAL1-CK2β binding while still bound to the BMAL1-CLOCK-CRY complex.
In vitro, CK2β can bind to BMAL1 in the absence of CRY and inhibit BMAL1-S90 phosphorylation by CK2α (Fig 2C and S6A Fig). Under these non-physiological conditions, inhibition of S90 phosphorylation may occur through (simultaneous) formation of CK2β-BMAL1 complexes that prevent CK2α from binding to BMAL1, and/or formation CK2α2β2 tetramers [20], which are probably incapable of phosphorylating BMAL1 [12]. However, as formation of a CK2α2β2 tetramer requires formation of a CK2β dimer that subsequently binds two CK2α monomers, and as in vitro BMAL1-S90 phosphorylation by CK2α is maximally inhibited at a CK2β:BMAL1 ratio of 1 (Fig 2C), interaction of 1 CK2β monomer rather than a tetramer with 1 BMAL1 molecule appears sufficient to inhibit S90 phosphorylation. In vivo, BMAL-CK2β association appears to block (BMAL1-bound) CK2α activity, rather than BMAL1-CK2α association, and requires the help of CRY proteins. Although we do not exclude a model in which CK2β is included in the BMAL1-CLOCK-CRY-CK2β-CK2α as a dimer or α2β2 tetramer complex, we consider inhibition of CK2α-mediated BMAL1-S90 phosphorylation by a CK2β monomer the most plausible option (step V). Taken together, inverse-phased circadian BMAL1-CLOCK-CRY-CK2β−CK2α complex formation might be the primary determinant for circadian CK2α-mediated BMAL1-S90 kinase activity. Next, S90 phosphorylated BMAL1 undergoes a SIRT1-mediated deacetylation step (step VI) [30] that likely liberates BMAL1 from the complex. Subsequent SUMOylation [3] and ubiquitination [34] of BMAL1 may target the protein for proteasomal degradation. In addition, non-degraded BMAL-S90 needs to be dephosphorylated by yet unknown phosphatases to initiate a new cycle through CK2α-mediated phosphorylation of BMAL1. BMAL1-S90 phosphorylation by the CK2α monomer most likely occurs at step I (Fig 8). S90-phosphorylation of BMAL1 takes place in the cytoplasm and triggers CLOCK binding and subsequent BMAL1-CLOCK nuclear accumulation [12]. We have shown that BMAL1-CK2α complexes exist throughout the circadian cycle (S7 Fig). This suggests that the CK2α monomer remains bound to the BMAL1-CLOCK complex up to step IV. Likely, CK2α remains catalytically active (though its substrate is no longer available) and only gets inactivated after CRYmediated binding of CK2β (step V).
In this study, we have unveiled the underlying mechanism for the cyclic CK2-mediated BMAL1 phosphorylation as a critical event in the mammalian circadian core clock machinery. BMAL1-P-BMAL1 loop forms a distinct interlocked loop in the clock machinery (step I, V, and VI) and have integral roles in the core circadian oscillator through periodic CRY-mediated negative feedback suppression. In this scenario, CRY proteins have a dual function. Strikingly, in addition to their known function as repressors of BMAL1-CLOCK-driven transcription, we found a novel role of CRY proteins as a repressor of CK2 protein kinase activity toward BMAL1-S90. Notably, we observed that the FAD binding region of CRY1, known to be essential for repression of BMAL1-CLOCK-driven transcription [28], is also critical for inhibition of CK2α-mediated BMAL1-S90 phosphorylation.
