Drosophila RSK Influences the Pace of the Circadian Clock by Negative Regulation of Protein Kinase Shaggy Activity

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.

Integrated omics in Drosophila uncover a circadian kinome

“…Under DD conditions, SLMB and CUL-3 exert effects on the clock by impinging on PER and TIM protein stability, whereas SGG times the clock by regulating PER/TIM nuclear translocation 25,41,42 . S6KII is believed to regulate the clock by targeting SGG and possibly PER, whereas NEJ binds with CLK/CYC to modulate transcription of clock genes 40,43 . Therefore, we first tested whether knocking down gskt affects PER and TIM protein level in fly heads.…”

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

“…The expression of S6KII mRNA in the whole fly head is rhythmic, peaking in the late night to early morning [396]. A null mutation of S6KII did not alter light-induced phase delays or advances but shortened the behavioral period by ~1 h [396,397]. Constitutively overexpressing S6KII in tim - or Pdf -positive cells rescued the period phenotype of S6KII-null flies, suggesting that the expression, but not the oscillation, of S6KII is important for the circadian function of this kinase [396].…”

“…Constitutively overexpressing S6KII in tim - or Pdf -positive cells rescued the period phenotype of S6KII-null flies, suggesting that the expression, but not the oscillation, of S6KII is important for the circadian function of this kinase [396]. Transcript and protein levels of per were decreased and increased, respectively, in whole head extracts of S6KII-null flies, suggesting that there may be enhanced feedback repression in the absence of S6KII [396,397]. Akten et al (2009) provided evidence to suggest that S6KII may be indirectly influencing the phosphorylation and stability of PER via CK2 [396].…”

“…These findings, along with the observation that S6KII physically associates with CK2, imply that S6KII is acting as an upstream inhibitor of CK2, affecting its ability to phosphorylate PER [396]. On the other hand, data from Beck et al (2018) suggest an alternate mechanism involving SGG [397]. The C-terminal kinase domain (CTKD) of S6KII was shown to phosphorylate SGG at the inhibitory residue, Ser9 [397].…”

Animal Models for Coffin-Lowry Syndrome: RSK2 and Nervous System Dysfunction

“…D-RSK mutants also show changes in circadian activity rhythms; instead of maintaining a 24-h rhythm in constant darkness, they display a 23-h periodicity (Akten et al, 2009 ). This function of D-RSK was mapped to master clock neurons in the brain and involves phosphorylation of the GSK3β homolog Shaggy as a key regulator of the Drosophila molecular clock (Beck et al, 2018 ).…”