Receptor Interaction with GTP-Regulatory Proteins

描述（由申请人提供）：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。该建议的重点是定义MAPK信号网络在控制综合生理反应中的作用。MAPK是由MAPK、MAP 2K和MAP 3 K组成的三激酶级联的成员。MAP 3 K整合响应于GTP酶的MAPK信号传导网络与用于控制生理反应的平行信号传导途径。MEKK 1，MAP 3 K，是网络中唯一编码具有E3连接酶活性的激酶结构域和RING结构域的激酶。MEKK 1也是所有基因组中唯一编码结合和泛素化蛋白底物的SWIM-RING结构域结构的激酶。MEKK 1是一种特异性底物磷酸化的激酶，也是一种具有独特SWIM-RING结构域结构的E3连接酶，用于特异性底物的泛素化。MEKK 1通过调节Fra-2和JunB的水平来调节AP-1转录复合物的组成。MEKK 1也能够靶向c-Jun进行泛素化和降解。我们的假设是MEKK 1的功能是刺激特定MAPK途径（主要是JNK和ERK 1/2）的活性，协调调节特定靶底物（例如，c-Jun，JunB，Fra 2），并控制AP-1组合物以改变特异性基因表达，从而综合控制包括迁移、侵袭、伤口愈合、炎症、组织重塑和转移在内的应答。拟议的研究是一种系统的方法来定义MEKK 1如何发挥作用来控制细胞和动物中复杂的调节反应，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。目的明确MEKK 1的SWIM和RING结构域在靶蛋白结合和泛素化中的功能。方法涉及使用定量质谱和泛素化测定来定义MEKK 1 SWIM-RING结构域的蛋白质底物。目的II将提供基因组分析MEKK 1调控的基因表达使用Chromatin-IP（ChIP）结合启动子平铺阵列和单分子DNA测序。这些研究将确定MEKK 1-/-细胞与野生型细胞中Fra 2和/或JunB差异结合的基因，以及其表达因MEKK 1对AP-1组成的调控丧失而选择性改变的基因集。目的3将定义需要E3连接酶活性和MEKK 1激酶活性的生理功能。在上一个资助期产生的MEKK 1的靶向敲除为MEKK 1野生型和突变蛋白的加回提供了零背景。为此，我们开发了灵敏的测定方法来测量MEKK 1在体外和体内的功能。累积起来，这项工作代表了一个重要的MAP 3 K，MEKK 1，这是在稳态控制的关键功能和调节的机制系统分析。MEKK 1在MAP 3 Ks中具有独特的位置，我们发现MEKK 1调节AP-1的组成而不仅仅是AP-1活性，这是MAP 3 Ks如何调节细胞生理学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。确定MEKK 1不仅对MAPK活性，而且对蛋白质降解和基因表达的全局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.