Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

细胞骨架蛋白调控组通过对哺乳动物和鱿鱼神经元中神经丝（NF）磷酸化的早期观察，一直在研究两个密切相关的项目。我们证明神经元中的细胞骨架蛋白磷酸化受地形调节；即，尽管激酶、调节因子和底物也存在于核周区，但许多lys-ser-pro （KSP）重复序列的NF尾结构域磷酸化仅限于轴突区。一个重要的问题是，由于放松管制导致神经退行性疾病（如阿尔茨海默病、肌萎缩侧索硬化症、帕金森病），这一过程通常是如何调节的；即，细胞骨架蛋白在神经元核周异常过度磷酸化。最初，我们关注的是负责NF蛋白尾部区域大量赖氨酸ser-pro （KSP）重复序列广泛磷酸化的激酶，并发现脯氨酸定向激酶（如cdk5和Erk1/2）主要参与其中。此外，我们发现NF头部结构域在细胞体中被第二信使蛋白激酶短暂磷酸化，而NF尾部结构域的脯氨酸导向丝氨酸/苏氨酸残基的轴突磷酸化是通过与胶质/轴突相互作用相关的严格调节的脯氨酸导向激酶信号转导级联发生的。目标1;细胞骨架蛋白磷酸化的地形调节；为了进一步研究这个问题，我们正在研究磷酸酶（酪氨酸和丝氨酸/苏氨酸磷酸酶），它们在哺乳动物和鱿鱼神经元中的鉴定，它们的定位和调控，并结合Pin1的作用的新线索，Pin1是一种肽基脯氨酸异构酶，它特异性地靶向和异构化磷酸化的脯氨酸导向的丝氨酸/苏氨酸残基来改变功能。目标2;cdk5在神经元存活和神经变性中的作用。Cdk5已被确定为神经系统中受到严格调控的多功能激酶，在发育过程中的神经元迁移中起重要作用，对生存至关重要。当cdk5在神经元应激（氧化、炎症等）中被解除调控时，将p35转化为更小的激活分子p25， p25与cdk5形成更稳定的复合物（cdk5/p25），异常地过度激活和解除cdk5活性，从而诱导特定的神经元病理，通常在阿尔茨海默病、帕金森病和ALS等神经退行性疾病中发现。这些疾病的小鼠模型可以通过基因诱导大脑中p25的高水平表达来产生，从而暗示过度活跃的cdk5是这些疾病病因学的主要因素。目前，大多数针对神经退行性疾病中失调的Cdk5/p25复合物的治疗方法主要集中在通过干扰激酶的ATP结合域来抑制Cdk5活性的药物，如罗斯科维汀。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。P5是一个24个氨基酸的肽，来源于p35激活子，在一个高度特异性的调节位点相互作用。基于cdk5/p25的晶体结构，我们设计了p25的截断片段，与p25/cdk5接口相互作用，抑制其活性。在许多体外和体内（细胞）实验中，P5肽显示出对cdk5/p25活性的最有效抑制，可以特异性地关闭失调的cdk5/p25活性，而不影响正常cdk5/p35复合物的活性，而正常cdk5/p35复合物对神经系统的发育和生存至关重要。此外，P5肽可以消除cdk5/p25活性失调引起的典型神经元病理和细胞死亡。这些戏剧性的结果提高了P5在AD和其他神经退行性疾病中具有巨大治疗潜力的可能性，这些疾病中失调的高活化Cdk5是一个病因。

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.