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

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, α-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. 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, α-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. Exocytosis is a highly regulated membrane fusion process consisting of multiple stages that culminate in the attachment of secretory vesicles to the plasma membrane followed by the opening of fusion pores (1Burgoyne R.D. Morgan A. Physiol. Rev... 2003; 83: 581-632Google Scholar). Many molecules belonging to, or associated with, the fusion machinery have been identified and characterized. These molecules include integral components of the vesicle (R-SNAREs) and of the plasma membrane (Q-SNAREs), in addition to soluble factors such as N-ethylmaleimide-sensitive factor (NSF), 2The abbreviations used are: NSF, N-ethylmaleimide-sensitive factor; AR, acrosome reaction; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; BoNT/B, botulinum toxin B; FITC-PSA, fluorescein isothiocyanate-coupled Pisum sativum agglutinin; IPTG, isopropyl β-d-thio-galactoside; NP-EGTA-AM, O-nitrophenyl EGTA acetoxymethyl ester; PTP, protein-tyrosine phosphatase; SLO, streptolysin O; TeTx, tetanus toxin; TPEN, N,N,N′,N′-tetrakis(2-pyridymethyl)ethylenediamine; DTT, dithiothreitol; WT, wild type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPγS, guanosine 5′-O-(thiotriphosphate). α-SNAP, Munc18, and complexin (2Sollner T.H. Mol. Membr. Biol... 2003; 20: 209-220Google Scholar, 3Jahn R. Lang T. Sudhof T.C. Cell.. 2003; 112: 519-533Google Scholar-4Jahn R. Scheller R.H. Nat. Rev. Mol. Cell Biol... 2006; 7: 631-643Google Scholar). SNAREs form a superfamily of proteins that assemble into tightly packed helical bundles (5Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature.. 1998; 395: 347-353Google Scholar). Assembly of Q- and R-SNAREs in trans (in opposite membranes) is thought to pull the fusing membranes closely together, which results in bilayer fusion. Disassembly of fusion-incompetent, cis (in the same membrane) SNARE complexes requires the concerted action of α-SNAP and NSF (6Zhao C. Slevin J.T. Whiteheart S.W. FEBS Lett... 2007; 581: 2140-2149Google Scholar, 7Collins K.M. Thorngren N.L. Fratti R.A. Wickner W.T. EMBO J... 2005; 24: 1775-1786Google Scholar). It was believed that NSF is constitutively active to guarantee the constant regeneration of free SNAREs. In the last few years, however, we have learned that NSF activity is regulated by post-translational modifications (8Huynh H. Bottini N. Williams S. Cherepanov V. Musumeci L. Saito K. Bruckner S. Vachon E. Wang X. Kruger J. Chow C.W. Pellecchia M. Monosov E. Greer P.A. Trimble W. Downey G.P. Mustelin T. Nat. Cell Biol... 2004; 6: 831-839Google Scholar, 9Matsushita K. Morrell C.N. Cambien B. Yang S.X. Yamakuchi M. Bao C. Hara M.R. Quick R.A. Cao W. O'Rourke B. Lowenstein J.M. Pevsner J. Wagner D.D. Lowenstein C.J. Cell.. 2003; 115: 139-150Google Scholar, 10Huang Y. Man H.Y. Sekine-Aizawa Y. Han Y. Juluri K. Luo H. Cheah J. Lowenstein C. Huganir R.L. Snyder S.H. Neuron.. 2005; 46: 533-540Google Scholar, 11Matveeva E.A. Whiteheart S.W. Vanaman T.C. Slevin J.T. J. Biol. Chem... 2001; 276: 12174-12181Google Scholar-12Liu Y. Cheng K. Gong K. Fu A.K. Ip N.Y. J. Biol. Chem... 2006; 281: 9852-9858Google Scholar). The acrosome is a large secretory vesicle that overlies the nucleus in the apical region of the mature sperm head (13Yanagimachi R. Mammalian Fertilization, The Physiology of Reproduction.