Phosphorylation of SNAP-23 Regulates Exocytosis from Mast Cells
2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês
10.1074/jbc.m412126200
ISSN1083-351X
AutoresRégine Hepp, Niti Puri, Anita C. Hohenstein, Garland L. Crawford, Sidney W. Whiteheart, Paul A. Roche,
Tópico(s)Retinal Development and Disorders
ResumoRegulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser95 and Ser120 as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that ∼10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser95 and Ser120 is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser120 and Ser95 modulates regulated exocytosis by mast cells. Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser95 and Ser120 as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that ∼10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser95 and Ser120 is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser120 and Ser95 modulates regulated exocytosis by mast cells. Regulated exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from intracellular stores into the extracellular space (1Burgoyne R.D. Morgan A. Physiol. Rev. 2003; 83: 581-632Crossref PubMed Scopus (540) Google Scholar). One common characteristic of regulated exocytosis, whether it be from neurons, cytotoxic lymphocytes, adipocytes, or mast cells, is that the cells response to this stimulus results in pre-formed intracellular granules moving toward and fusing with the plasma membrane. Secretory granule/plasma membrane fusion is the essence of regulated exocytosis, and there is an intense effort underway to identify the molecular mechanisms regulating this process in the hopes of identifying ways to modulate exocytosis. The RBL-2H3 1The abbreviations used are: RBL, rat basophilic leukemia; SNARE, SNAP receptor; SNAP, soluble NSF attachment protein; NSF, NEM-sensitive fusion protein; PKC, protein kinase C; DNP, dinitrophenol; GST, glutathione S-transferase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; VAMP, vesicle-associated membrane protein. 1The abbreviations used are: RBL, rat basophilic leukemia; SNARE, SNAP receptor; SNAP, soluble NSF attachment protein; NSF, NEM-sensitive fusion protein; PKC, protein kinase C; DNP, dinitrophenol; GST, glutathione S-transferase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; VAMP, vesicle-associated membrane protein. mast cell line has been extensively studied as a model not only for mast cell biology but also as a paradigm for regulated exocytosis from non-neuronal cells (2Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (483) Google Scholar). Stimulation of the high affinity IgE receptor, FcϵRI, on these cells by cross-linking initiates a signal transduction cascade that culminates in secretory granule fusion with the plasma membrane, thereby liberating a variety of inflammatory mediators (3Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1779) Google Scholar, 4Kinet J.P. Annu. Rev. Immunol. 1999; 17: 931-972Crossref PubMed Scopus (850) Google Scholar). Numerous proteins necessary for the tethering, docking, and fusion steps between various membrane compartments in eukaryotic cells have been described. Among those, SNAREs (soluble NSF-attachment protein receptors) are a large family of membrane-associated proteins essential for membrane-membrane fusion (5Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1014) Google Scholar, 6Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (418) Google Scholar). These proteins include members of the vesicle-associated synaptobrevin/VAMP family as well as members of the syntaxin and SNAP-23 families of "target" membrane SNAREs. The current model proposes that while vesicles are docked on the target membrane, SNAREs from the donor or vesicle membrane (v-SNAREs) form trans-SNARE complexes with their cognate