G Protein Signaling at the endosome

描述（由申请人提供）：G蛋白偶联受体是治疗心血管和精神疾病的主要靶点。这类受体将信号传递到多种效应酶，包括促分裂原活化蛋白激酶（MAPK）。该建议的重点是调节酵母中的两个MAPK，Fus 3和Kss 1。这两种MAPK都被相同的信息素刺激激活。然而，尽管Fus 3的激活需要激酶支架（Ste 5），但Kss 1不需要。 我们的研究揭示了Fus 3完全激活所需的另外四种蛋白质，而不是Kss 1;这些蛋白质包括两种磷脂酰肌醇4-激酶（Pik 1和Stt 4）和磷脂酰肌醇3-激酶复合物的两个亚基（Vps 15和Vps 34）。我们推测，这些酶促进支架MAP激酶的选择性激活。有三个目标来解决这个假设：目标1：确定Pik 1和Stt 4的信号功能。Pik 1和Stt 4是产生磷脂酰肌醇4-P（PI 4P）的磷脂酰肌醇4-激酶。已知PI 4P与MAPK支架Ste 5结合，但MAPK激活的后果尚不清楚。我们将检验Ste 5与PI 4P结合促进Fus 3催化活性活化的假设。 目的2：研究Vps 34和PI 3 P的信号转导功能。Vps 34是磷脂酰肌醇3-激酶并产生磷脂酰肌醇3-P（PI 3 P）。PI 3 P与具有PX或FYVE结构域的一组确定的蛋白质结合。我们将测试的假设，这些PI 3 P结合蛋白之一是专门需要Fus 3催化活性。 目的3：确定Vps 15的信号传导功能。Vps 15是Vps 34功能以及Fus 3激活所需的蛋白激酶。我们将测试的假设，Vps 15的自磷酸化是PI 3 P生产所需的。此外，我们将确定其他通路组件磷酸化的Vps 15，并确定磷酸化如何影响MAPK活性和底物特异性。 本提案研究了酵母中G蛋白和MAPK信号装置的新组分。在酵母中发现的机制通常在更复杂的生物体中重现，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。

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.