(Knobil, E., and Neill, J. D., eds) pp. Raven Press, New York. 1994; : 189-317Google Scholar). In response to physiological or pharmacological stimuli, the acrosome undergoes a special type of calcium-dependent exocytosis (the acrosome reaction, AR), which is a prerequisite for fertilization. The AR proceeds through a sequential set of events initiated when Rab3A is activated by calcium to trigger NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Interestingly, NSF appears to be dormant in resting cells, but its activity is de-repressed when sperm are challenged with AR inducers (14De Blas G.A. Roggero C.M. Tomes C.N. Mayorga L.S. PLoS Biol.. 2005; 3: e323Google Scholar). Later on, monomeric SNAREs re-associate in loose trans complexes until an efflux of calcium from the intra-acrosomal store promotes synaptotagmin- and SNARE-dependent membrane fusion (reviewed in Refs. 15Tomes C.N. Molecular Mechanisms of Exocytosis.(Regazzi, R., ed) pp. Landes Biosciences/Eurekah.com and Springer Science+Business Media, Austin, TX. 2006; : 117-147Google Scholar and 16Mayorga L. Tomes C.N. Belmonte S.A. IUBMB Life.. 2007; 59: 1-7Google Scholar). Tyrosine phosphorylation is controlled by the coordinated actions of protein-tyrosine kinases and phosphatases (PTPs). The widely expressed PTP1B is the prototype of the superfamily of PTPs and belongs to the non-transmembrane subfamily 1 of intracellular PTPs (17Tonks N.K. Nat. Rev. Mol. Cell Biol... 2006; 7: 833-846Google Scholar, 18Tonks N.K. Neel B.G. Curr. Opin. Cell Biol... 2001; 13: 182-195Google Scholar). PTP1B consists of a single N-terminal phosphatase domain (321 residues) and a regulatory C-terminal domain (114 residues). Using structural insights derived from biochemical and crystallographic data, scientists have identified several residues crucial for substrate recognition and catalysis. Two such residues are Cys215 and Asp181. 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 (19Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 1680-1685Google Scholar). These mutant forms are known as substrate traps. The precise role of protein tyrosine dephosphorylation on membrane fusion remains unexplored. We and others have inferred the modulation of the AR by PTPs from results achieved with pharmacological inhibitors (see Ref. 20Tomes C.N. Roggero C.M. De Blas G. Saling P.M. Mayorga L.S. Dev. Biol... 2004; 265: 399-415Google Scholar and references therein). However, data on the identity of the PTPs and substrates involved are lacking. We have determined the role and site of action of PTPs, particularly PTP1B, in secretion by employing antibodies, photosensitive inhibitors, and a substrate-trapping mutant in a functional assay using permeabilized human sperm as the model for exocytosis. 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 also explained the regulation of NSF activity in sperm through its interaction with PTP1B. Reagents—Recombinant streptolysin O (SLO) was obtained from Dr. Bhakdi (University of Mainz, Mainz, Germany). Spermatozoa were cultured in Human Tubal Fluid media (Irvine Scientific, Santa Ana, CA) supplemented with 0.5% bovine serum albumin (HTF media). The mouse monoclonal anti-PTP1B (clone FG6–1G, purified IgG2a) (21Schievella A.R. Paige L.A. Johnson K.A. Hill D.E. Erikson R.L. Cell Growth & Differ... 1993; 4: 239-246Google Scholar) was initially a gift from Dr. N. