The Ral small GTPase in C. elegans development

描述（由申请人提供：Ras 是人类癌症中最常见的突变癌基因，而 Raf-MEK-ERK 蛋白激酶级联是 Ras 下游效应器功能特征最明确的。然而，现在很清楚 Ras 功能不能简单地归因于一个效应器途径，并且 Ras 利用一系列功能多样的效应器，其中至少有 5 个与肿瘤发生有关。目前尚不清楚的是如何调节效应器的利用。最近的研究表明了一个关键的然而，我们对 RalGEF-Ral 和其他效应途径之间的相互作用知之甚少，而且我们对 RalGEF-Ral 活性在发育过程中的正常功能知之甚少。对线虫中 Ras 同源物 LET-60 的遗传分析阐明了 Raf-MEK-ERK 级联在癌症中的作用。由于基本上所有已知的 RalGEF-Ral 信号传导成分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，RGL-1-RAL-1 信号传导意外地发挥了作用。 RAL-1 与 Raf-MEK-ERK 信号拮抗而不是协同作用，诱导外阴 10 个细胞的命运，并且与 ERK 底物 LIN-31/HNF 转录因子并行或下游。此外，我们的结果表明，RAL-1 仅在与 10 个外阴细胞相邻的 20 个细胞中表达，表明 LET-60/Ras 在 10 个细胞中从使用一种效应器 (LIN-45/Raf) 转变。 20 个细胞中的另一个（RGL-1/RalGEF），并且 RAL-1 控制 Ras 依赖性细胞命运的平衡，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依据，这是 RalGEF-Ral 致癌功能的模型，也是我们所知的第一个研究 Ras 差异效应器使用的潜在关键调节机制的研究。利用遗传、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，我们提出的目标是 (1) 确定 RGL-1-RAL-1 模块相对于 Ras-Raf-MEK-ERK 途径的信号传导特性，(2) 确定 RGL-1-RAL-1 控制等价组命运决定的发育机制，以及 (3) 确定 RGL-1-RAL-1 模块的遗传和分子机制。 RGL-1-RAL-1 在 20 个细胞中的功能将解决形态发生素梯度的发育模式、Ras 和 Notch 信号之间的平衡以及发育和癌症中效应器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之间的关键交叉点。 公共健康相关性：叙述 Ras 是癌症中最常见的突变癌基因，重要的 Ras 效应子 RalGEF 的作用是激活 Ral 小 GTP 酶，促进肿瘤发生和转移。然而，关于 RalGEF-Ral 信号如何促进肿瘤发生，甚至 RalGEF-Ral 在动物中发挥什么功能，人们知之甚少。我们的研究将探讨 RalGEF-Ral 的体内功能及其与 Ras 在最著名的细胞命运诱导系统之一中的相互作用。这些研究将深入了解 RalGEF-Ral 信号传导（一个潜在的药物靶点）先前未表征的发育和分子机制。

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. PUBLIC HEALTH RELEVANCE: Narrative Ras is the most commonly mutated oncogene in cancer,and an important Ras effector, RalGEF, functions to activate the Ral small GTPase to promote oncogenesis and metastasis. However, little is known about how RalGEF-Ral signaling promotes tumorigenesis, or even what functions are performed by RalGEF-Ral in an animal. Our studies will address the in vivo function of RalGEF-Ral and its interactions with Ras in one of the best known cell fate induction systems. These studies will provide insights into the previously uncharacterized developmental and molecular mechanisms of RalGEF-Ral signaling, a potential pharmaceutical target.