Interestingly, CLOCK-mediated BMAL1-K537 acetylation [4], through sequential recruitment of CRYs and then CRY1/2-CK2β to the BMAL1-CLOCK complex, acts as a common molecular key for evoking CRY-mediated feedback inhibition of BMAL1-CLOCK transcription activity and CRY-dependent suppression of BMAL1 phosphorylation. Ultimate verification of our model ideally requires in vitro reconstitution of the CRY-driven circadian BMAL1-P-BMAL1 loop, as shown for cyanobacterial KaiC phosphorylation [35]. In a first experiment in which purified recombinant CRY1 was added to the in vitro BMAL1-S90 phosphorylation assay (as performed in Fig 2C), we observed that despite its ability to bind BMAL1 (S12A Fig),  CRY1 could not inhibit CK2α-mediated BMAL1-S90 phosphorylation (S12B Fig). This apparent difference markedly contrasts with the in vivo data, where CRY has been shown pivotal for CK2β-mediated inhibition of BMAL1-S90 phosphorylation by CK2α. Clearly, in vitro assays differ from the in vivo situation in that they do not take into account the effect of subcellular localization of the proteins studied, their interaction with DNA or chromatin, or the involvement of other protein partners. Moreover, in vitro synthesized proteins probably do not undergo posttranslational modification, leading us to hypothesize that CRY can only recruit CK2β to BMAL1 after acetylation of BMAL1-K537.
CK2 phosphorylates a large array of cellular proteins and is widely involved in regulating mammalian physiology [17]. However, temporal aspects of CK2 function are still elusive. Therefore, in addition to its role in the core clock, future investigations should focus on CK2-mediated circadian signaling as a regulator of various physiological and pathological pathways. A genome-wide phospho-proteomics study of periodic signaling systems focused on CK2 may help elucidate the chronobiological attributes of diverse physiological events and facilitate the development of therapies for circadian-system-related disorders [36], such as metabolic syndromes, cancer, and neuropsychiatric diseases. Recently, we demonstrated that CK2-BMAL1 kinase plays a critical role in controlling protective pathways evoked by reactive oxygen species and is crucial for preventing oxidative-stress-related diseases [37].
In vitro kinase assays were performed as described previously [12,13], using CK2 subunits, 1 mM ATP, and GST-BMAL1, with/without His-CRY1 (see below). CK2α, CK2β, and GST-BMAL1 were prepared as described previously [12]. Kinase activities were measured by immunoblot using an anti-P-BMAL1-S90 antibody, and quantified as described above. Immunoblot and kinase assay data were normalized to the control values.
GST-CK2α, α', and β subunits were expressed in bacteria, purified and analyzed by CBB staining. Mammalian clock proteins labeled with 35 S-methionine were produced using the TNT Quick Coupled Transcription/Translation system (Promega) with expression vectors for BMAL1 (donated by Dr. Ikeda) and V5-CRY1/2 (donated by Dr. Reppert). CK2 subunits and clock proteins were mixed (combinations as indicated in the text), incubated, and affinity-precipitated with glutathione Sepharose beads. Recombinant His (and V5)-tagged CRY1 protein was expressed in High Five insect cells using pIB/V5-His vector and InsectSelect System (Invitrogen, CA, US). His-CRY1 protein was purified using Talon-resin (Clontech, CA, US).
The peptides BMs90p (RRDKMNSFIDELAS, a 14 amino acid BMAL1 peptide centered around BMAL1 Serine 90) and BMa90p (RRDKMNAFIDELAS, a negative control peptide with serine 90 replaced by alanine) were custom-made by Gen Script (Piscataway, NJ, US). Peptides were dissolved in water and applied to actively growing cultured cells at 60% confluence. The RetroMax expression system (Imgenex, San Diego, CA, US) was used to produce retrovirus for the rescue experiment. Retroviral infection was performed as previously described [12]. Real time bioluminescence activities were monitored using the Kronos Dio system (ATTO, Tokyo, Japan) as previously described [37].
