Protein inhibitor of Raf-1 kinase

申请人简介：这项建议的目标是学习新的 确定了Raf-L激酶的抑制剂，命名为RKJP。英国皇家空军L是一名 胞浆丝氨酸/苏氨酸激酶的活性在 丝裂原活化蛋白激酶途径(MAPK)的调控。Raf-1激酶 活动受到积极和消极相互作用的严格控制 监管者。RKIP是Raf-L和MAPK信号通路的负调控因子 这是最近在我的实验室和 沃尔特·科尔奇和大卫·罗斯的实验室。在这份提案中，我概述了实验，以 研究RKJP函数的两个方面。在Aim One中，我将调查 详细说明RKJP对MAPK的调节机制 路径。具体地说，我将研究磷酸化在 Raf-RKTP相互作用的调节。有几条证据表明 RKJP与蛋白质上的磷酸化残基结合。我会就此展开调查 Raf-1的磷酸化状态是否在调节 RKIP-Raf-1相互作用。第二组实验是基于 观察到RKIP是体内的一种磷酸蛋白，是几种 体外试验中的激活酶。体外磷酸化的位置将被绘制出来。活体内 磷酸化位点将使用质量的组合来确定 光谱分析和代谢标记，然后是肽图和测序。 一旦确定了体内的位置，磷酸多肽特异的抗体将被 养大的。RKIP的体内磷酸化状态将在 各种已知的刺激MAPK活性的生理条件。在AIM 第二，我将研究RKIP在核因子-KB信号级联中的作用。我有过 获得的实验证据表明，RKIP表达水平的调节 影响核因子-kB的激活，我已经证明了RKIP与 核因子-kB级联的两个组件，NIK和TAK 1。我将采用组合 用遗传学和生化方法研究RKIP对核因子-kB的影响 发信号。这一系列调查的主要目标将是定义 RKIP在核因子-kB信号通路中的靶点，并启动对 他们是如何被监管的。总而言之，我相信这个实验 提案将为理解Raf-1信令提供一个新的句柄，并将 启动研究以揭示RKIP功能的全部范围。

APPLICANT'S DESCRIPTION: The goal of this proposal is to study the newly identified inhibitor of the Raf-l kinase designated RKJP. Raf-l is a cytoplasmic serine/threonine kinase whose activity plays a pivotal role in the control of the mitogen-activated protein kinase pathway (MAPK). Raf-1 kinase activity is tightly regulated by an interplay of positive and negative regulators. RKIP is a negative regulator of Raf-l and MAPK pathway signaling that was recently identified in a collaborative effort between my lab and the labs of Walter Kolch and David Rose. In this proposal I outline experiments to investigate two aspects of RKJP function. In Aim One I will investigate in detail the mechanism by which RKJP contributes to the regulation of the MAPK pathway. Specifically, I will investigate the role of phosphorylation in the regulation of the Raf-RKTP interaction. Several lines of evidence indicate that RKJP binds to phosphorylated residues on proteins. I will thus investigate whether the phosphorylation status of Raf- 1 plays a role in regulating the RKIP-Raf- 1 interaction. The second set of experiments is based on the observation that RKIP is a phosphoprotein in vivo and a substrate of several kinases in vitro. Sites of in vitro phosphorylation will be mapped. In vivo phosphorylation sites will be determined using a combination of mass spectrometry and metabolic labeling followed by peptide mapping and sequencing. Once in vivo sites are ascertained, phosphopeptide-specific antibodies will be raised. The in vivo phosphorylation status of RKIP will be examined under a variety of physiological conditions known to stimulate MAPK activity. In Aim Two I will investigate the role of RKIP in the NF-KB signaling cascade. I have obtained experimental evidence that modulation of RKIP expression levels affects NF-kB activation, and I have shown that RKIP physically interacts with two components of the NF-kB cascade, NIK and TAK 1. I will employ a combination of genetic and biochemical methods to study the effects of RKIP on NF-kB signaling. The primary goal of this line of investigation will be to define the targets of RKIP in the NF-kB signaling pathway, and to initiate studies into how they are regulated. In summary, I believe that the experiments in this proposal will provide a new handle for understanding Raf- 1 signaling, and will initiate studies to expose the full spectrum of RKIP functions.