PHOSPHORYLATION OF THE CYTOSKELETON IN NEURONAL GROWTH

这个项目将继续我们对蛋白质作用的研究 磷酸化在神经功能调节中的作用生化机制 在NGF处理的PC12细胞中，将研究控制轴突延伸的问题。 这个实验室的最新研究已经确定了一种新的由脯氨酸引导的 由NGF处理激活的蛋白激酶。最小识别序列 该激酶为--X-Ser/Thr-Pro-X-。许多神经元特有的结构 蛋白质包括突触素、tau、微管相关蛋白和 神经丝蛋白包含这个共同的序列，有几个 已被鉴定为这种新的激酶的体外底物。因为这些 蛋白质是不止一种蛋白激酶的底物，包含 多个磷酸化位点，特定的磷酸化位点将 被分离并测序。磷酸化位点将定位于 具有已知序列的蛋白质。 这些研究还将调查该蛋白的原位磷酸化 轴突生长过程中的细胞骨骼结构。32P-正磷酸盐 标记的PC12细胞将被NGF和关键细胞骨架蛋白处理 将进行磷酸盐掺入检查。特定的蛋白质包括 TH、突触素、Tau、MAP和神经丝蛋白将被检测 它们吸收磷酸盐的能力。因为这些相同的蛋白质 NGF治疗后原位磷酸化，特异性 将确定磷酸化位点，以研究PDPK在 介导NGF诱导神经突起生长。我们实验室的其他工作有 证实PK C的一种特异性抑制剂鞘氨醇可以阻断神经突起 分机。PKC在调节轴突延伸中的确切作用将是 关于特定底物和部位的进一步研究 在NGF诱导的轴突生长过程中被磷酸化。两国关系 PKC和PDPK介导的蛋白磷酸化在调控中的作用 将检查轴突延伸。 了解神经元如何使用蛋白质磷酸化机制形成 神经突起可能确定了构成神经细胞的基本分子机制。 阿尔茨海默病的发病机制，因为PDPK底物蛋白 在神经原纤维缠结中发现有异常的磷酸化 模式。

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.