Mechanisms of Regulated Membrane Targeting

调控膜靶向的机制

• 批准号: 8050702
• 负责人: JOSEPH J FALKE
• 金额: $ 31.63万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050702
• 关键词: 1,2-diacylglycerol; AKT1 gene; Accounting; Affinity; Binding; Binding Proteins; Biosensor; C2 Domain; Cell membrane; Cells; Chemotaxis; Complex; DAG/PE-Binding Domain; Diffuse; Diffusion; Diglycerides; Docking; Electrostatics; Ensure; Enzymes; Equilibrium; Event; Exhibits; Fluorescence Resonance Energy Transfer; Goals; Health; Immune response; In Vitro; Inflammation; Kinetics; Length; Leukocytes; Life; Lipid Bilayers; Lipid Binding; Lipids; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Proteins; Methods; Microscopic; Modeling; Molecular; Movement; Mutation; Oncogenic; PH Domain; Pathway interactions; Penetration; Peripheral; Phosphotransferases; Play; Property; Protein Binding; Protein Kinase C; Proteins; Reaction; Relaxation; Role; Second Messenger Systems; Side; Signal Transduction; Signaling Protein; Site; Specificity; Speed; Spin Labels; Surface; System; Tertiary Protein Structure; Testing; Time; Work; carcinogenicity; cell motility; design; human disease; innovation; insight; mutant; novel; polarized cell; research study; response; second messenger; sensor; single molecule

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.

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，其响应于第二信使驱动膜对接。当被第二信使信号激活时，含有这些结构域的信号蛋白停靠在特定的靶膜上，并控制广泛的基本细胞通路。这个持续的项目研究了极化细胞前沿的复杂信号通路，其中C2和PH结构域通过将数十种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性普遍需要的：目前的重点是驱动初级免疫应答的白细胞的前沿。优化给定C2或PH结构域的膜靶向反应，以确保结构域以精确的特异性以及适当的亲和力和速度对接到其靶膜。随后，脂质结合域在双层平面中随机扩散总的时间和距离需要进行生产性碰撞与效应分子。最后，结构域解离以终止活性。这个持续的提议的一个广泛的目标是阐明关键前沿C2和PH结构域的膜靶向反应的基本分子机制。具体目标开始与分离的C2和PH结构域的体外研究，然后移动到全长蛋白质的体外研究，最后到活细胞中的结构域和蛋白质的研究。目的1采用自旋标记EPR测量来阐明PH结构域的第一个膜对接几何结构。目的2分析了具有代表性的C2和PH结构域的膜靶向机制，并测试了控制靶向的蛋白质-脂质相互作用的新工作模型。目的3采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，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。这些目标的完成将揭示细胞迁移，免疫反应炎症和癌症中膜识别的关键原则。公共卫生关系：该提案调查了普遍存在的C2和PH结构域基序，其在广泛的细胞途径中调节信号蛋白的膜靶向。某些C2和PH结构域也在人类疾病如炎症和癌症中发挥核心作用;例如，AKT 1 PH结构域的高度致癌性E17 K突变通过未鉴定的机制驱动组成性质膜靶向，产生激酶超活化，导致其致癌性。初步结果表明，E17 K突变通过改变PH结构域的靶向脂质特异性来增强膜靶向，从而定义了这种重要致癌突变的分子机制。