title: Phosphorylation of the transcription activator CLOCK regulates progression through a ∼ 24-h feedback loop to influence the circadian period in Drosophila.
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.
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Guruswamy
authors.initials: G
authors.lastname: Mahesh
authors.affiliation: the Department of Brain Science, Ajou University School of Medicine, Suwon 443-380, Korea.
authors.forename: EunHee
authors.initials: E
authors.lastname: Jeong
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Fanny S
authors.initials: FS
authors.lastname: Ng
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Yixiao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Kushan
authors.initials: K
authors.lastname: Gunawardhana
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843.
authors.forename: Jerry H
authors.initials: JH
authors.lastname: Houl
authors.affiliation: the Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, Piscataway New Jersey 08854.
authors.forename: Evrim
authors.initials: E
authors.lastname: Yildirim
authors.affiliation: MS Bioworks, LLC, Ann Arbor, Michigan 48108, and.
authors.forename: Ravi
authors.initials: R
authors.lastname: Amunugama
authors.affiliation: MS Bioworks, LLC, Ann Arbor, Michigan 48108, and.
authors.forename: Richard
authors.initials: R
authors.lastname: Jones
authors.affiliation: MS Bioworks, LLC, Ann Arbor, Michigan 48108, and.
authors.forename: David L
authors.initials: DL
authors.lastname: Allen
authors.affiliation: the Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, Piscataway New Jersey 08854.
authors.forename: Isaac
authors.initials: I
authors.lastname: Edery
authors.affiliation: the Department of Brain Science, Ajou University School of Medicine, Suwon 443-380, Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.affiliation: From the Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, Texas 77843, phardin@bio.tamu.edu.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Hardin
mesh_terms: D056930:ARNTL Transcription Factors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D056926:CLOCK Proteins / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology
chemical_list: D056930:ARNTL Transcription Factors; C120526:Clk protein, Drosophila; D029721:Drosophila Proteins; C539040:PER protein, Drosophila; D056950:Period Circadian Proteins; C112805:cyc protein, Drosophila; C096445:tim protein, Drosophila; D056926:CLOCK Proteins
keywords: Behavioral Rhythms; CLOCK Protein; Circadian Clock; Drosophila Genetics; Feedback Loop; Mass Spectrometry (MS); Protein Phosphorylation; Transcription Regulation