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，Raf-MEK-ERK蛋白激酶级联是Ras最具特征的下游效应功能。然而，现在很清楚，Ras的功能不能简单地归因于一种效应途径，Ras利用了一系列功能多样的效应物，其中至少有五种与肿瘤发生有关。目前尚不清楚的是如何调节效应剂的利用。最近的研究表明，鸟嘌呤核苷酸交换因子作为Ras在癌症中的效应物，在Ral小gtp酶（RalGEFs）中起着关键作用。然而，我们对RalGEF-Ral与其他效应通路之间的相互作用知之甚少，对发育过程中RalGEF-Ral活性的正常功能了解得更少。对秀丽隐杆线虫LET-60中Ras同源基因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信号传导的机制提供了重要线索。由于基本上所有已知的RalGEF-Ral信号的成分在秀丽隐杆线虫中都是保守的，这促使我们有兴趣阐明进化保守的秀丽隐杆线虫RGL-1 （RalGEF）和Ral -1 （Ral）在let -60调控的发育中的作用。我们的初步结果表明，出乎意料的是，RGL-1-RAL-1信号在外阴诱导10个细胞命运的过程中是拮抗而不是与Raf-MEK-ERK信号合作的，并且与ERK底物LIN- 31/HNF转录因子平行或下游。此外，我们的研究结果表明，RAL-1仅在10个外阴细胞附近的20个细胞中表达，这表明LET-60/Ras在10个细胞中从使用一种效应物（LIN-45/Raf）切换到使用另一种效应物（RGL-1/RalGEF），在20个细胞中，RAL-1控制着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基础，这是一个研究RalGEF-Ral致癌功能的模型，据我们所知，这是第一个研究Ras差异效应物使用的潜在关键调控机制的发育模型。我们建议研究将采用多方面的方法，利用遗传、生化和细胞生物学方法来阐明LET-60动态调节这两种不同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通路的信号特性，(2)确定RGL-1-RAL-1控制等效群命运决定的发育机制，以及(3)确定RGL-1-RAL-1功能在20个细胞中的遗传和分子机制。我们的研究将解决发育模式响应形态梯度的突出问题，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，所有这些都处于发育生物学和癌症之间的关键交叉点。

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. 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.