Functional analysis of Casein Kinase 1 in a minimal circadian system.

Gerben Van Ooijen,John S O'Neill,Andrew J Millar,François-Yves Bouget,Martin Barrios-Llerena,Frédéric Sanchez,Sarah F Martin,Thierry Le Bihan,Matthew Hindle

doi:10.1371/journal.pone.0070021

Abstract

The Earth’s rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1) is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcus tauri , a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.

Highlights

The circadian clock is a timekeeping mechanism that evolved to adapt to, and anticipate the predictable daily changes associated with the Earth’s rotation
None of Casein Kinase 1 (CK1)’s identified animal or fungal TTFL protein targets PER, FRQ, Clock, and WC2 are conserved in O. tauri, nor in higher plants evolved from this branch
The position of O. tauri CK1 in a phylogenetic tree (Figure 1) indicates that this homolog is closer to the animal and fungal isoforms with known clock function than other plant CK1 proteins are, in line with diversification along the green lineage

Summary

Introduction

The circadian clock is a timekeeping mechanism that evolved to adapt to, and anticipate the predictable daily changes associated with the Earth’s rotation. Genes have been identified whose products are rhythmically expressed and subsequently drive rhythms in the activity of other genes, that can directly or indirectly feed back to form loops that oscillate in a ~24 hour rhythm [1,2] These so-called transcriptional-translational feedback loops, or TTFLs, are identified in most model organisms whose clock has been studied in any detail, and were long believed to be the sole drivers of cellular circadian rhythmicity. In cyanobacteria, a circadian oscillator was identified that did not rely upon rhythmic transcription, and is based upon cycles of phosphorylation and de-phosphorylation of the protein kinase KaiC, regulated by two other proteins, KaiA and KaiB [3,4,5] These three protein continue to oscillate outside their cellular context, since cycles of KaiC phosphorylation can be reconstituted in vitro using only the recombinant Kai proteins with ATP [6]

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