1. Volume 60, Issue 1, Pages 77-88 (October 2015)
A Period2 Phosphoswitch Regulates and Temperature Compensates Circadian Period 
Min Zhou, Jae Kyoung Kim, Gracie Wee Ling Eng, Daniel B. Forger, David M. Virshup 
Molecular Cell 
Volume 60, Issue 1, Pages (October 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 60, 77-88DOI: (10.1016/j.molcel.2015.08.022)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
PER2 Degrades in Three Distinct Stages Regulated by Clock Time and CK1
(A) PER2 shows three-stage degradation when CHX is added during the rising phase of PER2 oscillation. Per2Luc MEFs were synchronized with dexamethasone and the abundance of PER2::LUC was assessed by measuring the luminescence intensity in the Lumicycle. At the indicated time points following dexamethasone shock, CHX (40 μg/ml) was added. The second or plateau stage, defined as the time when the instantaneous half-life is greater than 5 hr (see also Figure S1A and Experimental Procedures for details), is indicated by the shaded area.
(B) The duration of the plateau is dependent on the time of addition of CHX. Data from Figure 1A are represented as mean ± SD.
(C) Inhibition of CK1 largely eliminated both the first and the second stage of degradation. Per2Luc MEFs were synchronized as above, and either CHX (40 μg/ml) or CHX (40 μg/ml) and PF670 (1 μM) together was added 22 hr after dexamethasone. Data were normalized to PER2::LUC abundance immediately prior to drug addition.
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4. Figure 2
A Mathematical Model Predicts the Phosphoswitch of PER2 as the Mechanism for Three-Stage Decay of PER2
(A) In the model, the translated PER2 protein has two fates, depending on the site of initial phosphorylation. A phosphorylation at one site (red dot) by CK1 induces β-TrCP binding and rapid degradation (Eide et al., 2005). A phosphorylation at another site, known as the FASP site, by the priming kinase (orange dot) induces sequential phosphorylation at neighboring downstream sites by CK1 (purple dots), which stabilizes PER2 protein (Shanware et al., 2011; Vanselow et al., 2006; Xu et al., 2007). Finally, fully phosphorylated PER2 protein becomes an active transcriptional repressor and degrades via a CK1- and β-TrCP-independent mechanism. See also Tables S1 and S2.
(B) The model simulates the occurrence of three-stage decay of PER2 exclusively during its rising phase. It also predicts the second-stage plateau increases when protein synthesis is inhibited earlier in the rising phase. To allow time for CHX to enter cells, the protein translation rate is assumed to be exponentially decreased with a half-life (∼0.13 hr) after CHX treatment.
(C) When both translation and CK1 are inhibited during the rising phase, PER2 shows monophasic degradation after an initial spike. Here, to simulate the inhibition of phosphorylation by CK1, the binding rate of CK1 with PER2 is immediately reduced to 1% of the original binding rate.
(D) The predicted fate of specific PER2 species. The model predicts that phosphorylation at β-TrCP binding site(s) induces the initial rapid degradation of PER2. The second, plateau stage is generated by the sequential phosphorylation around the FASP site (FASP [Prime] and FASP [Complete]), which stabilizes PER2. Finally, fully phosphorylated PER2 protein becomes unstable, which induces the third degradation stage. Time 0 is 22 hr following the prior PER2 peak, as in Figure 1C.
(E) The model predicts that CK1 inhibition blocks the degradation of PER2 induced by β-TrCP site phosphorylation and promotes the accumulation of stable PER2 phosphorylated at the FASP site by the priming kinase. This causes the spikes immediately following CK1 inhibition.
See also Figure S2.
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5. Figure 3 Molecular Characterization of the PER2 Phosphoswitch
(A) PER2 S478 phosphorylation regulates the binding of PER2 to β-TrCP. PER2, β-TrCP (ΔF-box), and CK1ε were co-expressed in NIH 3T3 cells. Twenty-four hours after transfection, Myc-PER2 was immunoprecipitated and binding partners assessed by SDS-PAGE and immunoblotting. Mutation of S478A in PER2 markedly decreased β-TrCP binding. WCL, whole-cell lysate.
(B) PER2 FASP site S659A mutation promotes β-TrCP site phosphorylation. The indicated PER2 mutants were expressed in NIH 3T3 cells with or without CK1ε. β-TrCP binding site phosphorylation, detected with the pS478 antibody, was markedly increased in the presence of the FASP mutant (S659A). ∗ Non-specific bands used as loading controls.
(C) PER2 β-TrCP site mutant S478A increases while FASP site mutant S659A decreases PER2 stability. The indicated mutants were transiently expressed (10 ng of PER2 plasmids per 35 mm dish) in unsynchronized NIH 3T3 cells, and their abundance was assessed in a Lumicycle after the addition of CHX. Mean PER2 half-life is shown ± SD.
(D) Limited proteolysis shows that PER2 S659A is more susceptible than WT PER2 to trypsin digest, indicating the S659A mutant causes a more open structure of PER2. The indicated PER2 wild-type and mutants were immunoprecipitated and digested with 1 μg/ml trypsin for 1 min on ice. PER2 abundance was assessed by SDS-PAGE and immunoblotting.
