The functional significance of SUMOylierung in biological timing

地球上大多数生物的生理和行为活动与昼夜的环境周期同步。这种同步是由转录因子组成的分子生物钟实现的，转录因子形成互锁的转录/翻译反馈环。果蝇和哺乳动物同源生物钟的中心是由转录因子时钟(CLK)和周期(Cyc)组成的复合体，它们从代谢、免疫反应、细胞增殖和神经元活动等一系列生命活动中控制着关键调控因子的全基因组转录。越来越多的证据表明，CLK的翻译后调节对生物钟的功能和生物钟转录的时间至关重要。我们最近发现了环核苷酸/PKA、钙/CaMK II和RAS/MAPK对CLK/Cyc活性的调节，其中包括CaMK II和MAPK对CLK的直接磷酸化。此外，酪蛋白激酶I也被证明可以磷酸化CLK。我们确定了控制CLK核定位和活性的特定磷酸化位点。此外，我们表征了功能性核定位和输出信号，证明了CLK的核质穿梭。有趣的是，除了CLK的SUMO化外，我们还发现了区室特异性的磷酸化状态。我们还确定了一个对核保留和CLK在核病灶中定位至关重要的SUMO化位点。我们的数据表明，在CLK的整个生命周期中，有一系列针对CLK的翻译后修饰。在这个建议中，我们旨在研究这一序列的修饰，特别是SUMO化和泛素化，以便在分子水平上了解CLK积累、核输入/输出、激活和失活以及最终降解的时间调控。这些研究将为泛素样蛋白在转录因子的翻译后调控中的作用，特别是对生物节律转录的时间选择提供重要的见解。

Most organisms on earth synchronize their physiological and behavioral activities with the environ-mental cycles of day and night. Such synchronization is achieved by a molecular circadian clock that is constituted by transcription factors, which form interlocked transcriptional/translational feedback loops. The center of the homologous circadian clock of Drosophila and mammals is formed by the complex of transcription factors CLOCK (CLK) and CYCLE (CYC), which control genome-wide tran-scription of key regulatory factors from a large array of vital activities including metabolism, immune-response, cell proliferation and neuronal activity. Increasing evidence has shown that posttranslational regulation of CLK is crucial for circadian clock function and timing of circadian transcription. We re-cently found a regulation of CLK/CYC activity by cyclic nucleotides/PKA, calcium/CaMK II and Ras/MAPK, which involves direct phosphorylation of CLK by CaMK II and MAPK. Also casein kinase I was shown to phosphorylate CLK. We identified specific phosphorylation sites that control CLK nuclear localization and activity. In addition, we characterized functional nuclear localization and export signals that demonstrated a nucleo-cytoplasmic shuttling of CLK. Interestingly, we found com-partment specific phosphorylation states in addition to SUMOylation of CLK. We also identified a SUMOylation site that is crucial for nuclear retention and localization of CLK in nuclear foci. Our data indicate a sequence of posttranlational modifications that target CLK through its life-cycle. In this proposal we aim to investigate this sequence of modifications, particularly SUMOylation and ubiquitination, in order to understand on the molecular level how the temporal regulation of CLK accumulation, nuclear import/export, activation and inactivation, and finally degradation is controlled. These studies will provide important insights into the role of ubiquitin like proteins for post-translational regulation of transcription factors in general and for biological timing of circadian transcription in particular.