Extended Data Fig. 2: CLOCK S106 phosphorylation is required for its nuclear exportation.
(e-i) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. All experiments were repeated three times independently with similar results. (a) Alignment of protein sequences spanning CLOCK S106 and the adjacent noncanonical NES from different species. (b, d) MD simulation of CLOCK bound with BMAL1 WT (b) or BMAL1 4Mut (D144A/D231A/D299A/E303A) (d). Evolution of the backbone root-mean-square-deviation (RMSD) of the CLOCK-BMAL1 complex from the initial frame in 250âns MD simulations. RMSD of the CLOCK-BMAL1 complex with and without CLOCK S106 phosphorylation are shown in red and black, respectively. (c) The conformation of the CLOCK-BMAL1 complex of the last frame extracted from 250âns MD simulation. CLOCK and BMAL1 structures are shown in green and blue, respectively. The red arrow indicates CLOCK S106. D144, D232, D299, and E303 of BMAL1, which are in a close proximity with CLOCK S106, are shown. (e) Bacterially purified WT GST-CLOCK or GST-CLOCK S106A was incubated with or without His-CK2Îħ in the presence of ATP for an in vitro kinase assay, followed by incubation with the indicated His-BMAL1 proteins. (f) Huh7 cells stably transfected with the indicated plasmids were treated with or without IGF1 for 1âh. Total cell lysates and cytosolic fractions were prepared. (g) Huh7 cells were pretreated with or without TBB for 30âmin before treatment with or without IGF1 for 1âh. (h) The indicated cells expressing CLOCK shRNA with reconstituted expression of the indicated CLOCK proteins were harvested. (i) Hep3B cells expressing CLOCK shRNA with reconstituted expression of Flag-rCLOCK proteins were treated with or without IGF1 for 1âh.