All animal experiments were approved by the Toho University Animal Committee, and carried out under the control with Guidelines for Proper Conduct of Animal Experiments by Science Council of Japan. mPER2 Luc mice (B6.129S6-Per2 tm1Jt /J) 21 were purchased from Jackson Laboratories (Bar Harbor, ME, US) and maintained at 25°C on a 12 h light/dark (LD) cycle (light: zeitgeber time [ZT] 0-12; dark: . Preparation of organotypic slices from 4-8 week old mice, real-time bioluminescence assay, and microscopic imaging analysis (using the LV200 Bioluminescence Imaging System; Olympus, Tokyo, Japan) and MetaMorph analysis (MetaMorph, Nashville, TN, US) were performed using previously published procedures [40], briefly described below. The reduction of the peak (Fig 1Bb)  Values obtained from bioluminescence analyses were normalized by the maximum peak intensities over time and further normalized by the averaged intensity over time, as described previously [37,41]. Real-time bioluminescence in cell cultures and organotypic slices treated with 0.2 mM Luciferin (Toyobo) were monitored using the Kronos Dio system with acquisition times of 2 (promoter-Luc assays) or 3 min (Split-Luc assays), according to the manufacturer's protocol. Values were obtained from each sample in a given experiment using the same detectors. The n-values indicated for each experiment refer to the number of samples analyzed with the same detectors in the same experiments. The y-axis label "Bioluminescence" indicates that the relative photo-counting values reflect arbitrary units (a.u.) from raw data; "RLU" (Relative Light Units) indicates that the relative photo-counting values were normalized by averaging intensity over time. The y-axis label "Deviation from the moving average" indicates that the values were detrended according to the Kronos Dio instrument protocol (ATTO). In many cases, as indicated in the figure legends, detrended values were further normalized by averaging intensity over time. The data in these graph labeled "deviation from the moving average" were further normalized using maximum circadian peak intensities over time. Real-time bioluminescence for microscopic imaging was monitored using the LV200 microscope with acquisition times of 48 min (EM-gain = 400) according to the manufacturer's protocol (Olympus). Values obtained from each tracked region of interest (ROI) surrounding neighboring small clusters of cell-areas were processed similarly.

Statistical Analyses
We used factorial design analysis of t test to analyze data as appropriate. The data presented in this study represent the average of multiple experiments, as specified in the figure legends.  Fig, except that data were further normalized using maximum circadian peak intensities over time. A reduction of rhythm amplitude (Fig 1Bb) after BMs90p-treatment was calculated from detrended data by comparing averaged differences between the peak and trough over 2 d before and after the treatment. Similarly, reduction of peak bioluminescence (Fig 1Cb) after BMs90p-treatment was calculated from raw data (Fig 1Ca) by comparing averaged peak differences over 2 d pre-and post-treatment. For each time point, BMA-L1-IPs and lysates from the nuclear and cytoplasmic fractions (prepared as previously described [38]) were subjected to IB analysis for P-BMAL1-S90 and BMAL1, using RNA polymerase II (Pol II) and actin as the loading controls. Red and black arrows indicate the position of major P-BMAL1-S90 (~90 kDa) and unmodified BMAL1 (~70 kDa), respectively. (EPS) S12 Fig. Effect of CRY1 protein on the in vitro CK2α-mediated BMAL1-S90 kinase activity. (A) Purified recombinant His-CRY1 protein expressed in High Five insect cells can bind to BMAL1. His-CRY1 expressed in High Five cells was purified and detected by CBB-staining and immunoblotting with anti-His antibody. By IB analysis for the His-immunoprecipitate (His-IP) for the mixture of the purified GST-BMAL1 and His-CRY1, GST-BMAL1 and His-CRY1 were detected with anti-BMAL1 and anti-His antibodies. Note that no apparent band with higher molecular weight than His-CRY1 was detected in the CBB-stained gel. (B) His-CRY1 does not show inhibitory effect on the in vitro BMAL1-S90 phosphorylation. CK2αmediated BMAL1-S90 kinase activity was measured by in vitro kinase assay with GST-BMAL1, CK2α, and differential doses of His-CRY1 and CK2β proteins, under the procedure for Fig 2C. (EPS) S1 Movie. Effect of BMs90p-treatment on Per2L-rhythm in the SCN organotypic slice. An organotypic slice of the SCN from mPER2 Luc mice was monitored by real-time bioluminescence microscopy (LV200, Olympus, Japan). Cultures were treated with 6 μM BMs90p around the trough phase (at time Zero after 2 d and 17 h) as indicated in Fig 1C and S4 Fig.  (MP4)