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New Mechanisms Regulating Ras and Protein Kinase A Signaling

调节 Ras 和蛋白激酶 A 信号转导的新机制

基本信息

批准号：
8496943
负责人：
Yuqi Wang
金额：
$ 30万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2018-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Ras proteins are key molecular switches that transmit signals to control a variety of cellular processes including proliferation, differentiation, and survival. Precise regulation of the intensity and duration of Ras-mediated signaling is critical fo normal physiological responses as well as for disease prevention. The goal of this proposal is to uncover novel and fundamentally important mechanisms for Ras regulation, using both the genetically tractable model organism yeast and the complimentary system of human cell biology. The main hypothesis is that Ras signaling is regulated via dynamic ubiquitination and deubiquitination of RasGAP. The hypothesis is based on recent observations that RasGAP (Ira2) is ubiquitinated and disrupting its interacting deubiquitinase (Ubp3) markedly enhances Ras activation. Three specific aims are proposed: Aim 1: How does Ubp3 inhibit Ras signaling? Disrupting Ubp3 leads to hyperactivation of Ras; Ubp3 interacts with Ira2, a negative regulator of Ras that undergoes ubiquitination and degradation. Our hypothesis is that Ubp3 inhibits Ras signaling via deubiquitinating Ira2 and protecting it from degradation. To test this, we will characterize the interaction between Ubp3 and Ira2, and examine whether Ubp3 deubiquitinates Ira2 and affects its abundance, stability, or subcellular localization. To identify other targets o Ubp3, we will systematically identify components in the pathway that are required for Ubp3 to exert its effect on signaling. Any component identified will be further characterized. Aim 2: What are the ubiquitinating enzymes that targets RasGAP? Despite the key role of RasGAP ubiquitination in regulating Ras, the responsible ubiquitin ligating enzymes remain elusive. To identify the enzymes that ubiquitinate Ira2, initially we will consider Gpb1, a protein known to play a role in Ira2 ubiquitination. To identify additional factors required for Ira2 ubiquitinationand normal signaling, we will systematically examine yeast strains lacking known or suspected ubiquitinating enzymes for loss of Ira2 ubiquitination and for an alteration of Ras-mediated signaling. Any proteins identified will be further analyzed using in vitro assay for their ability o ubiquitinate Ira2. Aim 3: Is human Ras signaling regulated by deubiquitination? Ubp3 has a human homolog Usp10, a protein implicated as a potential tumor suppressor. To determine if the mechanisms elucidated in yeast is conserved in humans and to provide a new perspective for considering the role of Usp10 in tumorigenesis, we will examine if Usp10 regulates Ras signaling in humans. Specifically, we will test whether silencing Usp10 expression affects Ras signaling in human cells, and whether it does so via deubiquitinating and protecting RasGAP neurofibromin (Ira2 homolog in human) from degradation.

描述(申请人提供)：RAS蛋白是关键的分子开关，它传递信号来控制包括增殖、分化和生存在内的各种细胞过程。精确调节RAS介导的信号强度和持续时间对于正常的生理反应以及疾病预防都是至关重要的。这项建议的目标是利用遗传上易驯化的模式生物酵母和人类细胞生物学的互补系统，揭示RAS调控的新的和根本上重要的机制。主要的假设是Ras信号是通过RasGAP的动态泛素化和去泛素化来调节的。这一假说是基于最近的观察，即RasGAP(IRA2)是泛素化的，破坏其相互作用的去泛素酶(Ubp3)显著增强了RAS的激活。提出了三个具体目标：目标1：Ubp3如何抑制RAS信号？破坏Ubp3导致RAS的过度激活；Ubp3与RAR2相互作用，IRA2是RAS的负调控因子，经历泛素化和降解。我们的假设是，Ubp3通过使IRA2去泛素化并保护其免受降解来抑制RAS信号。为了测试这一点，我们将表征Ubp3和IRA2之间的相互作用，并检查Ubp3去泛素化IRA2是否影响其丰度、稳定性或亚细胞定位。为了确定Ubp3的其他靶点，我们将系统地确定Ubp3在信号转导中发挥作用所需的途径成分。任何已确定的组件都将被进一步表征。目标2：什么泛素化的酶是针对RasGAP的吗？尽管RasGAP泛素化在调节RAS中起着关键作用，但负责的泛素连接酶仍然难以捉摸。为了确定使IRA2泛素化的酶，首先我们将考虑Gpb1，这是一种已知在IRA2泛素化中发挥作用的蛋白质。为了确定IRA2泛素化和正常信号转导所需的其他因素，我们将系统地检测缺乏已知或怀疑泛素化酶的酵母菌株IRA2泛素化的丢失和RAS介导的信号转导的改变。任何已鉴定的蛋白质都将通过体外实验进一步分析其泛素化IRA2的能力。目的3：人类RAS信号是否受去泛素化调节？Ubp3具有人类同源物Usp10，这是一种被认为是潜在的肿瘤抑制因子的蛋白质。为了确定酵母中阐明的机制是否在人类中保守，并为研究Usp10在肿瘤发生中的作用提供一个新的视角，我们将研究Usp10是否调节人类的RAS信号。具体地说，我们将测试沉默Usp10表达是否影响人类细胞中的RAS信号，以及它是否通过去泛素化和保护RasGAP神经纤维蛋白(人类中的IRA2同源物)免受降解而影响RAS信号。