SNARE partners on the opposing target membrane. Structurally, the exocytic SNARE complex is a trimolecular protein complex containing one member of the VAMP, syntaxin, and SNAP-23 family, each contributing to the formation of a four-helix coiled-coil bundle (7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1904) Google Scholar, 8Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Crossref PubMed Scopus (417) Google Scholar) whose formation is sufficient for in vitro membrane fusion (9Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2005) Google Scholar). Given that SNAREs play a central role in the membrane fusion process, it is likely that their function is modulated in vivo. In particular, protein kinases, which have been extensively associated with the regulation of exocytosis (10Rivera J. Beaven M.A. Parker P.J. Protein Kinase C. R. G. Landes Publishing Co., Austin, TX1997: 133-166Google Scholar), could participate in SNARE function by phosphorylating residues essential in SNARE complex assembly or the binding of SNARE regulatory proteins (6Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Crossref PubMed Scopus (418) Google Scholar, 11Marash M. Gerst J.E. EMBO J. 2001; 20: 411-421Crossref PubMed Scopus (56) Google Scholar, 12Klenchin V.A. Martin T.F. Biochimie (Paris). 2000; 82: 399-407Crossref PubMed Scopus (98) Google Scholar). Members of the syntaxin (13Foster L.J. Yeung B. Mohtashami M. Ross K. Trimble W.S. Klip A. Biochemistry. 1998; 37: 11089-11096Crossref PubMed Scopus (114) Google Scholar, 14Risinger C. Bennett M.K. J. Neurochem. 1999; 72: 614-624Crossref PubMed Scopus (148) Google Scholar, 15Pombo I. Martin-Verdeaux S. Iannascoli B. Le Mao J. Deriano L. Rivera J. Blank U. J. Biol. Chem. 2001; 276: 42893-42900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and SNAP-23/25 (14Risinger C. Bennett M.K. J. Neurochem. 1999; 72: 614-624Crossref PubMed Scopus (148) Google Scholar, 16Shimazaki Y. Nishiki T. Omori A. Sekiguchi M. Kamata Y. Kozaki S. Takahashi M. J. Biol. Chem. 1996; 271: 14548-14553Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 17Polgar J. Lane W.S. Chung S.H. Houng A.K. Reed G.L. J. Biol. Chem. 2003; 278: 44369-44376Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) family of proteins are substrates for various protein kinases in vitro, although precise SNARE substrates in vivo and the physiological consequences of SNARE phosphorylation are not clear. The plasma membrane localized target SNAREs, SNAP-23 and syntaxin 4, have been shown to be important mediators of granule/plasma membrane fusion in mast cells (18Guo Z. Turner C. Castle D. Cell. 1998; 94: 537-548Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 19Paumet F. Le Mao J. Martin S. Galli T. David B. Blank U. Roa M. J. Immunol. 2000; 164: 5850-5857Crossref PubMed Scopus (191) Google Scholar, 20Vaidyanathan V.V. Puri N. Roche P.A. J. Biol. Chem. 2001; 276: 25101-25106Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 21Martin-Verdeaux S. Pombo I. Iannascoli B. Roa M. Varin-Blank N. Rivera J. Blank U. J. Cell Sci. 2003; 116: 325-334Crossref PubMed Scopus (108) Google Scholar) and platelets (22Flaumenhaft R. Croce K. Chen E. Furie B. Furie B.C. J. Biol. Chem. 1999; 274: 2492-2501Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23Chen D. Bernstein A.M. Lemons P.P. Whiteheart S.W. Blood. 