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

ZARELLI, VALERIA E. P.; RUETE, MARIA C.; ROGGERO, CARLOS M.; MAYORGA, LUIS S.; TOMES, CLAUDIA N.

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2009 vol. 284 p. 10491 - 10503

0021-9258

The reversible phosphorylation of tyrosyl residues in proteinsis a cornerstone of the signaling pathways that regulate numerouscellular responses. Protein tyrosine phosphorylation is controlledthrough the concerted actions of protein-tyrosinekinases and phosphatases. The goal of the present study was tounveil the mechanisms by which protein tyrosine dephosphorylationmodulates secretion. The acrosome reaction, a specializedtype of regulated exocytosis undergone by sperm, is initiatedby calcium and carried out by a number of players,including tyrosine kinases and phosphatases, and fusion-relatedproteins such as Rab3A, -SNAP, N-ethylmaleimide-sensitivefactor (NSF), SNAREs, complexin, and synaptotagmin VI. Wereport here that inducers were unable to elicit the acrosomereaction when permeabilized human sperm were loaded withanti-PTP1B antibodies or with the dominant-negative mutantPTP1B D181A; subsequent introduction of wild type PTP1B orNSF rescued exocytosis. Wild type PTP1B, but not PTP1BD181A, caused cis SNARE complex dissociation during theacrosome reaction through a mechanism involving NSF. Unlikeits non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes.These results strengthen our previous observation thatNSF activity is regulated rather than constitutive during spermexocytosis and indicate that NSF must be dephosphorylated byPTP1B 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 tyrosineresidues prevents its SNARE complex dissociation activityand establish for the first time a role for PTP1B in the modulationof the membrane fusion machinery.