Mechanism of Gli3 processing in Hedgehog signaling

描述(申请人提供)：细胞间的通讯是精确控制细胞生长和分化的基本机制。这些过程中的缺陷会导致人类癌症和出生缺陷。Hedgehog(HH)家族分泌的信号蛋白控制细胞的生长和分化。HH信号通路活性的丧失或降低会导致严重的发育性出生缺陷。除了发育异常，HH信号通路的不适当激活也与人类癌症有关，包括基底细胞癌和髓母细胞瘤。我们的长期目标是了解HH信号转导的分子机制，并确定HH信号在模式指定和肿瘤形成中的作用。在果蝇中，HH信号是由Cubitus Interruptus(Ci)介导的，Ci是一个含有转录因子的锌指。在没有HH信号的情况下，很大一部分Ci蛋白被蛋白质降解处理以产生转录抑制因子。HH信号刺激阻断了CI的加工，激活了该通路。CI的加工需要它被PKA、CKI和GSK3磷酸化，以及被称为SCF复合体的新型泛素连接酶的一种成分SLimb的活性。Ci的脊椎动物同源物是Gli1、Gli2和Gli3。许多研究表明，Gill和Gli2在该途径中起积极作用，而Gli3起负面作用。与Gli3在该途径中的负面作用一致，大多数Gli3蛋白在没有HH信号的情况下被处理。HH信号抑制Gli3的加工，降低其RNA水平，从而调节Gli转录活性的净输出。Gli3/Ci加工的分子机制以及HH信号对其的调控在很大程度上是未知的。使用一种结合了生化、细胞生物学、遗传学和药理学方法的广泛的方法，建议的研究的目标是1)阐明CKI和GSK3，以及_TrCP，SLimb的脊椎动物同源物，在Gli3加工中的作用；2)了解蛋白酶体在Gli3加工中的作用；3)确定未加工和加工形式的Gli3蛋白在体内的作用。这项研究的完成将极大地促进我们对HH信号通路分子机制的理解。这也可能有助于揭示Gli3突变引起的人类先天性综合征的分子机制。此外，这项研究可能会为我们设计调节Gli3 Processin 9的治疗剂，以干预或治疗与HH信号通路调控不当有关的癌症。

DESCRIPTION (provided by applicant): Cell-cell communication is a fundamental mechanism by which cell growth and differentiation are precisely controlled. Defects in these processes cause human cancer and birth defects. The Hedgehog (Hh) family of secreted signaling proteins controls cell growth and differentiation. Loss of or decrease in the Hh signaling pathway activity results in severe developmental birth defects. In addition to developmental abnormalities, inappropriate activation of the Hh signaling pathway is also associated with human cancers including basal cell carcinoma and medulloblastoma. Our long-term goal is to understand the molecular mechanisms by which Hh signal is transduced and to determine the role of Hh signaling in pattern specification and tumor formation. In Drosophila, Hh signal is mediated by Cubitus Interruptus (Ci), a zinc finger containing transcription factor. In the absence of Hh signal, a significant fraction of Ci protein is proteolytically processed to generate a transcription repressor. Hh signal stimulation blocks the Ci processing and activates the pathway. Ci processing requires its phosphorylation by PKA, CKI, and GSK3 and the activity of Slimb, a component of the novel class of ubiquitin ligase called SCF complex. Vertebrate homologs of Ci are Glil, Gli2, and Gli3. Many studies suggest that Gill and Gli2 act positively, whereas Gli3 plays a negative role in the pathway. Consistent with a negative role of Gli3 in the pathway, the majority of Gli3 protein is processed in the absence of Hh signaling. Hh signaling inhibits Gli3 processing and reduces its RNA levels, thus regulating the net output of Gli transcriptional activities. Molecular mechanisms of Gli3/Ci processing and its regulation by Hh signaling are largely unknown. Using a broadly-based approach that incorporates biochemical, cell biological, genetic, and pharmacological methods, the objectives of the proposed study are to 1) Elucidate the role of CKI and GSK3, and _TrCP, the vertebrate homolog of Slimb, in Gli3 processin.q; 2) Understand the role of the proteasome in Gli3 processin,q; 3) Determine the role of unprocessed and processed forms of Gli3 protein in vivo. The completion of the proposed study will significantly advance our understanding of the molecular mechanisms of the Hh signaling pathway. It may also shed light on the molecular mechanisms of human congenital syndromes caused by Gli3 mutations. In addition, this research may give us insights into the design of therapeutic agents that modulate Gli3 processin 9 to intervene or remedy cancer related to misregulation of the Hh signaling pathway.