ELECTROSTATIC POTENTIALS AND BIOLOGICAL MEMBRANES

描述：长期目标是了解物理因素 例如静电学和降维，产生 通过钙/磷脂第二信使系统传递信息。 这 该提案的重点是MARCKS，一种普遍存在的蛋白激酶C的主要底物 (PKC)与钙调蛋白、肌动蛋白和细胞膜可逆结合。 两 将对假设进行检验。 第一个假设是， MARCKS需要将其N-末端肉豆蔻酸酯插入到内部 以及其碱性残基簇与酸性残基簇的相互作用 脂质。 丝氨酸残基的PKC磷酸化减弱了静电 相互作用并产生MARCKS从膜到细胞质的易位。 第二个假设是MARCKS可以自发地自组装成 具有单价酸性脂质侧域;另一个，不太普遍 该第二信使系统的组分（例如PKC、Src和PIP 2）将 加入域，因为静电的考虑;这些 信号转导结构域具有重要的生理功能。 的 三个具体目标是通过确定MARCKS如何测试这些假设 与膜结合，如何与酸性脂质形成结构域，以及 这些领域的重要性。 使用原子模型的理论计算 将结合实验测量， 实现这些目标。 活性、荧光和膜的测量 将使用重构的信号转导系统进行结合 由磷脂囊泡和纯化的蛋白质（异三聚体G 蛋白质亚基、磷脂酶C、PKC、Src和MARCKS）。 马克是 医学上很重要，因为它涉及分泌，细胞 运动性、细胞周期调节和转化。 研究还 与其他医学上重要的蛋白质有关， 碱性残基结合酸性磷脂，例如K-Ras 4 B、Src和 HIV-1基质蛋白。 最后，初步数据表明， 通过MARCKS和酸性脂质PIP 2产生一种新的信号放大 基于正反馈概念的转导导致“所有或 无”的回答。

DESCRIPTION: The long term objective is to understand how physical factors such as electrostatics and reduction of dimensionality produce a flow of information through the calcium/phospholipid second messenger system. This proposal focuses on MARCKS, a ubiquitous major substrate of protein kinase C (PKC) that binds reversibly to calmodulin, actin, and membranes. Two hypotheses will be tested. The first hypothesis is that membrane binding of MARCKS requires both insertion of its N-terminal myristate into the interior of the bilayer and interaction of its cluster of basic residues with acidic lipids. PKC phosphorylation of serine residues weakens the electrostatic interaction and produces translocation of MARCKS from membrane to cytoplasm. The second hypothesis is that MARCKS can spontaneously self assemble into lateral domains with monovalent acidic lipids; that other, less prevalent components of this second messenger system (e.g. PKC, Src, and PIP2) will join the domains because of electrostatic considerations; and that these signal transduction domains have important physiological functions. The three specific aims are to test these hypotheses by determining how MARCKS binds to membranes, how it forms domains with acidic lipids, and the significance of these domains. Theoretical calculations using atomic models of membranes and peptides will be combined with experimental measurements to achieve these aims. Measurements of activity, fluorescence, and membrane binding will be performed using a reconstituted signal transduction system composed of phospholipid vesicles and purified proteins (heterotrimeric G protein subunits, phospholipase C, PKC, Src, and MARCKS). MARCKS is medically important because it has been implicated in secretion, cell motility, regulation of the cell cycle and transformation. The studies also are relevant to other medically important proteins that use clusters of basic residues to bind to acidic phospholipids, e.g. K-Ras 4B, Src, and HIV-1 matrix protein. Finally, preliminary data suggest that domains formed by MARCKS and the acidic lipid PIP2 produce a novel amplification of signal transduction based on the concept of positive feedback leading to an "all or none" response.