PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

Valeria E.P Zarelli,Maria C Ruete,Carlos M Roggero,Luis S Mayorga,Claudia N Tomes

doi:10.1074/jbc.m807614200

Abstract

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Highlights

Leimide-sensitive factor (NSF),2 ␣-SNAP, Munc18, and complexin (2– 4)
protein-tyrosine kinases and phosphatases (PTPs) Activity Is Required after Rab3 and before N-ethylmaleimide-sensitive factor (NSF) during the AR—We resorted to the photosensitive PTP Inhibitor I to determine the steps governed by protein tyrosine dephosphorylation during the AR
C, streptolysin O (SLO)-permeabilized human sperm were treated for 10 min at 37 °C in the presence of 300 nM PTP1B D181A or 3.3 nM anti-PTP1B antibodies

Summary

Introduction

Leimide-sensitive factor (NSF),2 ␣-SNAP, Munc, and complexin (2– 4). SNAREs form a superfamily of proteins that assemble into tightly packed helical bundles (5). Of fusion-incompetent, cis (in the same membrane) SNARE complexes requires the concerted action of ␣-SNAP and NSF (6, 7). The catalytic activity of PTP1B is lost or severely diminished, without detrimental effects on its affinity for substrates, when Cys215 is mutated to Ser/Ala or Asp181 to Ala (19). These mutant forms are known as substrate traps. We identified a new role for PTP1B, that of indirectly catalyzing cis SNARE complex disassembly through the dephosphorylation of NSF. In defining this role, we explained the regulation of NSF activity in sperm through its interaction with PTP1B

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Conclusion