The period of the circadian oscillator is primarily determined by the balance between casein kinase 1 and protein phosphatase 1 Hyeong-min Leea,1,2, Rongmin Chena,1, Hyukmin Kima, Jean-Pierre Etchegarayb,3, David R. Weaverb, and Choogon Leea,4 aDepartment of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306; and bDepartment of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605-2324 Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 30, 2011 (received for review May 4, 2011) Mounting evidence suggests that PERIOD (PER) proteins play a PAGE—occurs progressively over several hours (12, 19), which is central role in setting the speed (period) and phase of the circadian critical for stretching the feedback loop to ∼24 h. However, PER2 clock. Pharmacological and genetic studies have shown that changes can be maximally phosphorylated by CK1ε in vitro kinase reac- in PER phosphorylation kinetics are associated with changes in tions within 30 min (20, 21), suggesting that PER phosphorylation circadian rhythm period and phase, which can lead to sleep disorders must be counterbalanced by phosphatases in vivo. such as Familial Advanced Sleep Phase Syndrome in humans. We Because the phase and period of the clock are primarily de- and others have shown that casein kinase 1δ and ε (CK1δ/ε) are termined by temporal regulation of PER phosphorylation (12, 15, essential PER kinases, but it is clear that additional, unknown mech- 21–26), the characterization of PER kinases and phosphatases is anisms are also crucial for regulating the kinetics of PER phosphor- vital to understanding the circadian clock mechanism. CK1δ and ε ylation. Here we report that circadian periodicity is determined have been shown to be PER kinases and are important for clock primarily through PER phosphorylation kinetics set by the balance function, but PER phosphorylation is largely intact in several between CK1δ/ε and protein phosphatase 1 (PP1). In CK1δ/ε-defi- CK1δ or CK1ε mutants (12, 21, 23, 27). Furthermore, PER can be cient cells, PER phosphorylation is severely compromised and non- phosphorylated by several other kinases in vitro, including CK1α rhythmic, and the PER proteins are constitutively cytoplasmic. and γ, CK2, and GSK3β (28–32). Thus, it remains unclear which However, when PP1 is disrupted, PER phosphorylation is dramati- kinases are responsible for the majority of mobility-shifting, pro- cally accelerated; the same effect is not seen when PP2A is disrupted. gressive phosphorylation of PER in vivo. In this study, we show Our work demonstrates that the speed and rhythmicity of PER phos- δ ε δ ε that CK1 and are the major PER kinases and that they are phorylation are controlled by the balance between CK1 / and PP1, largely redundant with each other, such that PER phosphorylation which in turn determines the period of the circadian oscillator. Thus, and the circadian clock’s function can endure the loss of just one our findings provide clear insights into the molecular basis of how kinase. Moreover, we identify PP1 as the major PER phosphatase NEUROSCIENCE the period and phase of our daily rhythms are determined. that counterbalances CK1δ/ε and other PER kinases in vivo. dynamic regulation of phosphorylation | stoichiometry | Results period determination PER Phosporylation Is Severely Compromised and the Molecular Clock Is Not Functional in CK1δ/ε-Deficient Fibroblasts. We previously he circadian clock controls so much of mammalian physiology provided evidence for the importance of CK1δ/ε for clock function Tthat dysregulation of the clock contributes to medical con- by combining CK1δ gene deficiency with dominant negative CK1ε ditions such as jet lag, shift work sleep disorder, metabolic dis- expression (21). However, the dominant negative CK1ε may have ’ eases, and mood disorders (1, 2). In most of the body s cells, from interfered not only with wild-type CK1ε (wtCK1ε) activity, but also the neurons of the suprachiasmatic nucleus (SCN) to hepatocytes, with other isoforms of CK1 as suggested by Hirota et al. (29). To fi lung epithelial cells, and even broblasts, the clock is present and definitively test how CK1δ/ε contribute to temporal PER phos- – its molecular mechanism is essentially the same (3 6). The back- phorylation in vivo, we assessed circadian rhythms and the molec- bone of the oscillator mechanism is a transcriptional negative fi fi δ ε – ular clock in mouse broblasts de cient in both CK1 and . feedback loop driven by positive and negative elements (7 10). Because CK1δ deletion results in embryonic lethality, we derived The positive elements are three bHLH/PAS-containing tran- the fibroblasts from mice with floxed CK1δ/ε genes, so that the scription factors, CLOCK, NPAS2, and BMAL1; the CLOCK (or genes could be deleted conditionally using the Cre-loxP recom- NPAS2):BMAL1 heterodimer binds to E-box enhancer motifs bination system (27). Cre recombinase was expressed using an and activates transcription of the negative element genes, Per1 and 2, and Cryptochrome (Cry)1 and 2. The PER:CRY complexes com- plete the feedback loop by inhibiting CLOCK:BMAL1. Author contributions: H.