Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis

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

doi:10.1074/jbc.m109.095752

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.

Highlights

With this fast release [3]
We wondered whether the early steps accomplished when acrosomal exocytosis was initiated in the presence or absence of calcium were the same
When exocytosis has progressed to the stage blocked by Nitrophenyl EGTA-acetoxymethyl ester (NP-EGTA), the system becomes resistant to the addition of inhibitors that affect early steps of the process, and secretion is completed in the presence of these inhibitors when NP-EGTA is inactivated [6]

Summary

Introduction

With this fast release [3]. Synaptotagmins carry two C2 domains (C2A and C2B) that bind acidic phospholipids in the presence of calcium. We have previously observed that synaptotagmin VI is dephosphorylated at an early stage of the acrosome reaction, before the release of calcium from the acrosome [9] and that phosphorylation profoundly affects the biological activity of this protein [15].

Objectives

Results

Conclusion