title: Phosphorylation of the cryptochrome 1 C-terminal tail regulates circadian period length.
abstract: The Cryptochrome (CRY) proteins are critical components of the mammalian circadian clock and act to rhythmically repress the activity of the transcriptional activators CLOCK and BMAL1 at the heart of the clock mechanism. The CRY proteins are part of a large repressive complex, the components of which are not completely known. Using mass spectroscopy, we identified the catalytic subunit of DNA-dependent protein kinase as a CRY-interacting protein and found that loss or inhibition of this kinase results in circadian rhythms with abnormally long periods. We then identified serine 588 in the C-terminal tail of mouse CRY1 as a potential DNA-PK phosphorylation site but surprisingly found that the phosphomimetic mutation S588D also results in long period rhythms, similar to the loss of DNA-PK. Consistent with this, we found that phosphorylation of this site is increased in cells lacking DNA-PK, suggesting that DNA-PK negatively regulates the phosphorylation of this site most likely through indirect means. Furthermore, we found that phosphorylation of this site increases the stability of the CRY1 protein and prevents FBXL3-mediated degradation. The phosphorylation of this site is robustly rhythmic in mouse liver nuclei, peaking in the middle of the circadian day at a time when CRY1 levels are declining. Therefore, these data suggest a new role for the C-terminal tail of CRY1 in which phosphorylation rhythmically regulates CRY1 stability and contributes to the proper circadian period length.
authors.affiliation: From the Department of Neuroscience, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390.
authors.forename: Peng
authors.initials: P
authors.lastname: Gao
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Yoo
authors.forename: Kyung-Jong
authors.initials: KJ
authors.lastname: Lee
authors.forename: Clark
authors.initials: C
authors.lastname: Rosensweig
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Takahashi
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Chen
authors.forename: Carla B
authors.initials: CB
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D057906:Circadian Clocks / Q000235:genetics / Q000502:physiology*; D056931:Cryptochromes / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry
chemical_list: C109198:CIB1 protein, human; C538963:CRY1 protein, human; D002135:Calcium-Binding Proteins; C538964:Cry1 protein, mouse; C538967:Cry2 protein, mouse; D056931:Cryptochromes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012694:Serine; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse
keywords: Circadian Clock; Phosphorylation; Protein Kinases; Protein Stability; Transcription Repressor