The network of molecules that makes up the circadian clock, which governs the timing of cellular processes in most living things, has been well studied. But thus far, technological limitations have prevented researchers from elucidating exactly how the clocks orchestrate these processes beyond the level of gene transcription. In a recent study in Cell Metabolism, biologists report progress toward this end. Working in mice and using advanced mass spectrometry, they found that the clock exerts greater control via protein phosphorylation, which modulates protein activity, than by altering either gene expression or protein abundance.

The circadian clock’s molecular mechanism drives predictable oscillations in cellular processes in accordance with an approximately 24-hour schedule, even in the absence of environmental stimuli such as light or temperature. And it affects behavioral, physiological, and metabolic oscillations such as sleep cycles, hormone production, body temperature, appetite, and brain wave activity.

While previous studies have indicated that protein phosphorylation plays a role in controlling the circadian clock’s molecular mechanism, scientists didn’t know how big. Seeking answers, Maria Robles, a chronobiologist at the Max Planck Institute of Biochemistry in Planegg Germany, and colleagues used high-resolution mass spectrometry to detect fluctuations in the amounts of phosphorylated proteins in mouse liver cells over a 24-hour period.

To their surprise, 40% of phosphorylated proteins in the mouse liver displayed distinct circadian oscillations. In contrast, only about 10-20% of genes in mouse liver tissue display such patterns, suggesting that circadian clocks regulate physiology primarily by controlling protein phosphorylation rather than by oscillating levels of gene expression. “This is how tissues face temporal changes – by means of phosphorylation,” says Robles.

The researchers’ estimation of phosphorylated protein abundance in the mouse liver tissue, known as its phosphoproteome, marks the first evidence of circadian oscillations found in any tissue’s phosphoproteome.

Further cementing protein phosphorylation’s prominent role, the researchers found that the average circadian fluctuation of phosphorylated proteins in the mouse liver tissue was four-fold higher than fluctuations in gene expression and five-fold higher than fluctuations in protein abundance.

Robles attributes phosphorylation’s outsized effects to its greater speed and efficiency. “It’s much easier to create this big amplitude change on the phosphorylation level than on the protein level,” she says. Protein phosphorylation can occur in seconds whereas changes in gene expression occur on a scale of minutes to hours. And only one ATP is required to phosphorylate a protein while approximately 1,200 ATPs are required to build a protein.

The phosphoproteome also revealed new phosphorylation sites on core genes comprising the circadian clock, and identified oscillating activation of sets of kinases that mediate protein phosphorylation. Both are additional control mechanisms for circadian clocks.

The group’s discovery of a circadian-controlled phosphoproteome is a major milestone and the study provides the most comprehensive picture to date of how extensively the circadian clock regulates protein activity, says Joseph Takahashi, a Howard Hughes Medical Institute Investigator at the University of Texas Southwestern Medical Center who was not involved with the study. But more work remains, he notes. The researchers discovered phosphorylation sites on 4,461 liver tissue proteins, but more than 10,000 proteins typically exist in any given cell, each likely to have phosphorylation sites yet to be discovered, Takahashi notes.

Robles already has plans to broaden the scope of her work. “We want to do a comparative phosphoproteome across different tissues,” she says. “By looking at temporal phosphorylation changes, we might understand more how the circadian clock controls [their] physiology. This is just a very tiny tip of the iceberg.”