The right place at the right time: regulation of daily timing by phosphorylation

Circadian clocks regulate processes from gene expression to behavior, and have been observed in all phyla. The clock mechanism is based on a network of genes (clock genes) that function as a transcriptional–translational negative feedback loop, with at least one protein feeding back to inhibit the transcriptional activator of its own gene. The circadian period derives from negative feedback formed by a collection of incremental processes, incorporating delays that include subcellular localization, protein production, and degradation, which all can be controlled by post-translational modification via phosphorylation. Clock proteins in all genetic model systems show striking and temporally distinct phosphorylation patterns. Very often the phosphorylation is a signal for a subsequent degradation of the target protein, followed by production of new (unphosphorylated) species. Genetic (both forward and reverse) and pharmacological experiments confirm the fundamental nature of phos- phorylation in circadian timing Dynamic regulation of reversible phosphorylation plays a fundamental role in most signal transduction pathways connected with essential cellular processes, ranging from cell cycle to differentiation to apoptosis. The phosphorylation state and even the activity of many proteins is fine-tuned by a balance between kinases and phosphatases. By mapping out the phosphorylome, sets of formulaic interactions between kinases and substrates become apparent, based on known biological activities. We inspected the most common regulatory modules and determined that the molecular clock network resembles a “kinase–substrate pair feedback loop II module”, suggesting that clock components could also form a metabolic, non transcriptional feedback loop, a phoscillator in mammals. We conclude by proposing an alternative hypothesis, namely that an extended “kinase–substrate pair feedback loop II module” is also part of the core clock mechanism generating the ∼24-h oscillation at the molecular level. This would be an ancestral clock mechanism, with the specialized transcription/translation regulatory components evolving uniquely for animals, plants, and fungi after the branches in the tree of life were established.