Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Sassone-Corsi P (2010) Regulation of BMAL1 Protein Stability and Circadian Function by GSK3b-Mediated Phosphorylation. PLoS ONE 5(1): e8561. doi:10.1371/journal.pone.0008561 Regulation of BMAL1 Protein Stability and Circadian Function by GSK3b-Mediated Phosphorylation Saurabh Sahar 0 Loredana Zocchi 0 Chisato Kinoshita 0 Emiliana Borrelli 0 Paolo Sassone-Corsi 0 Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America 0 1 Department of Pharmacology, University of California Irvine, Irvine, California, United States of America, 2 Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, United States of America, 3 Unite 904 INSERM 'Epigenetics and Neuronal Plasticity', University of California Irvine , Irvine, California , United States of America 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-GSK3b signaling pathway regulates BMAL1 protein stability and activity. Principal Findings: GSK3b phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3b-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3b 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 GSK3b 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. - Funding: S.S. was partially supported by a post-doctoral fellowship from American Heart Association, Western States Affiliates. L.Z. was supported by an American-Italian Cancer Foundation post-doctoral fellowship. Work in the laboratories is supported by the National Institute of Health (R01-DA024689; R01GM081634-01) and the Institut National de la Sante et de la Recherche Medicale (U904) (France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Circadian (from the Latin circa diem meaning about a day) rhythms occur with a periodicity of about 24 hours and enable organisms to adapt and anticipate environmental changes. Circadian control provides an evolutionary advantage to organisms in adapting their behavior and physiology to the appropriate time of day [1,2]. Feeding behavior, sleep-wake cycles, hormonal levels and body temperature are just a few examples of physiological circadian rhythms. From a molecular standpoint, circadian rhythms are regulated by transcriptional and post-translational feedback loops generated by a set of interplaying clock proteins. The positive limb of the mammalian clock machinery is comprised of CLOCK and BMAL1, which are transcription factors that heterodimerize through the PAS domain and induce the expression of clock-controlled genes by binding to their promoters at E-boxes. Cryptochromes (Cry 1, Cry2) and Period genes (Per1, Per2, Per3) are clock-controlled genes that encode proteins that form the negative limb of the circadian machinery. PER and CRY proteins are classically thought to translocate into the nucleus to inhibit CLOCK:BMAL1 mediated transcription, thereby closing the negative feedback loop [1]. Various core clock proteins undergo post-translational modifications (PTMs), a feature that is likely to contribute significantly to the plasticity of the circadian system. PTMs have been shown to regulate distinct functions, including transcriptional activation and intracellular localization. PTMs have been proposed to participate in controlling the timing between the activation and the repression of circadian transcription [3]. Among the clock proteins, BMAL1 undergoes an extensive repertoire of PTMs, including phosphorylation [4,5,6], acetylation [7], sumoylation [8,9] and ubiquitylation [10]. Yet, the signaling pathways controlling the stability of the BMAL1 protein have not been deciphered. In the present study we have identified GSK3b as a critical regulator of BMAL1 stability and activity. GSK3b is a ubiquitous kinase which regulates various cellular functions, ranging from glucose homeostasis, to cell survival and cell-fate specification [11]. GSK3b has been linked to various pathological conditions such as diabetes, Alzheimers, cancer and bipolar disorder [12,13,14,15]. The role of GSK3b in circadian control has been reported. The first evidence was based on the observation that shaggy (sgg), the Drosophila ortholog of GSK3, controls the period of circadian locomotor activity by phosphorylating TIMELESS and regulating nuclear translocation of the PERIOD/TIMELESS heterodimer [16,17]. Recently a high-throughput approach demonstrated that inhibition of GSK3b leads to shortening of the period in cultured mammalian cells [18]. In mammals, GSK3b has been reported to phosphorylate PER2, CRY2 and Rev-erba. It has also been reported that the kinase activity of GSK3b oscillates in the central mammalian clock, suprachiasmatic nucleus (SCN) and in the peripheral clocks (liver and fibroblasts) [19]. GSK3b mediated phosphorylation appears to have differential effect on the stability of the targeted substrates, since it has been shown to induce the degradation of CRY2 [20] and the stabilization of Rev-erba [21]. Here we show that GSK3b specifically phosphorylates BMAL1 and primes it for ubiquitylation, followed by proteasomal degradation. This control mechanism significantly influences the efficacy and amplitude of circadian gene expression. GSK3b Phosphorylates BMAL1 and Primes It for Ubiquitylation BMAL1 is a phosphoprotein targeted by various kinases [4,5,6]. We noted that BMAL1 contains 15 sites with the consensus T/ SXXXS/T phosphoacceptor sequence for GSK3b. This prompted us to determine whether GSK3b can phosphorylate BMAL1. We performed in vitro kinase assays on bacterially purified GSTBMAL1 or GST alone by incubating them with recombinant GSK3b in presence of c32P-ATP. Our results demonstrate that GST-BMAL1 was readily phosphorylated by GSK3b, whereas GST alone, although expressed at much greater levels, was not phosphorylated under equivalent conditions (Fig. 1A). Thus, BMAL1 appears to be an efficient substrate of GSK3b. Furthermore, to detect physical association between BMAL1 and GSK3b, HEK 293 cells were transiently transfected wit (...truncated)