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); we subsequently purchased the same antibody from Oncogene Science, Inc. (Cambridge, MA). The mouse monoclonal anti-phosphotyrosine antibody (clone 4G10, subclass IgG2b) was from Upstate Biotechnology Inc. (Lake Placid, NY). The rabbit polyclonal anti-NSF (whole serum) and the mouse monoclonal anti-synaptobrevin 2 (clone 69.1, purified IgG) were from Synaptic Systems (Göttingen, Germany). An anti-Rab3A antibody (rabbit polyclonal, purified IgG) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat antimouse IgG was from Kierkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Jackson ImmunoResearch (West Grove, PA). The Escherichia coli strain TKB1, which harbors an inducible Elk tyrosine kinase domain under the control of the tryptophan operon, was from Stratagene (La Jolla, CA). O-Nitrophenyl-EGTA-acetoxymethyl ester (NP-EGTA-AM) and BAPTA-AM were from Molecular Probes (Eugene, OR). α-Bromo-4-hydroxyacetophenone (PTP Inhibitor I) was from Calbiochem (La Jolla, CA). Prestained molecular weight markers were from Boston BioProducts Inc. (Worcester, MA). Glutathione-Sepharose was from GE Healthcare and nickel-nitrilotriacetic acid-agarose from Qiagen GmbH (Hilden, Germany). All other chemicals were reagent or analytical grade and were purchased from Sigma or ICN Biochemicals, Inc. (Aurora, OH). Recombinant Proteins—Two expression plasmids encoding amino acids 1–321 of PTP1B were kindly provided by Dr. N. Tonks: the substrate-trapping mutant PTP1B D181A fused to glutathione S-transferase was in pGEX-3X (GE Healthcare) and the wild type PTP1B fused to His6 was in pET21b (Stratagene). Two plasmids encoding NSF were used for protein expression: pQE9 (Qiagen) was generously provided by Dr. S. Whiteheart (University of Kentucky, Lexington, KY) and pET28a (Stratagene) was a kind gift from Dr. D. Fasshauer (Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany). To obtain tyrosine-phosphorylated NSF in E. coli, NSF in pET28a was transformed into TKB1 cells; protein expression and tyrosine phosphorylation were carried out following Stratagene's instructions. A pQE9 construct encoding α-SNAP was also from Dr. S. Whiteheart. Plasmid pGEX-2T containing the cDNA-encoding human Rab3A was provided by Dr. P. Stahl (Washington University, St. Louis, MO). The light chain of wild type TeTx and BoNT/B fused to His6 (pQE3, Qiagen) were generously provided by Dr. T. Binz (Medizinische Hochschule Hannover, Hannover, Germany). The light chain of catalytically dead TeTx fused to His6 (TeTx E234Q in pET28a) was a gift from Dr. F. Zilly (Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany). The plasmid constructs pQE9 encoding His6-NSF and pQE3 encoding His6-BoNT/B were transformed into E. coli M15pRep4 (Qiagen) and protein expression was induced 4 h at 30 °C with 1 mm isopropyl β-d-thiogalactoside (IPTG). DNAs encoding wild type His6-TeTx and α-SNAP were transformed into E. coli XL-1 Blue (Stratagene) and induced overnight at 20 °C with 0.2 mm IPTG. All proteins encoded by cDNAs contained in pET vectors were expressed in E. coli BLR(DE3) (Stratagene) by inducing with 0.25 mm IPTG (3 h at 25 °C for TeTx E234Q, 3 h at 30 °C for NSF), except PTP1B wild type that was induced with 0.1 mm IPTG overnight at 22 °C. Purification of His6-tagged recombinant proteins was carried out according to Qiagen's instructions, except that 0.5 mm ATP, 5 mm MgCl2, and 2 mm β-mercaptoethanol were added to all buffers involved in the purification of His6-NSF. The His6 tag was cleaved from NSF by incubation with thrombin during dialysis to remove imidazole. Thrombin activity was stopped with 2 mm phenylmethylsulfonyl fluoride. DNAs encoding Rab3A and PTP1B D181A were transformed into E. coli BL21 (Stratagene). We induced the expression of Rab3A with 0.5 mm IPTG overnight at 22 °C, and that of PTP1B D181A with 0.1 mm IPTG overnight at 22 °C. Recombinant proteins were purified on glutathione-Sepharose following standard procedures. Purified Rab3A was prenylated and loaded with guanosine 5′-O-3-thiotriphosphate as described (22Yunes R. Michaut M. Tomes C. Mayorga L.S. Biol. Reprod... 