Neuronal Phosphorylation/Regulation Of Cytoskeleton

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是地形调控。在正常条件下，激酶、磷酸酶、细胞骨架蛋白底物和调节剂在细胞体中合成，但细胞骨架蛋白的磷酸化，特别是例如中分子量（NF-M）和高分子量（NF-H）尾结构域，在轴突运输期间限于轴突隔室。在几种神经退行性疾病中，例如阿尔茨海默病（AD）和肌萎缩性侧索硬化症（ALS），在细胞体中发现细胞骨架蛋白的异常磷酸化。地形调节和去调节的机制还没有很好地理解，本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。我们提出了以下假说来解释细胞骨架蛋白的拓扑调控：1）细胞骨架蛋白在细胞内生物合成后，在N端被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（Cdk 5，MAPK）在C-末端结构域的磷酸化被抑制。2)来自靶组织或周围轴突相关胶质细胞的外源性信号激活脯氨酸定向激酶，其广泛磷酸化轴突隔室中的脯氨酸定向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，并且已经发表了支持这些建议的证据。为了解决第三个假设，我们正在使用鱿鱼巨纤维系统。我们研究了细胞体和轴突区室的差异磷酸酶活性，这可能是负责地形细胞骨架蛋白磷酸化。我们已经发现，在鱿鱼巨轴突系统，酪氨酸蛋白磷酸酶活性显着升高的perikarya（细胞体）相比，轴突室。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，我们的实验室继续研究细胞周期蛋白依赖性激酶5（Cdk 5）在神经系统发育和功能中的调节和作用。cdk 5是神经系统中磷酸化细胞骨架蛋白的主要激酶之一，对生存至关重要。我们以及其他实验室已经证明Cdk 5是一种多功能蛋白激酶。这种激酶的不同作用是基于其磷酸化多种底物的能力，调节其他激酶，如MEK 1，JNK 3，CPRK和GSK 3b和？串话与其他信号转导级联如Rac信号通路。已经提出，通过异常产生p25（其调节剂p35的截短的、更有活性的片段）来解除脑中Cdk 5活性的调节，可以导致过度磷酸化的tau，这是AD的病理学特征。我们对Cdk 5和其调节剂的截短形式之间的位点特异性相互作用的研究揭示了称为CIP的中心125个氨基酸片段，其对Cdk 5/p25复合物具有高亲和力并抑制其体外活性。我们已经表明，CIP特异性地抑制Cdk 5/p25在转染的非神经元和原代神经元培养物中的活性。此外，CIP还降低了tau的过度磷酸化。值得注意的是，CIP不影响Cdc 2激酶的活性，也不影响Cdk 5/p35的活性，而Cdk 5/p35对神经元存活至关重要。此外，我们已经表明，CIP特异性抑制Cdk 5在原代神经元的过度活跃。现在的问题是CIP是否可以抑制AD和ALS小鼠模型中tau和NF蛋白的过度磷酸化，以及过表达p25的小鼠。如果CIP确实在这些转基因条件下抑制体内Cdk 5活性，那么我们将预测CIP/p25双转基因和CIP/AD或CIP/ALS模型的脑中tau和NF过度磷酸化的水平将被消除或显著降低。这些研究的结果可能表明CIP是否可以作为神经病理学的治疗剂，涉及增加Cdk 5/p25活性和神经元细胞骨架蛋白的过度磷酸化。

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.