Annexin A7 interactions with SNARE protein SNAP23 are regulated by calcium and protein phosphorylation during surfactant secretion in alveolar type II cells

Abstract

We have previously postulated that annexin A7 (A7) facilitates the obligatory membrane fusion between lamellar bodies and plasma membrane during surfactant secretion in alveolar type II (T2) cells. Recently, we reported that several secretagogues of lung surfactant promote protein kinase‐dependent co‐localization of A7 with the lamellar body marker protein ABCA3 in T2 cells. We now report that secretagogues also promote association of A7 with SNAP23, one of the SNARE proteins. Confocal immuno‐fluorescence microscopy of T2 cells showed a time‐dependent increase in co‐localization of A7 and SNAP23 in T2 cells stimulated with PMA or calcium ionophore A23187. The increased co‐localization was prevented by pre‐treatment of T2 cells with PKC inhibitor, BisI. Supporting evidence for interactions of A7 and SNAP23 was derived from in vitro binding studies. The binding of bacterially expressed A7 with GST‐SNAP23 was Ca2+‐dependent. The binding was observed at 0.2μM Ca2+, which increased further in Ca2+ concentration‐dependent manner. Thus, physiologic conditions causing calcium elevation would increase interaction of A7 with SNAP23. In vitro phosphorylation of A7 with PKC also increased the Ca2+‐dependent binding to GST‐SNAP23. We conclude that calcium elevation and increased protein phosphorylation that occur in secretagogue‐stimulated T2 cells would facilitate A7 interactions with SNAP23 during surfactant secretion. We speculate that A7 phosphorylation and association with lamellar bodies and SNARE proteins is to facilitate the membrane fusion process.