COMPLEX REGULATION OF CI/GLI PROTEINS IN HEDGEHOG SIGNAL TRANSDUCT

描述（由申请人提供）：由Hedgehog（Hh）家族分泌蛋白介导的细胞信号传导在动物发育和人类疾病中起着至关重要的作用。Hh途径在从昆虫到人类的生物体中以类似的方式运作。果蝇一直是研究Hh信号机制的强大模式生物，因为复杂的遗传，分子和生化工具可用于在整个生物体以及培养细胞中剖析这一重要途径。我的实验室的长期目标是描绘复杂的调控网络，管理Hh信号转导，以了解分级Hh信号是如何转导产生多种发育输出。Hh通过保守的信号级联发挥其生物学影响，该信号级联在控制转录因子Ci/Gli（CiA/GliA和CiR/GliR）的激活子和阻遏子形式之间的平衡中达到高潮。本研究的目的是调查控制CI活动的多方面调节机制。我们最近的研究揭示了Ser/Thr激酶融合（Fu）在调节CiR的产生和CIA的活性中的双重作用，并揭示Fu通过其激活环的二聚化介导的磷酸化而被激活。这些发现提供了一个关键的进展到一个机械解剖的CI激活。我们将探索Fu促进Ci成熟为CiA的机制，并研究Hh梯度如何转化为Ci活性梯度（目的1）。在遗传 修饰物筛选，我们发现SUMO途径可以调节Hh信号传导活性，并确定Ci为SUMO底物。我们将进一步描述这种新的翻译后修饰的Ci，以探索其作用和机制的Hh信号（目的2）。腐乳抑制Ci的分子机制仍然知之甚少。我们已经发现了一个以前未被识别的核定位信号（NLS），与腐乳结合域在Ci重叠。我们将进一步研究这个NLS的功能及其调节（目的3）。最后，Ci如何在细胞核中发挥作用以控制Hh靶基因的表达还没有完全探索。我们已经鉴定了CiR介导的抑制所需的多个结构域。确定与这些结构域相互作用的辅因子，并研究它们在Hh信号传导中的作用，应该为CI如何调节其靶基因表达提供重要的启示。因此，我们将进行蛋白质-蛋白质相互作用筛选和体内RNAi筛选以鉴定C1共阻遏物（Aim 4）。这项研究将为Hh信号转导机制提供更深入的了解，并为Hh信号如何被转化为不同的发育结果提供新的线索。

DESCRIPTION (provided by applicant): Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins plays crucial roles in animal development and human diseases. The Hh pathway is operating in a similar way among organisms ranging from insects to human. Drosophila has been a powerful model organism to study Hh signaling mechanisms, as sophisticated genetic, molecular, and biochemical tools are available to dissect this important pathway in whole organisms as well as in cultured cells. The long-term goal of my laboratory is to delineate the complex regulatory network that governs Hh signal transduction in order to understand how graded Hh signal is transduced to generate multiple developmental outputs. Hh exerts its biological influence through a conserved signaling cascade that culminates in controlling the balance between the activator and repressor forms of the transcription factor Ci/Gli (CiA/GliA and CiR/GliR). The goal of this research is to investigate the multifaceted regulatory mechanisms that control Ci activity. Our recent study has uncovered a dual role of the Ser/Thr kinase Fused (Fu) in the regulation of both the production of CiR and the activity of CIA, and revealed that Fu is activated through dimerization-mediated phosphorylation of its activation loop. These findings provide a critical inroad into a mechanistic dissection of Ci activation. We will explore the mechanism by which Fu promotes the maturation of Ci into CiA and investigate how the Hh gradient is translated into a Ci activity gradient (Aim 1). In a genetic modifier screen, we have discovered that the SUMO pathway can modulate Hh signaling activity and identified Ci as a SUMO substrate. We will further characterize this new post-translational modification of Ci to explore its role and mechanism of action in Hh signaling (Aim 2). The molecular mechanism by which Sufu inhibits Ci is still poorly understood. We have uncovered a previously unidentified nuclear localization signal (NLS) that overlaps with the Sufu binding domain in Ci. We will further study the function of this NLS and its regulation (Aim 3). Finally, how Ci functions in the nucleus to control Hh target gene expression has not been fully explored. We have identified multiple domains required for CiR-mediated repression. Identifying cofactors that interact with these domains and investigating their roles in Hh signaling should shed important lights into how Ci regulates its target gene expression. Therefore, we will carry out protein- protein interaction screen and in vivo RNAi screen to identify Ci co-repressors (Aim 4). The proposed study should provide a much deeper understanding of the Hh signal transduction mechanism and shed new light into how graded Hh signals are translated into different developmental outcomes.