Hosoi T, Uchiyama M, Okumura E, Saito T, Ishiguro K, Uchida T, Okuyama A, Kishimoto T, Hisanaga S
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology.
J Biochem. 1995 Apr;117(4):741-9. doi: 10.1093/oxfordjournals.jbchem.a124771.
The major kinase capable of phosphorylating tau in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tau exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau 1 antibodies in tau proteins , while cdk5 phosphorylated the site recognized by SMI33 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau 1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.
在猪脑提取物中，能够使tau蛋白磷酸化的主要激酶被认为是一种脑内类似cdc2的激酶，称为cdk5。在猪脑提取物中利用ATP组装的微管中的tau蛋白成分被磷酸化至更高水平，并且与利用GTP组装的tau蛋白相比，其电泳迁移率更慢。在与ATP孵育的脑提取物中发生的这种磷酸化和电泳迁移率变化，大部分被丁内酯I抑制，丁内酯I是cdc2激酶和cdk5的特异性抑制剂。此外，丁内酯I可将外源性添加到脑提取物中的tau蛋白的磷酸化抑制约70%。从猪脑中纯化的cdk5通过体外将去磷酸化的tau蛋白磷酸化，使其电泳迁移率降至与利用ATP聚合的微管中存在的水平相同。从海星卵母细胞中纯化的cdc2激酶也能使tau蛋白磷酸化，并使其电泳迁移率的变化程度大于cdk5所导致的变化。蛋白质免疫印迹分析表明，cdc2激酶可使tau蛋白中被SMI31、33、34和tau 1抗体识别的表位磷酸化，而cdk5可使被SMI33识别的位点（对应于最长的人类tau异构体中Ser235位点的磷酸化）磷酸化，并部分使tau 1位点磷酸化。在脑提取物中，在冈田酸存在的情况下对tau蛋白进行的磷酸化实验表明，存在作用于磷酸化Ser235的冈田酸敏感和不敏感的磷酸酶。断头后立即制备的大鼠tau蛋白显示出与利用ATP聚合的微管中的tau蛋白相似的磷酸化状态，这表明tau蛋白在体内相对处于磷酸化状态。
