DEPHOSPHORYLATION OF SYNAPTOTAGMIN VI DURING ACROSOMAL EXOCYTOSIS IS MEDIATED BY CALCINEURIN

CASTILLO BENNETT, JIMENA; ROGGERO, CARLOS M.; DE BLAS, GERARDO A.; MAYORGA, LUIS S.

Rosario, Argentina

Congreso; XLII Reunión Anual de la Soc. Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Acrosomal reaction (RA) is a calcium regulated exocytosis that is essential for fertilization. Synaptotagmins (syt) are transmembrane proteins with two cytoplasmatic domains that binds calcium and phospholipids (C2A y C2B). Syt have been involucrated in exocytosis as calcium sensors. We have demonstrated that syt VI is required for RA. This protein is phosphorylated in resting sperm, and become dephosphorylated after stimulation. Our aim is to unveil the phosphatase involved in this process. We have used the streptolysin O permeabilizated sperm model and have evaluated RA by staining with fluorescein-isothiocyanate-coupled Pisum sativum (FITC-PSA). To detect the phosphorylated state we have used an antibody antiphosphosynaptotagmin (antiPP). The antiPP inhibition is lost after calcium stimulation that indicate dephosphorylation of endogenous syt. When sperm were stimulated with Rab3A, antiPP conserved the inhibiting effect like in resting sperm. This indicate that dephosphorylation of syt requires calcium. So, calcineurin, a calcium/calmodulin-activated serine/threonine protein phosphatase, could be implicated in this process. We incubated sperm with Cyclosporin A and FK506, two calcineurin inhibitors, and we observated that both inhibited the RA. Our results indicate that dephosphorylation of syt require calcium and this process is mediated by calcineurin.