Complex regulation of Ci/Gli proteins in Hedgehog signal transduction

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通路在从昆虫到人类的各种生物中也以类似的方式运作。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解剖整个生物体以及培养细胞中的这一重要途径。我的实验室的长期目标是描绘控制Hh信号转导的复杂调控网络。本研究的重点是研究控制Hh通路、Ci/Gli蛋白转录效应的多方面调控机制。我们之前的研究确定了三种激酶，PKA， GSK3和CKI，以及由F-box蛋白Slimb/(3TRCP)组成的泛素连接酶，作为Ci蛋白水解过程中产生其抑制形式的重要调节因子。我们提供的证据表明，激酶样蛋白Cos2作为一个支架，将Ci连接到其激酶，以实现有效的磷酸化。此外，我们发现多个Ci/Cos2/Fu/Sufu蛋白复合物的形成阻碍了Ci核易位，Sufu进一步抑制了Ci在细胞核中的活性。最近，我们发现了一种由BTB蛋白HIB组成的泛素连接酶，它通过降解活性形式的Ci，在负反馈回路中调节Hh信号反应。有趣的是，HIB在眼盘的形态发生沟后特异性表达，在那里它可以阻止异常的Hh信号活动，以确保正常的眼睛发育。关于Ci/Gli蛋白是如何调控的几个重要问题仍然存在。例如，素复如何抑制细胞核中Ci的转录活性？Cos2是否有额外的脚手架作用？Hh信号如何抑制Cos2的支架活性？介导HIB降解Ci/Gli的降解因子是什么？HIB是否参与Ci监管的其他方面？在组织特异性方式中调节Hh/Ci信号的其他机制是什么？我们将通过提出以下具体目标来解决这些问题：1)进一步定义Cos2在Ci调节中的支架作用；2)研究素复调控细胞核内Ci转录活性的机制；3)明确HIB调控Ci降解和亚细胞定位的机制；4)鉴定和表征Hh/Ci信号的新型组织特异性调节因子。提出的研究应该为Hh信号如何在转录效应水平上转导以及Hh信号反应如何被干扰Ci/Gli活性的保守机制调节提供新的见解。由于不受约束的Gli活性与许多类型的人类恶性肿瘤有关，包括脑癌、肺癌、胰腺癌和前列腺癌，我们的研究可能为改善这些毁灭性人类疾病的诊断和治疗提供新的途径。

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. The focus of this proposal is to investigate the multifaceted regulatory mechanisms that control the transcriptional effectors of the Hh pathway, Ci/Gli proteins. Our previous studies identified three kinases, PKA, GSK3, and CKI, and an ubiquitin ligase consisting of the F-box protein Slimb/(3TRCP, as essential regulators of Ci proteolytic processing to generate its repressor form. We provided evidence that the kinesin-like protein Cos2 acts as a scaffold to bridge Ci to its kinases for efficient phosphorylation. In addition, we showed that the formation of multiple Ci/Cos2/Fu/Sufu protein complexes impedes Ci nuclear translocation and that Sufu further inhibits Ci activity in the nucleus. More recently, we identified an ubiquitin ligase consisting of the BTB protein HIB, which acts in a negative feedback loop to fine-tune Hh signaling responses by degrading the active forms of Ci. Interestingly, HIB is specifically expressed posterior to the morphogenetic furrow in eye discs where it prevents aberrant Hh signaling activity to ensure normal eye development. Several important questions remain regarding how Ci/Gli proteins are regulated. For example, how does Sufu inhibit Ci transcriptional activity in the nucleus? Does Cos2 have additional scaffolding role? How does Hh signaling inhibit the scaffolding activity of Cos2? What are the degrons that mediate HIB degradation of Ci/Gli? Does HIB participate in other aspects of Ci regulation? What are the other mechanisms that regulate Hh/Ci signaling in tissue-specific manners? We will address these questions by proposing the following specific aims: 1) to further define the scaffolding role of Cos2 in Ci regulation; 2) to investigate the mechanism by which Sufu regulates Ci transcriptional activity in the nucleus; 3) to define the mechanisms by which HIB regulates Ci degradation and subcellular localization; 4) to identify and characterize novel tissue specific regulators of Hh/Ci signaling. The proposed study should provide novel insights into how the Hh signal is transduced at the level of transcriptional effector and how Hh signaling responses are modulated by conserved mechanisms that interfere with Ci/Gli activity. As unconstrained Gli activity contributes many types of human malignancy, including brain, lung, pancreas, and prostate cancers, our study may provide new avenues for improving diagnosis and therapeutics of these devastated human diseases.