Fig. 3. 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 CK1δ 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 CK1δ 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.