Neuronal Phosphorylation/Regulation Of Cytoskeleton

蛋白质磷酸化与神经系统细胞骨架的调节 神经元细胞骨架蛋白的磷酸化受地形图调节。在正常情况下，激酶、磷酸酶、细胞骨架蛋白底物和调节剂都是在细胞体中合成的，但细胞骨架蛋白的磷酸化，特别是中等分子质量(NF-M)和高分子质量(NF-H)尾部区域，在轴突运输过程中仅限于轴突间隔。在一些神经退行性疾病中，如阿尔茨海默病(AD)和肌萎缩侧索硬化症(ALS)，在细胞体中发现了细胞骨架蛋白的异常磷酸化。地形调节和解除调节的机制还不是很清楚，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。我们提出了以下假设来解释细胞骨架蛋白的地形调节：1)细胞骨架蛋白在细胞体中生物合成后，被PKA/PKC瞬时地在N-末端区域磷酸化。通过这种磷酸化引起的构象变化，Pro导向的激酶(CDK5，MAPKs)在C-末端区域的磷酸化被抑制。2)来自靶组织或周围轴突相关胶质细胞的外源信号激活了Pro导向的蛋白激酶，它广泛地磷酸化了Pro导向的轴突间隔中的S/T残基。3)胞体中磷酸酶活性高于轴突，抑制了细胞骨架蛋白的磷酸化。与第一个和第二个假设相关的实验已经进行，并发表了支持这些建议的证据。为了解决第三个假设，我们使用了鱿鱼巨型纤维系统。我们研究了细胞体和轴突间的差异磷酸酶活性，这可能与细胞骨架蛋白的地形性磷酸化有关。我们发现，在鱿鱼巨型轴突系统中，与轴突相比，核周(细胞体)中的酪氨酸蛋白磷酸酶活性显著升高。未来的研究方向是确定酪氨酸磷酸酶是否在调节细胞体磷酸化方面发挥作用。 此外，我们的实验室还在继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节和作用。CDK5是神经系统中使细胞骨架蛋白磷酸化的主要蛋白之一，对生存至关重要。我们和其他实验室一样，已经证明了CDK5是一种多功能的蛋白激酶。该激酶的不同作用是基于其对多种底物的磷酸化能力，对其他激酶如MEK1、JNK3、CPRK和GSK3b的调节以及串扰？与其他信号转导通路，如RAC信号通路。已经有人提出，通过异常产生p25来解除大脑中CDK5活性的调控，p25是其调节因子p35的一个截断的、更活跃的片段，可能导致tau过度磷酸化，这是AD的病理特征。我们对CDK5与其调节因子截短形式的位点特异性相互作用的研究发现，一个中心125个氨基酸的片段称为CIP，与CDK5/p25复合体具有高亲和力并抑制其体外活性。我们已经证明，在非神经元和原代神经元培养中，CIP特异性地抑制CDK5/p25的活性。此外，CIP还可以减少tau蛋白的过度磷酸化。值得注意的是，CIP不影响cdc2激酶的活性，也不影响对神经元生存至关重要的cdk5/p35。此外，我们还发现CIP可以特异性地抑制初级神经元中CDK5的过度活动。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p25基因的过度表达。如果在这些转基因条件下，CIP确实在体内抑制了CDK5的活性，那么我们可以预测，CIP/p25双转基因和CIP/AD或CIP/ALS模型脑中tau和NF的过度磷酸化水平将被取消或显著降低。这些研究的结果可能表明CIP是否可以作为一种治疗药物来治疗涉及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。

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.