AMP-Activated Protein Kinase Regulates Circadian Rhythm by Affecting CLOCK in Drosophila.

The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In the Drosophila TTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. We aimed to identify factors that regulate CLK by performing tandem affinity purification combined with mass spectrometry using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4Aγ, a homolog of mammalian AMP-activated protein kinase γ (AMPKγ), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKα and two regulatory subunits, AMPKß and AMPKγ, directly phosphorylated purified CLK in vitro Locomotor behavior analysis in Drosophila revealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKß reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes, such as period and vrille Finally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKß knockdown. Thus, we conclude that AMPK, a central regulator of cellular energy metabolism, regulates the Drosophila circadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription.SIGNIFICANCE STATEMENT Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPKγ subunit of AMPK, a central regulator of cellular metabolism, copurifies with the CLK/CYC complex in Drosophila S2 cells. Furthermore, the AMPK holoenzyme directly phosphorylates CLK in vitro This study demonstrates that AMPK activity regulates the core clock in Drosophila by activating CLK, which enhances circadian transcription. In mammals, AMPK affects the core clock by downregulating circadian repressor proteins. It is intriguing to note that AMPK activity is required for core clock regulation through circadian transcription enhancement, whereas the target of AMPK action is different in Drosophila and mammals (positive vs negative element, respectively).

Pacemaker-neuron-dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation.

Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657-707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657-707 deletion in the Clock (Clk(out)) genetic background (p{dClk-Δ};Clk(out)), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657-707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clk(out) flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clk(out) flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clk(out) flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions.