The Regulation of Smoothened in Hedgehog Signaling

Hedgehog信号中Smoothened的调节

• 批准号: 8628290
• 负责人: Jianhang Jia
• 金额: $ 30.71万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8628290
• 关键词: Address; Basal cell carcinoma; Beta-Adrenergic Receptor Kinase 1; Binding; Biochemical; Biological; Biological Models; Cancer Etiology; Cell surface; Cells; Complex; Congenital Abnormality; Cyclic AMP-Dependent Protein Kinases; Development; Diagnostic; Dimerization; Drosophila genus; Embryonic Development; Endocytosis; Erinaceidae; Event; Family member; Feedback; Functional disorder; G-Protein-Coupled Receptors; Genetic; Goals; Health; High Pressure Liquid Chromatography; Homologous Gene; Insecta; Integral Membrane Protein; Investigation; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Molecular; Pathway interactions; Pattern; Pattern Formation; Phospholipids; Phosphorylation; Phosphotransferases; Physiological; Play; Preventive; Process; Protein Tyrosine Kinase; Protein phosphatase; Proteins; Publishing; RNA Interference; Recruitment Activity; Regulation; Research; Role; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Technology; Tertiary Protein Structure; Testing; Therapeutic; USP8 gene; Ubiquitin; Ubiquitination; Wing; base; basolateral membrane; cancer type; casein kinase I; casein kinase II; cell growth; design; fly; hedgehog signal transduction; human SMO protein; insight; medulloblastoma; morphogens; novel; phosphatidylinositol 4-phosphate; prevent; protein complex; protein function; receptor; response; small molecule; smoothened signaling pathway; tool; trafficking; ubiquitin-protein ligase

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.

描述（由申请人提供）：Hedgehog（Hh）蛋白作为形态发生素发挥作用，在模式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诱导不同类型的癌症。在昆虫和哺乳动物中，Hh信号的转导都需要七次跨膜蛋白Smoothened（Smo）。我们的策略是使用果蝇作为一个简单的和遗传学上易处理的模型系统来探索Hh信号转导的受体复合物的机制。我们研究的长期目标是阐明Hh信号是如何被感知和传递的，以控制最终控制细胞生长和模式的下游生物事件。虽然Hh通路中的许多组分已被鉴定，但Hh信号如何通过Patched（Ptc）转导到Smo仍不清楚。我们已经发现Smo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（CK 1）、酪蛋白激酶2（CK 2）和G蛋白偶联受体激酶2（Gprk 2），并且磷酸化导致Smo细胞表面水平和信号传导活性增加。我们发现Smo通过直接募集下游融合Costal 2（Fu-Cos 2）复合物来转导Hh信号，并且Fus-Cos 2蛋白复合物促进Smo的过度磷酸化，揭示了最佳Hh途径活化所必需的反馈机制。此外，我们已经确定蛋白磷酸酶PP 4作为Smo的负调节剂。我们的研究结果表明，Smo的细胞表面积累和细胞内运输在调节Hh诱导的Smo活化中起着关键作用。然而，关于控制Smo本地化和贩运的机制仍存在许多问题。为了开始解决这些问题，我们最近发现去泛素化酶USP 8通过阻止Smo泛素化和调节Smo亚细胞定位来促进Smo信号传导活性。结合本项目的前期研究，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，Smo激活下游的Hh是严格控制其亚细胞定位，其中一种新的激酶和小分子促进Hh信号通过增强Smo的细胞表面积累，而泛素失活Smo通过促进Smo的内吞和降解。我们将在三个特定目标中使用遗传和生物化学方法的组合：1）确定新鉴定的激酶如何调节Smo磷酸化和基底外侧膜定位; 2）研究小分子响应Hh刺激激活Smo的分子机制; 3）确定Hh如何通过Smo的新型E3连接酶调节泛素介导的Smo内体分选。