2000; 95: 921-929Crossref PubMed Google Scholar). Pombo et al. (15Pombo I. Martin-Verdeaux S. Iannascoli B. Le Mao J. Deriano L. Rivera J. Blank U. J. Biol. Chem. 2001; 276: 42893-42900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) found that syntaxin 4 was constitutively phosphorylated in RBL mast cells and the extent of syntaxin 4 phosphorylation was not altered during secretion. By contrast, using human platelets Chung et al. (24Chung S.H. Polgar J. Reed G.L. J. Biol. Chem. 2000; 275: 25286-25291Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) found that thrombin stimulation resulted in phosphorylation of this same SNARE protein. In neither of these studies were the syntaxin 4 phosphorylation sites identified or the physiological importance of syntaxin 4 phosphorylation in regulated exocytosis directly examined. In addition to identifying syntaxin 4 as a substrate for protein kinases, it has recently been reported that the plasma membrane SNARE SNAP-23 is phosphorylated during human platelet activation (17Polgar J. Lane W.S. Chung S.H. Houng A.K. Reed G.L. J. Biol. Chem. 2003; 278: 44369-44376Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Using mass spectroscopy these authors identified Ser23/Thr24 and Ser161 as targets of SNAP-23 phosphorylation but were unable to ascribe a functional consequence to this. In the current study we have investigated in detail the extent and kinetics of SNARE phosphorylation in mast cells. We find that SNAP-23 undergoes rapid, stimulus-induced phosphorylation and the kinetics of phosphorylation mirror that of degranulation. In contrast to the results of Polgar et al. (17Polgar J. Lane W.S. Chung S.H. Houng A.K. Reed G.L. J. Biol. Chem. 2003; 278: 44369-44376Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), we demonstrate that the induced phosphorylation sites in rodent SNAP-23 are Ser95 and Ser120. By using phosphopeptide mapping studies, site-directed mutagenesis, and phosphorylation site-specific antibodies we show that these sites are phosphorylated in vivo in RBL mast cells, bone marrow-derived mast cells, and activated mouse platelets. Overexpression of SNAP-23 phosphorylation mutants inhibits the extent of regulated exocytosis from RBL mast cells. We also find that phosphorylated SNAP-23 preferentially associated with syntaxin 4 and VAMP-2 after exocytosis was triggered. These data suggest that SNAP-23 phosphorylation is an important post-translational modification of a critical SNARE that regulates mast cell exocytosis. Antibodies and Cell Culture—Rabbit antisera recognizing the SNAP-23 carboxyl terminus have been described (25Low S.H. Roche P.A. Anderson H.A. van Ijzendoorn S.C.D. Zhang M. Mostov K.E. Weimbs T. J. Biol. Chem. 1998; 273: 3422-3430Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). A polyclonal rabbit antisera recognizing rat syntaxin 3 (26Fujita H. Tuma P.L. Finnegan C.M. Locco L. Hubbard A.L. Biochem. J. 1998; 329: 527-538Crossref PubMed Scopus (70) Google Scholar) was the generous gift of Dr. Ann Hubbard (Johns Hopkins University, Baltimore, MD). A polyclonal rabbit antisera recognizing rat syntaxin 4 was generated by immunizing rabbits with a GST fusion protein of rat syntaxin 4 (amino acids 1–274). This antiserum showed no detectable cross-reactivity with rat syntaxin 2 or rat syntaxin 3 and was used in all immunoprecipitation and immunoblotting studies. Antisera recognizing phospho-SNAP-23-Ser120 was generated by immunizing rabbits with the synthetic peptide Val-Ser-Lys-Gln-Pro-phospho-Ser-Arg-Ile-Thr-Ans-Gly-Gln (corresponding to amino acids 115–126 of rat SNAP-23). Antisera recognizing phospho-SNAP-23-Ser95 was generated by immunizing rabbits with the synthetic peptide Thr-Lys-Asn-Phe-Glu-phospho-Ser-Gly-Lys-Asn-Tyr-Lys-Ala-Thr (corresponding to amino acids 90–102 of rat SNAP-23). The VAMP-2 monoclonal antibody cl 69.1 was obtained from Synaptic Systems (Goettingen, Germany). Unless indicated, anti-DNP-IgE (clone SPE-7) was from Sigma. For the experiments shown in Figs. 8, 9, 10, anti-DNP-IgE (clone TIB-142) was obtained from the American Type Culture Collection (Manassas, VA). Biotinylated anti-FLAG monoclonal antibody M5 was from Sigma and anti-GFP monoclonal antibody clone A.v. was from Clontech.Fig. 9Overexpression