Complex regulation of Ci/Gli proteins in Hedgehog signal transduction

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。这项建议的重点是研究控制HH途径转录效应因子Ci/Gli蛋白的多方面调控机制。我们以前的研究发现，PKA、GSK3和CKI三种激酶以及由F-box蛋白SLimb/(3TRCP)组成的泛素连接酶是Ci蛋白降解过程中产生其抑制物形式的重要调节因子。我们提供的证据表明，激动素样蛋白Cos2作为一种支架，将Ci连接到其激酶上，从而实现有效的磷酸化。此外，我们还发现，多种Ci/Cos2/FU/Sufu蛋白复合体的形成阻碍了Ci核的移位，而Sufu进一步抑制了Ci在细胞核内的活性。最近，我们发现了一种由BTB蛋白HIB组成的泛素连接酶，它在负反馈环中通过降解Ci的活性形式来微调HH信号反应。有趣的是，HIB在眼盘的形态发生皱纹之后特异表达，在那里它阻止异常的HH信号活动，以确保眼睛的正常发育。关于Ci/Gli蛋白是如何调控的，仍有几个重要的问题。例如，Sufu如何抑制Ci在细胞核中的转录活动？Cos2有没有额外的脚手架作用？HH信号如何抑制Cos2的支架活性？介导Ci/Gli HIB降解的降解物是什么？HIB是否参与CI监管的其他方面？以组织特异性的方式调节HH/CI信号的其他机制是什么？我们将通过以下具体目标来解决这些问题：1)进一步确定Cos2在Ci调控中的支架作用；2)研究Sufu调控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)寻找和表征HH/Ci信号的新的组织特异性调控因子。这项研究将为HH信号如何在转录效应分子水平上被转导，以及HH信号响应如何被干扰Ci/Gli活性的保守机制所调制提供新的见解。由于不受限制的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毁灭性的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.