title: Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts.
abstract: BACKGROUND
  Recent discoveries of clock proteins have unveiled an important part of the mammalian circadian clock mechanism. However, the molecular clockwork that cause these fundamental feedback loops to stably oscillate with a approximately 24 h-periodicity remain unclear.
  
  
  RESULTS
  Serum-shocked fibroblasts were used as a cellular clock model. Circadian changes in the subcellular localization and phosphorylation of BMAL1 protein in these cells were assessed by immunocytochemistry and immunoblotting. A significant time lag between Bmal1 transcription and the cytoplasmic/nuclear accumulation of BMAL1 was observed. After its nuclear accumulation, BMAL1 accumulated in the cytoplasm again, mainly by nucleoexport, before the increase of Bmal1 transcripts. Nuclear accumulation of BMAL1 matched nuclear accumulation of CLOCK and the peak of Per1 transcription. Nuclear BMAL1 was gradually phosphorylated and then dephosphorylated in a temporally regulated manner, although cytoplasmic BMAL1 was not. In serum-shocked mCry1/mCry2 (CRY)-deficient fibroblasts, which lack a functional clock, both the cytoplasmic and nuclear BMAL1 were only present as hyperphosphorylated forms and their circadian nucleocytoplasmic shuttling was absent.
  
  
  CONCLUSIONS
  We propose that the nucleocytoplasmic shuttling and phosphorylation states of BMAL1 are regulated by circadian clock, and that this temporally regulated and time-delayed nuclear entry of BMAL1 is important in the maintenance of a stably oscillating clock.
authors.affiliation: Department of Physiology, Toho University School of Medicine, 5-21-16 Ohmori-nishi Ohta-ku, Tokyo 143-8540, Japan. tetamaru@med.toho-u.ac.jp
authors.forename: Teruya
authors.initials: T
authors.lastname: Tamaru
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Isojima
authors.forename: Gijsbertus T J
authors.initials: GT
authors.lastname: van der Horst
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Takei
authors.forename: Katsuya
authors.initials: K
authors.lastname: Nagai
authors.forename: Ken
authors.initials: K
authors.lastname: Takamatsu
mesh_terms: D056930:ARNTL Transcription Factors; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002940:Circadian Rhythm; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D005231:Fatty Acids, Unsaturated; D012333:RNA, Messenger; D014157:Transcription Factors; C538961:Bmal1 protein, mouse; C038753:leptomycin B