of SNAP-23 phosphorylation mutants inhibits exocytosis from RBL mast cells.A, RBL cells expressing FLAG-tagged wild-type SNAP-23, SNAP-23 S95A/S120A, or SNAP-23 S95D/S120D were co-transfected with a trace amount of human growth hormone secretion reporter plasmid as described in the text. The cells were sensitized with DNP-specific IgE and mock-stimulated or stimulated with DNP-BSA. Cell supernatants were harvested after 15 min and the extent of degranulation was determined by measuring the amount of human growth hormone released from the cells. The amount of secretion from cells in each experimental condition was expressed as a percentage of the total amount of human growth hormone present in the cells before stimulation. Data are from three independent experiments, and asterisks indicate statistically significant differences in the percentage of human growth hormone released from each mutant as compared to FLAG-tagged wild-type SNAP-23 (*, p < 0.05 and **, p < 0.0005). B, equivalent amounts of cell lysate from mock-transfected RBL mast cells or RBL cells expressing FLAG-tagged wild-type SNAP-23, SNAP-23 S95A/S120A, or SNAP-23 S95D/S120D that were mock-stimulated or stimulated with DNP-BSA were analyzed by immunoblot analysis using a SNAP-23 carboxyl terminus antibody. This antibody detects endogenous SNAP-23 and FLAG-SNAP-23 equally well. The mobility of endogenous rat SNAP-23 and transfected FLAG-SNAP-23 are indicated by arrows. A representative blot is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 10After mast cell stimulation most syntaxin 4- and VAMP-2-associated SNAP-23 is phosphorylated.A, RBL mast cells were sensitized with DNP-specific IgE and mock-stimulated or stimulated with DNP-BSA for 10 min. The cells were lysed in Triton X-100 and equal portions of each lysate were analyzed by immunoprecipitation using a control (preimmune) antiserum, an antiserum that recognizes total SNAP-23, or an antiserum that recognizes phospho-SNAP-23-Ser120. Aliquots of each immunoprecipitate were analyzed by SDS-PAGE and immunoblotting using antibodies that recognize rat syntaxin 4 (upper panels) or VAMP-2 (lower panels). B, the percent of all SNAP-23·syntaxin 4 complexes containing phospho-SNAP-23-Ser120 was calculated by expressing the amount of syntaxin 4 in a phospho-SNAP-23-Ser120 immunoprecipitate relative to the total amount of syntaxin 4 bound to all SNAP-23 in the sample. Each data point represents a mean ± S.D. of four independent experiments. The percent of all SNAP-23·VAMP-2 complexes containing phospho-SNAP-23-Ser120 was calculated by expressing the amount of VAMP-2 in a phospho-SNAP-23-Ser120 immunoprecipitate relative to the total amount of VAMP-2 bound to all SNAP-23 in the sample. Each data point represents a mean ± S.D. of two independent experiments. Analysis of the supernatant after each immunoprecipitation confirmed that the SNAP-23 antibody and phospho-SNAP-23-Ser120 antibody removed essentially all SNAP-23 and phospho-SNAP-23-Ser120 from the sample, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RBL-2H3 mast cells were obtained from Dr. Juan Rivera (National Institutes of Health) and were grown in equal parts minimal essential medium and Iscove's medium containing 20% fetal calf serum (HyClone Laboratories), 25 mm Hepes (pH 7.5), and 50 μg/ml gentamicin. Cells were maintained as subconfluent monolayers at 37 °C in a humidified atmosphere containing 5% CO2 and passaged with trypsin. Cells were sensitized with 1 μg/ml anti-DNP-IgE for 24 h prior to stimulation with 100 ng/ml DNP-BSA in phenol red-free RPMI 1640 medium. Sensitized cells were mock-stimulated using 100 ng/ml BSA in the same medium. HeLa cells were grown in DMEM supplemented with 10% fetal calf serum, 20 mm Hepes, and 50 μg/ml gentamicin. The cells were maintained as subconfluent monolayers at 37 °C in a humidified atmosphere containing 5% CO2 and passaged with trypsin. In some experiments transfected HeLa cells were stimulated using 10 nm phorbol myristate acetate and 1 μm ionomycin. Plasmids and Recombinant Proteins—Rat SNAP-23 was subcloned into pcDNA3 (Invitrogen) using EcoRI and XhoI sites introduced by PCR using a previously described full-length rat SNAP-23 cDNA (27St-Denis J.F. Cabaniols J.P. Cushman S.W. Roche P.A. Biochem. J. 1999; 338: 709-715Crossref PubMed Scopus (38) Google Scholar). GST-rat SNAP-23 was generated by subcloning rat SNAP-23 from pcDNA3 into pGEX-4T1 (Amersham Biosciences). Ala mutants of all Ser and Thr residues present in rat SNAP-23 were generated with the QuikChange Site-directed Mutagenesis kit (Stratagene) using pGEX-4T1-rat SNAP-23 as the template according to the manufacturer's instructions. The double mutant of S95A/S120A was generated using rat SNAP-23 S120A DNA as a template. The double mutant replacing both Ser95 and Ser120 with Asp residues (S95D/S120D) was generated using the QuikChange Multi-Mutagenesis kit (Stratagene) as directed by the manufacturer. The inserts of wild-type SNAP-23 and the mutants of interest were subcloned into the pCMV-Tag 2B vector (Stratagene) to generate amino-terminal FLAG-tagged proteins. The integrity of all PCR-amplified products and all mutants were confirmed by automated sequence analysis using an ABI sequencer. Transfections—Exponentially growing RBL cells were re-suspended in serum-free and antibiotic-free DMEM and 10 × 106 cells were transfected by electroporation (310 mV, 960 microfarads, Bio-Rad GenePulser) using 10–20 μg of empty vector, FLAG-tagged wild-type SNAP-23, or FLAG-tagged mutant rat-SNAP-23 as described previously (20Vaidyanathan V.V. Puri N. Roche P.A. J. Biol. Chem. 2001; 276: 25101-25106Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Transfected cells were immediately diluted in antibiotic-free medium and allowed to adhere to plastic culture dishes for 5–6 h before adding anti-DNP IgE to the medium overnight. The next day, cells were triggered for exocytosis as described above. HeLa cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Metabolic Labeling of Cells—RBL cells were plated at 1.5 × 106 cells per 10-cm dish. IgE-sensitized cells were starved the next day for 15 min in phosphate-free DMEM containing 3% dialyzed calf serum (Hy-Clone Laboratories), 20 mm Hepes (pH 7.5), and gentamicin. Cells were labeled with 0.5 mCi of [32P]orthophosphate (PerkinElmer Life Sciences) for 5 h in 1 ml of phosphate-free DMEM. Cells were then stimulated for secretion with DNP-BSA as described above. Cells were washed with ice-cold Hanks' balanced salt solution and tissue culture plate-bound cells were frozen until further use. In Vitro Phosphorylation Reactions—GST and GST-SNAP-23 proteins were purified from isopropyl 1-thio-β-d-galactopyranoside-induced BL21 Escherichia coli using standard protocols. Briefly, GST and GST-SNAP-23 were isolated from 200-μl bacteria lysates with 10 μl (1:1) of glutathione-Sepharose beads (Amersham Biosciences). GST proteins were eluted with 30 μl of 10 mm reduced glutathione and phosphorylated for 30 min at 30 °C with 25 ng of purified PKC (Promega) in a reaction buffer of 20 mm Hepes (pH 7.4), 1.67 mm CaCl2, 1 mm dithiothreitol, 10 mm MgCl2, 20 μm ATP, and 2 μCi of [32P]ATP. After 30 min the entire reaction mixture was boiled in SDS-PAGE sample buffer and analyzed by SDS-PAGE. Immunoprecipitation and Electrophoresis—Immunoprecipitation of proteins from cell lysates was carried out as described previously (28Anderson H.A. Roche P.A. J. Immunol. 