In mammalian circadian clockwork, the CLOCK-BMAL1 heterodimer activates E-box-dependent transcription, while its activity is suppressed by circadian binding with negative regulators, such as CRYs. Here, we found that the CLOCK protein is kept mostly in the phosphorylated form throughout the day and is partly hyperphosphorylated in the suppression phase of E-box-dependent transcription in the mouse liver and NIH 3T3 cells. Coexpression of CRY2 in NIH 3T3 cells inhibited the phosphorylation of CLOCK, whereas CIPC coexpression markedly stimulated phosphorylation, indicating that CLOCK phosphorylation is regulated by a combination of the negative regulators in the suppression phase. CLOCK-BMAL1 purified from the mouse liver was subjected to tandem mass spectrometry analysis, which identified Ser38, Ser42, and Ser427 as in vivo phosphorylation sites of CLOCK. Ser38Asp and Ser42Asp mutations of CLOCK additively and markedly weakened the transactivation activity of CLOCK-BMAL1, with downregulation of the nuclear amount of CLOCK and the DNA-binding activity. On the other hand, CLOCK⌬19, lacking the CIPC-binding domain, was far less phosphorylated and much more stabilized than wild-type CLOCK in vivo. Calyculin A treatment of cultured NIH 3T3 cells promoted CLOCK phosphorylation and facilitated its proteasomal degradation. Together, these results show that CLOCK phosphorylation contributes to the suppression of CLOCK-BMAL1-mediated transactivation through dual regulation: inhibition of CLOCK activity and promotion of its degradation.Many physiological activities of living organisms show rhythmic changes, with a period of approximately 24 h, even under constant conditions without any external time cues. These daily variations, called circadian rhythms, are generated by the circadian clock, a cell-autonomous time-measuring system that has evolved in a wide variety of organisms, from cyanobacteria to higher plants and humans (9). In mammals, a master clock is located in the hypothalamic suprachiasmatic nucleus, while self-sustaining molecular clocks reside even in the peripheral tissues, such as the liver (18,40,45). In these central and peripheral clock cells, the clock genes form a transcription/ translation-based negative-feedback loop to generate the molecular oscillation with 24-h periodicity. CLOCK and BMAL1 are basic helix-loop-helix (bHLH)-PAS transcription factors, and the CLOCK-BMAL1 heterodimer binds to CACGTG E-box (14) or E-box-like (25, 61) sequences for the transactivation of negative regulatory genes, such as Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes. Newly translated PERs and CRYs enter the nuclei and bind to the CLOCK-BMAL1 heterodimer, leading to suppression of its transactivation (30).In the mouse liver, the abundances and the phosphorylation states of both PER1 and PER2 show striking temporal changes (32). The total amount of each PER protein shows the lowest level at CT6 (CT is circadian time, with CT0 under the constant-dark [DD] condition corresponding to the lights-on time in the...
In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoterdriven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1. Transcription assays with a 2.1-kb mPer1 promoter revealed that CRY2 inhibited the transactivation mediated by BMAL1-CLOCK more strongly than that by BMAL2-CLOCK. In contrast, PER2 showed a stronger inhibitory effect on BMAL2-CLOCK than on BMAL1-CLOCK. The molecular link between BMAL2 and PER2 was further strengthened by the fact that PER2 exhibited a greater affinity for BMAL2 than for BMAL1 in co-immunoprecipitation experiments. These results indicate a functional partnership between BMAL2 and PER2 and reemphasize the negative role of PER2 in the circadian transcription. As a broad spectrum function, BMAL2-CLOCK activated transcription from a variety of SV40-driven reporters harboring various E/E-box-containing sequences present in the upstream regions of clock and clock-controlled genes. Importantly, the efficiencies of BMAL2-CLOCK-mediated transactivation relative to that achieved by BMAL1-CLOCK were dependent heavily on the E-box-containing sequences, supporting distinguishable roles of the two BMALs. Collectively, it is strongly suggested that BMAL2 plays an active role in the circadian transcription.A variety of organisms from bacteria to humans show circadian rhythms in physiology and behavior under the regulation of endogenous circadian clocks oscillating with an ϳ24-h periodicity (1, 2). In mammals, the central clock is located in the hypothalamic suprachiasmatic nucleus, whereas peripheral clocks with self-sustainable oscillation machinery are located in many peripheral tissues (3). Even cultured fibroblasts were shown to retain the cellular clocks (4), and hence they have been used for studies on the oscillatory mechanism of peripheral clocks. The molecular clockwork in mammals centers on transcription/translation-based autoregulatory feedback loops of clock genes, to which bHLH 3 -PAS proteins, BMAL1 and CLOCK, contribute as positive regulators of the transcription (2, 5). BMAL1-CLOCK complex activates transcription through CACGTG-type E-box and its related sequences found in promoter regions of clock genes and clock-controlled genes such as Per1 (6), Per2 (CACGTT E-box-like or EЈ-box sequence; see Ref. 7), plasminogen activator inhibitor-1 (PAI-1) (8), and Rev-Erb␣/ (9, 10). The E-box-dependent transactivation mediated by the BMAL1-CLOCK complex is suppressed by an expanding number of negative regulators, including PER1 (11, 12), PER2 (13-15), PER3 (16,1...
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