PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

神经元系统中蛋白质磷酸化和细胞骨架的调节

• 批准号: 2579565
• 负责人: H C PANT
• 金额: --
• 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/2579565
• 关键词: animal tissue; antibody; binding proteins; cerebellum; cyclins; cytoskeleton; developmental neurobiology; enzyme activity; gene expression; hippocampus; immunocytochemistry; neural transmission; neurofilament proteins; neurons; nucleic acid repetitive sequence; phosphorylation; protein kinase; protein purification; protein structure function; secretion; tissue /cell culture; western blottings

Our process in understanding the phosphorylation mechanisms of neurofilament protein, (NF-H) is as follows: Analysis of in vivo phosphorylated sites of tail domain of rat NF-H showed that most of the Ser -residues in the Lys-Ser-Pro (KSP) repeats of rat NF-H are phosphorylated. The structural analysis of these repeat sequences suggests that multiple kinases are involved in their phosphorylation. One of the kinases phosphorylating KSPXKX repeats is neuronal cyclin- dependent kinase-5 (cdk5). Although Cdk5 is associated with G1 cyclins (cyclin D) in mitotic cells, the kinase activity is found only in mature neuronal cells. We demonstrated that, neuronal cdk5 activity is regulated by a protein of 67kd (P67). Peak activity correlated with the maximum levels of p67 and cdk5. p67 is neurospecific, present in both CNS and PNS neurons. It is expressed in axons of hippocampal cell cultures where it colocalized with phosphorylated NF-H (P-NF-H). In addition to its role as a putative regulator of cdk5, p67 is also a syntaxin binding protein that is thought to play a role in synaptic transmission and secretion. To further characterize the role of p67 in neural tissue, we carried out an immunoblot and immunohistochemical analysis of the developing rat postnatal cerebellum using antibodies to cdk5, p67, syntaxin and P-NF-H. The immunoblots showed that all antigens were developmentally regulated, increasing in expression from PN2 to the adult, with p67 and cdk5 showing a close temporal correlation. Immunohistochemically, however, cdk5 and P-NF-H showed strong colocalization whereas syntaxin and p67 antigens were tightly colocalized in regions undergoing vigorous synaptogenesis. In fiber bundles in the deep cerebellum, however, p67, cdk5 and P-NF-H were colocalized at several stages. The results suggest that p67 may have more than one function in different regions of the developing cerebellum. Recently, another regulator protein molecule 35kd (p35), of cdk5 has been reported but its expression is restricted to the CNS. It is not clear whether both regulator molecules are required for maximal activity, or the kinase specificity is directed to different substrates by regulators. To understand the specific roles of p67 and p35 in cdk5 regulations, we have purified large quantities of bacterially expressed cdk5, p67 and p35. A quantitative evaluation of kinase activity in the presence of p67 and or p35 and phosphorylation of different neuron specific substrates molecules with KSPXKX motifs will provide some of the answers to the above questions.

我们在理解磷酸化机制的过程中， 神经丝蛋白（NF-H）的体内分析如下： 大鼠NF-H尾部结构域的磷酸化位点显示， 大鼠NF-H的Lys-Ser-Pro（KSP）重复序列中的Ser残基是 磷酸化 这些重复序列的结构分析 表明多种激酶参与了它们的磷酸化。 磷酸化KSPXKX重复序列的激酶之一是神经元细胞周期蛋白， 依赖性激酶5（CDK5）。 虽然Cdk5与G1细胞周期蛋白相关， 在有丝分裂细胞中，激酶活性仅在成熟的细胞周期蛋白D中发现。 神经元细胞 我们证明，神经元cdk 5活性是 受67kd蛋白（P67）调控。峰值活性与 p67和cdk5的最高水平。p67是神经特异性的，存在于两个CNS PNS神经元它在海马细胞培养物的轴突中表达 在那里它与磷酸化NF-H（P-NF-H）共定位。除了 它作为CDK 5、p67的假定调节因子的作用也是突触融合蛋白结合 被认为在突触传递中起作用的蛋白质， 分泌物。 为了进一步描述p67在神经组织中的作用，我们 进行了免疫印迹和免疫组织化学分析， 使用CDK5，p67， syntaxin和P-NF-H。 免疫印迹显示所有抗原均为 发育调节，从PN2到 成人，与p67和cdk5显示密切的时间相关性。 免疫组化结果显示，cdk5和P-NF-H表达较强， 共定位，而突触融合蛋白和p67抗原紧密共定位 突触发生活跃的区域。 在纤维束中 小脑深部，p67，cdk5和P-NF-H共定位于 几个阶段。结果表明，p67可能有不止一个 在发育中的小脑的不同区域发挥作用。最近， 另一个调节蛋白分子35kD（p35），cdk 5已被报道 但其表达仅限于CNS。 目前尚不清楚是否 这两种调节分子都是最大活性所必需的，或者激酶 特异性通过调节剂针对不同的底物。 到 了解p67和p35在cdk5规则中的具体作用，我们有 纯化大量细菌表达的CDK5、p67和p35。 在存在p67和p53的情况下， 或p35和不同神经元特异性底物的磷酸化 具有KSPXKX基序的分子将提供一些答案， 以上问题。