2000; 62: 1084-1089Google Scholar). Recombinant protein concentrations were determined by the Bio-Rad Protein assay in 96-well microplates. Bovine serum albumin was used as a standard and the results were quantified on a Bio-Rad 3550 Microplate Reader. Human Sperm Sample Preparation for AR Assays and Western Blotting—Human semen samples were obtained from normal healthy donors. Semen was allowed to liquify for 30–60 min at 37 °C. We used a swim-up protocol to isolate highly motile sperm. Sperm concentrations were adjusted to 5–10 × 106/ml before incubating for at least 2 h under capacitating conditions (HTF, 37 °C, 5% CO2, 95% air). Permeabilization was accomplished as described (22Yunes R. Michaut M. Tomes C. Mayorga L.S. Biol. Reprod... 2000; 62: 1084-1089Google Scholar). Briefly, washed spermatozoa were resuspended in cold phosphate-buffered saline containing 1.5 units/ml SLO for 15 min at 4 °C. Cells were washed once with phosphate-buffered saline and resuspended in ice-cold sucrose buffer (250 mm sucrose, 0.5 mm EGTA, 20 mm Hepes-K, pH 7) containing 2 mm DTT. For AR assays, we added inhibitors and stimulants sequentially as indicated in the figures legends, and incubated for 10–15 min at 37 °C after each addition. When indicated, we preloaded SLO-permeabilized sperm with photo-inhibitable reagents before incubating in the presence of inhibitors and/or calcium, carrying out all procedures in the dark. Photolysis was induced after the last incubation by exposing twice (1 min each time) to an UV transilluminator, mixing, and incubating for 5 min at 37 °C. Sperm were spotted on Teflon-printed slides, air dried, and fixed/permeabilized in ice-cold methanol for 1 min. Acrosomal status was evaluated by staining with FITC-coupled Pisum sativum (FITC-PSA) according to Ref. 23Mendoza C. Carreras A. Moos J. Tesarik J. J. Reprod. Fertil... 1992; 95: 755-763Google Scholar. At least 200 cells were scored using an upright Nikon microscope equipped with epifluorescence optics. Basal (no stimulation, "control") and positive (0.5 mm CaCl2, corresponding to 10 μm free Ca2+, "Ca2+") controls were included in all experiments. Acrosomal exocytosis indexes were calculated by subtracting the number of spontaneously reacted spermatozoa from all values and expressing the results as a percentage of the AR observed in the positive control. Data were evaluated using one-way analysis of variance. The Tukey-Kramer post hoc test was used for pairwise comparisons. Differences were considered significant at the p < 0.05 level. For Western blotting, SLO-permeabilized sperm (5–10 × 106 cells) were treated or not with 300 nm PTP1B D181A, 27 nm wild type PTP1B or both, and incubated for 30 min at 37 °C. Cells were lysed in cold 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5 mm Na3VO4, 2 mm EDTA, a protease inhibitor mixture (P2714, Sigma), 5 μg/ml trypsin inhibitor, 0.5 mm phenylmethylsulfonyl fluoride, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS. We sonicated sperm (2 × 15 s) and let proteins diffuse into the lysis buffer for 30 min at 4 °C. These whole cell detergent extracts were clarified by centrifugation at 12,000 × g for 5 min and electrophoresed immediately or stored at –20 °C. Disassembly of the SNARE Complex—Brain cortices from adult male Sprague-Dawley rats were homogenized in 5 mm Hepes buffer, pH 7.4, containing 0.32 m sucrose and 1 mm EDTA using a glass Teflon homogenizer (8 strokes at 800 rpm). The homogenates were centrifuged at 1,000 × g for 5 min and the pellets re-extracted in 1 volume of buffer. Following a second centrifugation at 1,000 × g for 5 min, the supernatants were pooled and spun at 17,500 × g for 15 min. The crude synaptosomal pellets were washed once by centrifugation and stored at –20 °C until use (24Valdez S.R. Patterson S.I. Ezquer M.E. Torrecilla M. Lama M.C. Seltzer A.M. Synapse.. 