Investigating KA1 domain-mediated regulation of MARK/PAR1 kinases

 描述(申请人提供)：Mark/PAR1激酶在高等生物体的发育中占据着关键的信号节点，并与包括自闭症、阿尔茨海默氏症、癌症和心脏病在内的多种疾病状态有关。这项建议的目的是阐明位于Mark/PAR1及其相关激酶C末端的KA1结构域的结构和功能。该模块最近被证明可以与磷脂和蛋白质配体相互作用，但也被提出为连接的激酶结构域提供自我抑制功能。初步研究证实，单独纯化的KA1模块确实与Mark1激活域结合，并能在体外抑制活性。我们假设，当KA1结构域与脂质和/或蛋白质配体结合时，激酶自抑制被解除，并计划严格评估Mark/PAR1激酶的这些特征。本项目的目的是(I)通过一系列的结构和生物物理方法来充分表征Mark1结构域和KA1结构域之间的相互作用，以及(Ii)通过体外动力学和基于细胞的方法来确定KA1结构域在Mark1活性中的功能作用，包括确定暴露于该结构域的脂质或蛋白质配体是否破坏自身抑制并激活该激酶。这些研究将阐明来自脂质或蛋白质配体的不同输入如何导致激酶调节，并将为设计特定的Mark/PAR1激酶变构抑制剂奠定基础。

 DESCRIPTION (provided by applicant): MARK/PAR1 kinases occupy critical signaling nodes in the development of higher organisms, and have been linked to a plethora of disease states including Autism, Alzheimer's, cancer, and heart disease. The goal of this proposal is to elucidate the structural and functional role of the kinase-associated 1 (KA1) domain, which resides at the C-terminus of MARK/PAR1 and related kinases. This module has recently been shown to interact with both phospholipid and protein ligands, but has also been proposed to provide an autoinhibitory function for the attached kinase domain. Preliminary studies have established that a separately purified KA1 module does bind the MARK1 kinase domain and can inhibit activity in vitro. We hypothesize that kinase autoinhibition is relieved upon binding o the KA1 domain to lipid and/or protein ligands and plan to rigorously assess these features of MARK/PAR1 kinases. The aims of this project are to (i) fully characterize the interaction between the MARK1 kinase and KA1 domains through a battery of structural and biophysical methods and, and (ii) establish a functional role for the KA1 domain in MARK1 activity by in vitro kinetic and in vivo cell-based methods, including determination of whether exposure to lipid or protein ligands of this domain disrupt autoinhibition and activate the kinase. These studies will shed light onto how varied inputs from lipid or protein ligands can lead to kinase regulation, and will provide the groundwork for design of specific allosteric inhibitors of MARK/PAR1 kinases.