New Mechanisms Regulating Ras and Protein Kinase A Signaling

描述（由申请人提供）：Ras蛋白是传递信号以控制多种细胞过程（包括增殖、分化和存活）的关键分子开关。精确调节Ras介导的信号传导的强度和持续时间对于正常的生理反应以及疾病预防是至关重要的。该提案的目标是使用遗传上易于处理的模式生物酵母和人类细胞生物学的互补系统来揭示Ras调节的新的和根本上重要的机制。主要的假设是Ras信号通过RasGAP的动态泛素化和去泛素化来调节。该假说基于最近的观察，即RasGAP（Ubp 2）是泛素化的，并且破坏其相互作用的去泛素化酶（Ubp 3）显著增强Ras激活。提出了三个具体目标：目标1：Ubp 3如何抑制Ras信号转导？破坏Ubp 3导致Ras的超活化; Ubp 3与Ras的负调节因子β 2相互作用，β 2经历泛素化和降解。我们的假设是Ubp 3通过去泛素化β 2并保护其免于降解来抑制Ras信号传导。为了验证这一点，我们将描述Ubp 3和BMP 2之间的相互作用，并检查Ubp 3是否去泛素化BMP 2并影响其丰度，稳定性或亚细胞定位。为了鉴定Ubp 3的其他靶点，我们将系统地鉴定Ubp 3对信号传导发挥作用所需的通路中的组分。将进一步表征识别出的任何组件。 目标2：什么 是靶向RasGAP的泛素化酶吗尽管RasGAP泛素化在调节Ras中起着关键作用，但负责泛素连接酶仍然难以捉摸。为了确定泛素化β 2的酶，首先我们将考虑Gpb 1，一种已知在β 2泛素化中起作用的蛋白质。为了确定额外的因素所需的β 2泛素化和正常的信号，我们将系统地检查酵母菌株缺乏已知的或怀疑的泛素化酶的β 2泛素化的损失和Ras介导的信号的改变。将使用体外测定法进一步分析鉴定的任何蛋白质的泛素化β 2的能力。 目的3：去泛素化是否调节人Ras信号转导？Ubp 3有一个人类同源物Usp 10，一种被认为是潜在的肿瘤抑制因子的蛋白质。为了确定在酵母中阐明的机制是否在人类中保守，并为考虑Usp 10在肿瘤发生中的作用提供新的视角，我们将研究Usp 10是否调节人类的Ras信号传导。具体来说，我们将测试沉默Usp 10表达是否会影响人类细胞中的Ras信号传导，以及它是否会通过去泛素化和保护RasGAP神经纤维蛋白（人类中的RasGAP 2同源物）免受降解来实现这一点。

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.