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 affmity purification combined with mass spectrometry using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4A gamma, a homolog of mammalian AMP-activated protein kinase gamma (AMPK gamma), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPK alpha and two regulatory subunits, AMPK beta and AMPK gamma, 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 AMPK beta 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 AMPK beta 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.