Balance between DBT/CKIε kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein
Balance between DBT/CKIε kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein
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DOI：
10.1073/pnas.0511215103
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发表时间：
2006-04-18
影响因子：
11.1
通讯作者：
Edery, I
中科院分区：
文献类型：
--
作者：
Kim, EY;Edery, I
第一个被发现的与昼夜节律相关的激酶是果蝇的DOUBLE-TIME (DBT)，它是脊椎动物CKl - epsilon的同源物，在动物体内调节PERIOD （PER）蛋白的进行性磷酸化和稳定性。在一个负反馈回路中，PER直接抑制时钟周期（CLK-CYC）异二聚体的转录活性，这对果蝇分子节律的产生和时钟的正常进程至关重要。我们发现DBT活性是体内CLK阶段特异性超磷酸化所必需的，这一事件与每RNA水平的最大抑制时间相关。在培养的果蝇细胞中，DBT能够使CLK过度磷酸化，增强其降解，并适度抑制昼夜节律启动子元件对CLK依赖性的转激活。有趣的是，DBT似乎与per相关的蛋白磷酸酶2A密切合作，导致CLK低磷酸化和高磷酸化亚型之间的动态平衡。这种平衡机制可以稳定CLK的极限水平，以对抗随机波动，最大限度地减少反馈电路中“分子噪声”的传播。此外，在PER的存在下，CLK的亚细胞定位从主要的细胞核变为强烈的细胞质染色。这些结果表明，与哺乳动物时钟相反，果蝇的昼夜节律转录抑制涉及染色质中积极因子的移位。这些结果还表明，DBT可以靶向昼夜节律反馈回路中的负因子和正因子，并支持在直接调节昼夜节律转录因子活性的可逆磷酸化动态调控中起保守作用。
The first circadian-relevant kinase to be identified was DOUBLE-TIME (DBT) in Drosophila, a homolog of vertebrate CKl epsilon, which regulates the progressive phosphorylation and stability of PERIOD (PER) proteins in animals. A negative feedback loop wherein PER directly inhibits the transcriptional activity of the CLOCK-CYCLE (CLK-CYC) heterodimer is central to the generation of molecular rhythms and normal progression of the clock in Drosophila. We show that DBT activity is required for the phase-specific hyper-phosphorylation of CLK in vivo, an event that correlates with times of maximal repression in per RNA levels. The ability of DBT to hyperphosphorylate CLK, enhance its degradation, and evoke modest inhibition of CLK-dependent transactivation from circadian promoter elements was directly shown in cultured Drosophila cells. Intriguingly, DBT seems to function in close partnership with the PER-relevant protein phosphatase 2A, resulting in dynamic equilibrium between hypo- and hyperphosphorylated isoforms of CLK. This balancing mechanism might act to stabilize the limiting levels of CLK against stochastic fluctuations minimizing the propagation of "molecular noise" in the feedback circuitry. Also, the subcellular localization of CLK was altered from predominately nuclear to strong cytoplasmic staining in the presence of PER. These results suggest that, in contrast to mammalian clocks, circadian transcriptional inhibition in Drosophila involves displacement of the positive factors from chromatin. These results also demonstrate that DBT can target both negative and positive factors in circadian feedback loops and support a conserved role for dynamic regulation of reversible phosphorylation in directly modulating the activities of circadian transcription factors.