Calcineurin-mediated dephosphorylation of synaptotagmin VI is necessary for acrosomal exocytosis

Jimena Castillo Bennett, Carlos M. Roggero, Franco E. Mancifesta, Luis S. Mayorga

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.

Original languageEnglish (US)
Pages (from-to)26269-26278
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number34
DOIs
StatePublished - Aug 20 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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