Mechanism of Gli3 processing in Hedgehog Signaling

描述(申请人提供)：Hedgehog(HH)分泌信号分子家族在细胞命运指定、细胞增殖和分化中发挥重要作用。HH信号异常会导致人类胚胎发育异常和多种癌症的发生，因此，了解HH信号转导的分子机制对于预防和治疗由HH信号异常引起的出生缺陷和癌症具有重要意义。在小鼠中，Gli2和Gli3锌指转录因子是HH信号的主要介导者。在没有HH信号的情况下，大多数全长Gli3蛋白(Gli3FL)被蛋白分解为转录抑制物(Gli3Rep)。Gli3的加工需要蛋白激酶A(PKA)、糖原合成酶激酶3(GSK3)和酪蛋白激酶1(CK1)的C末端磷酸化，以及随后SCFTrCP E3泛素连接酶的泛素化。多泛素化的Gli3然后由蛋白酶体处理。Gli3FL主要定位于细胞质，穿梭于细胞质和胞核之间，而Gli3Rep仅存在于胞核。HH信号通过一种未知的机制抑制Gli3的处理，并将Gli3FL转化为激活剂Gli3Act，从而解除对HH靶基因的抑制。Gli3和Gli2的稳定性也受到严格控制。到目前为止，在分子水平上对HH信号如何调节Gli3处理、核转位或Gli2和Gli3稳定性知之甚少。遗传和有限的生化证据表明，HH信号通过各种效应器分子调节这些分子事件，尽管这些效应器与Gli蛋白之间的联系尚不清楚。这些效应器中有许多是脊椎动物特有的，尽管有些在果蝇中是保守的。我们的目标是确定这些效应器在HH信号中的作用以及它们调节Gli2和Gli3功能的分子机制。具体地说，目标1将了解Dzip1在Gli3功能调节中的作用，目标2将利用鞭毛内转运(IFT)和Dynein基因突变体阐明Gli3加工的分子机制，目标3将了解素福突变中HH途径激活的分子基础。这些研究的完成将极大地促进我们对Gli3蛋白在HH信号中的调节和功能的理解。公共卫生相关性：Hedgehog(HH)分泌信号蛋白家族在细胞命运指定以及细胞增殖和分化中发挥重要作用。HH信号的丢失会导致广泛的出生缺陷，而HH通路的异常激活会导致几种类型的人类癌症。了解HH信号转导的分子机制可能有助于设计治疗药物来调节HH信号通路的活性，从而预防或治疗由于HH信号转导活性的错误调节而导致的出生缺陷和癌症。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) family of secreted signaling molecules plays an important role in cell fate specification, cell proliferation and differentiation. Dysfunction in Hh signaling results in abnormal embryonic development and several types of cancer in human; thus, understanding the molecular mechanisms of Hh signal transduction is of great significance for the prevention and remedy of the birth defects and cancers caused by aberrant Hh signaling. In mice, Gli2 and Gli3 zinc-finger-containing transcription factors are the primary mediators of Hh signaling. In the absence of a Hh signal, most full-length Gli3 protein (Gli3FL) is proteolytically processed to a transcriptional repressor (Gli3Rep). Gli3 processing requires C-terminal phosphorylation by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1), and subsequent ubiquitination by SCFTrCP E3 ubiquitin ligase. Polyubiquitinated Gli3 is then processed by the proteasome. Gli3FL is predominantly localized in the cytoplasm and shuttles between the cytoplasm and nucleus, while Gli3Rep is exclusively found in the nucleus. Hh signaling counters Gli3 processing through an unknown mechanism and converts Gli3FL into an activator, Gli3Act, thus derepressing Hh target genes. The stability of Gli3, as well as Gli2, is also tightly controlled. To date, little is known at the molecular level about how Hh signaling regulates Gli3 processing, nuclear translocation, or Gli2 and Gli3 stability. Genetic and limited biochemical evidence indicates that Hh signaling regulates these molecular events through various effector molecules, though the connections between these effectors and Gli proteins are unknown. Many of these effectors are vertebrate specific, although some are conserved in Drosophila. Our objectives are to determine the role of these effectors in Hh signaling and the molecular mechanisms by which they regulate Gli2 and Gli3 function. Specifically, Aim 1 will understand the role of Dzip1 in the regulation of Gli3 function, Aim 2 will elucidate the molecular mechanism of Gli3 processing using intraflagellar transport (IFT) and Dynein gene mutants, and Aim 3 will understand the molecular basis of the Hh pathway activation in Sufu mutant. The completion of the proposed studies will significantly advance our understanding of the regulation and function of Gli3 protein in Hh signaling. PUBLIC HEALTH RELEVANCE: The Hedgehog (Hh) family of secreted signaling proteins plays fundamental roles in cell fate specification and cell proliferation and differentiation. Loss of Hh signaling results in a wide range of birth defects, whereas aberrant activation of the Hh pathway causes several types of human cancer. Understanding the molecular mechanism of Hh signaling may provide insights into the design of therapeutic agents to modulate Hh pathway activity and therefore prevent or treat birth defects and cancers caused by misregulation of the Hh signaling activity.