2007; 61: 124-137Google Scholar). For a typical disassembly reaction, synaptosomal protein (2–4 μg) was incubated in a 20-μl volume containing 20 mm Hepes-NaOH, pH 7.8, 100 mm KCl, 1% (v/v) glycerol, 1 mm DTT, 2 μm TeTx or BoNT/B, 8 μm α-SNAP, 600 nm NSF or phospho-NSF, 2.5 mm ATP, and 2 mm MgCl2. In control reactions, 10 mm EDTA was added to chelate the MgCl2 and thus prevent ATP hydrolysis by NSF. Reactions were carried out for 15 min at 37 °C and terminated by addition of SDS sample buffer containing 50 mm Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (w/v) glycerol, 100 mm DTT, and 0.01% (w/v) Serva Blue G and incubated for an additional 5 min at 95 °C before separation by SDS-PAGE (25Schagger H. Von Jagow G. Anal. Biochem... 1987; 166: 368-379Google Scholar) and immunoblotting with the anti-synaptobrevin antibody. SDS-PAGE and Western Blots—Prior to electrophoresis, sperm proteins were dissolved in SDS sample buffer containing 0.0625 m Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, and 0.05% bromphenol blue by incubating for 5 min at 95 °C, separated on polyacrylamide slab gels (26Laemmli U.K. Nature.. 1970; 227: 680-685Google Scholar), and transferred to 0.22-μm nitrocellulose membranes (Hybond, GE Healthcare). Nonspecific reactivity was blocked by incubation for 1 h at room temperature with 3% gelatin dissolved in washing buffer (phosphate-buffered saline, pH 7.6, 0.1% Tween 20). Blots were incubated with the anti-phosphotyrosine antibody (0.1 μg/ml in blocking solution) for 2 h at room temperature or overnight at 4 °C. Anti-NSF blots were processed similarly to the anti-phosphotyrosine, except that the blocking solution contained 3% skim milk and 1% polyvinylpyrrolidone (40,000 average Mr, ICN) instead of gelatin, and that the primary antibody was diluted 1:6,000 in 3% skim milk. For brain samples, blots were blocked in 5% skim milk and the anti-synaptobrevin 2 antibody was used at 0.05 μg/ml in blocking solution. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgGs were used as secondary antibodies (0.25 μg/ml) with 1-h incubations. Excess first and second antibodies were removed by washing 5 × 7 min in washing buffer. Detection was accomplished with a chemiluminescence system (Western Lightning, PerkinElmer Life Sciences, Boston, MA) and subsequent exposure to HyperFilm™ ECL High performance (GE Healthcare) or CL-XPosure (Pierce, Tecnolab, Buenos Aires, Argentina) chemiluminescence film for 1–10 min. Phospho-NSF Dephosphorylation by PTP1B—NSF was tyrosine phosphorylated in E. coli TKB1 following a two-step protocol. First, IPTG induced the expression of NSF; second, indoleacrylic acid elicited the synthesis of the Elk tyrosine kinase, which phosphorylated NSF in vivo. His6-phospho-NSF was purified with nickel-nitrilotriacetic acid-agarose; tyrosine phosphorylation was confirmed by Western blot. Phospho-NSF (310 nm) was incubated with 1 μg/ml wild type or D181A PTP1B in a buffer containing 50 mm Hepes-Na, pH 7.4, 150 mm NaCl, 1 mm EDTA, 3 mm DTT, and protease inhibitors in a final reaction volume of 30 μl. Incubations were carried out at 30 °C in a water bath. At the times indicated reactions were stopped with SDS-sample buffer and the products analyzed by anti-phosphotyrosine and anti-NSF Western blotting. Two-dimensional Gel Electrophoresis—Recombinant phospho-NSF (10–20 μg) and frozen sperm pellets (150 × 106 cells) were suspended in a rehydration solution containing 8 m urea, 2% CHAPS, 0.002% bromphenol blue, 0.5 mm Na3VO4, and a protease inhibitor mixture. Each sample was sonicated at 4 °C using 2 × 15-s bursts; insoluble material was removed by centrifugation. Aliquots (150 μl) containing ∼150 μg of solubilized sperm protein were mixed