Protein Phosphorylation and Regulation of cytoskeleton in Neuronal System

神经系统中蛋白质磷酸化和细胞骨架的调节

• 批准号: 8940039
• 负责人: HARISH C PANT
• 金额: $ 36.76万
• 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/8940039
• 关键词: Accounting; Actin-Binding Protein; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Antibodies; Antigens; Axon; Biochemical; Biological Assay; Chromatography; Collaborations; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Cytoskeleton; Development; Epitopes; FMRFamide; Fiber; Genes; Incubated; Isomerism; Laboratories; Lobe; MAP Kinase Regulation Pathway; MARCKS gene; Metabolic; Mitogen-Activated Protein Kinases; Molecular; Muscle Development; Nervous system structure; Neurodegenerative Disorders; Neurofilament Proteins; Neurons; PKA inhibitor; Parkinson Disease; Pathway interactions; Pattern; Peptides; Peptidylprolyl Isomerase; Perikaryon; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Play; Procedures; Process; Proline; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Regulation; Role; Site; Squid; Structure of stellate ganglion; System; Tail; Testing; Tyrosine; Western Blotting; axoplasm; base; cross reactivity; enzyme activity; myristoylated alanine-rich C kinase substrate; neurofilament; neuron loss; neuronal cell body; protein complex; protein kinase A kinase

We continue to investigate further the molecular basis of topographic phosphorylation of cytoskeletal proteins using squid giant axon system. To understand the mechanisms whereby compartment-specific metabolic patterns are regulated in the highly asymmetric neuron with its distinct soma, axonal and dendritic compartments, the ideal system is the giant fiber system in the squid. Here, for biochemical studies, it is possible to separate pure axoplasm from giant axons from the large cell bodies in the giant fiber lobe from which they originate. This cannot be done in the much smaller mammalian neurons. For several years our laboratory, using the squid giant fiber system, has focussed on the kinases responsible for the phosphorylation of neurofilament proteins and has shown dramatic differences in the level and pattern of phosphorylation in the two compartments. Recently we have turned to the phosphatases, that also play a key role in regulation. Accordingly, our efforts each summer are to: (a) use Western and ICC analyses to identify the compartment-specific kinases/phosphatases involved using specific antibodies; (b) study the cross-talk interactions between kinases and phosphatases and (c) explore the factors that regulate the activities of these enzymes in each cellular compartment. Among these is the role of Pin1, a peptidyl-prolyl cis/trans isomerase that isomerizes phospho Ser/thr-proline residues of which there are many in squid neurofilaments. In addition to these studies we are investigating the development of the squid nervous system so as to (1) study the development of the IIIrd order giant fibers in the stellate ganglion; (2) establish the neuroanatomical substrate in which to analyze the expression of neuronal-specific genes (in collaboration with Dr. Peter Burbach, Utrecht), and (3) to explore the developmental expression of neuronal regulatory peptide FMRF-amide, recently cloned from the squid stellate ganglion . In addition we continue to study the neuron specific kinase Cdk5, which in the squid hatchling, shows extensive expression in the mantle musculature, suggesting a role in muscle development. As we have done in previous years, our biochemical studies compare whole giant axons (or extruded axoplasm) and the giant fiber lobe (GFL) which contains the cell bodies that, by axonal fusion, generate the giant axons. In previous studies we have established the following ; (1) Axoplasm extracts display much higher levels of protein phosphorylation than GFL extracts with many more proteins serving as substrates. (2) Axonal neurofilament proteins, particularly the high molecular weigh NF220, are highly phosphorylated at multiple KSP repeat sites in the tail domain. On the other hand, NFs from cell bodies are not phosphorylated. (3) Cell bodies and axons share the same active kinases, NF sub unit proteins and regulators yet phosphorylation of the tail domain is restricted to the axonal compartment. The obvious question is why, and of course, how? (4) P13 chromatography of cell body and axoplasm lysates (a procedure that extracts Cdc2-like kinases and associated proteins) revealed that the axonal multimeric complex of proteins was significantly more active than the cell body complex, although both, for the most part, shared similar kinases and substrates. (4) GFL extracts express higher levels of tyrosine phosphatase activity than axoplasm extracts suggesting that this difference may, in part, account for the different patterns of protein phosphorylation in each compartment. To determine the role of tyrosine phosphatase activity we are studying "cross-talk" regulation of the MAP kinase pathway by tyrosine kinases/phosphatases. To test this, we are comparing the level of MAP kinase activity in GFL and axoplasm extracts since Erk1/2, a key kinase in the pathway, is known to phosphorylate KSP repeats in NFs. The hypothesis predicts that higher tyrosine phosphatase levels in the GFL should down-regulate the Erk1/2 pathway. Meanwhile, we continue to explore by Western blotting and ICC assays, the expression of other kinases (PKA and PKC) as well as tyrosine and ser/thr phosphatases in GFL and axoplasm, to identify the "players" that may be involved in each compartment. Since the antibodies used are primarily those prepared from mammalian antigens, the results are difficult to interpret unless proper negative controls (pre-incubation of antibodies with specific blocking peptides) are used to eliminate the possibility of cross reactivity with non-specific squid epitopes. In subsequent studies we detected a dramatic difference in the phosphorylation of MARCKS protein, an actin binding protein that is regulated by PKC phosphorylation. GFL lysate phosphorylation of MARCKS substrate was considerably greater than that in axoplasm, which is virtually inactive. We also demonstrated a significant cross-talk interaction between PKA and PKC activities. This suggests an important regulatory role for PKC and PKA in the GFL and we plan to explore this further by (1) comparing PKC and PKA activities in each compartment using various substrates (2) comparing the level of expression of PKC isomers in Western blots and ICC of stellate ganglia (3) Incubating GFL in PKC and PKA inhibitors and activators to determine their effects on protein phosphorylation in each compartment. These studies will provide a better understanding of topographic neuronal cytoskeletal protein phosphorylation.

