Receptor Interaction with GTP-Regulatory Proteins

描述(申请人提供)：许多人类疾病是由受体和GTP酶控制的细胞中的信号网络调节不当引起的。这项建议的重点是确定MAPK信号网络在控制综合生理反应中的作用。MAPK是由MAPK、MAP2K和MAP3K组成的三个激酶级联的成员。MAP3K将响应GTP酶的MAPK信号网络与控制生理反应的并行信号通路整合在一起。MEKK1是一种MAP3K，是该网络中唯一编码具有E3连接酶活性的激活域和环域的激酶。MEKK1也是所有基因组中唯一编码游泳环结构域结构的蛋白底物结合和泛化的激酶。MEKK1既是一种用于特定底物磷酸化的激酶，也是一种具有独特的游泳环结构域结构的E3连接酶，用于特定底物的泛素化。MEKK1通过调节FrA-2和JunB的水平来调节AP-1转录复合体的组成。MEKK1还能够针对c-jun进行泛素化和降解。我们的假设是，MEKK1的功能是刺激特定的MAPK通路(主要是JNK和ERK1/2)的活性，协调调节特定靶底物(如c-Jun、JunB、Fra2)的降解，并控制AP-1的组成以改变特定的基因表达，从而对包括迁移、侵袭、伤口愈合、炎症、组织重塑和转移在内的反应进行综合控制。拟议的研究是一种系统的方法，以确定MEKK1如何在细胞和动物中控制复杂的调节反应，我们相信这将导致治疗涉及组织重塑的疾病的新的治疗策略。目的确定MEKK1的游泳区和环区与靶蛋白的结合和泛素化的功能。方法包括使用定量质谱学和泛素化分析来定义MEKK1游泳环结构域的蛋白质底物。目的利用染色质IP芯片结合启动子芯片和单分子DNA测序技术对MEKK1调控基因的表达进行基因组分析。这些研究将确定MEKK1-/-细胞中Fra2和/或JunB与野生型细胞的差异结合基因，以及其表达因失去MEKK1对AP-1组成的调节而选择性改变的基因集。目标3将定义需要E3连接酶活性和MEKK1的激酶活性的生理功能。在上一个资助期产生的MEKK1的靶向敲除为MEKK1野生型和突变型蛋白的回补提供了一个零背景。为此，我们开发了灵敏的检测方法来检测MEKK1在体外和体内的功能。总的来说，这项工作代表了对一个重要的MAP3K，MEKK1的功能和调节的机械系统分析，MEKK1在动态平衡的控制中是关键的。MEKK1在MAP3K中具有独特的地位，我们的发现是MEKK1调节AP-1的组成而不仅仅是AP-1的活性，这是MAP3K调节细胞生理以控制复杂生物反应的范式转变。 公共卫生相关性：MAPK网络的失调与许多疾病有关，包括癌症、炎症和代谢紊乱。明确MEKK1不仅对MAPK活性，而且对蛋白质降解和基因表达的全球控制将确定治疗疾病的新治疗策略。

DESCRIPTION (provided by applicant): Many human diseases result from improper regulation of signaling networks in cells controlled by receptors and GTPases. The focus for this proposal is to define the role of the MAPK signaling network in controlling integrated physiological responses. MAPKs are members of a three kinase cascade composed of the MAPK, MAP2K and MAP3K. MAP3Ks integrate the MAPK signaling network in response to GTPases with parallel signaling pathways for control of physiological responses. MEKK1, a MAP3K, is the only kinase within the network to encode a kinase domain and a RING domain with E3 ligase activity. MEKK1 is also the only kinase in all genomes to encode a SWIM-RING domain architecture for binding and ubiquitinating protein substrates. MEKK1 is both a kinase for phosphorylation of specific substrates and an E3 ligase with a unique SWIM-RING domain architecture for ubiquitination of specific substrates. MEKK1 regulates the composition of the AP-1 transcription complex by regulating the levels of Fra-2 and JunB. MEKK1 is also capable of targeting c-Jun for ubiquitination and degradation. Our hypothesis is that MEKK1 functions to stimulate the activity of specific MAPK pathways (primarily JNK and ERK1/2), coordinately regulate the degradation of specific target substrates (e.g., c-Jun, JunB, Fra2), and controls AP-1 composition to alter specific gene expression for the integrated control of responses including migration, invasion, wound healing, inflammation, tissue remodeling and metastasis. The proposed studies are a systems approach to define how MEKK1 functions to control complex regulatory responses in cells and animals, which we believe will lead to novel therapeutic strategies for the treatment of diseases that involve tissue remodeling. Aim I will define function for the SWIM and RING domains of MEKK1 for binding and ubiquitination of target proteins. Approaches involve the use of quantitative mass spectrometry and ubiquitination assays to define protein substrates for the MEKK1 SWIM-RING domains. Aim II will provide genomic analysis of MEKK1-regulated gene expression using Chromatin-IP (ChIP) combined with promoter tiling arrays and single molecule-based DNA sequencing. The studies will define genes with differential binding of Fra2 and/or JunB in MEKK1-/- cells versus wild-type cells and the gene set whose expression is selectively altered by loss of MEKK1 regulation of AP-1 composition. Aim 3 will define physiological functions requiring E3 ligase activity and the kinase activity of MEKK1. The targeted knockout of MEKK1 generated in the last funding period provides a null background for add back of MEKK1 wild-type and mutant proteins. To this end, we have developed sensitive assays to measure MEKK1 function both in vitro and in vivo. Cumulatively, this work represents a mechanistic systems analysis of the function and regulation of an important MAP3K, MEKK1, which is critical in the control of homeostasis. MEKK1 has a unique position among the MAP3Ks and our discovery that MEKK1 regulates composition of AP-1 not just AP-1 activity, is a paradigm shift in how MAP3Ks regulate cellular physiology for the control of complex biological responses. PUBLIC HEALTH RELEVANCE: Dysregulation of the MAPK network is associated with many diseases including cancer, inflammation and metabolic disorders. Defining the global control not just of MAPK activity but protein degradation and gene expression by MEKK1 will identify novel therapeutic strategies for treating disease.