Fig 1: The PER-CK1 interaction is required for CLOCK hyperphosphorylation which can repress CLOCK-BMAL1 activity.a Western blot analysis showing that hPER2 expression results in hyperphosphorylation of CLOCK but L730G mutation of hPER2 blocks CLOCK hyperphosphorylation. CLOCK, BMAL1, CRY1 and CK1d expression plasmids were cotransfected with PER2 or PER2L730G in HEK293 cells. 200 ng of BMAL1, 20 ng of CRY1,400 ng of CLOCK, 400 ng of CK1d and 400 ng of PER2 or PER2L730G plasmids per 35 mm dish of HEK293 cells. The hyperphosphorylated CLOCK was indicated by the top arrow. Note that the endogenous CLOCK protein was not detected due to its low expression level. Five independent experiments were performed to validate the results. b A diagram depicting the model that PER2 acts as the CK1 scaffold to promote the phosphorylation of PER and CLOCK proteins in the PER-CRY-CLOCK-BAML1 complex. c Hyperphosphorylation of CLOCK is PER expression dose-dependent. The indicated different amounts of hPER2 expression plasmids were used in the transfection. The hyperphosphorylated CLOCK was indicated by the top arrow. Three independent experiments were performed to validate the results. d Mutation of CLOCK phosphorylation sites abolished the PER-dependent CLOCK hyperphosphorylation. The previously identified phosphorylation CLOCK sites were mutated in the M1 (S427/431/436/437/440/441 to glycine) and M2 (S38/42/S427/431/436/437/440/441 to glycine) mutants. Three independent experiments were performed to validate the results. e Mutations of CLOCK phosphorylation sites result in increased expression of the CLOCK/BMAL1-driven E-box controlled luciferase reporter gene. ***p value < 0.001 (two-side t-test). Data are represented as mean ± SD, n = 5. The western blot results of the same experiment were shown above.
Fig 2: The stable CK1d-PER interaction is required for hPER phosphorylation by CK1d and promotes PER degradation.a, b V729G/L730G or L730G mutations of hPER2 abolishes PER2 phosphorylation by CK1d in cells. Upshift of hPER2 mobilities indicate its phosphorylation by CK1d. Indicated samples were treated with lambda phosphatase to confirm the mobility shifts were due to phosphorylation. 1.5 µg of hPER2 and 1 µg of CK1d plasmids were transfected per 35 mm dish of HEK293 cells. The low PER2 levels in lane 5 and 7 are due to variations in transfect experiments. Three independent experiments were performed to validate the results. c CHX treatment showing that the L730G mutation dramatically slows down hPER2 turnover rate. Western blot result of a representative experiment showing the levels of hPER2 after the addition of CHX (10 µg/ml) (top). (n = 3). Densitometric analysis of three independent experiments of the Western blot results (bottom). * and *** indicate p value < 0.05 and 0.001 (two-sided t-test), respectively, at the indicated time points. Data are represented as mean ± SD. d Western blot analysis showing the hPER2 L730G mutation abolished hPER2 S662 phosphorylation. Three independent experiments were performed to validate the results. e Immunoprecipitation assays and western blot analysis showing that hPER2L730G can be phosphorylated by CK1d when hPER1 was co-expressed. The co-expression of hPER1 results in immunoprecipitation of hPER2L730G with CK1d. The phosphorylated hPER2 species are indicated by the arrow. See also Fig. S2. Three independent experiments were performed to validate the results. f A diagram showing the model that CK1 uses PER as a scaffold to phosphorylate different PER proteins in the complex.
