DEVELOPMENTAL EXPRESSION OF CAM KINASE II ISOFORMS

蛋白激酶对细胞骨架的修饰可能在细胞凋亡中起重要作用。 在神经元再生和可塑性中的作用。 ii型 钙/钙调蛋白依赖性激酶（CaM激酶II）是一种被提出的介导剂 由于其磷酸化细胞骨架功能的能力， 微管相关蛋白（MAP-2），它与微管相关蛋白（MAP-2）共定位。 树突中的激酶。 成年大鼠钙调素激酶Ⅱ的主要形式 前脑是500-730 kDa的全酶，含有两个亚基（50 kDa/60 kDa kDa）以3/1的摩尔比。 有证据表明， 激酶的同种型存在于大脑中， 控制 MAP-2由三种同源蛋白质（a/B/c）组成，它们也是 发展规律。 所有三种MAP-2变体都促进微管蛋白 组装成微管，而只有高分子量MAP-2a/B 另外促进树突细胞骨架的稳定， 与其他细胞元素相互作用。 这个的主要目标 我们的建议是研究钙调蛋白激酶II亚型之间的关系， 和MAP a/B/c底物，以便深入了解 其中CaM激酶II调节细胞骨架动力学。 两个模型系统将被用来比较激酶同工酶的表达 和MAP-2变体在培养的神经元和神经元样细胞中。 （一） 几个独特的异构体钙调素激酶II目前在大鼠脑将是 使用阳离子交换色谱分离。 发展差异 在这些亚型和MAP-2变异体中，将进行检查。 (b)的 神经母细胞瘤/神经胶质瘤细胞系（NG 108 -15）含有独特的钙调素亚型 激酶II和激酶在细胞分化期间被诱导。 细胞分化的特征是电化学的发展。 兴奋性，形成功能性突触的能力，以及 充满细胞突起的神经突 因此，该细胞系是一种有希望的 模型系统研究激酶调控的细胞骨架。 以下 本文将讨论以下问题：（1）CaM激酶II的亚型是什么 负责磷酸化的未成熟和成熟的变异体， 微管相关蛋白MAP-2a、MAP-2b、MAP-2c？ (2)这些是 同工酶和它们各自的细胞骨架蛋白底物 同时在完整的老鼠大脑中 (3)Is这些共同发展 在培养的NG 108中观察到的同工酶和相应的细胞骨架底物 细胞？

Modification of the cytoskeleton by protein kinases may play an important role in neuronal regeneration and plasticity. A type II calcium/calmodulin-dependent kinase (CaM kinase II) is a proposed mediator of cytoskeletal function due to its ability to phosphorylate the microtubule-associated protein (MAP-2) which is co-localized with the kinase in dendrites. The predominant form of CaM kinase II in adult rat forebrain is a 500-730 kDa holoenzyme containing two subunits (50 kDa/60 kDa) in a 3/1 molar ratio. Suggestive evidence indicates that several isoforms of the kinase are present in brain and under developmental control. MAP-2 consists of three homologous proteins (a/b/c) that are also developmentally regulated. All three MAP-2 variants promote tubulin assembly into microtubules, whereas only the high-molecular weight MAP-2a/b additionally promote stabilization of the dendritic cytoskeleton by interacting with other cellular elements. The primary goal of this proposal is to examine the relationship between isoforms of CaM kinase II and MAP a/b/c substrates in order to gain insights into the mechanism in which CaM kinase II regulates cytoskeletal dynamics. Two model systems will be sued to compare the expression of kinase isozymes and MAP-2 variants in neurons and neuron-like cells in culture. (a) several unique isoforms of CaM kinase II present in rat brain will be separated using cation-exchange chromatography. Developmental differences in these isoforms and MAP-2 variants will be examined. (b) The neuroblastoma/ glioma cell line (NG108-15) contains a unique isoform of CaM kinase II and kinase is induced during cellular differentiation. Differentiation of cells is characterized by development of electrical excitability, ability to form functional synapses, and elaboration of cytoskeleton-filled neurites. Therefore, this cell line is a promising model system to study kinase regulation of cytoskeleton. The following questions will be addressed: (1) What are the isoforms of CaM kinase II responsible for the phosphorylation of immature and mature variants of the microtubule-associated proteins MAP-2a, MAP-2b, MAP-2c? (2) Are these isozymes and their respective cytoskeletal protein substrates developed concurrently in intact rat brains? (3)Is the co-development of these isozymes and respective cytoskeletal substrates observed in cultured NG108 cells?