Annexin A7 and SNAP23 interactions in alveolar type II cells and in vitro: a role for Ca(2+) and PKC.
Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.
Department of Pediatrics, Stony Brook University Medical, Stony Brook, NY 11794, USA.
Tudevdagva
T
Gerelsaikhan
Pavan Kumar
PK
Vasa
Avinash
A
Chander
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D056809:Alveolar Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D017310:Annexin A7 / Q000276:immunology / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D017310:Annexin A7; D000906:Antibodies; D047428:Protein Kinase Inhibitors; D050600:SNARE Proteins; C498858:Snap23 protein, rat; D033921:Vesicular Transport Proteins; D000001:Calcimycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium