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

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, ␣-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.

Membrane Fusions During Mammalian Fertilization

“…Zona binding evokes Ca 2+ entry (see Section 5.3.1) and this causes both the dissociation of the MUPP1/CaMKII fusion clamp [64] and a calcineurin-mediated dephosphorylation of synaptotagmin VI [63]. The dephosphorylation of synaptotagmins also appear to be essential for the acrosome reaction [59, 62,117,63] (unpublished observation). The role of Rab3A [62] and Rab2A (unpublished observation) in the formation of the cis ternary SNARE complex conformation is not yet clear.…”

Protein-tyrosine Phosphatase 1B Modulates Early Endosome Fusion and Trafficking of Met and Epidermal Growth Factor Receptors

“…Recently, the ATPase NSF was identified as a substrate for PTP1B (42). NSF activity is tightly modulated by its phosphorylation status; dephosphorylation promotes its interaction with endosomal components and results in endosome fusion.…”

“…PTP-MEG2 is located on the cytoplasmic face of secretory vesicles where it regulates vesicle size by promoting homotypic vesicle fusion via tyrosine dephosphorylation of NSF (41). Recently, PTP1B has been identified as a PTP that dephosphorylates NSF in acrosomes (42). Additionally, PTP1B has been implicated in the dephosphorylation and inactivation of internalized RTKs, including Met and EGFR (26,30), and in MVB maturation in response to EGF (39).…”

“…After vesicle fusion, the SNARE complex, which includes ␣-soluble N-ethylmaleimide-sensitive factor (NSF)-associated protein (␣-SNAP), must be disassembled via ATP hydrolysis of its ␣-SNAP subunit by the NSF; inefficient disassembly of the SNARE complex halts further vesicle fusion. The activity of NSF is regulated via its phosphorylation status; the kinases Fes and Ca 2ϩ /calmodulin-dependent protein kinase II (40) and the phosphatases PTP-MEG2 (41) and PTP1B (42) have been shown to control NSF phosphorylation. PTP-MEG2 is located on the cytoplasmic face of secretory vesicles where it regulates vesicle size by promoting homotypic vesicle fusion via tyrosine dephosphorylation of NSF (41).…”

Molecular Machinery Regulating Exocytosis

“…This has facilitated the functional study of individual proteins as targets of particular protein kinases, whilst uncovering the significance of phosphorylation/dephosphorylation events in the regulation of exocytosis, such as in: synaptic transmission and cell plasticity (Amin, et al, 2008;Barclay, et al, 2003;Boczan, et al, 2004), neuronal morphogenesis (Chernyshova, et al, 2011), insulin secretion (Butelman, 1990;Sugawara, et al, 2009;Wang & Thurmond, 2010), insulin stimulated GLUT4 transport (Aran, et al, 2011;X. W. Chen, et al, 2011a;Sano, et al, 2011), mast cell and platelet degranulation (Fitzgerald & Reed, 1999;Foger, et al, 2011), exocytosis of factors required for neutrophil adhesion (Fu, et al, 2005), acrosomal exocytosis in sperm (Castillo Bennett, et al, 2010;Zarelli, et al, 2009) and lung surfactant exocytosis (Gerelsaikhan, et al, 2011). It has now become obvious that phospho-regulation of exocytosis is a complex and dynamic process implicated at almost all points along the exocytic route, from recruitment and transport of vesicles to their ultimate fusion at the plasma membrane ( Figure 1&2).…”