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

1)为了研究钙调素依赖性蛋白激酶II(CaK II)在神经分化中的生理作用，我们将小鼠CaM激酶IIα亚基的基因转入PC12细胞。我们已经建立了组成性表达CaM激酶II的亚克隆细胞系。在这些细胞系中，微管相关蛋白2(MAP2)的磷酸化水平高于野生型细胞，并且被离子霉素处理后增强。2)两种针对tau的抗磷酸肽抗体表明，tau在周围神经系统和阿尔茨海默病脑中高度磷酸化，分别位于丝氨酸199、丝氨酸202和丝氨酸396.3。4)我们制备了抗磷酸肽抗体，该抗体与CaM激酶II在1562位丝氨酸发生磷酸化的MAP2反应。5)经离子霉素处理后，小脑培养细胞中MAP2的磷酸化程度增加。这些结果表明，CaM激酶II调节细胞内MAP2的功能，并且在阿尔茨海默病脑中，tau的磷酸化水平增加。MAP2的抗磷酸肽抗体可用于研究细胞内CaM激酶II对MAP2的磷酸化作用。

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.