AMP-activated protein kinase regulates circadian rhythm by affecting CLOCK in Drosophila

Abstract

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 SNF4A␥, a homolog of mammalian AMP-activated protein kinase ␥ (AMPK␥), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPK␣ and two regulatory subunits, AMPK␤ and AMPK␥, 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␤ 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␤ 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. Hardin (Texas A&M) for generously providing anti-CLK and anti-VRI antibodies; Thomas Kusch (Rutgers University) for providing the pMT-HA/FLAG plasmid; and Joungkyeong Chung (Seoul National University) for providing the AMPK null mutants.Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPK␥ 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).

A Symphony of Signals: Intercellular and Intracellular Signaling Mechanisms Underlying Circadian Timekeeping in Mice and Flies

“…A recent study by Cho et al (2019) implicated AMPK in the regulation of circadian rhythms in Drosophila [402]. The AMPK holoenzyme consists of a catalytic α subunit and two regulatory β and γ subunits, each encoded by a single gene in the fly genome.…”

“…The AMPK holoenzyme consists of a catalytic α subunit and two regulatory β and γ subunits, each encoded by a single gene in the fly genome. Knocking down each subunit in tim -expressing cells altered locomotor activity rhythms [402]. AMPKα and AMPKβ knockdown delayed the phase of evening anticipatory activity under LD and lengthened the period under DD [402].…”

“…Knocking down each subunit in tim -expressing cells altered locomotor activity rhythms [402]. AMPKα and AMPKβ knockdown delayed the phase of evening anticipatory activity under LD and lengthened the period under DD [402]. AMPKγ knockdown advanced the evening peak under LD and resulted in arrhythmicity under DD [402].…”

“…AMPKα and AMPKβ knockdown delayed the phase of evening anticipatory activity under LD and lengthened the period under DD [402]. AMPKγ knockdown advanced the evening peak under LD and resulted in arrhythmicity under DD [402]. The arrhythmic phenotype of AMPKγ mutant flies was attributed to a severe defect in the morphological integrity of the s-LN v s, which appeared to be missing the dorsally projecting neurites [402].…”

“…AMPKγ knockdown advanced the evening peak under LD and resulted in arrhythmicity under DD [402]. The arrhythmic phenotype of AMPKγ mutant flies was attributed to a severe defect in the morphological integrity of the s-LN v s, which appeared to be missing the dorsally projecting neurites [402]. Adult-restricted silencing of AMPKγ reduced rhythmicity and shortened the period without affecting s-LN v integrity, indicating that AMPKγ has a role in the adult clock in addition to a developmental function [402].…”