Abstract
Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.
Highlights
CLOCK phosphorylation coincides with circadian rhythms in transcription
Because CLK is hyperphosphorylated in cultured Schneider 2 (S2) cells when co-expressed with DBT [19], S2 cells were used to produce sufficient quantities of hyperphosphorylated CLK for mass spectrometry
S2 cells that express only tetra-CLK drive ϳ3-fold higher levels of per promoter-driven luciferase activity than cells that co-express tetra-CLK, PER, and DBT (Fig. 1c), demonstrating that epitope-tagged CLK transcriptional activity is subject to the same regulation as native CLK
Summary
Results: CLOCK phosphorylation sites are identified that regulate the timing and level of transcriptional activity and influence circadian period. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. We found that five of the eight Ser to Ala mutants rescued behavioral rhythms having short periods, but did not alter CLK levels or phosphorylation state in fly heads One of these mutants, S859A increased CLK activity, thereby accelerating transcriptional feedback, whereas the other mutants appear to accelerate or bypass a step that delays transcriptional activation or repression. These results indicate that phosphorylation decreases CLK activity or imposes a delay to lengthen circadian period, thereby serving as a determinant for setting the pace of the circadian oscillator
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