The Ral small GTPase in C. elegans development

描述(申请人提供：RAS是人类癌症中最频繁突变的癌基因，Raf-MEK-ERK蛋白激酶级联是RAS下游效应功能的最佳表征。然而，现在清楚的是，RAS的功能不能简单地归因于一个效应途径，而且RAS利用了一系列功能不同的效应分子，其中至少有五个参与了肿瘤的发生。目前尚不清楚的是效应器的使用是如何受到监管的。最近的研究表明，鸟嘌呤核苷酸交换因子作为RAS的效应因子(RalGEF)在癌症中具有重要作用。然而，我们对Ralgef-ral和其他效应器通路之间的相互作用知之甚少，我们更不知道Ralgef-ral活动在发育过程中的正常功能。对线虫Ras同源物LET-60的遗传分析已经阐明了Raf-MEK-ERK级联在发育中的作用，并为理解哺乳动物细胞中Ras信号的机制提供了关键线索。由于基本上所有已知的Ralgef-ral信号在线虫中都是保守的，这促使我们有兴趣阐明进化上保守的线虫rgl-1(Ralgef)和ral-1(Ral)在let-60调节的发育中的作用。我们的初步结果表明，出乎意料的是，Rgl-1-ral-1信号对抗而不是协同Raf-MEK-ERK信号诱导外阴10个细胞的命运，并且平行或下游于ERK底物LIN-31/HNF转录因子。此外，我们的结果表明，只有在与10个外阴细胞相邻的20个细胞中才有ral-1的表达，这表明let-60/RAS在10个细胞中从使用一个效应器(LIN-45/Raf)切换到在20个细胞中使用另一个效应器(Rgl-1/Ralgef)，并且Ral-1控制着RAS依赖的细胞命运的平衡。这些观察结果为我们分析Rgl-1-ral-1信号在细胞命运诱导中的作用提供了理论基础，这是一个Ralgef-ral致癌功能的模型，据我们所知，也是第一个研究RAS不同效应器使用的潜在关键调控机制的发展模型。我们建议的研究将应用多方面的方法，利用遗传学、生化和细胞生物学方法来阐明let-60动态调节这两个不同的效应信号网络的机制和作用。我们的目标是(1)确定Rgl-1-Ral-1模块相对于Ras-Raf-MEK-ERK通路的信号特性，(2)确定Rgl-1-Ral-1控制等价基团命运决定的发育机制，以及(3)确定Rgl-1-Ral-1在20个细胞中发挥作用的遗传和分子机制。我们的研究将解决与形态原梯度反应的发育模式、RAS和Notch信号之间的平衡以及发育和癌症中效应物的差异使用等突出问题，所有这些都位于发育生物学和癌症的关键交叉点。

DESCRIPTION (provided by applicant: Ras is the most frequently mutated oncogene in human cancers and the Raf-MEK-ERK protein kinase cascade is the best characterized downstream effector function of Ras. However, it is now clear that Ras function cannot be ascribed simply to one effector pathway, and that Ras utilizes a spectrum of functionally diverse effectors, with at least five implicated in oncogenesis. What remains unclear is how effector utilization is regulated. Recent studies have implicated a critical role for guanine nucleotide exchange factors for the Ral small GTPases (RalGEFs) as effectors of Ras in cancer. Yet we know little about the interplay between RalGEF-Ral and other effector pathways, and we know even less about the normal functions of RalGEF-Ral activity during development. Genetic analyses of the Ras homolog in C. elegans, LET-60, have elucidated the role of the Raf-MEK-ERK cascade in development and provided critical clues for understanding mechanisms of Ras signaling in mammalian cells. Since essentially all known components of RalGEF-Ral signaling are conserved in C. elegans, this prompted our interest in elucidating a role for the evolutionarily conserved C. elegans RGL-1 (RalGEF) and RAL-1 (Ral) in LET-60-regulated development. Our preliminary results indicate that, unexpectedly, RGL-1-RAL-1 signaling antagonized rather than cooperated with the Raf-MEK-ERK signal in inducing 10 cell fate in the vulva, and does so in parallel or downstream to the ERK substrate, the LIN- 31/HNF transcription factor. Additionally, our results suggest that RAL-1 is expressed only in 20 cells neighboring 10 vulval cells, suggesting that LET-60/Ras switches from the use of one effector (LIN-45/Raf) in 10 cells to another (RGL-1/RalGEF) in 20 cells, and that RAL-1 controls the balance of Ras-dependent cell fates. These observations provide the rationale for our analysis of the role of RGL-1-RAL-1 signaling in cell fate induction, a model for RalGEF-Ral oncogenic functions, and to our knowledge the first developmental model to study the potentially critical regulatory mechanism of differential effector usage by Ras. We propose studies that will apply a multi-faceted approach that utilizes genetic, biochemical and cell biological methods to elucidate the mechanisms and roles by which LET-60 dynamically regulates these two distinct effector- signaling networks. We propose aims to (1) determine the signaling properties of the RGL-1-RAL-1 module relative to the Ras-Raf-MEK-ERK pathway, (2) determine the developmental mechanisms by which RGL-1- RAL-1 controls equivalence group fate decisions, and (3) determine the genetic and molecular mechanism of RGL-1-RAL-1 function in 20 cells. Our studies will address outstanding questions of developmental patterning in response to a morphogen gradient, balance between Ras and Notch signaling, and differential use of effectors in development and cancer, all of which lie at a critical intersection between developmental biology and cancer.