Neuronal Phosphorylation/Regulation Of Cytoskeleton

神经元磷酸化/细胞骨架的调节

• 批准号: 6990036
• 负责人: HARISH C PANT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990036
• 关键词: axon; biological signal transduction; conformation; cytoskeletal proteins; cytoskeleton; developmental neurobiology; enzyme inhibitors; intermolecular interaction; laboratory mouse; laboratory rat; neurofilament proteins; neurons; neuroregulation; phosphoprotein phosphatase; phosphorylation; posttranslational modifications; protein kinase; protein localization; protein structure function; squid; tau proteins; tissue /cell culture

Protein Phosphorylation And regulation of Cytoskeleton in Nervous system In the nervous system: Neuronal cytoskeletal protein phosphorylation is topographically regulated. Under normal conditions, kinases, phosphatases, cytoskeletal protein substrates, and regulators are synthesized in cell bodies but the phosphorylation of cytoskeletal proteins, particularly medium molecular mass (NF-M) and high molecular mass (NF-H) tail domains, for example, is restricted to the axonal compartment during axon transport. In several neurodegenerative disorders, such as Alzheimer's disease (AD) and Amyotrophic Lateral Sclerosis (ALS), an aberrant phosphorylation of cytoskeletal proteins is found in the cell body. The mechanisms of topographic regulation and deregulation are not well understood and the major focus of this laboratory has been to study of the factors that regulate the phosphorylation of the cytoskeletal proteins. We proposed the following hypotheses to explain the topographic regulation of cytoskeletal proteins; 1) after biosynthesis in the cell bodies, the cytoskeletal proteins are transiently phosphorylated in the N-terminal domains by PKA/PKC. By virtue of conformational changes induced by this phosphorylation, the phosphorylation in the C-terminal domains by proline directed kinases (Cdk5, MAPKs) is inhibited. 2) Exogenous signals, either from the target tissues or from surrounding axon-associated glia, activate the proline-directed kinases, which extensively phosphorylate the proline-directed S/T residues in the axonal compartment. 3) Higher phosphatase activity in the cell body compared to axonal compartment inhibits cytoskeletal protein phosphorylation. Experiments related to the 1st and 2nd hypotheses have been conducted and supporting evidence for these proposals have been published. To address the 3rd hypothesis we are using the squid giant fiber system. We studied the differential phosphatase activities in cell body and axonal compartments that may be responsible for the topographic cytoskeletal protein phosphorylation. We have found that in the squid giant axon system, tyrosine protein phosphatase activity is significantly elevated in the perikarya (cell body) compared to the axonal compartment. Future studies are directed to determine whether tyrosine phosphatases play a role in regulating cell body phosphorylation. In addition, our laboratory has continued to study the regulation and role of Cyclin-dependent kinase 5 (Cdk5) in nervous system development and function. Cdk5 is one of the major kinases that phosphorylate the cytoskeletal proteins in the nervous system and is essential for survival. We, as well as other laboratories, have shown that Cdk5 is a multifunctional protein kinase. The diverse roles of this kinase are based upon its ability to phosphorylate a diverse array of substrates, regulation of other kinases such as MEK1, JNK3, CPRK and GSK3b and ?cross-talk? with other signal transduction cascades such as the Rac signaling pathway. It has been proposed that deregulation of Cdk5 activity in the brain, by abnormal production of p25, a truncated, more active fragment of its regulator p35, can lead to hyperphosphorylated tau, a pathology characteristic of AD. Our study of site specific interactions between Cdk5 and truncated forms of its regulator have revealed a central 125 amino acid fragment termed CIP, has a high affinity for and inhibits the in vitro activity of the Cdk5/p25 complex. We have shown that CIP specifically inhibits Cdk5/p25 activity in transfected non-neuronal and primary neuronal cultures. Additionally, CIP also reduces the hyperphosphorylation of tau. It is important to note that CIP does not affect the activity of Cdc2 kinase nor Cdk5/p35 which is essential for neuronal survival. In addition, we have shown that CIP specifically inhibits Cdk5 hyperactivity in primary neurons. Now the question arises whether CIP can inhibit the hyperphosphorylation of tau and NF proteins in mouse models of AD and ALS, as well as in mice over expressing p25. If CIP does indeed inhibit Cdk5 activity in vivo under these transgenic conditions, then we would predict that the level of tau and NF hyperphosphorylation in the brains of CIP/p25 double transgenics and CIP/AD or CIP/ALS models would be abolished or significantly lower. The outcome of these studies may indicate whether CIP may serve as a therapeutic agent for neuropathologies involving increased Cdk5/p25 activity and the hyperphosphorylation of neuronal cytoskeletal proteins.

神经系统中蛋白质磷酸化和细胞骨架的调节在神经系统中： 神经元细胞骨架蛋白磷酸化受地形调节。在正常条件下，激酶、磷酸酶、细胞骨架蛋白底物和调节因子在细胞体内合成，但细胞骨架蛋白的磷酸化，特别是中等分子量 (NF-M) 和高分子质量 (NF-H) 尾部结构域，例如，在轴突运输过程中仅限于轴突区室。在几种神经退行性疾病中，例如阿尔茨海默氏病 (AD) 和肌萎缩侧索硬化症 (ALS)，细胞体内发现细胞骨架蛋白的异常磷酸化。拓扑调节和失调的机制尚不清楚，该实验室的主要重点是研究调节细胞骨架蛋白磷酸化的因素。我们提出以下假设来解释细胞骨架蛋白的拓扑调节； 1) 在细胞体内生物合成后，细胞骨架蛋白的 N 末端结构域被 PKA/PKC 瞬时磷酸化。由于这种磷酸化诱导的构象变化，脯氨酸定向激酶（Cdk5、MAPK）在 C 末端结构域中的磷酸化受到抑制。 2)来自靶组织或周围轴突相关神经胶质细胞的外源信号激活脯氨酸定向激酶，其广泛磷酸化轴突区室中脯氨酸定向的S/T残基。 3) 与轴突区室相比，细胞体中较高的磷酸酶活性抑制细胞骨架蛋白磷酸化。与第一和第二假设相关的实验已经进行，并且这些提议的支持证据已经发表。为了解决第三个假设，我们正在使用鱿鱼巨型纤维系统。我们研究了细胞体和轴突区室中可能导致细胞骨架蛋白磷酸化的差异磷酸酶活性。我们发现，在鱿鱼巨轴突系统中，与轴突区室相比，核周（细胞体）中的酪氨酸蛋白磷酸酶活性显着升高。未来的研究旨在确定酪氨酸磷酸酶是否在调节细胞体磷酸化中发挥作用。 此外，我们实验室还持续研究细胞周期蛋白依赖性激酶5（Cdk5）在神经系统发育和功能中的调控和作用。 Cdk5 是磷酸化神经系统中细胞骨架蛋白的主要激酶之一，对于生存至关重要。我们以及其他实验室已经证明 Cdk5 是一种多功能蛋白激酶。该激酶的多种作用基于其磷酸化多种底物的能力、对其他激酶（如 MEK1、JNK3、CPRK 和 GSK3b）的调节以及“串扰”的能力。与其他信号转导级联，例如 Rac 信号通路。有人提出，由于 p25（其调节因子 p35 的一个截短的、活性更高的片段）的异常产生，导致大脑中 Cdk5 活性的失调，可导致 tau 蛋白过度磷酸化，这是 AD 的病理特征。我们对 Cdk5 与其调节剂截短形式之间的位点特异性相互作用的研究揭示了一个称为 CIP 的中心 125 个氨基酸片段，它对 Cdk5/p25 复合物具有高亲和力并抑制其体外活性。我们已经证明，CIP 特异性抑制转染的非神经元和原代神经元培养物中的 Cdk5/p25 活性。此外，CIP 还可以减少 tau 蛋白的过度磷酸化。值得注意的是，CIP 不会影响 Cdc2 激酶或 Cdk5/p35 的活性，而后者对于神经元存活至关重要。此外，我们还发现 CIP 特异性抑制初级神经元中的 Cdk5 过度活跃。现在的问题是，在 AD 和 ALS 小鼠模型以及过度表达 p25 的小鼠中，CIP 是否可以抑制 tau 和 NF 蛋白的过度磷酸化。如果在这些转基因条件下 CIP 确实抑制体内 Cdk5 活性，那么我们可以预测 CIP/p25 双转基因和 CIP/AD 或 CIP/ALS 模型大脑中 tau 和 NF 过度磷酸化的水平将被消除或显着降低。这些研究的结果可能表明 CIP 是否可以作为涉及 Cdk5/p25 活性增加和神经元细胞骨架蛋白过度磷酸化的神经病理学的治疗剂。