1998; 160: 4850-4858PubMed Google Scholar). Briefly, cells were lysed for 1 h in ice-cold lysis buffer, 10 mm Tris (pH 7.4), 150 mm NaCl containing 1% Triton X-100 as well as protease inhibitors (5 mm iodoacetamide, 50 mm phenylmethylsulfonyl fluoride, and 0.1 mmNα-p-tosyl-l-lysine chloromethyl ketone) and phosphatase inhibitors (5 mm EDTA, 5 mm EGTA, 50 mm NaF, 10 mm Na4P2O7, and 1 mm Na3VO4). Specific immunoprecipitations were performed for 2 h at 4 °C by incubating pre-cleared lysates with specific antibodies bound to protein A-Sepharose beads (Sigma). Proteins from the immunoprecipitates were separated on 12.5% SDS-PAGE gels. Radiolabeled molecules were visualized by autoradiography and/or PhosphorImager analysis. The extent of phosphorylation was quantitated by PhosphorImager analysis. For phosphopeptide mapping studies, proteins were transferred to 0.2-μm nitrocellulose membranes (Schleicher & Schuell) and the location of phosphoproteins on the membrane was determined after autoradiography. For immunoblot analysis proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and proteins were visualized by enhanced chemiluminescence using Western Lightning (PerkinElmer Life Sciences). Band intensity was determined by automatic integrating densitometry using a Amersham Biosciences densitometer. For quantitative experiments, statistical analyses were carried out by using a Student's t test. Results were considered significant when a p value of less then 0.05 was obtained. Tryptic Phosphopeptide Mapping—Nitrocellulose membranes containing excised SNAP-23 were incubated for 1 h at 37 °C in 1% polyvinylpyrrolidone-40 (Sigma), washed twice with 0.4% NH4HCO3 (pH 8.0), and incubated overnight with 0.5 mg/ml trypsin at 37 °C in the same buffer. The released peptides were dried in a Speed-vac, washed with H2O several times, and resuspended in 5 μl of H2O. Peptides were spotted at the bottom of a 20 × 20-cm thin layer chromatography plate (#5577, E. M. Science), dried, and subjected to electrophoresis for 75 min at 1000 V in a buffer of 2.5% formic acid, 7.5% acetic acid. After electrophoresis, the plates were dried for at least 1 h and subjected to analysis in a second dimension of thin layer chromatography using a solvent containing 62.5% isobutyric acid, 4.8% pyridine, 1.9% butanol-1, 2.9% acetic acid until the solvent front migrated to within 1 cm of the top of the TLC plate (generally overnight). The plates were dried and analyzed with a PhosphorImager. Subcellular Fractionation—RBL cells were harvested by trypsinization and washed in phosphate-buffered saline. The cells were re-suspended in hypotonic buffer (10 mm Tris, 10 mm KCl, 1 mm EGTA, 0.5 mm MgCl2, pH 7.4) and were disrupted by repeated passage of cells through a 25-gauge syringe. Nuclei and unbroken cells were removed by centrifugation at 1000 × g and the post-nuclear supernatant was subjected to centrifugation at 100,000 × g for 1 h at 4°C to isolate membranes (pellet) and cytosol (supernatant). In some experiments RBL cells were transfected with pEGFP (Clontech) to allow expression of the cytosolic marker protein GFP. The membrane pellet and cytosolic supernatant were brought to the same volume in hypotonic buffer and each was adjusted to a final concentration of 1% Triton X-100. Equal portions of each fraction were analyzed by SDS-PAGE and immunoblotting. Mast Cell Exocytosis Assay—The degranulation in RBL-2H3 cells was monitored by measuring the β-hexosaminidase activity released from cells grown in 6- or 12-well plates. IgE-sensitized cells were washed twice with RPMI and mock-stimulated or stimulated by the addition of DNP-BSA. The cell supernatant was collected and the cells were lysed in phenol red-free RPMI containing 0.2% Triton X-100 to determine the total enzyme content. A colorimetric assay with p-nitrophenyl-N-acetyl-β-d-glucosaminide (Sigma) as the substrate was used to measure the amount of β-hexosaminidase released into the medium