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. 2004 May;24(10):4184–4195. doi: 10.1128/MCB.24.10.4184-4195.2004

FIG. 7.

FIG. 7.

The F-X-X-X-F motif also serves as a CK1 docking site in the PER circadian-rhythm proteins. (A) Sequences of F-X-X-X-F motifs in NFAT and PER proteins thought to interact with CK1. (B) Mutation of the F-X-X-X-F motif in mPER2 abolishes binding to CK1ɛ. Mutations targeting the phenylalanines shown in panel A were introduced into GST fusion proteins containing mPER2 residues 583 to 765, which encompass most of the CK1 binding domain. Fusion proteins were incubated with HEK cell lysates and analyzed for the ability to interact with endogenous CK1 by Western blotting (WB) using an antibody against CK1ɛ. GST fusion proteins containing the wild-type CK1 binding domain of mPER2 (amino acids 583 to 765) or NFAT1 (amino acids 1 to 100) were able to associate with CK1ɛ, while GST alone or fusion proteins carrying mutations in the F-X-X-X-F motif were unable to interact with the kinase. (C) Processive phosphorylation by CK1. See Discussion for details.