Role of calmodulin kinases on the regulation of Ca^<2+> signaling of central nervous systems

神经元Ca^2+/钙调蛋白依赖性蛋白激酶II（CaM激酶II）调节重要的神经元功能，包括神经递质合成和神经递质释放、离子通道活性调节、突触可塑性和基因表达。本研究取得了以下结果：1.将CaM激酶II导入神经元细胞后，神经突起生长受到刺激，表明CaM激酶II在神经突起生长中起刺激作用;在Thr-286处自磷酸化的激酶的Ca^<2+>非依赖性活性参与神经突生长; 3.在突触后致密区（postsynaptic density，PSD）发现了28种以上的CaM激酶II底物蛋白，并通过蛋白质测序和质谱鉴定了几乎所有的蛋白质; 4.当研究培养细胞神经分化过程中激酶的发育变化时，发现CaM激酶II在神经元分化过程中上调;细胞类型不仅在大鼠脑组织发育过程中，而且在培养的神经元细胞分化过程中，δ异构体的剪接模式也发生了明显的变化，表明CaM激酶II δ异构体的选择性剪接变体在神经分化过程中表达; 6.当我们用β特异性插入的缺失突变体研究α和β CaM激酶II在酶学性质上的差异时，发现β特异性插入对β CaM激酶II的细胞分布起重要作用; 7克隆了编码大鼠Ca^2+/钙调素依赖性蛋白激酶II β亚型的基因，并测定其结构和外显子-内含子组织; 8.当使用神经元和非神经元细胞分析该基因的启动子活性时，在该基因的5 '上游区域发现神经元细胞类型特异性启动子活性。

Neuronal Ca^<2+> /calmodulin-dependent protein kinase II (CaM kinase II) regulates important neuronal functions, including neurotransmitter synthesis and neurotransmitter release, modulation of ion channel activity, synaptic plasticity, and gene expression. The following results were obtained in this study; 1. when CaM kinase II was introduced in neuronal cells, neurite outgrowth was stimulated, indicating that CaM kinase II played a role stimulation of neurite outgrowth; 2. Ca^<2+> independent activity of the kinase autophosphorylated at Thr-286 involved in neurite outgrowth; 3. more than 28 protein substrates of CaM kinase II were found in the postsynaptic density (PSD), and almost all of these proteins were identified by protein sequencing and mass spectrometry; 4. when the developmental change of the kinase was investigated during the neural differentiation of cultured cells, it was found that CaM kinase II was upregulated during the neuronal differentiation; 5. cell type distinctive changes of splicing pattern of δ isoform were found not only during development of rat brain tissue, but also during differentiation of cultured neuronal cells, indicating that alternatively spliced variants of δ isoform of CaM kinase II were expressed during neural differentiation; 6. when we used the deletion mutants for β-specific insertions to explore the difference between α and β CaM kinase II in enzymatic properties, it was found that β specific insertion of β CaM kinase II played an important role in the cellular distribution of the kinase; 7 The gene encoding the β isoform of rat Ca^<2+>/calmodulin-dependent protein kinase II was cloned, and its structure and exon-intron organization were determined; 8. When the promoter activity of the gene was analyzed using nueronal and non-neuronal cells, neuronal cell type-specific promoter activity was found in the 5'-upstream region of the gene.