Mechanisms of Vertebrate Hedgehog Signaling

描述（由申请人提供）：Hedgehog （Hh）信号通路在胚胎发育、成体干细胞维持和许多癌症中起关键作用。在脊椎动物中，Hh信号是从初级纤毛开始的，从那里信号传递到细胞质，然后传递到细胞核，最终控制特定靶基因的表达，由Gli转录因子介导。在没有刺激的情况下，Gli蛋白通过至少三种抑制机制被阻止：1)直接结合融合抑制因子（SuFu）；2)抑制因子形式的部分降解和3)蛋白激酶A （PKA）的抑制。当细胞接收到Hh信号时，这些抑制被克服，允许Gli激活。尽管Hh信号的重要性，但我们仍然不了解在Hh通路的静息状态和刺激状态下抑制和激活Gli蛋白的许多关键机制。我们发现Hh刺激将SuFu-Gli复合物招募到初级纤毛并使其解离，导致Gli激活；我们发现PKA能拮抗这一过程。这为Gli蛋白的活化和PKA的抑制作用提供了新的机制。我们还发现，抑制蛋白酶体可以有效地阻断Gli活性，这表明Gli的周转和转录激活是密切耦合的。我们建议采用生物化学和细胞生物学相结合的方法来阐明脊椎动物Hh信号通路中以下关键事件的机制：a) Hh信号通路如何激活Gli和抑制SuFu？B)在Hh信号中，纤复- gli向纤毛的募集有何作用，PKA如何阻断纤复- gli向纤毛的募集？C)抑制蛋白酶体如何阻断Gli蛋白的转录激活，Gli蛋白的部分蛋白水解是如何调节的？这些研究具有以下重要意义：1)它们将阐明控制关键Gli蛋白的基本机制，从而促进我们对Hh信号传导的理解；2)他们将发现在癌症中抑制Hh的新靶点；3)我们发现蛋白酶体抑制剂能有效阻断Hh信号传导，这可能对癌症有直接的治疗意义，特别是因为蛋白酶体抑制剂硼替佐米是fda批准的治疗多发性骨髓瘤的药物。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling pathway has critical roles in embryonic development, adult stem cell maintenance and in many cancers. In vertebrates, Hh signaling is initiated at the primary cilium, from where the signal is relayed to the cytoplasm and then nucleus, to ultimately control expression of specific target genes, mediated by the Gli transcription factors. In the absence of stimulation, Gli proteins are kept off by at least three inhibitory mechanisms: 1) direct binding of Suppressor of Fused (SuFu); 2) partial degradation to repressor forms and 3) inhibition by protein kinase A (PKA). When cells receive an Hh signal, these inhibitions are overcome, allowing Gli activation. In spite of the importance of Hh signaling, we still do not understand many of the critical mechanisms involved in inhibiting and in activating Gli proteins, in the resting and stimulated states of the Hh pathway, respectively. We discovered that Hh stimulation recruits SuFu-Gli complexes to primary cilia and causes their dissociation, resulting in Gli activation; we found that this process is antagonized by PKA. This provided a novel mechanism for activation of Gli proteins and for the inhibitory effect of PKA. We also discovered that inhibition of the proteasome potently blocks Gli activity, suggesting that Gli turnover and transcriptional activation are intimately coupled. We propose to use a combination of biochemistry and cell biology, to elucidate the mechanism of the following critical events in vertebrate Hh signaling: A) How does Hh signaling activate Gli and inhibit SuFu? B) What is the function of SuFu-Gli recruitment to cilia in Hh signaling, and how does PKA block SuFu-Gli recruitment to cilia? C) How does inhibition of the proteasome block transcriptional activation by Gli proteins and how is the partial proteolysis of Gli proteins regulated? These studies are important for the following reasons: 1) They will elucidate basic mechanisms that control the critical Gli proteins, thus advancing our understanding of Hh signaling; 2) They will identify novel targets for Hh inhibition in cancer; and 3) Our finding that proteasome inhibitors potently block Hh signaling could have immediate therapeutic implications in cancer, particularly since the proteasome inhibitor bortezomib is an FDA-approved drug for the treatment of multiple myeloma.