Mechanisms of Regulated Membrane Targeting

描述(由申请人提供)：C2和PH结构域是普遍存在的真核靶向基序，驱动膜对接以响应第二信使。当被第二信使信号激活时，包含这些结构域的信号蛋白对接到特定的靶膜上，并控制广泛的必要细胞通路。这个继续进行的项目研究了极化细胞前沿的复杂信号电路，其中C2和PH结构域通过将数十个信号蛋白靶向适当的膜表面而发挥主导作用。由此产生的靶向是细胞迁移和趋化所普遍需要的：目前的焦点是驱动初级免疫反应的白细胞的前沿。对给定C2或PH结构域的膜靶向反应进行优化，以确保该结构域以精致的特异性以及适当的亲和力和速度对接到其目标膜上。随后，脂质结合区域在双层平面内随机扩散，总时间和距离需要与效应器分子发生有效碰撞。最后，域解除关联以终止活动。这一持续提案的一个主要目标是阐明关键前沿C2和PH结构域的膜靶向反应背后的基本分子机制。具体目标从分离的C2和PH结构域的体外研究开始，然后转移到全长蛋白质的体外研究，最后转移到活细胞中的结构域和蛋白质的研究。目的1利用自旋标记EPR测量来阐明PH结构域的第一个膜对接几何构型。目的2分析具有代表性的C2和PH结构域的膜靶向性机制，并测试控制靶向性的蛋白质-脂类相互作用的新工作模型。目的3利用创新的单分子方法剖析全长蛋白质中多个膜对接结构域的作用，并分析全长蛋白质的调控机制。目的4验证活细胞体外研究的结论，并开发新的生物传感器来分析极化细胞前沿的第二信使信号。完成这些目标将揭示膜识别在细胞迁移、免疫反应、炎症和癌症中的关键原理。与公共健康相关：这项建议调查了在广泛的细胞通路中调节信号蛋白的膜靶向的普遍存在的C2和PH结构域基序。某些C2和PH结构域也在人类疾病中发挥核心作用，如炎症和癌症；例如，AKT1 PH结构域的高度致癌突变E17K以一种未知的机制驱动结构性质膜靶向，产生导致其致癌的激酶过度激活。初步结果显示，E17K突变通过改变PH域的靶脂质特异性来增强膜靶向性，从而确定了这一重要致癌突变的分子机制。

DESCRIPTION (provided by applicant): C2 and PH domains are ubiquitous eukaryotic targeting motifs that drive membrane docking in response to a second messenger. When activated by second messenger signals, signaling proteins containing these domains dock to specific target membranes and control a wide array of essential cellular pathways. This continuing project investigates the complex signaling circuit at the leading edge of polarized cells, where C2 and PH domains play a dominant role by targeting dozens of signaling proteins to the appropriate membrane surface. The resulting targeting is universally required for cell migration and chemotaxis: the present focus is the leading edge of leukocytes that drive the primary immune response. The membrane targeting reaction of a given C2 or PH domain is optimized to ensure that the domain docks with exquisite specificity, as well as the appropriate affinity and speed, to its target membrane. Subsequently, the lipid-bound domain diffuses randomly in the bilayer plane for the total time and distance needed to undergo productive collisions with effector molecules. Finally, the domain dissociates to terminate activity. One broad goal of this continuing proposal is to elucidate the fundamental molecular mechanisms underlying the membrane targeting reactions of key leading edge C2 and PH domains. The Specific Aims begin with in vitro studies of isolated C2 and PH domains, then move to in vitro studies of full length proteins, and finally to studies of domains and proteins in live cells. Aim 1 employs spin label EPR measurements to elucidate the first membrane docking geometries of PH domains. Aim 2 analyzes the membrane targeting mechanisms of representative C2 and PH domains, and tests a new working model for the protein-lipid interactions that control targeting. Aim 3 employs innovative single molecule methods to dissect the contributions of multiple membrane docking domains in full length proteins, and to analyze the regulatory mechanisms of full length proteins. Aim 4 tests the conclusions of in vitro studies in live cells, and develops new biosensors to analyze second messenger signals at the leading edge of polarized cells. Completion of these Aims will reveal key principles of membrane recognition in cell migration, the immune response inflammation, and cancer. PUBLIC HEALTH RELEVANCE: This proposal investigates the ubiquitous C2 and PH domain motifs that regulate the membrane targeting of signaling proteins in a wide array of cellular pathways. Certain C2 and PH domains also play central roles in human diseases such as inflammation and cancer; for example, the highly oncogenic E17K mutation of AKT1 PH domain drives constitutive plasma membrane targeting by an unidentified mechanism, yielding kinase superactivation that accounts for its carcinogenicity. Preliminary results reveal that the E17K mutation enhances membrane targeting by changing the target lipid specificity of the PH domain, thereby defining the molecular mechanism of this important oncogenic mutation.