G Protein Signaling at the endosome

描述(申请人提供)：G蛋白偶联受体是治疗心血管和精神疾病的主要靶点。这类受体向多种效应酶传递信号，包括丝裂原活化蛋白激酶(MAPKs)。这项建议的重点是调控酵母中的两个MAPK，Fus3和Kss1。这两种MAPK都被相同的信息素刺激激活。然而，虽然Fus3的激活需要一个激酶支架(Ste5)，但Kss1并不需要。我们的研究揭示了Fus3而不是Kss1完全激活所需的另外四种蛋白质；其中包括两个磷脂酰肌醇4-激酶(Pik1和Stt4)和磷脂酰肌醇3-激酶复合体的两个亚单位(Vps15和Vps34)。我们推测，这些酶促进支架MAP激酶的选择性激活。有三个目的可以解释这一假说：目的1：确定Pik1和Stt4的信号功能。Pik1和Stt4是磷脂酰肌醇4-激酶，可产生磷脂酰肌醇4-P(PI4P)。已知PI4P与MAPK支架Ste5结合，但MAPK激活的后果尚不清楚。我们将检验Ste5与PI4P结合促进Fus3催化活性激活的假设。目的2：确定Vps34和PI3P的信号功能。Vps34是一种磷脂酰肌醇3-激酶，可产生磷脂酰肌醇3-P(PI3P)。PI3P与一组具有PX或FYVE结构域的蛋白质结合。我们将检验这样的假设，即这些PI3P结合蛋白中的一个是Fus3催化活性所特需的。目的3：确定Vps15的信号转导功能。Vps15是Vps34功能和Fus3激活所需的一种蛋白激酶。我们将检验这一假设，即PI3P的产生需要Vps15的自动磷酸化。此外，我们还将确定Vps15磷酸化的其他途径成分，并确定磷酸化如何影响MAPK活性和底物特异性。这项建议研究了酵母中G蛋白和MAPK信号转导装置的新成分。在酵母中发现的机制通常在更复杂的生物体中重述，因此这里阐明的活动可能适用于人类的激素和神经递质功能。 与公共健康相关：在这项提案中，我们将定义细胞对激素信号做出反应的新方式。对这些新信号蛋白的详细分析将为了解人类疾病的病因和最有效的治疗方法奠定基础。

DESCRIPTION (provided by applicant): G protein-coupled receptors are major targets for the treatment of cardiovascular and psychiatric disorders. Receptors of this class transmit signals to a variety of effector enzymes including mitogen activated protein kinases (MAPKs). This proposal is focused on the regulation of two MAPKs in yeast, Fus3 and Kss1. Both of these MAPKs are activated by the same pheromone stimulus. However while activation of Fus3 requires a kinase scaffold (Ste5) Kss1 does not. Our investigations have revealed four additional proteins required for full activation of Fus3 but not Kss1; these include two phosphatidylinositol 4-kinases (Pik1 & Stt4) and two subunits of the phosphatidylinositol 3-kinase complex (Vps15 & Vps34). We postulate that these enzymes promote the selective activation of scaffolded MAP kinases. There are three aims that address the hypothesis: Aim 1: Determine the signaling function of Pik1 and Stt4. Pik1 and Stt4 are phosphatidylinositol 4- kinases, which produce phosphatidylinositol 4-P (PI4P). PI4P is known to bind to the MAPK scaffold Ste5, but the consequences for MAPK activation are not known. We will test the hypothesis that Ste5 binding to PI4P promotes the activation of Fus3 catalytic activity. Aim 2: Determine the signaling function of Vps34 and PI3P. Vps34 is a phosphatidylinositol 3-kinase and produces phosphatidylinositol 3-P (PI3P). PI3P binds to a defined set of proteins having PX or FYVE domains. We will test the hypothesis that one of these PI3P-binding proteins is specifically required for Fus3 catalytic activity. Aim 3: Determine the signaling function of Vps15. Vps15 is a protein kinase required for Vps34 function, as well as for activation of Fus3. We will test the hypothesis that autophosphorylation of Vps15 is required for PI3P production. In addition, we will identify other pathway components phosphorylated by Vps15, and determine how phosphorylation affects MAPK activity and substrate specificity. This proposal investigates new components of the G protein and MAPK signaling apparatus in yeast. Mechanisms discovered in yeast are typically recapitulated in more complex organisms, so the activities elucidated here will likely apply to hormone and neurotransmitter function in humans. PUBLIC HEALTH RELEVANCE: In this proposal we will define new ways that cells respond to hormone signals. Detailed analysis of these new signaling proteins will lay the foundation for understanding the causes and the most effective treatments of human disease.