Novel Mechanisms Regulating G Protein and MAP Kinase Signaling

描述(申请人提供)：G蛋白是关键的分子开关，调节细胞对各种信号的反应，包括光、气味、味道、激素和神经递质。精确调节G蛋白介导的信号强度和持续时间对于正常的生理反应和疾病预防至关重要。这项建议的目标是利用遗传易驯化的生物酵母作为模型系统，揭示G蛋白调节的新的和根本上重要的机制。主要的假设是G蛋白信号是通过G蛋白亚基的泛素化来调节的。这一假说是基于最近在酵母中观察到的G(Gpa1)和G(Ste4)亚基都是泛素化的，而阻断G蛋白泛素化的突变体会深刻地影响信号转导。提出了三个具体目标：目标1：G(Ste4泛素化)的功能作用是什么？抗泛素化的Ste4截断突变体对信息素刺激高度敏感。我们的假设是，依赖于刺激的Ste4泛素化是一种下调信息素信号的机制。为了测试这一点，我们将确定Ste4泛素化所需的关键残基，并产生专门阻止Ste4泛素化的点突变。然后，我们将通过对野生型Ste4和阻断其泛素化的Ste4的点突变的信号、亚细胞定位、结合能力和稳定性的详细比较来确定Ste4泛素化的后果。目标2：G(Ste4)如何以泛素化为目标？步骤4泛素化是在信息素刺激下诱导的。我们的假设是信息素诱导Ste4的磷酸化或它与G(Gpa1)的解离触发了它的泛素化。为了测试这一点，我们将检测Ste4磷酸化位点突变体、缺乏负责Ste4磷酸化的激酶的突变体和缺乏G(Gpa1)的突变体中的Ste4泛素化。或者，信息素刺激可以直接激活泛素化酶。为了测试这一点，我们将确定信息素处理是否在体外和体内改变了相关的E2和E3酶的活性。目的3：为什么激活G蛋白信号需要Rsp5？在酵母中破坏E3连接酶Rsp5会严重减弱G蛋白步骤中的信号。Gpa1是由Rsp5单一泛素化的，在内体具有积极的信号转导作用。我们的假设是，Rsp5催化的单一泛素化是靶向Gpa1到内体从而发出信号所必需的。这一假设将通过确定干扰Rsp5对Gpa1的内体定位和信号转导的影响来检验。Rsp5催化的泛素化可能调节G蛋白激活的替代可能性也将被测试。 公共卫生相关性：G蛋白介导的信号通路缺陷可导致多种疾病和紊乱，包括心脏病、高血压、失明和内分泌紊乱。该项目将揭示调节G蛋白信号的新机制，这将为理解疾病机制和帮助开发新的治疗方法提供重要的见解。

DESCRIPTION (provided by applicant): G proteins are key molecular switches that mediate cellular responses to a wide variety of signals including light, odor, taste, hormones, and neurotransmitters. Precise regulation of the intensity and duration of G protein-mediated signaling is critical for normal physiological responses as well as for disease prevention. The goal of this proposal is to uncover novel and fundamentally important mechanisms for G protein regulation, using the genetically tractable organism yeast as a model system. The main hypothesis is that G protein signaling is regulated via ubiquitination of G protein subunits. The hypothesis is based on recent observations in yeast that both G( subunit (Gpa1) and G( subunit (Ste4) are ubiquitinated, and mutants that block G protein ubiquitination profoundly affect signaling. Three specific aims are proposed: Aim 1: What is the functional role of G( Ste4 ubiquitination? A truncation mutant of Ste4 that is resistant to ubiquitination is hypersensitive to pheromone stimulation. Our hypothesis is that stimulus- dependent ubiquitination of Ste4 serves as a mechanism to down-regulate pheromone signaling. To test this, we will identify critical residues required for Ste4 ubiquitination and generate point-mutants that specifically block Ste4 ubiquitination. We will then determine the consequences of Ste4 ubiquitination by a detailed comparison of signaling, subcellular localization, binding ability and stability of wild type Ste4 versus point mutants of Ste4 that block its ubiquitination. Aim 2: How is G( Ste4 targeted for ubiquitination? Ste4 ubiquitination is induced upon pheromone stimulation. Our hypothesis is that pheromone-induced phosphorylation of Ste4 or its dissociation from the G( Gpa1 triggers its ubiquitination. To test this, we will examine Ste4 ubiquitination in Ste4 phosphorylation-sites mutants, mutants that lack kinase responsible for Ste4 phosphorylation, and mutants that lack the G( Gpa1. Alternatively, pheromone stimulation may directly activate the ubiquitinating enzymes. To test this, we will determine whether pheromone treatment alters activity of responsible E2 and E3 enzymes in vitro and in vivo. Aim 3: Why is Rsp5 required for the activation of G protein signaling? Disrupting E3 ligase Rsp5 in yeast severely diminishes signaling at the step of G protein. Gpa1 is mono-ubiquitinated by Rsp5 and has a positive signaling role at endosome. Our hypothesis is that Rsp5-catalyzed mono- ubiquitination is required for targeting Gpa1 to endosome to signal. This hypothesis will be tested by determining the effect of disrupting Rsp5 on endosomal localization and signaling of Gpa1. Alternative possibility that Rsp5-catalyzed ubiquitination may regulate G protein activation will also be tested. PUBLIC HEALTH RELEVANCE: Defects in G protein-mediated signaling pathways can lead to a variety of diseases and disorders including heart diseases, hypertension, blindness and endocrine disorders. This project will reveal novel mechanisms that regulate G protein signaling, which will provide significant insights for the understanding of disease mechanisms and aid in the development of new therapeutics.