Regulation of MAP Activity by EGFR Endocytosis

描述（由申请人提供）：丝裂原活化蛋白激酶（MARK）是主要的效应蛋白，参与细胞增殖、分化和凋亡。MARK信号的改变导致各种病理生理状况的发展。通过mapk的信号主要由生长因子控制，特别是EGF。EGF受体（EGFR）激活触发信号转导事件网络，同时加速EGF受体复合物的内吞作用。有人提出，内吞运输调节了MARK信号的强度和持续时间，但这种调节的机制尚不清楚。此外，这两个特征（信号持续时间和强度）都依赖于MARK支架蛋白的适当功能，但它们在信号传播中的作用尚未完全阐明。该项目的主要目标是确定内吞作用在激活MARK级联中的作用，并确定内吞作用和信号传导的调节机制，这对癌症的发生和进展至关重要。我们将分析EGFR激活和内吞运输如何提供MARK通路信号的空间和时间控制。我们将使用高分辨率显微镜的定量方法，荧光共振能量转移（FRET），这是我们实验室开发的，以前曾用于证明活细胞内体中EGFR信号复合物的存在。在这里，我们提出了一种新的siRNA敲低和拯救（KDAR）策略来识别参与MARK激活的内吞机制。我们实验室开发的一些试剂和新方法将使我们能够在生理表达水平条件下及时表征MAPK1/2模块的几个成分在活细胞中的定位。

DESCRIPTION (provided by applicant): Mitogen-activated protein kinases (MARK) are major effector proteins, involved in cell proliferation, differentiation and apoptosis. Alterations in MARK signaling lead to development of various patho-physiological conditions. Signaling through MAPKs is predominantly controlled by growth factors, in particular, EGF. EGF receptor (EGFR) activation triggers a network of signal transduction events along with accelerated endocytosis of EGF-receptor complexes. It is proposed that endocytic trafficking regulates the intensity and duration of MARK signaling, but the mechanisms of this regulation are not understood. Moreover, both characteristics (signaling duration and intensity) have been shown to depend on the proper function of MARK scaffold proteins, yet their role in signal propagation has not been fully elucidated. The main goal of this project is to identify the role of endocytosis in activation of the MARK cascade and to define the regulatory mechanisms of endocytosis and signaling that are critical for cancer development and progression. We will analyze how EGFR activation and endocytic trafficking provides spatial and temporal control of MARK pathway signaling. We will use a quantitative method of high-resolution microscopy, fluorescence resonance energy transfer (FRET), that has been developed in our lab and has been previously used to demonstrate the presence of EGFR signaling complexes in endosomes of living cells. Here we propose a novel siRNA Knock Down And Rescue (KDAR) strategy to identify involvement of endocytic machinery in MARK activation. A number of reagents and new methodologies developed in our lab will allow us to characterize the localization of several components of MAPK1/2 module in time in living cells under conditions of physiological expression levels. The research proposed here will provide new insight into the specific mechanisms of regulation of the signal-transduction events by endocytic trafficking. The purpose of this research is to understand the regulatory mechanisms of the molecular cascade that affect tumor progression. Results from this study will define requirements of this intracellular signaling pathway for normal cell function, which will lead to development of new diagnostic markers work described in the Research Proposal will be conducted at the University of Colorado Health Sciences Center, Aurora Colorado