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Protein kinase activation is required for secretagogue‐stimulated membrane‐association of fusion promoting protein annexin A7 in alveolar type II cells
Author(s) -
Chander Avinash,
Gerelsaikhan Tudevdagva,
Holbrook Kelly,
Hayrapetyan Yedema
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.612.23
Subject(s) - lamellar granule , secretion , annexin , protein kinase c , chemistry , microbiology and biotechnology , protein kinase a , cytosol , fusion protein , western blot , secretagogue , pulmonary surfactant , a549 cell , kinase , flow cytometry , biochemistry , biology , in vitro , recombinant dna , enzyme , gene
The secretion of lung surfactant in alveolar type II (T2) cells requires fusion of lamellar bodies (LB) with the plasma membrane. Our in vitro studies have shown a role for Annexin A7 (A7) in such membrane fusion, since purified A7 binds to LB and plasma membranes and promotes their fusion. Using antibodies against recombinant A7, we have shown that secretagogues of lung surfactant promote membrane‐association of endogenous A7 in isolated T2 cells. We now show that activation of protein kinase is required for such membrane‐association. Isolated rat T2 cells were treated for 30min with phorbol myristate acetate (PMA), A23187, ATP or isoproterenol, and the A7 levels in the cytosol and membrane fractions were analyzed by immuno‐blot analysis. All agents increased relative proportion of A7 in the membranes when compared with the control cells. Immuno‐fluorescence staining of T2 cells showed that secretagogues promoted relocation of A7 to membranes including LB, as verified by co‐staining for ABCA3. Because of the significance of protein kinase activation in regulation of surfactant secretion, we evaluated the effect of protein kinase inhibitors on A7 relocation in treated cells. The PMA‐induced relocation was inhibited by pretreatment with bis‐indolylmaleimide I, a specific inhibitor of PKC, and the isoproterenol‐induced relocation was blocked with H‐89, a specific inhibitor of PKA. Thus, our studies provide further correlative evidence for the role of A7 in lung surfactant secretion.