抄録
The molecular clockwork that cause the fundamental feedback loops to stably oscillate with a ~24 hr-periodicity remain unclear. 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. Similar to the fibroblast, circadian regulated nucleocytoplasmic shuttling of BMAL1:CLOCK were also observed in the SCN. 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. [Jpn J Physiol 54 Suppl:S207 (2004)]