and remaining in the cells as described previously (20Vaidyanathan V.V. Puri N. Roche P.A. J. Biol. Chem. 2001; 276: 25101-25106Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The β-hexosaminidase activity released was expressed as a percentage of the activity released into the medium relative to the total activity (released plus cell-associated). In some experiments, RBL cells were transfected by electroporation with an expression vector of human growth hormone (2 μg) together with empty pCMV vector or pCMV-FLAG-SNAP-23 (20 μg). After overnight culture, the cells were sensitized with IgE and mock-stimulated or stimulated with DNP-BSA as described above. The amount of human growth hormone released into the medium or remaining cell associated was determined using a human growth hormone enzyme-linked immunosorbent assay (Roche Diagnostics Corp.) as described previously (29Puri N. Kruhlak M.J. Whiteheart S.W. Roche P.A. J. Immunol. 2003; 171: 5345-5352Crossref PubMed Scopus (65) Google Scholar). For quantitative experiments, statistical analyses were carried out by using a Student's t test. Results were considered significant when a p value of less than 0.05 was obtained. Platelet Preparation and Measurement of Thrombin-induced Secretion—Mouse platelets were isolated as described previously (30Schraw T.D. Rutledge T.W. Crawford G.L. Bernstein A.M. Kalen A.L. Pessin J.E. Whiteheart S.W. Blood. 2003; 102: 1716-1722Crossref PubMed Scopus (42) Google Scholar). Briefly, blood was obtained from the right ventricle of the heart of sacrificed C57BL/6 mice. The blood was mixed with 1.8% sodium citrate (pH 7.4) to a final concentration of 0.18%. Platelet-rich plasma was prepared by centrifugation at 100 × g for 10 min. Care was taken to harvest only the platelet-containing upper layer to avoid the buffy coat layer of nucleated cells above the red blood cells. Platelets were assayed for dense core release of [3H]5-hydroxytryptamine, lysosomal release of β-hexosaminidase, and α-granule release of platelet factor IV, as described previously (31Rutledge T.W. Whiteheart S.W. Methods Mol. Biol. 2004; 272: 109-120PubMed Google Scholar). Platelets were activated by the addition of thrombin (0.5 units/ml; Chronolog) in a Hepes/Tyrode's release buffer containing 0.7 mm CaCl2 at 25 °C for the indicated times. The data were tabulated as the percent release compared with the total present in each reaction. The background release of each marker in unstimulated platelets was subtracted from that obtained after thrombin stimulation to yield net granule marker release. A parallel set of reactions was solubilized in SDS-PAGE sample buffer for analysis by immunoblotting. SNAP-23 Is Phosphorylated in Stimulated RBL Cells—RBL cell exocytosis can be triggered by cross-linking of surface FcϵRI receptors for IgE by the appropriate antigen (3Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1779) Google Scholar, 4Kinet J.P. Annu. Rev. Immunol. 1999; 17: 931-972Crossref PubMed Scopus (850) Google Scholar). To determine whether SNAREs were phosphorylated during physiological stimulation in mast cells, RBL-2H3 cells were metabolically labeled with [32P]orthophosphate prior to cross-linking surface Fc receptors for DNP-specific IgE using DNP-BSA. The cells were then mock-stimulated (using unconjugated BSA) or stimulated for 20 min with DNP-BSA and the phosphorylation status of the SNARE proteins was analyzed by immunoprecipitation, SDS-PAGE, and PhosphorImager analysis (Fig. 1A). In agreement with published results (15Pombo I. Martin-Verdeaux S. Iannascoli B. Le Mao J. Deriano L. Rivera J. Blank U. J. Biol. Chem. 2001; 276: 42893-42900Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), syntaxin 4 was phosphorylated in mock-treate
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