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Phosphorylation of Syntaphilin by cAMP-dependent Protein Kinase Modulates Its Interaction with Syntaxin-1 and Annuls Its Inhibitory Effect on Vesicle Exocytosis

2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês

10.1074/jbc.m400496200

1083-351X

Judit Boczan, A. G. Miriam Leenders, Zu‐Hang Sheng,

Calcium signaling and nucleotide metabolism

cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilin-transfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca2+-dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K+-induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway. cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilin-transfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca2+-dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K+-induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway. Neurotransmitter release involves a series of protein interactions between the membranes of synaptic vesicles and presynaptic terminals, culminating in the calcium-dependent fusion of the two membranes (for reviews see Refs. 1Jahn R. Südhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Google Scholar, 3Bajjalieh S.M. Scheller R.H. J. Biol. Chem. 1995; 270: 1971-1974Google Scholar, 4Südhof T.C. Nature. 1995; 375: 645-653Google Scholar). The synaptic vesicle-associated protein synaptobrevin interacts with two membrane-associated proteins, SNAP 1The abbreviations used are: SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; SNARE, SNAP receptor; PKA, cAMP-dependent protein kinase; hGH, human growth hormone; GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TBS, Tris-buffered saline; PIPES, 1,4-piperazinedi-ethanesulfonic acid; BIMPS, Sp-5,6-DCl-cBIMPS. -25 and syntaxin, to form a stable SNARE complex (5Hilfiker S. Greengard P. Augustine G.J. Nat. Neurosci. 1999; 2: 104-106Google Scholar, 6Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 85: 4538-4542Google Scholar, 7Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Google Scholar, 8Yoshida A. Oho C. Omori A. Kuwahara R. Ito T. Takahashi M. J. Biol. Chem. 1992; 267: 24925-24928Google Scholar, 9Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Google Scholar, 11Calakos N. Bennett M.K. Peterson K. Scheller R.H. Science. 1994; 263: 1146-1149Google Scholar, 12Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Google Scholar). Formation of the SNARE complex has been proposed to bring the synaptic vesicle and plasma membranes into close apposition and provide the energy that drives the mixing of the two lipid bilayers (6Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 85: 4538-4542Google Scholar, 7Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Google Scholar). Calcium influx into the presynaptic terminal triggers the calcium sensor of the fusion machinery, upon which the SNARE core complex binding matures from a trans-state to a cis-state, resulting in the complete fusion of the two membranes and the release of neurotransmitter. The very stable cis-SNARE core complex is then dissociated by the action of α-SNAP and the ATPase N-ethylmaleimide-sensitive factor (10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Google Scholar). The assembly and disassembly of the SNARE complex must be highly regulated to gain plasticity in neurotransmitter release. Recent studies have made significant progress in understanding this regulatory mechanism by the isolation of the SNARE complex interacting proteins, including Munc18/nSec1/rbSec1, complexins, Munc-13, tomosyn, cysteine string protein, snapin, and syntaphilin (13Pevsner J. Hsu S. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1445-1449Google Scholar, 14McMahon H.T. Missler M. Li C. Südhof T.C. Cell. 1995; 83: 111-119Google Scholar, 15Brose N. Hofmann K. Hata Y. Südhof T.C. J. Biol. Chem. 1995; 270: 25273-25280Google Scholar, 16Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Google Scholar, 17Ilardi J.M. Mochida S. Sheng Z.-H. Nat. Neurosci. 1999; 2: 119-124Google Scholar, 18Lao G. Scheuss V. Gerwin C.M. Su Q. Mochida S. Rettig J. Sheng Z.-H. Neuron. 2000; 25: 191-201Google Scholar, 19Wu M.N. Fergestad T. Lloyd T.E. He Y. Broadie K. Bellen H.J. Neuron. 1999; 23: 593-605Google Scholar), which bind to individual SNARE proteins and regulate their availability to form functional SNARE complexes at release sites. Second messenger regulation of the protein interactions underlying neurotransmission is one mechanism by which cellular events modulate synaptic transmission (4Südhof T.C. Nature. 1995; 375: 645-653Google Scholar). Although the time course between action potential arrival at the nerve terminal and synaptic vesicle fusion is too short for protein phosphorylation/dephosphorylation to exact a direct and acute role in a single round of vesicle exocytosis, protein kinases and phosphatases may have significant effects on subsequent neurotransmitter release events. It is reasonable to speculate that the phosphorylation/dephosphorylation states of synaptic proteins that mediate vesicle exocytosis could regulate the biochemical pathways leading from synaptic vesicle docking to fusion. Activation of cAMP-dependent protein kinase (PKA) has been shown to facilitate synaptic transmission at many synapses (20Dixon D. Atwood H.L. J. Neurosci. 1989; 9: 4246-4252Google Scholar, 21Chavez-Noriega L.E. Stevens C.F. J. Neurosci. 1994; 14: 310-317Google Scholar, 22Klein M. Neuron. 1994; 13: 159-166Google Scholar, 23Weisskopf M.G. Castillo P.E. Zalutsky R.A. Nicoll R.A. Science. 1994; 265: 1878-1882Google Scholar, 24Capogna M. Gahwiler B.H. Thompson S.M. J. Neurosci. 1995; 15: 1249-1260Google Scholar). Application of the adenylate cyclase activator forskolin on adult rat and mouse hippocampal slices or hippocampal neuron cultures increased the frequency of spontaneous miniature excitatory postsynaptic currents without affecting their amplitude (21Chavez-Noriega L.E. Stevens C.F. J. Neurosci. 1994; 14: 310-317Google Scholar, 25Yasuda H. Barth A.L. Stellwagen D. Malenka R.C. Nat. Neurosci. 2003; 6: 15-16Google Scholar, 26Bouron A. Eur. J. Neurosci. 1999; 11: 4446-4450Google Scholar). In hippocampal neurons, activation of PKA increases neurotransmitter release by directly acting on the exocytotic apparatus (27Trudeau L.E. Emery D.G. Haydon P.G. Neuron. 1996; 17: 789-797Google Scholar). The number of functional presynaptic boutons of hippocampal neuron cultures increased after long term exposure to the cAMP analogue (28Ma L. Zablow L. Kandel E.R. Siegelbaum S.A. Nat. Neurosci. 1999; 2: 24-30Google Scholar, 29Kohara K. Ogura A. Akagawa K. Yamaguchi K. Neurosci. Res. 2001; 41: 79-88Google Scholar). However, there was no change in the number of functioning terminals and nor in the number of docked vesicles within individual synapses after 3 min of treatment of cultured hippocampal neurons with forskolin (27Trudeau L.E. Emery D.G. Haydon P.G. Neuron. 1996; 17: 789-797Google Scholar), suggesting that the short term effects of cAMP may affect the secretory machinery directly. Thus, identifying the PKA target(s) that regulates assembly/disassembly of the fusion machinery and the priming of docked vesicles is critical to the elucidation of the molecular mechanisms underlying synaptic transmission and presynaptic plasticity. Syntaphilin is a brain-enriched protein that we first characterized as a binding partner of syntaxin-1 (18Lao G. Scheuss V. Gerwin C.M. Su Q. Mochida S. Rettig J. Sheng Z.-H. Neuron. 2000; 25: 191-201Google Scholar). Binding of syntaphilin to syntaxin inhibits the binding of syntaxin to SNAP-25 and thus prevents the formation of the SNARE core complex. Functionally, overexpression of syntaphilin in cultured hippocampal neurons inhibits neurotransmitter release; furthermore, injection of the syntaphilin syntaxin-binding peptide into the presynaptic cell body of superior cervical ganglion neurons in culture results in the inhibition of neurotransmission, suggesting that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex and thereby regulates synaptic vesicle exocytosis. Syntaphilin also binds to dynamin-1 and inhibits its interaction with amphiphysin, suggesting an inhibitory role for this protein in dynamin-mediated endocytosis (30Das S. Gerwin C. Sheng Z.-H. J. Biol. Chem. 2003; 278: 41221-41226Google Scholar). The expression of syntaphilin is developmentally regulated, and it is more prominently expressed in the mature rat brain in areas undergoing synaptic plastic changes (31Das S. Boczan J. Gerwin C. Zald P.B. Sheng Z.