Protein Phosphorylation And Regulation Of Cytoskeleton I

蛋白质磷酸化和细胞骨架调控 I

• 批准号: 7143852
• 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/7143852
• 关键词: JUN kinase; Lentivirus; amyloid proteins; apoptosis; axon; biological signal transduction; conformation; cyclin dependent kinase; cytoskeletal proteins; enzyme activity; enzyme inhibitors; intermolecular interaction; mitogen activated protein kinase; neurofilament; neuronal transport; neuroregulation; phosphoprotein phosphatase; phosphorylation; protein localization; protein structure function; soma; squid; tau proteins; tissue /cell culture; transfection

Protein Phosphorylation And regulation of Cytoskeleton in Nervous system The topographic regulation by phosphorylation of neuronal cytoskeleton proteins is tightly regulated in the nervous system. Cytoskeletal proteins are selectively phosphorylated in the axonal compartment in lys/ser/pro (KSP) motifs of their carboxy tail domains but not in the cell body. Deregulation of this post translational modification has been implicated in various neurodegenerative diseases. We have proposed the following hypotheses to explain the topographic regulation of cytoskeletal proteins in the nervous system under physiological conditions. After biosynthesis in the cell bodies, CaMK/ PKA/PKC transiently phosphorylate the cytoskeletal proteins in the N-terminal domains. By virtue of conformational change induced by this phosphorylation, the phosphorylation in the C-terminal domains by proline directed kinases (Cdk5, MAPKs) is inhibited. 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; and the differential expression of kinases and phosphatase activities in the cell body and axonal compartments. Our in situ and in vitro experiments have provided the evidence that as long as head domains are phosphorylated, the phosphorylation of their tail domains on KSP motifs is inhibited. In vivo and in vitro studies have shown that glial / axonal interaction is responsible for MAPK activation and tail domain KSP phosphorylation; and to evaluate the kinase and phosphatase activities, we have used the squid giant fiber system where the axonal compartment can be separated from the cell body. We have found that the kinase and ser/thr phosphatase activities are similar in both compartments however, protein tyrosine phosphatase activity is significantly higher in the cell body compared to the axonal compartment. This may be one of the factors responsible for low phosphorylation of the cytoskeletal proteins in the cell body compartment in anormal system. The extracellular aggregation of amyloid b (Ab) peptides and the intracellular hyperphosphorylation of tau, a neuronal cytoskeletal protein at specific epitopes, are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator which is produced by the proteolytic cleavage of the physiological Cdk5 activator, p35, upon exposure to Ab and other neurotoxic insults. We showed that lentiviral infections of cortical neurons with a Cdk5 Inhibitory Peptide, CIP (a 125 amino acid peptide truncated form of p35) selectively inhibited p25/Cdk5 activity and suppressed the aberrant tau hyperphosphorylation in cortical neurons. Furthermore, Ab1-42-induced apoptosis of cortical neurons was also reduced by co-infection with CIP. Of particular importance, is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, which is required for normal nervous system function and survival. CIP also did not inhibit endogenous Cdc2 activity, nor did it inhibit the other mitotic cyclin dependent kinases such as Cdk2, Cdk4 and Cdk6. It is highly specific for the hyperactive p25/Cdk5 complex seen in abnormally stressed neurons. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

神经系统中蛋白质磷酸化和细胞骨架的调节 神经元细胞骨架蛋白磷酸化的拓扑调节在神经系统中受到严格调节。细胞骨架蛋白在轴突区室中其羧基尾结构域的 lys/ser/pro (KSP) 基序中被选择性磷酸化，但在细胞体中则不然。这种翻译后修饰的失调与多种神经退行性疾病有关。我们提出以下假设来解释生理条件下神经系统中细胞骨架蛋白的拓扑调节。 在细胞体内生物合成后，CaMK/PKA/PKC 瞬时磷酸化 N 端结构域中的细胞骨架蛋白。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。来自靶组织或周围轴突相关神经胶质细胞的外源信号会激活脯氨酸定向激酶，从而广泛磷酸化轴突区室中脯氨酸定向的 S/T 残基；以及细胞体和轴突区室中激酶和磷酸酶活性的差异表达。我们的原位和体外实验提供了证据，只要头部结构域被磷酸化，KSP基序上的尾部结构域的磷酸化就会受到抑制。体内和体外研究表明，神经胶质/轴突相互作用负责 MAPK 激活和尾部结构域 KSP 磷酸化；为了评估激酶和磷酸酶活性，我们使用了鱿鱼巨纤维系统，其中轴突室可以与细胞体分离。我们发现两个区室中的激酶和丝氨酸/苏氨酸磷酸酶活性相似，然而，与轴突区室相比，细胞体中的蛋白酪氨酸磷酸酶活性显着更高。这可能是导致异常系统中细胞体区室中细胞骨架蛋白低磷酸化的因素之一。 b 淀粉样蛋白 (Ab) 肽的细胞外聚集和 tau（一种位于特定表位的神经元细胞骨架蛋白）的细胞内过度磷酸化，是阿尔茨海默氏病 (AD) 等神经退行性疾病的病理标志。当 Cdk5 与 p25 结合时，它会磷酸化 AD 特异性磷酸化表位上的 tau 蛋白。 p25 是一种截短的激活剂，在暴露于 Ab 和其他神经毒性损伤后，通过生理学 Cdk5 激活剂 p35 的蛋白水解裂解产生。我们发现，用 Cdk5 抑制肽 CIP（p35 的 125 个氨基酸肽截短形式）慢病毒感染皮质神经元可选择性抑制 p25/Cdk5 活性，并抑制皮质神经元中异常的 tau 过度磷酸化。此外，与 CIP 共感染也减少了 Ab1-42 诱导的皮质神经元凋亡。特别重要的是，我们发现 CIP 不会抑制内源性或转染的 p35/Cdk5 活性，而这是正常神经系统功能和生存所必需的。 CIP 也不抑制内源性 Cdc2 活性，也不抑制其他有丝分裂周期蛋白依赖性激酶，如 Cdk2、Cdk4 和 Cdk6。它对异常应激神经元中高度活跃的 p25/Cdk5 复合物具有高度特异性。因此，这些结果提供了一种解决并可能改善神经退行性疾病病理学的策略，神经退行性疾病可能是 Cdk5 异常 p25 激活的结果，而不影响“正常”Cdk5 活性。