Regulation of BMAL1 protein stability and circadian function by GSK3beta-mediated phosphorylation

Abstract

Background: Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3beta signaling pathway regulates BMAL1 protein stability and activity.

Principal findings: GSK3beta phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3beta-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3beta pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.

Conclusions: These findings uncover a previously unknown mechanism of circadian clock control. The GSK3beta kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock.

Figure 1. GSK3β phosphorylates BMAL1 and primes it for ubiquitylation.

(A) In vitro Kinase assay. Bacterially purified GST and GST-BMAL1 were incubated with recombinant GSK3β in presence of γ³²P-ATP. Top panel shows the autoradiogram; bottom panel shows the coomassie blue staining for the same gel. (B) HEK 293 cells were transfected with plasmids expressing Flag-myc-BMAL1, myc-CLOCK with or without HA-GSK3β. Total lysates were prepared and subjected to immunoprecipitation using anti-BMAL1 antibody. Immunoprecipitated proteins were detected by Western blotting (WB) using indicated antibodies. (C) Ubiquitylation assay. HEK 293 cells were transfected with plasmids expressing HIS-Ubiquitin, Flag-myc-BMAL1, myc-CLOCK, with or without the constitutively active (CA) or kinase dead (KD) mutants of HA- tagged GSK3β. Ubiquitylated BMAL1 was detected by Western analysis using anti-Flag antibody. Input samples were probed with anti-Flag (to detect BMAL1) and anti-GSK3β antibodies.

Figure 2. GSK3β regulates BMAL1 stability.

(A) HEK 293 cells were co-transfected with plasmids expressing Flag-myc-BMAL1, myc-CLOCK with or without CA-Akt. 48 hours post-transfection nuclear lysates were prepared and resolved by SDS-PAGE. CLOCK and BMAL1 levels were detected by Western analysis using anti-Myc antibody; phospho- and total GSK3β and HSC70 (loading control) were detected by using specific antibodies. (B) WT and GSK3β^−/− MEFs were transfected with plasmids expressing Flag-myc-BMAL1, myc-CLOCK and myc-GFP. Nuclear lysates were resolved by SDS-PAGE followed by Western analysis. BMAL1 and GFP levels were detected by anti-Myc antibody. Total GSK3β was detected using a specific antibody. (C) Same as in (B) except that indicated lanes were also transfected with a plasmid expressing HA-tagged GSK3β. (D) WT and GSK3β^−/− MEFs were treated with 50 µg/ml Cycloheximide (CHX), total lysates were prepared at indicated times and resolved by SDS-PAGE. Endogenous BMAL1 and GAPDH levels were detected by Western analysis using specific antibodies. Data shown is representative of three independent experiments.

Figure 3. GSK3β phosphorylates Ser 17 and Thr 21 on BMAL1.

(A) Amino acid sequence around two putative GSK3β consensus sites on mBMAL1. (B) Sequence alignment of the two putative GSK3β consensus sites on BMAL1 showing conservation across species. (C) HEK 293 were transfected with plasmids expressing myc-CLOCK and WT or mutant Flag-myc-BMAL1. 48 hours post-transfection total lysates were prepared and resolved by SDS-PAGE. BMAL1 and CLOCK levels were detected by anti-Myc antibody. Slower migrating phosphorylated BMAL1 band is indicated by an arrow. (D) Bar graph representing phosphorylation levels of WT and S17,T21/A BMAL1 as analyzed by In vitro kinase assay (as in Fig. 1A). Data is representative of three independent experiments. (E) WT or S17,T21/A mutant of Flag-myc-BMAL1, along with myc-GFP were transiently expressed in JEG3 cells and treated with 50 µg/ml CHX. Total lysates were prepared at indicated times and resolved by SDS-PAGE. BMAL1 and GFP levels were detected by Western analysis using anti-myc antibody. Data shown is representative of three independent experiments. (F) Line graph showing quantitation of BMAL1 levels, normalized to GFP levels, from Fig. 3E.

Figure 4. Circadian defects in GSK3β^−/− MEFs.

WT and GSK3β^−/− MEFs were synchronized by 2 hour treatment with 100nM dexamethasone (DEX). (A) Total lysates were prepared at indicated times post synchronization and resolved by SDS-PAGE followed by Western analysis. BMAL1 and α-tubulin levels were detected by specific antibodies. (B) RNA was prepared at indicated times, reverse transcribed, and real-time PCR was performed using primers for Bmal1 and 18S rRNA. Data is represented as relative levels of Bmal1 normalized to 18S rRNA. (C) Same as in (A), except phospho-GSK3α/β and total GSK3β levels were detected by specific antibodies. (D) RNA was prepared at indicated times, reverse transcribed, and real-time PCR was performed using primers for Dbp and 18S rRNA. Data is represented as relative levels of Dbp normalized to 18S rRNA.

Figure 5. BMAL1 mRNA and protein levels in WT and D2R^−/− mice.

Mice entrained in 12 hr Light – 12 hr Dark cycles were sacrificed at indicated times and their striatum was dissected out. (A) RNA was prepared at indicated times, reverse transcribed, and real-time PCR was performed using primers for Bmal1 and 18S rRNA. Data is represented as relative levels of Bmal1 normalized to 18S rRNA. (B) Total lysates were prepared and resolved by SDS-PAGE. BMAL1 and α-tubulin levels were detected by Western analysis using specific antibodies. (C,D) Same as in (A), except real-time PCR was performed using primers for (C) Dbp and (D) Per2.