COMPLEX REGULATION OF CI/GLI PROTEINS IN HEDGEHOG SIGNAL TRANSDUCT

描述(申请人提供)：由Hedgehog(HH)分泌蛋白家族介导的细胞信号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各种有机体中也以类似的方式运作。果蝇已经成为研究HH信号机制的强大模式生物，因为复杂的遗传、分子和生化工具可以在整个生物体以及培养的细胞中剖析这一重要的途径。我的实验室的长期目标是描绘管理HH信号转导的复杂调控网络，以便了解分级HH信号是如何被转导以产生多种发育输出的。HH通过一个保守的信号级联来发挥其生物学影响，最终控制转录因子Ci/Gli(CIA/Glia和Cir/Glir)的激活型和抑制型之间的平衡。本研究的目的是探讨控制CI活动的多方面调控机制。我们最近的研究揭示了丝氨酸/苏氨酸激酶融合(FU)在调节Cir的产生和CIA活性方面的双重作用，并揭示了FU是通过二聚化介导的激活环的磷酸化来激活的。这些发现为对CI激活的机械性剖析提供了关键的切入点。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活动梯度(目标1)。在一种基因上 经过修饰筛选，我们发现相扑通路可以调节HH信号活性，并确定Ci为相扑底物。我们将进一步描述Ci的这种新的翻译后修饰，以探索其在HH信号转导中的作用和作用机制(目标2)。关于腐乳抑制Ci的分子机制目前还不甚清楚。我们发现了一个先前未知的核定位信号(NLS)，该信号与Ci中的Sufu结合结构域重叠。我们将进一步研究这一NLS的功能及其调节(目标3)。最后，Ci如何在细胞核中控制HH靶基因的表达还没有被充分探索。我们已经确定了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信号中的作用，将有助于了解Ci如何调节其靶基因的表达。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，以确定Ci共抑制物(目标4)。这项研究将提供对HH信号转导机制的更深入的理解，并为HH信号如何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.