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

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.

Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons.

Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.

Light-dependent phosphorylation of the drosophila transient receptor potential ion channel.

The Drosophila phototransduction cascade terminates in the opening of an ion channel, designated transient receptor potential (TRP). TRP has been shown to become phosphorylated in vitro, suggesting regulation of the ion channel through posttranslational modification. However, except for one phosphorylation site, Ser(982), which was analyzed by functional in vivo studies (Popescu, D. C., Ham, A. J., and Shieh, B. H. (2006) J. Neurosci. 26, 8570-8577), nothing is known about the role of TRP phosphorylation in vivo. Here, we report the identification of 21 TRP phosphorylation sites by a mass spectrometry approach. 20 phosphorylation sites are located in the C-terminal portion of the channel, and one site is located near the N terminus. All 21 phosphorylation sites were also identified in the inaC(P209) mutant, indicating that phosphorylation of TRP at these sites occurred independently from the eye-enriched protein kinase C. Relative quantification of phosphopeptides revealed that at least seven phosphorylation sites were predominantly phosphorylated in the light, whereas one site, Ser(936), was predominantly phosphorylated in the dark. We show that TRP phosphorylated at Ser(936) was located in the rhabomere. Light-dependent changes in the phosphorylation state of this site occurred within minutes. The dephosphorylation of TRP at Ser(936) required activation of the phototransduction cascade.