The functional significance of SUMOylierung in biological timing

地球上大多数生物的生理和行为活动都与昼夜的环境循环同步。这种同步是由转录因子构成的分子昼夜节律钟实现的，这些转录因子形成了互锁的转录/翻译反馈回路。果蝇和哺乳动物同源生物钟的中心是由转录因子clock （CLK）和CYCLE （CYC）的复合体构成的，它们控制着来自代谢、免疫反应、细胞增殖和神经元活动等一系列重要活动的关键调节因子的全基因组转录。越来越多的证据表明，CLK的翻译后调控对昼夜节律钟功能和昼夜节律转录的定时至关重要。我们最近发现了环核苷酸/PKA、钙/CaMK II和Ras/MAPK对CLK/CYC活性的调控，这涉及到CaMK II和MAPK对CLK的直接磷酸化。酪蛋白激酶I也被证明能磷酸化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-胞质穿梭。有趣的是，除了CLK的SUMOylation外，我们还发现了细胞室特异性磷酸化状态。我们还发现了一个SUMOylation位点，它对细胞核保留和CLK在核病灶中的定位至关重要。我们的数据表明，在CLK的整个生命周期中，存在一系列针对CLK的翻译后修饰。在本文中，我们的目标是研究这一系列的修饰，特别是SUMOylation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是如何被控制的。这些研究将为研究泛素样蛋白在转录因子翻译后调控中的作用，特别是在昼夜节律转录的生物定时中的作用提供重要的见解。

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.