Figure 3. Phosphorylation of the human PER2 FASP region inhibits CK1 activity.

a-d, NMR kinase assay for the WT FASP or indicated mutant peptides monitoring the reaction kinetics of priming phosphorylation at S662 by NMR. e, Schematic of FASP alanine mutations and resulting discrete phosphostates. f, Plot of priming rate constant (k_prime) as a function of the possible successive phosphosites in the FASP. Error bars represent SEM from fits in panels a-d. g, ADP-Glo kinase assay with titration of FASP mutant peptides with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% C.I. of the fit. h, Data from panel f normalized by V_max values calculated from the preferred kinetic model (see Supplementary Figure 2, Table 1). i, ADP-Glo kinase assay of hPER2 PAS-degron peptide (see Supplementary Figure 2b) with titration of pFASP peptides corresponding to 2 (2p) or 3 (3p) phosphoserines (see Supplementary Figure 3a) with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% C.I. of the fit. j-k, Western blot and quantification of the phosphorylation of the FASP priming site (pS659 in mouse PER2). Full-length mouse PER2 was immunoprecipitated from transfected HEK293T cells, dephosphorylated, and subjected to an in vitro kinase assay with 200 ng CK1 in the presence and absence of unphosphorylated mouse PER2 FASP or human PER2 4pFASP peptides as indicated.