Understanding how ciliary gene mutations affect the processing and activity of Gli2 and Gli3 transcription factors

摘要 初级纤毛是一个孤立的微管细胞器，从细胞表面伸出，是 存在于大多数脊椎动物细胞中。它在肾脏和视网膜等器官中充当感觉器官，并 也在细胞外信号转导中发挥作用，如Hedgehog(HH)，一种分泌的信号分子， 是胚胎发育、细胞增殖和分化所必需的。纤毛结构和纤毛缺陷 纤毛功能与一系列不同的发育异常有关，统称为“纤毛疾病”。 脊椎动物中的HH信号发生在初级纤毛中，主要由Gli2和Gli3锌指介导。 含有转录因子。Gli2主要是一个激活物，而Gli3主要是一个抑制物，尽管它也 表现出弱的激活剂作用。与它们的功能一致，大多数全长Gli3(Gli3FL)是蛋白水解性的 在没有HH信号的情况下被处理以产生C末端截断的抑制子，而只有一小部分 对Gli2FL的一部分进行了加工。Gli2/Gli3的加工是由六个中的前四个的磷酸化诱导的 C末端的丝氨酸/苏氨酸残基由蛋白激酶A(PKA)决定，然后由糖原合成酶3决定 (GSK3)和酪蛋白激酶1(CK1)。然后，多磷酸化的Gli2/Gli3被泛素化并部分 被蛋白酶体降解。HH信号通过抑制PKA介导的Gli2/Gli3途径抑制Gli2/Gli3的加工 通过抑制前四个PKA位点的磷酸化而激活Gli2FL/Gli3FL 它们C-末端的第五和第六个PKA位点。 纤毛结构和功能的缺陷主要影响HH信号转导。纤毛的一个近乎普遍的特征 基因突变在分子水平上是对Gli2/Gli3的还原处理。然而，有趣的是， 纤毛基因突变体的Gli2/Gli3活性因纤毛基因突变的不同而不同。这 这表明纤毛蛋白调节HH信号的机制是多样和复杂的。而当 纤毛突变对HH信号的影响是公认的，其背后的分子机制 到目前为止，影响还不得而知。这项提议的目的是阐明 在纤毛基因突变体中，Gli2/Gli3的加工、稳定性和活性发生了改变。

Abstract The primary cilium is a solitary microtubule-based organelle that protrudes from the cell surface and is found on most vertebrate cells. It serves as a sensory organelle in organs such as the kidney and retina and also functions in transducing extracellular signals such as Hedgehog (Hh), a secreted signaling molecule that is essential for embryo development and cell proliferation and differentiation. Defects in cilia structure and function are associated with a diverse array of developmental abnormalities, collectively termed “ciliopathies”. Hh signaling in vertebrates occurs in primary cilia and is primarily mediated by Gli2 and Gli3 zinc finger- containing transcription factors. Gli2 is primarily an activator, whereas Gli3 is mostly a repressor, though it also exhibits a weak activator function. Consistent with their functions, most full-length Gli3 (Gli3FL) is proteolytically processed to generate a C-terminally truncated repressor in the absence of Hh signaling, while only a small fraction of Gli2FL is processed. Gli2/Gli3 processing is induced by phosphorylation of the first four of the six serine/threonine residues at their C-termini by protein kinase A (PKA) and then by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). The multi-phosphorylated Gli2/Gli3 are then ubiquitinated and partially degraded by the proteasome. Hh signaling inhibits Gli2/Gli3 processing by suppressing PKA-mediated phosphorylation of the first four PKA sites and also activates Gli2FL/Gli3FL by inhibiting the phosphorylation of the fifth and sixth PKA sites in their C-termini. Defects in cilia structure and function mostly affect Hh signaling. One near universal hallmark of ciliary gene mutants at the molecular level is the reduced Gli2/Gli3 processing. Interestingly, however, the levels of Gli2/Gli3 activity in ciliary gene mutants vary from none to elevated depending on mutated ciliary genes. This suggests that the mechanism by which ciliary proteins regulate Hh signaling is diverse and complex. While the effect of ciliary mutations on Hh signaling is well established, the molecular mechanisms underlying these effects are thus far unknown. The goal of this proposal is to elucidate the molecular mechanisms by which Gli2/Gli3 processing, stability, and activity are altered in ciliary gene mutants.