Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts
Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts
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DOI：
10.1046/j.1365-2443.2003.00686.x
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发表时间：
2003-12-01
期刊：
影响因子：
2.1
通讯作者：
Takamatsu, K
中科院分区：
文献类型：
--
作者：
Tamaru, T;Isojima, Y;Takamatsu, K
背景：生物钟蛋白是哺乳动物生物钟机制的重要组成部分。然而，导致这些基本的反馈回路以类似于24 h周期性稳定振荡的分子时钟机制尚不清楚。通过免疫细胞化学和免疫印迹法评估这些细胞中BMAL 1蛋白的亚细胞定位和磷酸化的昼夜节律变化。观察到Bmal 1转录和BMAL 1的细胞质/细胞核积累之间存在显著的时间滞后。BMAL 1在细胞核中积累后，在BMAL 1转录本增加之前，BMAL 1主要通过核输出在细胞质中积累。BMAL 1的核积累与CLOCK的核积累和Per 1转录的峰值相匹配。细胞核BMAL 1逐渐磷酸化，然后以时间调节的方式去磷酸化，但细胞质BMAL 1则不然。在缺乏功能时钟的血清休克mCry 1/mCry 2（CRY）缺陷型成纤维细胞中，细胞质和细胞核BMAL 1仅以过度磷酸化形式存在，不存在昼夜节律的核质穿梭。我们认为BMAL 1的核质穿梭和磷酸化状态受生物钟的调节，并且BMAL 1的这种时间调节和时间延迟的核进入对于维持稳定振荡的时钟是重要的。
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 similar to24 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.