Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

细胞骨架蛋白调节组一直在研究两个项目，这两个项目与哺乳动物和鱿鱼神经元中神经丝（NF）磷酸化的早期观察密切相关。我们证明了神经元中的细胞骨架蛋白磷酸化是地形调节的;即，许多lys-ser-pro（KSP）重复序列的NF尾域磷酸化仅限于轴突区室，尽管激酶、调节剂和底物也存在于核周区室中并具有活性。一个重要的问题是，由于失调导致神经变性（例如阿尔茨海默病、肌萎缩性侧索硬化症、帕金森病），该过程通常如何调节;即，细胞骨架蛋白在神经元胞体中异常过度磷酸化。最初，我们专注于NF蛋白尾部结构域中大量lys-ser-pro（KSP）重复序列的广泛磷酸化的激酶，并发现脯氨酸定向激酶如cdk 5和Erk 1/2主要参与其中。此外，我们发现，NF头部结构域是瞬时磷酸化的细胞体隔室的第二信使蛋白激酶，而轴突磷酸化的脯氨酸指导的丝氨酸/苏氨酸残基在NF尾部结构域发生密切调控的脯氨酸指导的激酶信号转导级联与胶质细胞/轴突的相互作用。目标1;细胞骨架蛋白磷酸化的地形调控;为了进一步研究这个问题，我们正在研究磷酸酶（酪氨酸和Ser/Thr磷酸酶），它们在哺乳动物和鱿鱼神经元中的识别，它们的定位和调节，结合一个新的线索，关于Pin 1的作用，一种肽基脯氨酰异构酶，其特异性靶向和异构化磷酸化脯氨酸指导的Ser/Thr残基，以改变功能。目的2：cdk 5在神经元存活和神经元变性中的作用。cdk 5已被确定为神经系统中的一种严格调节的多功能激酶，在发育过程中的神经元迁移中很重要，并且对生存至关重要。当cdk 5在神经元应激（氧化、炎症等）中失调时，p35转化为较小的激活分子p25，其与cdk 5形成更稳定的复合物（cdk 5/p25），异常地过度激活和失调cdk 5活性，其诱导通常在诸如阿尔茨海默氏症、帕金森氏症和ALS的神经退行性疾病中发现的特定神经元病理。这些疾病的小鼠模型可以通过遗传诱导p25在大脑中的高水平表达来产生，从而暗示过度活跃的cdk 5是这些疾病的病因学中的主要因素。目前，针对神经退行性疾病中失调的Cdk 5/p25复合物的大多数治疗方法主要集中在通过干扰激酶的ATP结合结构域来抑制cdk 5活性的药物，如roscovitine。然而，大多数这些药物缺乏所需的特异性，因为大多数cdk激酶（和大多数激酶）在ATP结合位点是脆弱的。然而，P5是一种24个氨基酸的肽，来源于p35激活剂，在高度特异性的调节位点相互作用。基于cdk 5/p25的晶体结构，我们设计了与p25/cdk 5界面相互作用并抑制其活性的p25截短片段。在许多体外和体内（在细胞中）实验性试验中显示，P5（表现出对cdk 5/p25活性的最有效抑制的肽）特异性地关闭失调的cdk 5/p25活性，而不影响正常cdk 5/p35复合物的活性，正常cdk 5/p35复合物对于神经系统发育和存活是必需的。此外，典型的神经元病理和细胞死亡诱导的失调cdk 5/p25活性被废除的P5肽治疗。这些戏剧性的结果提出了P5对AD和其他神经退行性疾病具有巨大治疗潜力的强烈可能性，其中失调的过度激活的Cdk 5是致病因素。

The Cytoskeletal Protein Regulation Section has been studying two programs closely linked by an early observation on neurofilament (NF) phosphorylation in mammalian and squid neurons. We demonstrated that cytoskeletal protein phosphorylation in neurons is topographically regulated; i.e., NF tail domain phosphorylation of the many lys-ser-pro (KSP) repeats is restricted to the axonal compartment although kinases, regulators and substrates are also present and active in the perikaryal compartment. An important question is, how is this process normally regulated since deregulation results in neurodegeneration (e.g. Alzheimers Disease, Amyotrophic lateral Sclerosis, Parkinson); i.e., cytoskeletal proteins are aberrantly hyperphosphorylated in neuronal perikarya. Initially we focused on the kinases responsible for the extensive phosphorylation of the numerous lys-ser-pro (KSP) repeats in tail domains of NF proteins and found that the proline directed kinases such as cdk5 and Erk1/2 are primarily involved. In addition, we found that the NF head domains are transiently phosphorylated in the cell body compartment by second messenger protein kinases while axonal phosphorylation of proline-directed ser/thr residues in NF tail domains occurs by tightly regulated proline-directed kinase signal transduction cascades associated with glial / axonal interaction. Aim 1;Topographic regulation of cytoskeletal protein phosphorylation; To further examine this problem, we are studying phosphatases (tyrosine and ser/thr phosphatases), their identification in the mammalian and squid neurons, their localization and regulation, combined with a new lead concerning the role of Pin1, a peptidyl-prolyl isomerase which specifically targets and isomerizes phosphorylated proline-directed ser/thr residues to change function. Aim 2; Role of cdk5 in neuronal survival and neurodegeneration. Cdk5 has been identified as a tightly regulated multifunctional kinase in the nervous system, important in neuronal migration during development, and essential for survival. When cdk5 is deregulated in neuronal stress (oxidative, inflammatory, etc) converts p35 into a smaller activator molecule, p25, which forms a more stable complex with cdk5 (cdk5/p25), abnormally hyperactivates and deregulates cdk5 activity which induces a specific neuronal pathology typically found in such neurodegenerative disorders as Alzheimer, Parkinson and ALS. Mouse models of these diseases can be produced by genetically inducing the expression of high levels of p25 in the brain, thereby implicating hyperactive cdk5 as a major factor in the etiology of these disorders. Currently, most therapeutic approaches targeting the deregulated Cdk5/p25 complex in neurodegenerative disorders have focused primarily on drugs like roscovitine that inhibit cdk5 activity by interfering with the ATP binding domain of the kinase. Most of these drugs, however, lack the specificity required since most cdk kinases (and most kinases) are vulnerable at the ATP binding site. The P5, a 24 amino acid peptide, however, derived from the p35 activator, interacts at a highly specific regulator site. Based on the crystal structure of cdk5/p25 we designed truncated fragments of p25 that interact with the p25/cdk5 interface and inhibit its activity. P5, the peptide exhibiting the most effective inhibition of cdk5/p25 activity, was shown, in a number of in vitro and in vivo (in cells) experimental trials to specifically shut down deregulated cdk5/p25 activity without affecting the activity of the normal cdk5/p35 complex, which is essential for nervous system development and survival. Moreover, the typical neuronal pathology and cell death induced by deregulated cdk5/p25 activity was abrogated by treatment with the P5 peptide. These dramatic results raise the strong possibility that P5 has great therapeutic potential for AD and other neurodegenerative diseases in which deregulated hypperactivated Cdk5 is an etiological factor.