Investigating KA1 domain-mediated regulation of MARK/PAR1 kinases

 描述（由申请人提供）：MARK/PAR 1激酶在高等生物体的发育中占据关键的信号节点，并且已经与包括自闭症、阿尔茨海默氏症、癌症和心脏病在内的多种疾病状态相关联。该提案的目标是阐明激酶相关1（KA 1）结构域的结构和功能作用，该结构域位于MARK/PAR 1和相关激酶的C-末端。该模块最近已被证明与磷脂和蛋白质配体相互作用，但也被提出为所连接的激酶结构域提供自抑制功能。初步研究已经确定，一个单独纯化的KA 1模块结合MARK 1激酶结构域，并能抑制体外活性。我们假设激酶自身抑制在KA 1结构域与脂质和/或蛋白质配体结合后得到缓解，并计划严格评估MARK/PAR 1激酶的这些特征。该项目的目的是（i）通过一系列结构和生物物理方法充分表征MARK 1激酶和KA 1结构域之间的相互作用，以及（ii）通过体外动力学和体内基于细胞的方法确定KA 1结构域在MARK 1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会破坏自抑制并激活激酶。这些研究将阐明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MARK/PAR 1激酶的特异性变构抑制剂提供基础。

 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.