ROLE OF SHOC2 PROTEIN IN SPATIO-TEMPORAL REGULATION OF ERK KINASE CASCADE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 表皮生长因子（EGF）受体的激活触发信号转导事件的网络，包括激活MAPK级联沿着加速EGF-受体复合物的内吞作用。MEK和ERK蛋白定位于内体，然而内体靶向的精确机制和作用尚不清楚。此外，据报道，Ras-RAF-MEK-ERK级联的成员被组织成由支架蛋白调控的信号转导模块。支架蛋白的建议作用是通过将激酶-底物伴侣引入这些模块来增强相互作用的特异性和选择性。我们试图阐明内吞参与调节MAPK信号通路的支架蛋白Shoc 2。 Shoc 2是ERK 1/2激活和信号传导的正调节因子。我们证明了Shoc 2在EGFR激活后重新定位到一个内体亚组。这种蛋白质在其控制MAPK活化的能力方面是独特的，MAPK活化也受到内吞运输的调节。然而，人们对这一机制知之甚少。从本质上讲，我们的目标将是通过利用各种生化测定和先进的荧光显微镜方法来确定Shoc 2的内体定位对MAPK活性的功能重要性。我们将分析Shoc 2的空间调控机制，并在肿瘤细胞中重组Shoc 2，研究Shoc 2对肿瘤细胞MAPK激活的贡献。一些试剂和新开发的方法将使我们能够实现这一目标。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Activation of Epidermal Growth Factor (EGF) Receptor triggers a network of signal transduction events including activating the MAPK cascade along with accelerating endocytosis of EGF-receptor complexes. MEK and ERK proteins are localized on endosomes, however the precise mechanism and the role of endosomal targeting are yet to be understood. In addition, members of the Ras-RAF-MEK-ERK cascade are reported to be organized into signal-transducing modules regulated by scaffold proteins. The suggested role of scaffold proteins is to enhance the specificity and the selectivity of interactions by bringing kinase-substrate partners into these modules. We seek to elucidate endocytosis involvement in regulating of the MAPK signaling pathway by the scaffold protein Shoc2. Shoc2 is a positive regulator of ERK1/2 activation and signaling. We demonstrated that Shoc2 relocates to a subset of endosomes upon EGFR activation. This protein is unique in its ability to control MAPK activation that is also regulated by endocytic trafficking. However, the mechanism is poorly understood. Essentially, our goal will be to define the functional importance of endosomal localization of Shoc2 for MAPK activity by utilizing various biochemical assays and advanced methods of fluorescence microscopy. We will analyze the mechanism of the spatial regulation of Shoc2 and reconstitute Shoc2 in cancer cells to study the contribution of Shoc2 to MAPK activation in tumor cells. A number of reagents and newly developed methodologies will allow us to pursue this goal.