Regulation of cytoskeleton by annexin VI

为了阐明膜联蛋白VI在脑中的生理意义，我们建立了一种检测膜联蛋白VI结合蛋白的方法，在SDS聚丙烯酰胺凝胶电泳后将大鼠脑蛋白电泳转移到硝化纤维素片上，利用^<125 bb0 i -膜联蛋白VI检测膜联蛋白VI结合蛋白。我们发现了几种膜联蛋白VI结合蛋白，它们以Ca^<2+>-和磷脂依赖的方式与膜联蛋白VI相互作用。其中，240K和80K蛋白分别被鉴定为calspectin和synapsin I。在天然状态下也观察到膜联蛋白VI与这些蛋白的结合。在Ca^<2+>和ps存在的情况下，Annexin VI通过与calspectin结合而抑制了calspectin与F-actin的相互作用。calspectin中Annexin VI的结合位点定位在靠近actin结合位点的b-calspectin的n端区域。另一方面，膜联蛋白VI与突触蛋白I的n端头区结合，这种结合被camp依赖性蛋白激酶或钙调素依赖性蛋白激酶Ca^<2+>磷酸化的突触蛋白I所抑制。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。这些蛋白的相互作用是高亲和力的，Kd约为10 mm，结合被camp依赖性蛋白激酶或钙调素依赖性蛋白激酶Ca^<2+>磷酸化的突触蛋白I所抑制。这些结果表明，膜联蛋白vi结合蛋白在突触前区神经递质释放调控中起重要作用。

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.