Phosphorylation /dephosphorylation of synaptotagmin 6 during acrosomal exocytosis.

CASTILLO BENNETT J, ROGGERO CM, MANCIFESTA F, MAYORGA LS.

Carlos Paz

Congreso; SAIB XLIV; 2008

Sociedad Argentina de Investigaciones en bioquímica y Biología Molecular

The acrosomal exocytosis (AE) is a calcium regulated exocitosisessential for fertilization. We have demonstrated thatsynaptotagmin 6 (Syt 6) is phosphorylated in resting sperm, andthat its C2B domain presents a polybasic region which is a target forPKC. As we predicted that threonine 419 (T419) has moreprobability to be phosphorylated by this kinase, we created aphosphomimetic mutant (T419E). Using a FRET assay wedemonstrated that T419E lost the ability to bind liposomes in aCa2+-dependent way and its inhibitory effect in an AE assay. Theseresults suggest that the PKC mediated regulation of C2Bwt activitydepends on T419 phosphorylation. However, T419 is not the onlytarget for PKC; the T419E mutant was still able to incorporate 32P inan in vitro phosphorylation assay. We previously demonstrated thatSyt 6 is dephosphorylated after sperm stimulation. Calcineurin(CaN), a calcium/calmodulin-activated protein phosphatase that ispresent in sperm and is required for AE, could be responsible of Syt6 dephosphorylation. In an in vitro phosphorylation/dephosphorylation assay with 32P, using a constitutively active formof CaN, we showed that the Syt 6 C2B domain wasdephosphorylated by CaN. We conclude that Syt 6 activity isregulated by PKC phosphorylation at T419 and needs to bedephosphorylated by CaN to participate in AE.