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。我实验室的长期目标是描绘出控制Hh信号转导的复杂调控网络。该建议的重点是研究控制Hh通路的转录效应子Ci/Gli蛋白的多方面调控机制。我们先前的研究鉴定了三种激酶PKA、GSK 3和CKI，以及由F-盒蛋白Slim/（3 TRCP）组成的泛素连接酶，作为Ci蛋白水解加工产生其阻遏物形式的必需调节剂。我们提供的证据表明，驱动蛋白样蛋白Cos 2作为一个支架桥Ci到其激酶有效的磷酸化。此外，我们发现，多个Ci/Cos 2/Fu/Sufu蛋白复合物的形成阻碍了Ci核转位，并且Sufu进一步抑制了细胞核中的Ci活性。最近，我们鉴定了由BTB蛋白HIB组成的泛素连接酶，其在负反馈回路中起作用以通过降解活性形式的Ci来微调Hh信号传导反应。有趣的是，HIB特异性地表达在视盘中的形态发生沟的后面，在那里它阻止异常的Hh信号传导活性以确保正常的眼睛发育。关于Ci/Gli蛋白如何被调节的几个重要问题仍然存在。例如，腐乳如何抑制细胞核中Ci的转录活性？Cos 2是否有额外的脚手架作用？Hh信号如何抑制Cos 2的支架活性？介导Ci/Gli的HIB降解的降解决定子是什么？HIB是否参与CI监管的其他方面？还有哪些机制以组织特异性方式调节Hh/Ci信号传导？我们将通过提出以下具体目标来解决这些问题：1）进一步确定Cos 2在Ci调节中的支架作用; 2）研究Sufu调节细胞核中Ci转录活性的机制; 3）确定HIB调节Ci降解和亚细胞定位的机制; 4）鉴定和表征Hh/Ci信号传导的新型组织特异性调节剂。拟议的研究应提供新的见解Hh信号是如何在转录效应子的水平上转导，以及Hh信号转导反应是如何通过干扰Ci/Gli活性的保守机制来调节的。由于不受约束的Gli活性导致许多类型的人类恶性肿瘤，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破坏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.