-H. Mol. Brain Res. 2003; 116: 38-49Google Scholar). Our finding that syntaphilin expression is limited to a subset of synapses led us to ask whether it might be an expression-limited modulator of synaptic activity. Syntaphilin is serine-rich (12% of total amino acid content) and contains numerous consensus sites for protein phosphorylation, suggesting that its functional roles on synaptic vesicle recycling are further regulated through phosphorylation. In this study we show that syntaphilin can be phosphorylated by PKA both in vitro and in vivo and that this phosphorylation inhibits its binding to syntaxin-1A. Furthermore, mutation of the phosphorylation site serine 43 to aspartic acid (mimicking a constitutive phosphorylation) annuls the inhibitory effect of syntaphilin on Ca2+-dependent exocytosis in PC12 cells. Thus, PKA activation could act as an "off" switch for syntaphilin by blocking its inhibitory functions at the nerve terminal. Preparation of Fusion Proteins and Site-directed Mutagenesis—Full-length and truncated mutants of syntaphilin were subcloned into the hexahistidine-tagged fusion protein vectors pET28A (Novagen) and pcDNA 3.1 His-A (Invitrogen). Wild type syntaphilin 1–469 was subcloned into the pGEX-4T-1 vector (Amersham Biosciences). Syntaxin-1A 1–265 was subcloned into pET-28A vector. His-tagged proteins in pcDNA vectors were prepared as lysates of transfected HEK-293 cells using LipofectAMINE 2000 (Invitrogen). His-tagged proteins in pET28 and GST fusion proteins were prepared as crude bacterial lysates using BL21-competent cells (Star-DE3-pLysS One Shot; Invitrogen) and purified as previously described (17Ilardi J.M. Mochida S. Sheng Z.-H. Nat. Neurosci. 1999; 2: 119-124Google Scholar, 18Lao G. Scheuss V. Gerwin C.M. Su Q. Mochida S. Rettig J. Sheng Z.-H. Neuron. 2000; 25: 191-201Google Scholar). Site-directed mutations were generated using the QuikChange XL site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) according to the manufacturer's instructions. Generation of Phosphorylation State-specific Antibody—Rabbit polyclonal antibody pS43 was raised against the following cysteine-containing phosphopeptide Ac-LTRTH(pS)LMAC-amide (residues 38–45). Affinity purification was performed using dephospho- and phosphopeptides coupled to thiol coupling gels (BIOSOURCE Int., Hopkinton, MA). In Vitro Phosphorylation Assays—Recombinant His-tagged full-length syntaphilin was immunoprecipitated from HEK 293 cell lysate using anti-T7-His monoclonal antibody (Novagen) and protein A-Sepharose CL-4B resin (Amersham Biosciences). Equal amounts (∼7 pmol) of resin-bound proteins were incubated either with or without the catalytic subunit of PKA to a final concentration of 0.5 unit/μl or 100 μg of PKA inhibitor peptide PKI (Promega) in phosphorylation buffer (Buffer A: 40 mm Tris-HCl, pH 7.4, 20 mm MgCl2, 0.1 mm ATP) containing 20 μm [γ-32P]ATP for 1 h at 30 °C. The 25-μl reactions were terminated by boiling with 12.5 μl of Tricine sample buffer (Bio-Rad) containing 4.5% 2-mercaptoethanol and 45 mm dithiothreitol (Sigma). Phosphorylation products were separated by 10–20% Tricine-SDS-PAGE; the gels were stained with Coomassie Blue, dried, and exposed to x-ray film. Bacterially expressed His-tagged syntaphilin 1–130, 130–205, and 203–469 (50 pmol of each) was bound to nickel-nitrilotriacetic acid beads and phosphorylated as described for the full-length protein. 1 μg of syntaphilin mutants of affinity-purified GST-syntaphilin 1–469 were phosphorylated by the same method. For back-phosphorylation assays, glutathione-Sepharose bound in vitro phosphorylated GST-syntaphilin 1–469 and His-syntaphilin immunoprecipitated from HEK 293 cells was subjected to back-phosphorylation by incubating with PKA catalytic subunit (to a final concentration of 0.5 unit/μl) and 0.125 mm [γ-32P]ATP in Buffer A for 1 h at 30 °C. The products were separated by 10–20% Tricine-SDS-PAGE, and then the gels were stained with Coomassie Blue, dried, and exposed to x-ray film. Stoichiometric Measurements—For phosphorylation time course experiments, 60 pmol of purified recombinant GST-syntaphilin 1–469 was phosphorylated by 80 units of PKA catalytic subunit in Buffer A at 30 °C. 6 μl of each sample (containing 7.5 pmol of GST-syntaphilin) was removed from the reaction mixture between 5 and 240 min, and these reactions were terminated by the addition of SDS sample buffer and boiling. The products were separated by SDS-PAGE, stained with Coomassie Blue, and exposed to x-ray film. The gel slices containing syntaphilin were excised and scintillation-counted, and the molar ratio of inorganic phosphate incorporated per mol of syntaphilin was calculated and