Coordinated regulation of MAP kinase signaling by ubiquitination

描述（由申请人提供）：泛素化是一种翻译后修饰，可诱导蛋白酶体降解其靶蛋白。近年来，泛素化的功能已经扩展到包括膜运输，蛋白-蛋白相互作用，基因转录和激酶激活的作用。三种酶介导泛素化：E1泛素活化酶、E2泛素结合酶和E3泛素连接酶，这决定了泛素化的特异性。Skp1/Cullin/F-box E3泛素连接酶复合物（SCF）的突变与许多人类疾病有关，包括多种形式的癌症、免疫疾病如糖尿病和狼疮、神经系统疾病如帕金森病、病毒感染和心肌病。我们的实验室最近证明，一个功能性的SCF复合体是需要适当的信息素反应在出芽酵母酿酒酵母。酵母信息素反应途径是表征最明确的信号转导途径之一，由典型的G蛋白偶联受体（GPCR）、异源三聚体G蛋白和丝裂原活化蛋白激酶（MAPK）信号级联组成。此外，该信号通路已被用于鉴定几乎所有真核生物中保守的多种细胞信号传导机制。我们和其他人已经证明，在信息素反应途径中，SCF复合物是多种信号成分泛素化所必需的，包括G1亚基Gpa1、MAPK激酶Ste7、支架蛋白Ste5和效应物Far1。此外，我们最近在实验室中获得的数据表明，泛素化改变了Ste7激活下游MAPKs Fus3和Kss1的选择性。基于这些数据，我们假设scf介导的Ste7泛素化调节其选择性激活下游信号蛋白的能力。我们进一步假设SCF是MAPK信号级联的主要调节器，同时作用于多个通路成分，以决定整个通路的输出。这一假设将通过以下具体目标来解决：目标#1：确定泛素化如何调节信息素途径MAPKK Ste7的活性和特异性。目标2：系统地识别信息素反应途径中被SCF E3泛素连接酶复合物泛素化的所有蛋白质。拟议的研究将定义泛素化如何影响靶蛋白的信号行为，确定泛素化对整个信号通路输出的贡献，并扩大我们对高度保守的MAPK级联是如何调节的理解。

DESCRIPTION (provided by applicant): Ubiquitination is a posttranslational modification that instigates proteasomal degradation of its target proteins. In recent years, the function of ubiquitination has expanded to include roles in membrane trafficking, protein-protein interactions, gene transcription, and kinase activation. Three enzymes mediate ubiquitination: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase, which determines the specificity of ubiquitination. Mutations of Skp1/Cullin/F-box E3 ubiquitin ligase complexes (SCF) have been linked to many human diseases, including multiple forms of cancer, immune disorders such as diabetes and lupus, neurological disorders like Parkinson's disease, viral infections, and cardiac myopathies. Our lab has recently demonstrated that a functional SCF complex is required for proper pheromone response in the budding yeast S. cerevisiae. The yeast pheromone response pathway is one of the best-characterized signal transduction pathways, consisting of a canonical G-protein coupled receptor (GPCR), heterotrimeric G- protein, and mitogen-activated protein kinase (MAPK) signaling cascade. Moreover, this signaling pathway has been used to identify multiple cell signaling mechanisms that are conserved in virtually all eukaryotes. We and others have demonstrated that the SCF complex is required for ubiquitination of multiple signaling components within the pheromone response pathway, including the G1 subunit Gpa1, the MAPK kinase Ste7, the scaffold protein Ste5, and the effector Far1. Further, we have recently generated data in our lab demonstrating that ubiquitination alters the selectivity of Ste7 activation of the downstream MAPKs Fus3 and Kss1. Based on these data, we hypothesize that SCF-mediated ubiquitination of Ste7 regulates its ability to selectively activate downstream signaling proteins. We further hypothesize that SCF is a master regulator of the MAPK signaling cascade, acting simultaneously on multiple pathway components to dictate overall pathway output. This hypothesis will be addressed through the following specific aims: Aim #1: Determine how ubiquitination modulates the activity and specificity of the pheromone pathway MAPKK Ste7. Aim #2: Systematically identify all proteins in the pheromone response pathway that are ubiquitinated by the SCF E3 ubiquitin ligase complex. The proposed studies will define how ubiquitination influences the signaling behavior of a target protein, determine the contribution of ubiquitination to overall signaling pathway output, and expand our understanding of how the highly-conserved MAPK cascade is regulated. PUBLIC HEALTH RELEVANCE: Cellular signaling pathways regulate how cells respond to their environment. Incorrect cellular signaling causes many human diseases including cancer, immune disorders like diabetes and lupus, and neurological disorders such as Parkinson's disease. This proposal aims to identify a new mechanism that regulates cellular signaling and possibly reveal new ways to modulate signaling pathways to combat disease.