PROTEIN PHOSPHORYLATION AND REGULATION OF CYTOSKELETON IN NEURONAL SYSTEMS

我们对磷酸化机制的理解过程 神经丝蛋白(NF-H)如下：体内分析 大鼠神经营养因子-H尾部结构域的磷酸化位点表明，大多数 大鼠核因子-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 提示多个激酶参与了它们的磷酸化。 其中一种使KSPXKX重复磷酸化的激酶是神经元周期蛋白- 依赖性激酶-5(CDK5)。尽管CDK5与G1期细胞周期蛋白相关 (细胞周期蛋白D)在有丝分裂细胞中，只有在成熟细胞中才有激酶活性 神经细胞。我们证明，神经元的CDK5活性是 受67kd的蛋白调控(P67)。峰值活跃度与 P67和CDK5的最高水平。P67是神经特异性的，存在于两个中枢神经系统 和三叉神经节神经元。它在培养的海马神经元轴突中表达 它与磷酸化的核因子-H(P-NF-H)共定位。除了……之外 它作为cdk5、p67的潜在调节因子，也是一种突触蛋白结合。 一种蛋白质，被认为在突触传递和 分泌物。为了进一步研究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 用CDK5、p67、CDK5、p67、CDK5、p67抗体发育大鼠生后小脑 Synaxin和P-NF-H。免疫印迹显示所有抗原都是 发育调节，从Pn2到Pn2的表达增加 成体，p67和CDK5呈时间相关关系。 然而，免疫组织化学显示CDK5和P-NF-H表达较强 共定位，而Synaxin和p67抗原紧密共定位 在经历旺盛的突触发生的区域。中的纤维束中 而在小脑深部，p67、CDK5和P-NF-H共定位于 有几个阶段。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， 另一种CDK5调节蛋白分子35kD(P35)已被报道。 但它的表达仅限于中枢神经系统。目前尚不清楚是否 这两个调节分子都是最大活性所必需的，或者说是激酶 特异性由调节剂针对不同的底物。至 了解p67和p35在CDK5调控中的具体作用，我们有 纯化了大量在细菌中表达的CDK5、p67和p35。 P67和p67联合检测蛋白激酶活性的定量研究 或p35与不同神经元特异性底物的磷酸化 带有KSPXKX基序的分子将提供一些答案 以上问题。

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.