plotted as a function of time. In Vitro Binding Experiments—Equal amounts (5 μg) of GST-syntaphilin 1–469 or GST alone were immobilized on glutathione-Sepharose beads (Amersham Biosciences). The resin-bound proteins were incubated either with or without the catalytic subunit of PKA (247.5 units/reaction) in Buffer A at 30 °C for 3 h under continuous agitation. After washing with TBST/PI buffer (50 mm TBS, pH 7.4, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, and 1 mm aprotinin), one-fifth of each phosphorylation reaction was subjected to back-phosphorylation to verify the initial phosphorylation. One-third of each reaction was incubated with equal volumes of bacterial lysates containing syntaxin-1A in TBST/PI on a microtube rotator at 4 °C for 3 h. After washing with TBST/PI, the bound complexes were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with anti-syntaxin-1 (10H5) antibody. The membranes were stripped in stripping buffer (62.5 mm Tris-HCl, pH 7.5, 20 mm dithiothreitol, and 1% SDS) at 60 °C for 20 min and blotted with anti-GST antibody (Pierce). Horseradish peroxidase-conjugated secondary antibodies and ECL chemiluminescence (Amersham Biosciences) were used to visualize the bands. For the binding dose-response curve, GST-syntaphilin 1–469 immobilized on glutathione-Sepharose beads was divided into two parts and incubated either with or without the catalytic subunit of PKA (410 units/reaction) in Buffer A at 30 °C for 3 h under continuous agitation. After washing with TBST/PI, each phosphorylation reaction was divided into seven equal aliquots, and each part was incubated with increasing amounts of purified His-syntaxin-1A (0, 15, 30, 50, 100, 150, and 450 pmol, respectively) and 200 μg of bovine serum albumin for blocking nonspecific binding in TBST/PI on a microtube rotator at 4 °C for 3 h. After washing with TBST/PI, the bound complexes were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with anti-syntaxin-1 (10H5) antibody, followed by stripping, and blotting with anti-GST antibody. For quantitative analysis, the binding intensities were measured with National Institutes of Health Image 1.61. Care was taken during exposure of the ECL film to ensure that all readings were in the linear range of a standard. The percentage of binding relative to the nonphosphorylated or wild type syntaphilin was calculated based on standard curves. Student's t tests were performed, and the results are presented as the means ± S.E. of three or four independent experiments. Preparation of Synaptosomes, Stimulation of PKA in Vivo, and Immunoprecipitation—Rat brain synaptosomes were prepared by differential and discontinuous Percoll gradient centrifugation (32Sheng Z.-H. Rettig J. Cook T. Catterall W.A. Nature. 1996; 379: 451-454Google Scholar). Briefly, whole rat brains were homogenized in ice-cold 21.9% sucrose buffer, pH 7.4. The homogenates were centrifuged in a Beckman GSR-6RHT centrifuge at 3,000 rpm for 10 min. The supernatant was placed on top of Percoll gradients (23, 15, 10, and 3% in sucrose buffer) and spun in an SS34 rotor at 17,250 rpm for 5 min. The synaptosome bands between the 15 and 23% gradients were collected, mixed with Wash Buffer (122.8 mm NaCl, 5 mm KCl, 1.15 mm NaH2PO4, 20 mm PIPES, 0.1% d(+)-glucose, pH 6.8), and then spun in an SS34 rotor at 11,000 rpm for 15 min. The synaptosomes were resuspended in Neurobasal medium supplemented with B-27 (both from Invitrogen), then divided into two equal volumes, and incubated in the presence of either 50 μm of the cAMP analogue Sp-5,6-DCl-cBIMPS (BIMPS) or Me2SO alone (vehicle control) at 37 °C for 1 h. The samples were then lysed in lysis buffer supplemented with phosphatase inhibitors (50 mm TBS, pH 7.4, 1% Triton X-100, 0.5% deoxycholic acid, 1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, 1 mm aprotinin, 1 mm EDTA, 1 mm EGTA, 1 mm β-glycerophosphate, 1 mm sodium vanadate, 100 nm cyclosporin A, 10 nm okadaic acid; all from Sigma) at 4 °C for 30 min and centrifuged by 13,000 rpm for 20 min. The protein concentrations were determined by the BCA protein assay (Pierce). Solubilized proteins from both BIMPS- and Me2SO-treated synaptosomes (300 μg each) were incubated with anti-syntaxin-1 (10H5) antibody or normal mouse IgG as control in 500 μl of TBST/PI supplemented with phosphatase inhibitors overnight at 4 °C. Immune complexes were resolved by SDS-PAGE, and co-precipitated