(E) PER2 is predicted to have an extended disordered domain between the β-TrCP site and the FASP site, as well as after the FASP site. Mouse PER2 protein sequence was analyzed using PONDR-FIT from DisProt.org using default parameters (Sickmeier et al., 2007). A region is considered as intrinsic disordered when the Disorder Disposition score is greater than 0.5. PER2 domains and the location of S478 and S659 are indicated on the top of the graph. PAS, PAS domain 1 (dark blue) and 2 (purple); CLD, cytoplasmic localization domain (lime); CK1, CK1 binding domain (navy); NLS, nuclear localization signal (blue); CRY, Cry binding site (green).
(F) Metabolic perturbation of the phosphoswitch affects the three-stage decay of PER2. ALX (10 mM) or PUG (200 μM) was added to PerLuc MEFs during the rising phase (5 hr before the peak), and degradation was assessed as above. These data were collected together with those shown in Figure 1C and the PF670+CHX curve is repeated here to aid in comparison.
See also Figure S3.
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6. Figure 4
CK1ε tau Mutation Destabilizes PER2 by Affecting the Phosphoswitch
(A) CK1εtau is a loss of function on the FASP site. The indicated amount of CK1ε or CK1εtau expression plasmid was cotransfected with plasmid encoding wild-type or S659A myc-PER2 into NIH 3T3 cells. Phosphorylation at the FASP site was assessed in whole-cell lysates by immunoblotting with pFASP antibodies. Specific PER2 phosphorylation was calculated as the ratio of pFASP to total PER2 immunoreactivity. β-TrCP (ΔF-box) was co-expressed to block PER2 degradation.
(B) CK1εtau is a gain of function on the β-TrCP S478 site, phenocopying the FASP mutation. Wild-type or tau mutant CK1ε (plasmid amount shown) was co-expressed with the indicated myc-PER2 variants as above. Phosphorylation on the β-TrCP binding site was assessed in whole-cell lysates by immunoblotting with pS478 antibodies. Mutation of the FASP site S659 minimizes the difference between CK1ε and CK1εtau activity on PER2.
(C) tau MEFs show a larger first rapid degradation stage and a shorter second plateau stage than wild-type Per2Luc MEFs. The three-stage degradation of the Per2Luc and CK1εtau MEFs was measured as in Figure 1. The three-stage decay (CHX was added three [−3] and five [−5] hours before the second peak) shown is the mean of four duplicates.
(D) The duration of plateau stage is shorter in Per2Luc/CK1εtau MEFs compare to Per2Luc MEFs. The plateau stage was quantified as in Figure 1B. Data are represented as mean ± SD.
See also Figure S4.
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7. Figure 5
PER2 Phosphoswitch Compensates Circadian Period in Response to Temperature Change
(A) Three scenarios for relative temperature sensitivity of the FASP site priming kinase and CK1. The mathematical model (Figure 2A) predicts that the observed temperature over-compensation occurs only when the priming kinase is more temperature sensitive than CK1 (Case 2: Q10 of priming kinase and CK1 are 4 and 1.33, respectively).
(B) The plateau stage of three-stage decay is markedly shorter when Per2Luc MEFs were cultured at 30°C while the initial rapid degradation stages are similar. The plateau stage was quantified as in Figure 1B.
(C) Temperature over-compensation mainly occurs during the rising phase. Per2Luc MEFs were cultured at either 30°C or 37°C. The duration of rising phase (bottom to peak) and falling phase (peak to bottom) were estimated with FFT-nonlinear least-squares analysis (Izumo et al., 2006). The differences in these stages in 30°C and 37°C were quantified as shown on the right. The rising phase was 1 hr longer at 37°C. Data are represented as mean ± SD.
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8. Figure 6 Temperature Compensation Requires the Phosphoswitch
(A) Circadian period is under-compensated in tau MEFs. Per2Luc and CK1εtau MEF cells were cultured in the Lumicycle as indicated to assess their temperature compensation. Period was calculated based on four replicates by Lumicycle data analysis software and represented as mean ± SD.
(B) Temperature compensation does not occur when CK1 is inhibited. PF670 (0.1 mM) or vehicle was added at the middle of the third peak (red arrow) while Per2Luc MEF cells were cultured at 30°C and 37°C.
(C) Half-life of PER2 becomes significantly shorter at low temperature (30°C) when CK1ε is coexpressed. However, this shortening of PER2 half-life does not occur when the phosphoswitch is disrupted with PF670 treatment, CK1εtau (Tau) coexpression, or mutation of the β-TrCP (S478A) or FASP (S659A) phosphorylation sites. Data are represented as mean ± SD.
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9. Figure 7 Model for Temperature Compensation with the Phosphoswitch
As temperature decreases, the PER2 phosphorylated on the β-TrCP binding site dominates the PER2 phosphorylated on the FASP site (Figure 5B). This shortens the plateau stage decay of PER2 during rising phase, which accelerates the rising phase and shortens period in the low temperature (Figure 5C). Domain architectures were shown by colors. PAS, PAS domain 1 (dark blue) and 2 (purple); CLD, cytoplasmic localization domain (lime); CK1, CK1 binding domain (navy); NLS, nuclear localization signal (blue); CRY, Cry binding site (green).
Molecular Cell  , 77-88DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions