Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
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DOI：
10.1042/bj20060827
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发表时间：
2007-03-15
影响因子：
4.1
通讯作者：
Cermakian, Nicolas
中科院分区：
文献类型：
--
作者：
Dardente, Hugues;Fortier, Erin E.;Cermakian, Nicolas
CLOCK和BMAL 1 [脑和肌肉ARNT（芳香烃受体核转运蛋白）样蛋白1]是哺乳动物分子钟的核心组分，属于bHLH（碱性螺旋-环-螺旋）/PAS [PER（Period）/ARNT/SIM（singleminded）]家族。它们的二聚化特征从未被研究过。在这里，我们证明了PAS结构域功能需要在短PAS核心重复序列延伸的区域。引人注目的是，虽然删除PAS核心重复不会明显影响二聚化，但它会消除异源二聚体的转录活性。有趣的是，这些缺失也消除了CLOCK和BMAL 1的共依赖性磷酸化，表明异二聚体的磷酸化状态与其反式激活潜力之间存在联系。我们证明，NPAS 2（神经元PAS结构域蛋白2）和BMAL 2也经历了类似的翻译后修饰，从而建立了作为生物钟中转录激活因子的共同特征的CLOCK-BMAL 1的机制。BMAL 2的两种新剪接变体的发现证实了PAS结构域的关键作用，并进一步加强了共依赖磷酸化具有功能意义的观点。与此一致，我们证明，CRY 1-2（隐花色素1-2）影响反式激活和磷酸化的时钟转录激活因子。此外，CRY蛋白稳定BMAL 1（BMAL 2）的非磷酸化形式，从而将磷酸化/非磷酸化比例向主要非磷酸化（转录失活）形式转移。相比之下，PER蛋白是弱阻遏物，没有作用。根据这些结果，我们提出了CRY 1-2抑制CLOCK（NPAS 2）-BMAL 1（BMAL 2）昼夜转录激活的一般机制。
CLOCK and BMAL 1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator) -like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (singleminded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish codependent phosphorylation of CLOCK and BMAL 1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY 1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1 (BMAL2) circadian transcriptional activation by CRY 1-2.