The Regulation of Smoothened in Hedgehog Signaling

描述(由申请人提供)：Hedgehog(HH)蛋白作为形态原发挥作用，并在图案形成和细胞生长控制中发挥关键作用。HH信号通路的失调和功能障碍会导致多种类型的癌症。昆虫和哺乳动物都需要七通跨膜蛋白Smoothens(Smo)来传递HH信号。我们的策略是使用果蝇作为一个简单和遗传上容易处理的模型系统来探索受体复合体转导HH信号的机制。我们研究的长期目标是阐明HH信号是如何被感知和传输的，以控制最终控制细胞生长和图案形成的下游生物事件。尽管HH途径中的许多成分已经被确定，但HH信号是如何通过Patted(PTC)转导到Smo的仍不清楚。我们发现，Smo的激活需要多种激酶的磷酸化，包括蛋白激酶A(PKA)、酪蛋白激酶1(CK1)、酪蛋白激酶2(CK2)和G蛋白偶联受体激酶2(Gprk2)，而磷酸化导致Smo细胞表面水平和信号活性的增加。我们发现Smo通过直接招募下游的融合核心蛋白2(FU-Cos2)复合体来传递HH信号，而Fus-Cos2蛋白复合体促进Smo的过度磷酸化，揭示了最佳HH途径激活所必需的反馈机制。此外，我们还发现蛋白磷酸酶PP4是Smo的负调控因子。我们的研究结果表明，Smo的细胞表面聚集和细胞内转运在调节HH诱导的Smo激活中起着关键作用。然而，关于控制Smo本地化和贩运的机制仍然存在许多问题。为了开始解决这些问题，我们最近发现去泛素酶USP8通过阻止Smo泛素化和调节Smo亚细胞定位来促进Smo信号活性。结合该项目的初步研究，我们的发现为研究SMO信号转导机制提供了新的工具和假设。在这个项目中，我们的中心假设是HH下游的Smo激活受到其亚细胞定位的严格控制，其中一种新的激酶和小分子通过促进Smo在细胞表面的积聚来促进HH信号的传递，而泛素通过促进Smo的内吞和降解来灭活Smo。我们将在三个特定目标中结合使用遗传和生化方法：1)确定一个新发现的激酶如何调节Smo的磷酸化和基底膜的定位；2)研究小分子激活Smo以响应HH刺激的分子机制；3)确定HH如何通过Smo的一个新的E3连接酶调节泛素介导的Smo的内体分选。

DESCRIPTION (provided by applicant): Hedgehog (Hh) proteins function as morphogens and play critical roles in pattern formation and cell growth control. Dysregulation and dysfunction of the Hh signaling pathway induces diverse types of cancers. The seven-pass transmembrane protein Smoothened (Smo) is required in both insects and mammals for transduction of the Hh signal. Our strategy is to use Drosophila as a simple and genetically tractable model system to explore the mechanisms of Hh signal transduction by the receptor complex. The long-term goal of our research is to elucidate how Hh signals are sensed and transmitted to control downstream biological events that ultimately govern cell growth and patterning. Although many components in the Hh pathway have been identified, how the Hh signal is transduced through Patched (Ptc) to Smo is still unclear. We have discovered that Smo activation requires phosphorylation by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), and G protein-coupled receptor kinase 2 (Gprk2), and that phosphorylation leads to increased Smo cell surface levels and signaling activity. We show that Smo transduces Hh signals by directly recruiting a downstream Fused-Costal2 (Fu-Cos2) complex, and that Fus- Cos2 protein complex promotes hyperphosphorylation of Smo, uncovering a feedback mechanism essential for optimal Hh pathway activation. Furthermore, we have identified protein phosphatase PP4 as a negative regulator of Smo. Results from our studies have suggested that the cell surface accumulation and intracellular trafficking of Smo play critical roles in regulating Hh-induced Smo activation. However, many questions persist regarding the mechanism controlling Smo localization and trafficking. To begin address these questions, we have recently discovered that the deubiquitinase USP8 promotes Smo signaling activity by preventing Smo ubiquitination and regulating Smo subcellular localization. Combined with the preliminary studies in the project, our findings provide new tools and hypotheses for investigating the mechanisms of Smo signaling. In this project, our central hypothesis is that Smo activation downstream of Hh is tightly controlled by its subcellular localization, in which a novel kinase and small molecules promotes Hh signaling by enhancing the cell surface accumulation of Smo whereas ubiquitin inactivates Smo by promoting Smo endocytosis and degradation. We will use a combination of genetic and biochemical approaches in three Specific Aims: 1) To determine how a newly identified kinase regulates Smo phosphorylation and basolateral membrane localization; 2) to investigate the molecular mechanisms by which small molecules activate Smo in response to Hh stimulation; 3) to determine how Hh regulates the ubiquitin-mediated endosomal sorting of Smo via a novel E3 ligase of Smo.