syntaphilin was detected by immunoblotting with syntaphilin polyclonal antibody. Phosphorylation of syntaphilin in Me2SO- and BIMPS-treated synaptosomes was detected by immunoblotting with the phospho-specific syntaphilin antibody anti-pS43. For semi-quantitative analysis, the intensity of the syntaphilin signals was measured with National Institutes of Health Image 1.61, and the relative amounts were calculated using a linear standard curve of syntaphilin from the synaptosomal lysates using a paired Student's t test for statistical analysis. High K+-stimulated Exocytosis in PC12 Cells—Exocytosis assays were generally conducted as previously described, with a few modifications (33Sugita S. Janz R. Südhof T.C. J. Biol. Chem. 1999; 274: 18893-18901Google Scholar). Briefly, PC12 cells (2 × 106 cells grown in 35-mm collagen-coated dishes (Corning Inc.)) were co-transfected the next day with a plasmid expressing human GH (pXGH5) along with 3.6 μg of empty pcDNA vector or pcDNA vector encoding either wild type syntaphilin or its mutant by using LipofectAMINE 2000 in OPTI-MEM (both from Invitrogen). After 48 h of incubation in RPMI culture medium supplemented with 10% horse serum, and 5% fetal bovine serum (all from ATCC) and 100 unit/ml penicillin G and 100 μg/ml streptomycin (both from Invitrogen) at 37 °C in 10% CO2, the cells were lifted and replated (split from one 35-mm dish into two 22-mm dishes). 24 h after replating, the cells were subjected to exocytosis assays by incubating in low K+ (basal) and high K+ (stimulated) release conditions. Release buffers consisted of low (5.6 mm) or high (56 mm) KCl solutions in 145 mm NaCl, 2.2 mm CaCl2, 0.5 mm MgCl2, 5.6 mm glucose, and 15 mm HEPES, pH 7.4, at room temperature for 30 min. The medium was then removed and centrifuged by 13,000 rpm at 4 °C for 5 min. Human growth hormone (hGH) in the supernatant was taken as secreted hGH. The cells from the dishes were harvested in Tris-buffered saline, pH 7.4, containing protease inhibitors, 1 mm EDTA, 0.5% Triton X-100, and added to the centrifuged pellet of the secretion medium. Following a 20-min incubation at 4 °C on a microtube rotator, the cell lysates were centrifuged at 13,000 rpm for 20 min. The supernatant of this step was taken as the intracellular hGH that was not secreted after K+ stimulation. hGH levels in the various samples were measured by the hGH enzyme-linked immunosorbent assay kit (Roche Applied Science) according to the manufacturer's instructions. The percentage of hGH released was calculated as the hGH in supernatant divided by the total hGH obtained from solubilized cells plus hGH in the supernatant. All of the experiments were carried out in duplicate or triplicate each time. The statistical analyses were performed with the Student's t test, and the results are presented as the means ± S.E. of five independent experiments. In Vitro Phosphorylation of Syntaphilin by PKA—Our previous studies indicate that syntaphilin is a developmentally regulated and expression-limited protein capable of inhibiting both synaptic vesicle exocytosis and endocytosis (18Lao G. Scheuss V. Gerwin C.M. Su Q. Mochida S. Rettig J. Sheng Z.-H. Neuron. 2000; 25: 191-201Google Scholar, 30Das S. Gerwin C. Sheng Z.-H. J. Biol. Chem. 2003; 278: 41221-41226Google Scholar, 31Das S. Boczan J. Gerwin C. Zald P.B. Sheng Z.-H. Mol. Brain Res. 2003; 116: 38-49Google Scholar). Syntaphilin is serine-rich (12% of total amino acid content) and contains numerous sites for protein phosphorylation, suggesting that its role is further regulated through phosphorylation. Because PKA activation induces vesicle cycling at both previously silent cerebellar granule cell synapses (34Chavis P. Mollard P. Bockaert J. Manzoni O. Neuron. 1998; 20: 773-781Google Scholar) and in synapses of hippocampal cultures (35Trudeau L.E. Fang Y. Haydon P.