KSR Scaffolding and the MAP Kinase Signaling Pathway

描述(由申请人提供)：RAS/MAP信号通路在细胞对生长因子和应激反应的反应中发挥核心作用。它在肿瘤的发展过程中也起着至关重要的作用。虽然在过去的十年里，人们对它进行了深入的研究，但许多重要的问题仍然没有得到回答。例如，虽然很明显RAS在质膜上被激活，但Raf、MEK和ERK在哪里以及何时被激活尚不清楚。第二个问题涉及信号通路的架构。虽然有人认为三激酶级联是为信号放大或“超灵敏信号”而设计的，但这些想法在很大程度上是未经检验的。最后，在信号转导方面的进展已经发现了与组织信号通路有关的支架，但它们对信号响应的大小和信号转导质量的影响尚不清楚。我们最近报道了一只基因敲除小鼠的表型，我们认为这是哺乳动物MAP激酶信号通路中的第一个支架。在这里，我们建议使用这种基因敲除的小鼠来探索支架在RAS/MAP激酶信号通路中的作用。因为我们对这些信号通路的理解将依赖于未知的生物物理参数，所以测量并确定通路每一步的这些参数将是重要的。我们的第一个目标是研究KSR在相互作用中的作用，以及对级联中每个激酶的动力学酶速率常数的影响。该项目主要涉及蛋白质的纯化以及亲和力和酶活性的生物物理测量。我们的第二个目标是研究KSR在体内的作用。首先，我们专注于确定KSR是否影响MAP激酶信号蛋白在体内的定位以及MAP激酶成分在哪里被激活。其次，我们重点研究了KSR在单细胞水平上对MAP激酶信号动力学的影响。这包括测量每个步骤之间的滞后时间、放大水平以及信号的数字/模拟质量。我们希望我们的研究将导致对信号通路的更全面的理解。由于这些通路对正常的细胞动态平衡非常关键，对这些通路的更好理解也应该有助于更好地理解当这些通路在癌症、糖尿病和自身免疫性疾病中出错时会发生什么。

DESCRIPTION (provided by applicant): The Ras/MAP kinase signaling pathway plays a central role in cell responses to growth factors as well as stress responses. It also plays a critical role in tumor progression. While it has been intensively studied over the last decade, many important questions remain unanswered. For example, while it is clear that Ras is activated at the plasma membrane, where and when Raf, MEK and ERK are activated is not known. A second issue concerns the architecture of the signaling pathway. While it has been suggested that the three-kinase cascade is designed for signal amplification or for "ultrasensitive signaling", these ideas are largely untested. Lastly, progress in signal transduction has seen the identification of scaffolds involved in organizing signaling pathways, but their effects on the magnitude of signaling responses and the quality of signal transduction is unknown. We have recently reported the phenotype of a knock-out mouse of what we believe to be the first scaffold in the mammalian MAP kinase signaling pathway. Here we propose to use this knockout mouse to explore the role of scaffolds in the Ras/MAP kinase signaling pathway. Because our understanding of these signaling pathways will rely on biophysical parameters that are not known, it will be important to measure and establish these parameters for each step of the pathway. Our first aim is to study the role of KSR in the interactions as well as on the kinetic enzymatic rate constants for each kinase in the cascade. This project mainly involves purification of proteins and biophysical measurements of affinity as well as enzymatic activity. Our second aim is to study the role of KSR in vivo. First, we focus on determining whether KSR effects the localization of MAP kinase signaling proteins in vivo as well as where MAP kinase components are activated. Secondly, we focus on effects of KSR on the kinetic of MAP kinase signaling at the single cell level. This includes measurements of the lag times between each step, the level of amplification as well as on the digital/analog quality of signaling. We hope that our studies will lead to a more comprehensive understanding of signaling pathways. Since these pathways are so critical to normal cell homeostasis, a better understanding of these pathways should also lead to a better understanding of what is happening when these pathways go awry in cancer, diabetes and autoimmune diseases.