DEVELOPMENTAL EXPRESSION OF CAM KINASE II ISOFORMS

蛋白激酶对细胞骨架的修饰可能起着重要的作用 在神经元再生和可塑性中的作用。A类II 钙/钙调蛋白依赖的激酶(CaM激酶II)是一种被认为的介体 对细胞骨架功能的影响，这是由于它能够磷酸化 微管相关蛋白(MAP-2)与 树突中的激酶。成年大鼠钙调素蛋白激酶II的主要形式 前脑是一个500-730 kDa的全酶，包含两个亚基(50 kDa/60 KDA)，摩尔比为3/1。提示性的证据表明，有几个 该激酶的亚型存在于大脑中，并处于发育阶段 控制力。MAP-2由三种同源蛋白(a/b/c)组成，它们也是 发育调节的。三种MAP-2变异体均促进微管蛋白 组装成微管，而只有高分子量的MAP-2a/b 此外，通过以下方式促进树突状细胞骨架的稳定 与其他细胞元素相互作用。这样做的主要目标是 建议研究CaM激酶II的亚型之间的关系 和映射a/b/c衬底，以便深入了解 哪一种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。 培养中的神经元和神经元样细胞中的MAP-2突变体。(A) 存在于大鼠脑中的几种独特的CaM激酶II亚型将是 用阳离子交换层析分离。发展差异 在这些异构体和MAP-2变异体中，将对其进行研究。(B) 神经母细胞瘤/胶质瘤细胞系(NG108-15)含有一种独特的CaM亚型 激酶II和激酶在细胞分化过程中被诱导。 细胞的分化以电的发育为特征 兴奋性，形成功能性突触的能力，以及阐述 充满细胞骨架的轴突。因此，该细胞系是一种很有前途的 研究细胞骨架蛋白激酶调控的模型系统。以下是 问题将被解决：(1)CaM激酶II的亚型是什么 负责未成熟和成熟变种的磷酸化 微管相关蛋白MAP-2a、MAP-2b、MAP-2c？(2)是 开发同工酶及其相应的细胞骨架蛋白底物 同时在完整的大鼠大脑中？(3)这些是共同发育的吗？ 培养的NG108细胞同工酶及其细胞骨架底物的观察 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?