Regulation of cytoskeleton by annexin VI

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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.