Abstract
Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin–Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli.
Highlights
Cell endogenous circadian clocks control daily oscillations in behavior, physiology, metabolism, and gene expression
The central feedback loop consists of the transcription factors Clock (CLK) and Cycle (CYC), which bind to promotor sequences of timeless and period
Ribosomal S6 Kinase (RSK), like the Drosophila GSK3β homolog SGG, is required for axonal transport, synaptic organization, and circadian behavior (Akten et al, 2009; Beck et al, 2015) indicating a direct interaction of both kinases. We confirmed this by transient expression of Myc-tagged RSK (Myc::RSK) in Schneider S2 cells and co-immunoprecipitation of Myc::RSK and endogenous SGG (Figure 1A)
Summary
Cell endogenous circadian clocks control daily oscillations in behavior, physiology, metabolism, and gene expression. In Drosophila melanogaster, the key cellular system for circadian timekeeping of sleep-wake cycles is a group of 150 clock neurons residing in the lateral and dorsal brain, which can be further subdivided into several functional distinct groups (Hermann-Luibl and Helfrich-Förster, 2015; Dubowy and Sehgal, 2017). All clock neurons harbor a molecular network comprised of several interconnected transcriptional-translational feedback loops where transcription factors induce expression of clock genes, which encode for proteins that act as regulators of their own expression. The central feedback loop consists of the transcription factors Clock (CLK) and Cycle (CYC), which bind to promotor sequences of timeless (tim) and period (per). Protein kinases Nemo, Doubletime [DBT, corresponding to vertebrate Casein Kinase (CK1)], Casein Kinase (CK2), Shaggy (SGG, the Drosophila ortholog of vertebrate GSK3β) and, at least in vitro, MAP kinases p38 (Dusik et al, 2014) and ERK (Ko et al, 2010) phosphorylate PER, whereas TIM is the target of CK2 and SGG (Top et al, 2016)
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