with 1 μl of carrier ampholytes (Pharmalyte pH 3–10) and loaded onto immobilized pH-gradient (IPG, Immobiline DryStrip gels, Amersham Biosciences) strips (7 cm, linear pH 3–10). The IPG strips were then covered in mineral oil and left to rehydrate for 15 h at 20 °C; 100 mm DTT was added just prior to the run. Isoelectric focusing was performed at 20 °C using an Ettan IPGphor isoelectric focusing system (Amersham Biosciences), with a program of stepped voltages (30 min at 500 V, 30 min at 1000 V, and 100 min at 5000 V). Following focusing, the IPG strips were immediately incubated in 10 ml of equilibration buffer (30% glycerol, 2% SDS, 6 m urea, 50 mm Tris-HCI, pH 8.8, and 0.002% bromphenol blue) supplemented with 100 mg of DTT for 15 min at room temperature, followed by fresh equilibration buffer (10 ml) containing 250 mg of iodoacetamide for a further 15 min at room temperature. The strips were then placed on top of 8% polyacrylamide slab gels (1 mm thick, 10 × 10 cm) and held in place using 0.5% agarose prepared in SDS-running buffer. Proteins were then resolved at constant current (10–15 mA), electrotransferred onto nitrocellulose membranes, and immunoblotted sequentially using anti-NSF and anti-phosphotyrosine antibodies. Blots were stripped prior to reprobing in a solution containing 0.1 n HCl (30 min at room temperature with vigorous shaking), followed by two 10-min washes in washing buffer, and a final 10-min wash in double-distilled water. PTP Activity Is Required after Rab3 and before NSF during the AR—We resorted to the photosensitive PTP Inhibitor I to determine the steps governed by protein tyrosine dephosphorylation during the AR. This compound binds covalently to the catalytic domain of SHP-1 and PTP1B, blocking their enzymatic activity. The inhibition is reversed by irradiation with UV light (27Arabaci G. Guo X.C. Beebe K.D. Coggeshall K.M. Pei D. J. Am. Chem. Soc... 1999; 121: 5085-5086Google Scholar). We reasoned that the AR can be modeled to fit in a linear signaling pathway operating through the sequence below (14De Blas G.A. Roggero C.M. Tomes C.N. Mayorga L.S. PLoS Biol.. 2005; 3: e323Google Scholar), where single arrows mean there is one step between the proteins connected, and double arrows mean that the number of steps taking place between the connected proteins is unknown: calcium →→ Rab3 →→ α-SNAP/NSF → SNAREs (cis complex disassembly → monomeric proteins →→ reassembly in trans) →→ intra-acrosomal calcium efflux →→ SNAREs (full zippering in trans) →→ exocytosis. If PTPs catalyzed any of those unknown steps, PTP Inhibitor I would halt the AR at the point when the activity of the first PTP was required, because the sequence is linear, for as long as the system was kept in the dark. Photoreversion of the inhibition caused by PTP Inhibitor I should resume exocytosis. Indeed, when permeabilized sperm were pre-loaded with PTP Inhibitor I, calcium failed to elicit exocytosis when the system was protected from light. Exocytosis was restored upon illumination (Fig. 1B). These data confirm that PTPs are required for sperm exocytosis and also demonstrate the applicability of PTP Inhibitor I to our study. PTP Inhibitor I serves to place the requirement for fusion-related factors to steps occurring before or after the first PTP Inhibitor I-sensitive step. Briefly, PTP Inhibitor I allows calcium to prepare the fusion machinery up to the point when the first PTP is required. An inhibitor of step X (see schematic in Fig. 1A) is then added and the tubes illuminated. Resistance to the X-blocker, reflected in unaffected exocytosis, implies that its target is required upstream of PTP activity (Fig. 1A, top). Sensitivity to the X-blocker, revealed by no exocytosis, means its