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Regulation of cytoskeleton by annexin VI

膜联蛋白 VI 对细胞骨架的调节

基本信息

批准号：
07680842
负责人：
INUI Makoto
金额：
$ 1.41万
依托单位：
Yamaguchi University (1996)Osaka University (1995)
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-07680842/
关键词：
Annexin Calspectin Synapsin I Calcium ion Phospholipid Phosphorylation

项目摘要

To elucidate the physiological significance of annexin VI in brain, we developed a method to detect annexin VI-binding proteins using ^<125> I-annexin VI on a nitrocellulose sheet to which rat brain proteins were electrophoretically transferred after SDS polyacrylamide gel electrophoresis.We found several annexin VI-binding proteins, which interact with annexin VI in a Ca^<2+>- and phospholipid-dependent manner. Of these, the 240K and the 80K proteins were identified to be calspectin and synapsin I,respectively. The bindings of annexin VI to these proteins were also observed in the native state. Annexin VI inhibited the interaction between calspectin and F-actin by binding to calspectin in the presence of Ca^<2+> and PS.An annexin VI-binding site in calspectin was localized to the N-terminal region of b-calspectin near the actin-binding site. On the other hand, annexin VI bound to the N-terminal head region of synapsin I.The binding was inhibited by phosphorylation of synapsin I by cAMP-dependent protein kinase or by Ca^<2+>, calmodulin-dependent protein kinase II.We further examined the direct interaction between these two annexin VI-binding proteins. The interaction of these proteins was high affinity one with Kd of about 10 mM.The binding was inhibited by phosphorylation of synapsin I by cAMP-dependent protein kinase or by Ca^<2+>, calmodulin-dependent protein kinase II.These results indicate that annexin VI-binding proteins play an important role in the regulation of neurotransmitter release in the presynaptic region.

为了阐明膜联蛋白VI在脑中的生理意义，我们建立了一种利用硝酸纤维素膜上的膜联蛋白VI结合蛋白的方法，大鼠脑蛋白质经SDS-聚丙烯酰胺凝胶电泳后被转移到硝酸纤维素膜上。我们发现了几种与膜联蛋白VI结合的蛋白，它们以钙和磷脂依赖的方式相互作用。其中，240K和80K蛋白分别被鉴定为钙调素和突触素I。在天然状态下，也观察到Annexin VI与这些蛋白的结合。在Ca~(2+)和PS存在下，Annexin VI通过与Calspectin结合来抑制Calspectin与F-肌动蛋白的相互作用。另一方面，膜联蛋白VI与突触素I的N-末端头部区域结合，cAMP依赖的蛋白激酶或钙调素依赖的蛋白激酶II对突触素I的磷酸化作用被抑制。我们进一步研究了这两种膜联蛋白VI结合蛋白之间的直接相互作用。这些蛋白之间的相互作用是高亲和力的，其Kd约为10 mM。结合被cAMP依赖的蛋白激酶或钙调素依赖的蛋白激酶II抑制突触素I的磷酸化。这些结果表明，Annexin VI结合蛋白在突触前区神经递质释放的调节中起重要作用。