Phosphorylation/dephosphorylation of synaptotagmin 6 during acrosomal exocytosis.

CASTILLO BENNETT, JIMENA; ROGGERO, CARLOS M.; MANCIFESTA, FRANCO E.; MAYORGA, LUIS S.

Villa Carlos Paz, Cordoba

Congreso; XLIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

SAIB

The acrosomal exocytosis (AE) is a calcium regulated exocitosis essential for fertilization. We have demonstrated that synaptotagmin 6 (Syt 6) is phosphorylated in resting sperm, and that its C2B domain presents a polybasic region which is a target for PKC. As we predicted that threonine 419 (T419) has more probability to be phosphorylated by this kinase, we created a phosphomimetic mutant (T419E). Using a FRET assay we demonstrated that T419E lost the ability to bind liposomes in a 2+ Ca -dependent way and its inhibitory effect in an AE assay. These results suggest that the PKC mediated regulation of C2Bwt activity depends on T419 phosphorylation. However, T419 is not the only 32 target for PKC; the T419E mutant was still able to incorporate P in an in vitro phosphorylation assay. We previously demonstrated that Syt 6 is dephosphorylated after sperm stimulation. Calcineurin (CaN), a calcium/calmodulin-activated protein phosphatase that is present in sperm and is required for AE, could be responsible of Syt 6 dephosphorylation. In an in vitro phosphorylation/ 32 dephosphorylation assay with P, using a constitutively active form of CaN, we showed that the Syt 6 C2B domain was dephosphorylated by CaN. We conclude that Syt 6 activity is regulated by PKC phosphorylation at T419 and needs to be dephosphorylated by CaN to participate in AE.