The roles in neuronal differentiation of Ca^<2+>/calmodulin-dependent phosphorylation of microtubule proteins

Ca^2/钙调蛋白依赖性微管蛋白磷酸化在神经元分化中的作用

• 批准号: 07680847
• 负责人: YAMAMOTO Hideyuki
• 金额: $ 1.47万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680847/
• 关键词: Alzheimer's disease; Ca^<2+>; Calmodulin; Neuronal differentiation; CaM kinase II; Microtubule-associated protein 2; PC12 cells; Antiphosphopeptide antibody; 細胞骨格蛋白質; 蛋白質燐酸化酵素; 分子生物学

1) In order to investigate the physiological roles of Ca^<2+>/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the alpha subunit of mouse CaM kinase II into PC12 cells. We have established the subclonal cell lines that constitutively express the transfected CaM kinase II.The phosphorylation of microtubule-associated protein 2 (MAP2) in these cell lines was higher than in the wild type cells, and was enhanced by treatment with ionomycin. 2) The two antiphosphopeptide antibodies to tau indicate that tau in the peripheral nervous system and Alzheimer's disease brain is highly phosphorylated at serine 199, serine 202 and serine 396.3) We synthesized a peptide (MAP2 peptide) which included serine 1562 of MAP2. MAP2 peptide was strongly phosphorylated by CaM kinase II.4) We prepared antiphosphopeptide antibody which reacted with MAP2 phosphorylated at serine 1562 by CaM kinase II.5) The phosphorylation of MAP2 in cerebellar culture cells was enhanced by treatment with ionomycin.These results suggest that CaM kinase II regulates the functions of MAP2 in the cells and that phosphorylation of tau is increased in Alzheimer's disease brain. The antiphosphopeptide antibody to MAP2 is useful for the investigation of phosphorylation of MAP2 by CaM kinase II in the cells.

1)为了研究Ca^2+/钙调素依赖性蛋白激酶II（CaM kinase II）在神经元分化中的生理作用，我们将小鼠CaM kinase II α亚基的cDNA转染到PC 12细胞中。我们已经建立了亚克隆细胞系，组成型表达转染CaM激酶II。微管相关蛋白2（MAP 2）在这些细胞系中的磷酸化高于野生型细胞，并增强处理离子霉素。2)两种针对tau的抗磷酸肽抗体表明，外周神经系统和阿尔茨海默病大脑中的tau在丝氨酸199、丝氨酸202和丝氨酸396处高度磷酸化。3）我们合成了一种肽（MAP 2肽），其中包含MAP 2的丝氨酸1562。4）制备了抗磷酸肽抗体，该抗体与被CaM激酶II磷酸化的MAP 2的丝氨酸1562位反应。5）用离子霉素处理小脑培养细胞，可增强MAP 2的磷酸化。MAP 2的抗磷酸肽抗体可用于研究细胞中CaM激酶II对MAP 2的磷酸化。

项目成果

K.Ikegami, T.Kimura, S.Katsuragi, T.Ono, H.Yamamoto, E.Miyamoto and T.Miyakawa: "Immunohistochemical examination of phosphorylated tau in granulovacuolar degeneration granules." Psy.Clin.Neurosci.50. 137-140 (1996)

K.Ikegami、T.Kimura、S.Katsuragi、T.Ono、H.Yamamoto、E.Miyamoto 和 T.Miyakawa：“颗粒空泡变性颗粒中磷酸化 tau 蛋白的免疫组织化学检查。”

X.Sun: "Preparation of tau from the periferal nerve : Presence of insoluble low molecular weight tau with high phosphorylation" Biochem.Biophys.Res.Commun.210. 338-344 (1995)

X.Sun：“从周围神经制备 tau 蛋白：存在高度磷酸化的不溶性低分子量 tau 蛋白”Biochem.Biophys.Res.Commun.210。

Y.He: "Modifications of neurofilament proteins by possible metabolites of allyl chloride in vitro" Drug and Chemical Toxicology. 18. 315-331 (1995)

Y.He：“烯丙基氯可能的体外代谢物对神经丝蛋白的修饰”药物和化学毒理学。

山本秀幸: "脳の細胞情報伝達機構に関する分子薬理学的研究" 日薬理誌. 107. 9-19 (1996)

Hideyuki Yamamoto：“脑细胞信息传递机制的分子药理学研究”日本药理学杂志107. 9-19（1996）。

Yasuharu Oishi: "The effects of 2,5-hexandione and acrylamide on myosin heavy chain isoforms of slow and fast skeletal muscles of the rat" Toxicol.Appl.Pahrmacol.139. 15-21 (1996)

Yasuharu Oishi：“2,5-己二酮和丙烯酰胺对大鼠慢速和快速骨骼肌肌球蛋白重链亚型的影响”Toxicol.Appl.Pahrmacol.139。