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7163-7168Google Scholar), we wondered whether PKA phosphorylation of syntaphilin could influence its inhibitory functions at the nerve terminal via the cAMP signaling pathway. To address this question, we examined the ability of recombinant syntaphilin to serve as a substrate for PKA. 7 pmol of immunoprecipitated His-tagged syntaphilin expressed in HEK 293 T cells was incubated with PKA catalytic subunit in the presence of [γ-32P]ATP. The reactions were terminated by adding SDS sample buffer and heating, and the products were separated by SDS-PAGE and stained with Coomassie Blue to verify that equal amounts of protein were loaded in each lane, and the gel was dried and exposed to x-ray film to detect 32P incorporation. As shown in Fig. 1A, syntaphilin functions as a substrate for PKA, and its phosphorylation is blocked by the pseudosubstrate PKA inhibitor peptide PKI. To map phosphorylation sites in syntaphilin, we examined the capacity of syntaphilin truncated mutants to serve as PKA substrates. We incubated 50 pmol of purified His-tagged syntaphilin truncated mutants including 1–130, 130–205, and 203–469 with PKA and found that syntaphilin amino-terminal domain (1–130) was efficiently phosphorylated in vitro by PKA (Fig. 1B, upper panel). In contrast, both the coiled-coil domain (130–205) and the carboxyl half (203–469) could not serve as a PKA substrate under our experimental conditions. Immunoblotting of the reaction mixtures with anti-T7-His antibody demonstrated that approximately equal amounts of proteins were used for in vitro phosphorylation (Fig. 1B, lower panel). We then conducted a sequence search against the Scansite Motif Scanner Program (36Yaffe M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Google Scholar), and several consensus PKA phosphorylation residues were predicted between amino acids 1 and 130. We focused on three serine residues at positions 43, 56, and 64 that show high potential as PKA targets. To identify the amino acid residue(s) phosphorylated by PKA, we used site-directed mutagenesis to generate syntaphilin mutants where the serine at positions 43, 56, or 64 was substituted with alanine. Approximately 1 μg of the GST-syntaphilin mutants were incubated with PKA catalytic subunit at 30 °C for 1 h, separated by SDS-PAGE, stained with Coomassie Blue (Fig. 2A, lower panel), and then exposed to x-ray film (Fig. 2A, upper panel). To quantitate 32P incorporation, the gel slices corresponding to phosphorylated syntaphilin bands were excised and scintillation counted. Although the S64A mutant was as efficiently phosphorylated as the wild type syntaphilin, the 32P incorporation into S43A and S56A mutants was significantly decreased. Double mutation of S43A and S56A resulted in further reduction of the phosphorylation signal, suggesting that both the Ser43 and Ser56 residues serve as primary PKA phosphorylation sites in syntaphilin. Furthermore, we confirmed the site-directed mutagenesis results by mass spectrometry analysis using the phosphorylated syntaphilin and found that serine 43 is indeed a primary target for PKA phosphorylation in vitro. The surrounding sequence of syntaphilin serine 43 (RTHS43) and serine 56 (RRTS56) match one of the consensus sequences for PKA (RRX(S/T), RX(S/T), and RX2(S/T)). To further confirm that PKA phosphorylates only two residues in syntaphilin, we performed stoichiometric analysis of phosphorylation for purified recombinant syntaphilin protein. The reactions included 60 pmol of GST-syntaphilin and [γ-32P]ATP. 6 μl of samples containing 7.5 pmol of GST-syntaphilin were removed from the reaction mixture at various time points between 5 and 240 min, and these reactions were terminated by the addition of SDS sample buffer and boiling. The products were separated by SDS-PAGE, and inorganic P incorporation was quantitated by scintillation counting of excised gel slices corresponding to phosphorylated syntaphilin bands. The stoichiometry values are expressed as the ratios of moles of inorganic phosphate (Pi) incorporated per mole of syntaphilin protein and plotted against reaction time (Fig. 2B). Under conditions optimized for maximal phosphorylation, we found that PKA phosphorylation of syntaphilin reached a plateau level after 120 min of incubation at 30 °C, and the maximal stoichiometry is nearly 2, consistent with the two primary PKA sites identified by the mutagenetic studies. In Vivo Phosphorylation of Syntaphilin by PKA—Although our biochemical experiments showed that PKA incorporates 32P into syntaphilin, the conditions used for in vitro phosphorylation may not reflect conditions found in the native cellular environment. In addition, treatment of proteins with detergent for solubilization may expose sites that are normally not available for phosphorylation in vivo. Therefore, to investigate in vivo phosphorylation, we performed back-phosphorylation assays. We transfected a T7-His-tag