target is located after the PTP Inhibitor I-sensitive step (Fig. 1A, bottom). In the control condition, the AR should be prevented when the X-blocker is added prior to the inducer (calcium in this case) and maintained throughout the experiment (not shown in schematic). We asked whether the PTP-dependent step takes place before or after Rab3A by blocking the function of this small G protein with a specific antibody. The anti-Rab3A antibody inhibited exocytosis when added before, but not after, challenging with calcium. In contrast, antibodies against NSF and synaptobrevin 2 were able to abrogate exocytosis even when added after calcium. The acrosome is a calcium store and intra-acrosomal calcium release is mandatory for exocytosis. The calcium chelator BAPTA conjugated to an AM group accumulates inside the acrosome in permeabilized sperm, preventing the AR. When combined with PTP Inhibitor I, BAPTA-AM displayed the same behavior than the anti-NSF and anti-synaptobrevin antibodies (Fig. 1B, black bars). These results suggest that PTP activity is required after Rab3A, but before NSF, SNAREs, and intra-acrosomal calcium, during the signaling cascade leading to the AR, allowing us to update the original sequence as follows: calcium →→ Rab3 →→ PTP → substrate-P → substrate →→ α-SNAP/NSF → SNAREs (cis complex disassembly → monomeric proteins →→ reassembly in trans) →→ intra-acrosomal calcium efflux →→ SNAREs (full zippering in trans) →→ exocytosis. PTP1B Is Required for Exocytosis in a Human Sperm AR Model—We have shown that PTP activity is necessary for the AR (Fig. 1 and Ref. 20Tomes C.N. Roggero C.M. De Blas G. Saling P.M. Mayorga L.S. Dev. Biol... 2004; 265: 399-415Google Scholar). Because one of the PTPs targeted by the photosensitive PTP Inhibitor I is PTP1B, and the enzyme has been detected in ejaculated human and epididymal mouse (20Tomes C.N. Roggero C.M. De Blas G. Saling P.M. Mayorga L.S. Dev. Biol... 2004; 265: 399-415Google Scholar) and rat (28Seligman J. Zipser Y. Kosower N.S. Biol. Reprod... 2004; 71: 1009-1015Google Scholar) sperm, we asked whether this phosphatase might modulate protein tyrosine phosphorylation in human sperm. Full-length PTP1B contains 435 amino acids, although a shorter variant containing the entire catalytic domain is typically used for biochemical studies. To test the effect of this domain, and that of the substrate-trapping mutant D181A, on the extent of tyrosine phosphorylation of proteins, we expressed these constructs in E. coli, introduced them into SLO-permeabilized sperm, and immunoblotted cell lysates with an anti-phosphotyrosine antibody (Fig. 2A). The level of tyrosine phosphorylation in cells treated with wild type PTP1B was comparable with that in untreated controls, as was the case when PTP1B was overexpressed in COS7 cells (19Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A... 1997; 94: 1680-1685Google Scholar). Overall protein tyrosine phosphorylation increased in response to PTP1B D181A; the increase dissapeared with the wild type enzyme. These data suggest that PTP1B D181A binds to and protects sperm proteins from dephosphorylation by endogenous PTP1B. They also suggest that recombinant, wild type PTP1B competes with the D181A mutant for sperm substrates. When added to permeabilized human sperm, PTP1B D181A prevented exocytosis in a dose-dependent fashion (Fig. 2B). The mutant PTP inhibits the AR because, although it binds to the substrates of PTP1B, it is unable to dephosphorylate them efficiently. Thus, mutant and substrate become locked in a stable, "dead-end" complex. Wild type PTP1B overcame the D181A mutant (Fig. 2C) and PTP Inhibitor I (see Fig. 1) protected from light (Fig. 2D) blocks on the AR, possibly by competiti