Fig 3: Disruption of the CK1d-PER interaction increases CRY1 enrichment at the E-box region.a Immunoprecipitation assay using mPER2 antibody showing the levels of CRY1 associated with mPER2 in the WT and Per1-/-; Per2m/m mouse livers. Three independent experiments were performed to validate the results. b CRY1 ChIP assay results showing the relative CRY1 enrichment at the E-box region of the indicated gene promoters at the indicated time points in the WT and Per1-/-; Per2m/m mice. Chromatin samples from mouse livers were analyzed by ChIP using CRY1 antibody. Error bars are standard deviations. *p value < 0.05 and **p value < 0.01 (two-sided t-test). c A model of the coupled CK1-dependent and CK1-independent negative feedback loops in the core mammalian circadian oscillator. During subjective days, the PER levels are low and CLOCK-BMAL is associated with E-box and active. During the subjective night, PER and CRY levels are high. The CK1-dependent negative feedback process (bottom) involving CK1, PER, and CRY that mediates efficient removal CLOCK-BMAL1 from the E-boxes by promoting PER-dependent phosphorylation of CLOCK by CK1. The CK1-independent process (top) results in the repression of the CLOCK-BMAL1 complex transcription activation activity by the PER-CRY complex on DNA. This process also causes slow removal of CLOCK-BMAL1 complex from E-boxes.
Fig 4: Constitutive unphosphorylated PER2 and hypophosphorylated CLOCK in the mPER2 PCD knock-in mice.a A diagram depicting domains of mPER2 protein and the knock-in (KI) mutations made in the mPER2 PCD domain. b Comparison of the DNA sequencing results in the wild-type (WT) and the Per2m/m mice. c Immunoprecipitation assays and Western blot results showing the levels and phosphorylation profiles of mPER2 and other proteins in the liver extracts of the indicated mouse strains. mPER2 antibody was used in the immunoprecipitation assays. Three independent experiments were performed to validate the results. d Western blot results showing the comparison of mPER2 phosphorylation profiles of the liver extracts collected at CT20 of the WT and Per1-/-; Per2m/m mice. The protein samples were treated with lambda phosphatase. Three independent experiments were performed to validate the results. e Western blot results comparing the mPER2 phosphorylation profiles of the liver extracts collected at CT20 of the WT, Per1-/-; Per2m/m and Per1-/-; Per2m/m; Per3-/- mice. Three independent experiments were performed to validate the results. f Western blot results showing the levels and phosphorylation profiles of mouse clock proteins at the indicated time points in constant darkness. Three independent experiments were performed to validate the results. g The quantification of mPER2 levels of Western blot results in the WT and Per1-/-; Per2m/m mice. Data are represented as mean ± SD, n = 3. h Western blot results showing the levels and phosphorylation profiles of the nuclear mPER2 protein at the indicated time points in constant darkness. See also Supplementary Fig. 3. i The quantification of the nuclear mPER2 levels of Western blot results in the WT and Per1-/-; Per2m/m mice. Data are represented as mean ± SD, n = 3.
Fig 5: Identification of the PER2 domains and residues required for PER2-CK1d interaction.a–c Western blot analyses of immunoprecipitation assays of FLAG-tagged PER constructs and GFP-tagged CK1d were expressed in HEK293 cells (3 µg of PER plasmid and 2 µg of CK1d plasmid transfected per 60-mm dish of cells) and showed that CK1d associates with hPER when co-expressed in HEK293 cells. The levels of PER2 proteins and CK1d were determined by western blot using anti-FLAG and GFP antibody, respectively. Analyses of the interaction of FLAG-tagged PER1, PER2, and PER3 with GFP-tagged CK1d (a). Identification of the region of hPER2 sufficient for the hPER2-CK1d interaction (b). Analyses of hPER2 deletion constructs (c). Three independent experiments were performed to validate the results. See also Fig. S1a–c. d Amino acid sequence alignment of PCD domains of hPER1-3 proteins. Yellow regions mark conserved residues in the a helical domain (top). Diagrams showing the secondary structure prediction of the hPER proteins (bottom). e Western blot analyses of immunoprecipitation assays of FLAG-tagged PER constructs with the indicated mutation and GFP-tagged CK1d were expressed in HEK293 cells. Three independent experiments were performed to validate the results. f Structure prediction of the wild-type hPER2 (top right) and hPER2 (V729G-L730G) (bottom right) region described in 1d by Alphafold. The L730 residue in the hPER2 was indicated by a red arrow in an enlarged picture on the left. g Amino acid sequence alignment of the PCD domains of nine indicated vertebrate PER2 homologs.
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