PHOSPHORYLATION OF THE CYTOSKELETON IN NEURONAL GROWTH

神经元生长中细胞骨架的磷酸化

• 批准号: 3415399
• 负责人: PHILIP Richard VULLIET
• 金额: $ 10.57万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415399
• 关键词: axon; cytoskeletal proteins; gel electrophoresis; high performance liquid chromatography; immunocytochemistry; laboratory mouse; laboratory rabbit; laboratory rat; microtubule associated protein; neurofilament proteins; neuronal guidance; neurotrophic factors; phosphorylation; proline; protein kinase; protein kinase C; protein sequence; radiotracer; tissue /cell culture

This project will continue our investigation of the role of protein phosphorylation in regulating neuronal function. The biochemical mechanisms controlling neurite extension will be examined in NGF treated PC12 cells. Recent research in this laboratory has identified a novel proline-directed protein kinase activated by NGF treatment. The minimal recognition sequence for this kinase is --X-Ser/Thr-Pro-X--. Many neuron specific structural proteins including synapsin, tau, microtubule associated proteins, and neurofilament proteins contain this consensus sequence and several have been identified as in vitro substrates of this novel kinase. Since these proteins are substrates for more than one protein kinase and contain multiple phosphorylation sites, the specific sites of phosphorylation will be isolated and sequenced. The phosphorylation sites will be localized in the proteins that have a known sequence. These studies also will investigate the in situ phosphorylation of the cytoskeletal apparatus during neurite outgrowth. 32p-orthophosphate labelled PC12 cells will be treated with NGF and key cytoskeletal proteins will be examined for phosphate incorporation. Specific proteins including TH, synapsin, Tau, MAP's, and neurofilament proteins will be examined for their ability to incorporate phosphate. Since these same proteins are phosphorylated in situ following NGF treatment, the specific phosphorylation sites will be identified to examine the role of PDPK in mediating NGF induced neurite outgrowth. Other work in our laboratory has established that sphingosine, a specific inhibitor of PK C, blocks neurite extension. The exact role of PK C in modulating neurite extension will be further investigated in regard to specific substrates and sites phosphorylated during NGF induced neurite outgrowth. The relationship between PK C and PDPK mediated protein phosphorylation in regulating neurite extension will be examined. Understanding how neurons use protein phosphorylation mechanisms to form neurites may identify fundamental molecular mechanisms that underlie the pathogenesis of Alzheimer's disease, since the PDPK substrate proteins found in the neurofibrillary tangles have abnormal phosphorylation patterns.

这个项目将继续我们对蛋白质作用的研究 磷酸化在调节神经元功能中的作用。的生化机制 将在NGF处理的PC 12细胞中检查控制神经突延伸的方法。 该实验室最近的研究已经确定了一种新的脯氨酸导向的 通过NGF处理激活的蛋白激酶。最小识别序列 这个激酶是-X-Ser/Thr-Pro-X-。许多神经元特异性结构 蛋白质，包括突触蛋白、tau、微管相关蛋白，以及 神经丝蛋白含有这种共有序列， 被鉴定为这种新型激酶的体外底物。由于这些 蛋白质是一种以上蛋白激酶的底物， 多个磷酸化位点，磷酸化的特定位点将 分离并测序。磷酸化位点将定位在 那些有已知序列的蛋白质 这些研究还将调查在原位磷酸化的 在神经突生长期间的细胞骨架装置。32磷正磷酸盐 标记的PC 12细胞将用NGF和关键的细胞骨架蛋白处理 将检查磷酸盐掺入。特定蛋白质包括 TH、突触蛋白、Tau、MAP和神经丝蛋白将被检查， 它们吸收磷酸盐的能力因为这些蛋白质 在NGF处理后原位磷酸化，特异性 将鉴定磷酸化位点以检查PDPK在以下中的作用： 介导NGF诱导的神经突生长。我们实验室的其他工作 确定了鞘氨醇，一种PK C的特异性抑制剂， 扩展名. PK C在调节神经突延伸中的确切作用将是 针对特定底物和位点进行进一步研究 在NGF诱导的神经突生长过程中磷酸化。的关系 PKC和PDPK介导的蛋白磷酸化在调节 将检查神经突延伸。 了解神经元如何利用蛋白质磷酸化机制形成 神经突可以识别基本的分子机制， 阿尔茨海默病的发病机制，因为PDPK底物蛋白 在神经纤维缠结中发现有异常磷酸化 模式.