我们继续利用鱿鱼巨轴突系统进一步研究细胞骨架蛋白局部磷酸化的分子基础。为了了解在高度不对称的神经元中调节隔室特异性代谢模式的机制，该神经元具有不同的胞体、轴突和树突间隔，理想的系统是鱿鱼中的巨型纤维系统。在这里，对于生化研究，有可能从巨大轴突和它们起源的巨大纤维叶中的大细胞体中分离出纯粹的轴浆。这在小得多的哺乳动物神经元中是不可能做到的。几年来，我们的实验室使用鱿鱼巨型纤维系统，一直专注于负责神经丝蛋白磷酸化的激酶，并在两个隔室显示出显著不同的磷酸化水平和模式。最近，我们转向了磷酸酶，它也在调控中发挥着关键作用。因此，我们每年夏天的工作是：(A)使用Western和ICC分析，使用特定的抗体来确定所涉及的隔室特定的激酶/磷酸酶；(B)研究激酶和磷酸酶之间的相互作用；以及(C)探索调节每个细胞隔室中这些酶活性的因素。其中包括Pin1的作用，它是一种多肽-脯氨酰顺/反式异构酶，可以异构化在鱿鱼神经细丝中存在的磷酸丝氨酸/酪氨酸残基。 除了这些研究外，我们还在研究鱿鱼神经系统的发育，以便(1)研究星状神经节中III级巨型纤维的发育；(2)建立分析神经元特异基因表达的神经解剖学基础(与乌得勒支的Peter Burbach博士合作)，以及(3)探索最近从鱿鱼星状神经节克隆的神经调节肽FMRF-AME的发育表达。此外，我们还在继续研究神经元特异性激酶CDK5，它在鱿鱼幼体中显示出在外套膜肌肉系统中的广泛表达，这表明它在肌肉发育中发挥了作用。 正如我们在前几年所做的那样，我们的生化研究比较了整个巨型轴突(或挤出的轴浆)和巨型纤维叶(GFL)，GFL包含通过轴突融合产生巨型轴突的细胞体。在以前的研究中，我们已经建立了以下结论：(1)轴质提取物比GFL提取物显示出更高的蛋白质磷酸化水平，并且有更多的蛋白质作为底物。(2)轴突神经丝蛋白，尤其是高相对分子质量的NF220，在尾区的多个KSP重复序列上高度磷酸化。另一方面，细胞体中的神经营养因子不被磷酸化。(3)细胞体和轴突具有相同的活性激酶、核因子亚单位蛋白和调节因子，但尾部结构域的磷酸化仅限于轴突间隔。显而易见的问题是，为什么，当然是如何的？(4)对细胞体和轴浆裂解物(提取类CD2蛋白和相关蛋白的过程)的P13层析显示，蛋白质的轴突多聚体复合体明显比细胞体复合体更活跃，尽管两者在大部分情况下共享相似的激酶和底物。(4)GFL提取物比轴浆提取物表达更高水平的酪氨酸磷酸酶活性，这一差异可能部分解释了不同隔室中蛋白质磷酸化的不同模式。为了确定酪氨酸磷酸酶活性的作用，我们正在研究酪氨酸激酶/磷酸酶对MAP激酶通路的“串扰”调节。为了测试这一点，我们比较了GFL和轴浆提取液中MAP激酶的活性水平，因为已知ERK1/2是该通路中的关键激酶，可以磷酸化NFSKSP重复序列。该假说预测，GFL中较高的酪氨酸磷酸酶水平应该下调ERK1/2途径。 同时，通过Western blotting和ICC分析，我们继续探索其他激酶(PKA和PKC)以及酪氨酸和丝氨酸/酪氨酸磷酸酶在GFL和轴浆中的表达，以确定可能参与每个隔室的“玩家”。由于所使用的抗体主要是从哺乳动物抗原中制备的抗体，除非使用适当的阴性对照(预先孵育抗体与特定的封闭肽)来消除与非特异性鱿鱼表位发生交叉反应的可能性，否则很难解释结果。 在随后的研究中，我们发现Marcks蛋白的磷酸化程度有显著差异，Marcks蛋白是一种受PKC磷酸化调控的肌动蛋白结合蛋白。Marcks底物的GFL裂解产物磷酸化程度明显高于轴浆中的GFL裂解产物磷酸化水平，后者几乎是不活跃的。我们还证明了PKA和PKC活动之间存在显著的相互作用。这表明PKC和PKA在GFL中具有重要的调节作用，我们计划通过以下方式进一步探讨这一点：(1)使用不同底物比较每个隔室的PKC和PKA活性(2)比较PKC异构体在Western blotts和星状神经节ICC中的表达水平(3)在PKC和PKA抑制剂和激活剂中孵育GFL，以确定它们对每个隔室蛋白质磷酸化的影响。这些研究将为更好地理解局部神经元细胞骨架蛋白的磷酸化提供依据。