Direct Observation of the Regulation of Microtubule Dynamics by MAP2 Phosporylation

直接观察 MAP2 磷酸化对微管动力学的调节

基本信息

批准号：
07680715
负责人：
ITOH Tomohiko
金额：
$ 1.47万
依托单位：
Nagoya 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

项目摘要

Phosphorylation-dependent regulation of microtubule-stabilizing activities of microtubule-associated protein 2 (MAP2) was examined using optical microscopy. MAP2, purified from mammalian brain, was phosphorylated by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. By PKA,15 mloles of phoshoryl groups was incorporated into one mole of MAP2, but about 70% was distributed to the projection region. By cdc2 kinase, 8-10 moles of phosphoryl groups was incorporated into one mole of MAP2 and more than 60% of the phosphates was distributed to the microtubule-binding region. Both phosphorylations similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules under a dark-field microscope showed that interconversion between polymerization phase and depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, phase transition from depolymerization to polymerization was difficult to take place, with the result that life-time of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous nucleation of microtubules under a dark-field microscope showed that PKA-phosphorylated MAP2 redued nucleating-activity, while cdc2 kinase-phosphorylated MAP2 completely abolished it. These observations show that cdc2 kinase-dependent phosphorylation inhibited both microtubule-stabilizing activity and microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affected only microtubule-nucleating activity of MAP2.

使用光学显微镜检查了微管相关蛋白2（MAP2）的微管稳定活性的磷酸化依赖性调节。从哺乳动物脑纯化的MAP2被CAMP依赖性蛋白激酶（PKA）或细胞周期蛋白B依赖性CDC2激酶磷酸化。通过PKA，将15个phoshoryl基团的1个mloles掺入了MAP2的一摩尔中，但将约70％分布到投影区域中。通过CDC2激酶，将8-10摩尔的磷酸基团掺入一摩尔的MAP2中，并将60％以上的磷酸盐分布在微管结合区域中。这两种磷酸化都类似地降低了MAP2对微管的结合活性。在暗场显微镜下对单个微管的直接观察表明，在未磷酸化和PKA磷酸化的MAP2中重复了聚合阶段和去聚合阶段之间的互连。然而，在Cdc2激酶磷酸化的MAP2中，从解聚到聚合的相变很难进行，结果是单个微管的寿命与没有MAP2的寿命一样短。在暗场显微镜下对微管的自发成核的检查表明，PKA磷酸化的MAP2重新核定活性，而Cdc2激酶磷酸化的MAP2完全废除了它。这些观察结果表明，CDC2激酶依赖性磷酸化抑制了MAP2的微管稳定活性和微管核活性，而PKA依赖性磷酸化仅影响了MAP2的微管 - 核核活性。