The Regulation of Smoothened in Hedgehog Signaling

描述（由申请人提供）： 刺猬（hh）家族成员控制着脊椎动物和无脊椎动物发育的许多方面。Hh信号传导与重要的生物学现象如图案形成、细胞生长和形态发生有关。已在几种类型的人类癌症中观察到该途径的异常激活。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上易处理的模型系统来探索Hh信号转导的机制。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。我们发现Smo通过直接募集Costal 2-Fused（Cos 2-Fu）复合物来转导Hh信号。我们还表明，Smo激活需要蛋白激酶A（PKA）和酪蛋白激酶I（CKI）的磷酸化，磷酸化导致Smo细胞表面水平和信号活性增加。我们最近发现了一种反馈机制，通过这种机制，Fu通过拮抗Cos 2促进Smo过度磷酸化和细胞表面积累。这些发现为研究Hh信号在细胞膜上的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细胞表面积累和信号传导活性受多种激酶调节，而磷酸酶作为抑制组分减弱Smo激活。为了验证我们的假设，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）进一步确定差异Smo磷酸化是否转导梯度Hh活性; 2）研究G蛋白偶联受体激酶2（GRK 2）参与Hh信号传导的分子机制; 3）确定磷酸酶在Smo去磷酸化和失活中的作用。健康相关性异常Smoothened（Smo）激活导致基底细胞癌（BCC）和髓母细胞瘤等癌症。Smo信号机制的研究将为基本的发育问题和开发癌症诊断工具和治疗方法提供新的途径。PHS 398/2590（2004年9月修订，2006年4月重新印发）

DESCRIPTION (provided by applicant): The hedgehog (hh) family members control many aspects of development in both vertebrates and invertebrates. Hh signaling is associated with important biological phenomena such as patterning, cell growth, and morphogenesis. Abnormal activation of this pathway has been observed in several types of human 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. 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. We showed that Smo transduces the Hh signals by directly recruiting a Costal2-Fused (Cos2-Fu) complex. We also showed that Smo activation requires phosphorylation by protein kinase A (PKA) and casein kinase I (CKI), and that phosphorylation leads to increased Smo cell-surface levels and signaling activity. We recently uncovered a feedback mechanism by which Fu promotes Smo hyperphosphorylation and cell-surface accumulation by antagonizing Cos2. These findings provide new tools and hypotheses to investigate the mechanisms of Hh signaling at the cell membrane. In this project, our central hypothesis is that Smo cell-surface accumulation and signaling activity are regulated by multiple kinases, while phosphatase(s) act as inhibitory components to attenuate Smo activation. To test our hypothesis, we will use a combination of genetic and biochemical approaches in three Specific Aims: 1) To further determine if differential Smo phosphorylation transduces gradient Hh activity; 2) to investigate the molecular mechanisms by which G protein-coupled receptor kinase 2 (GRK2) is involved in Hh signaling; and 3) to determine the role of phosphatase in Smo dephosphorylation and inactivation. HEALTH RELEVANCE Abnormal Smoothened (Smo) activation results cancers such as basal cell carcinoma (BCC) and medulloblastoma. Investigation of the Smo signaling mechanisms will provide both insights into fundamental developmental problems and new avenues for developing diagnostic tools and therapeutical treatments of cancers. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Project Narrative