-m.L., R.C., and C.L. designed research; H.-m.L., R.C., and H.K. Although all of these clock proteins are essential, PERIOD performed research; J.-P.E. and D.R.W. contributed new reagents/analytic tools; H.-m.L., (PER) is of special importance for clock regulation. PER is the R.C., and C.L. analyzed data; and H.-m.L., R.C., D.R.W., and C.L. wrote the paper. rate-limiting component for PER:CRY complex formation, and The authors declare no conflict of interest. thus the timing and duration of PER nuclear entry and accumu- This article is a PNAS Direct Submission. lation dictate the phase and period of the molecular clock (11–13). 1 ’ H.-m.L. and R.C. contributed equally to this work. Of all of the core clock proteins, only PER s expression is abso- 2Present address: Department of Pharmacology, University of North Carolina, Chapel Hill, lutely required to oscillate for the clock to function; constitutive NC 27514. high expression of PER completely disrupts circadian rhythms in 3Present address: Massachusetts General Hospital Cancer Center, Harvard Medical School, cells and mice (11). Phosphorylation of PER is a vital part of clock Boston, MA 02114. regulation, as it affects PER’s nuclear translocation, interaction 4To whom correspondence should be addressed. E-mail: [email protected]. – with other clock proteins, and timely degradation (14 18). In vivo, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PER phosphorylation—detectable as a mobility shift in SDS/ 1073/pnas.1107178108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1107178108 PNAS | September 27, 2011 | vol. 108 | no. 39 | 16451–16456 Downloaded by guest on September 26, 2021 CK1δfl/fl; CK1εfl/fl CK1δfl/fl; CK1εfl/fl (Fig. S3), these protein levels suggest that PER1 and PER2 are A B GFP Cre 0 3 4 days more stable in the mutant cells. Time (hr) 12 18 24 30 12 18 24 30 fi GFP Cre GFP Cre Our ndings are further supported when degradation rates were PER1 CK1δ measured after the cells were treated with cycloheximide. Both PER1 and PER2 were significantly more stable in the mutant PER2 cells compared with control cells (Fig. 1D). Nonphosphorylated CK1ε CLOCK was also more pronounced in the mutant cells (Fig. 1B), NS long which suggests that CLOCK is also a substrate of CK1δ/ε, likely CLOCK δ ε fi ACTIN via CK1 / :PER complexes. This nding is reminiscent of what short * – * has been found in Neurospora and Drosophila (33 36). Deletion of CK1δ/ε also caused PER1 and PER2 to accumu- CK1δ CK1δfl/fl; CK1εfl/fl + Cre late predominantly in the cytoplasm, whereas they were mostly C ε λ Pase - + + CK1 nuclear in control cells (Fig. 2A and Fig. S4 A and B). These vanadate -- + ACTIN results suggest that PER phosphorylation is required for nuclear PER1 translocation or accumulation, as has been reported in Dro- sophila (37). Our findings are consistent with data obtained from PER2 PER1 Cre 100 liver tissue, where hypophosphorylated PER species are cyto- ** GFP ** plasmic and not complexed with CK1δ/ε (12). 50 * fl/fl fl/fl Because previous research suggested that hyperphosphorylation D CK1δ ; CK1ε Cre+CHX GFP+CHX 0 targets PER for proteasomal degradation (22, 28, 38), we con- 0 3 6 9 12 hrs Time (hr) 0 3 6 9 12 0 3 6 9 12 sidered the possibility that the lack of hyperphosphorylated, nu- PER2 clear PER1/2 may have been due to rapid degradation as opposed PER1 Cre 100 GFP Relative abundance to reduced phosphorylation and lack of nuclear transport. To test * * * this possibility, we treated the cells with a mixture of proteasome PER2 50 * inhibitors (MG132 + PSI + lactacystin). In control cells, in- 0 hibition of proteasomal degradation increased levels of hyper- ACTIN 0 3 6 9 12 hrs phosphorylated PER1 and both hypo- and hyperphosphorylated CHX treatment PER2 levels, consistent with previous work (Fig. 2B) (21). The Fig. 1. PER1/2 do not oscillate, and their phosphorylation is severely com- same treatment in the CK1δ/ε-deficient cells did not result in the Δ Δ − − promised in CK1 double-mutant (CK1δ 2/ 2:CK1ε / ) fibroblasts. (A) CK1δ and ε detection of hyperphosphorylated forms, demonstrating that were successfully deleted by introducing adenoviral cre into the double- elimination of CK1δ/ε does prevent a substantial amount of PER fl oxed mutant cells. Cells were harvested 3 and 4 d after adenoviral cre or gfp phosphorylation. However, the treatment induced accumulation infection, and then subjected to immunoblotting for CK1δ/ε and ACTIN.