Protein inhibitor of Raf-1 kinase

申请人的描述：本提案的目标是研究新的 鉴定的Raf-I激酶抑制剂，命名为RKJP。Raf-l是一个 细胞质丝氨酸/苏氨酸激酶，其活性在 丝裂原活化蛋白激酶途径（MAPK）的控制。Raf-1激酶 活动受到积极和消极因素的相互作用的严格调节 监管部门RKIP是Raf-I和MAPK信号通路的负调节剂 这是最近在我的实验室和 Walter Kolch和大卫罗斯的实验室。在这份提案中，我概述了一些实验， 研究RKJP功能的两个方面。在目标一，我将调查， 详细说明RKJP促进MAPK调节的机制 通路具体来说，我将研究磷酸化在 调节Raf-RKTP相互作用。几条证据表明， RKJP与蛋白质上的磷酸化残基结合。因此我会调查 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 RKIP在体内是一种磷蛋白，是几种蛋白质的底物。 体外激酶。将绘制体外磷酸化位点。体内 磷酸化位点将使用质量 通过质谱法和代谢标记，随后进行肽图谱和测序。 一旦体内位点确定，磷酸肽特异性抗体将被释放。 提出... RKIP的体内磷酸化状态将在以下条件下检查： 已知刺激MAPK活性的各种生理条件。在Aim中 第二，我将研究RKIP在NF-κ B信号级联中的作用。我有 获得的实验证据表明，RKIP表达水平的调节 影响NF-kB激活，我已经证明RKIP与 NF-κ B级联的两个组分，NIK和TAK 1。我会使用一种组合 遗传学和生物化学方法研究RKIP对NF-kB的影响 发信号。这条调查路线的主要目标是确定 RKIP在NF-kB信号通路中的靶点，并启动研究 它们是如何受到监管的。总之，我相信这个实验 该提案将为理解Raf- 1信令提供一个新的解决方案，并将 开展研究，以揭示RKIP的全部功能。

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.