Correspondence to Yoshimi Takai: ytakai{at}molbio.med.osaka-u.ac.jp
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Introduction |
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Second messengers regulate proteinprotein interactions within the exocytotic machinery and, thereby, modulate neurotransmitter release (Evans and Morgan, 2003). 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 transiting from the docking to the fusion step. The activation of cAMP-dependent protein kinase (PKA) is known to promote neurotransmitter release from chromaffin cells, hippocampal mossy fiber synapses, and cerebellar parallel fiber synapses by directly acting on the exocytotic machinery (Lonart and Südhof, 1998; Lonart et al., 2003; Nagy et al., 2004). Thus, it is critical to identify the PKA targets that regulate assembly/disassembly of the SNARE complex and the priming of docked vesicles for the elucidation of the molecular mechanisms underlying neurotransmitter release.
Several presynaptic proteins serve as substrates for PKA: -SNAP (Hirling and Scheller, 1996), Cys string protein (Evans et al., 2001), synapsin I (Jovanovic et al., 2001), snapin (Chheda et al., 2001), syntaphilin (Boczan et al., 2004), RIM1 (Lonart et al., 2003), and SNAP-25 (Risinger and Bennett, 1999; Hepp et al., 2002). In chromaffin cells, two releasable (primed) vesicle pools (the slowly releasable pool [SRP] and the readily releasable pool [RRP]) are functionally defined, and their pool sizes are regulated by activation or inhibition of PKA. The PKA-catalyzed phosphorylation of SNAP-25 influences the size of SRP, possibly by altering the rate of vesicle priming. For example, the expression of Asp and Ala mutants of the phosphorylation site of SNAP-25 by PKA mirrors the effects of activation and inhibition of PKA, respectively, and selectively rescues the inhibitory effect of PKA inhibitors on the size of the SRP but not that of the RRP (Nagy et al., 2004). Therefore, for regulating RRP size, another PKA target is required. Neurons, like chromaffin cells, exhibit multiple kinetic components of release, although overall rates are approximately 10-fold faster than that for chromaffin cells (Mennerick and Matthews, 1996; Sakaba and Neher, 2001). Whether neurons have a vesicle pool that is analogous to the SRP is not known, but the high degree to which SNAREs are conserved among different secretory cell types suggests that RRP may be regulated by the phosphorylation of protein interacting with the SNARE complex in addition to SNAP-25. However, the key target protein for PKA remains elusive in neurons.
The upstream regulator of SNARE complex assembly, and not just the SNAREs themselves, also could be a PKA target. We have identified a novel syntaxin-1binding protein named tomosyn (Fujita et al., 1998). Tomosyn is abundantly expressed in the brain, where its distribution partly overlaps with that of syntaxin-1 on the presynaptic plasma membrane. Tomosyn is a 130-kD protein that contains a large NH2-terminal domain with WD40 repeats and a COOH-terminal domain that is homologous to vesicle-associated membrane protein (VAMP) 2. A large NH2-terminal region of tomosyn shares similarities with the Drosophila melanogaster tumor suppressor lethal giant larvae (Lgl), its recently characterized mammalian homologue Mlgl1, and the yeast proteins Sro7p and Sro77p (Lehman et al., 1999; Katoh and Katoh, 2004). Recent studies suggest that they play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane in yeast and epithelial cells (Lehman et al., 1999; Katoh and Katoh, 2004). The role of the Lgl family cannot be confined to an activator or inhibitor of membrane fusion. We have previously shown that tomosyn displaces Munc-18 from syntaxin-1 and binds to the SNARE motif of syntaxin-1 via its COOH-terminal VAMP homology region and forms a 10-S tomosyn complex with syntaxin-1, SNAP-25, and synaptotagmin (Fujita et al., 1998). Based on these data, we have postulated that tomosyn plays a positive role in neurotransmission by enhancing the availability of syntaxin-1 to form the SNARE complex (Fujita et al., 1998). Contrary to this hypothesis, the expression of tomosyn results in a reduction in Ca2+-dependent exocytosis from PC12 cells (Fujita et al., 1998; Hatsuzawa et al., 2003). Tomosyn interacts with the t-SNAREs syntaxin-4 and SNAP-23 and inhibits insulin-stimulated fusion of GLUT4-containing vesicles with the plasma membrane in 3T3-L1 adipocytes (Widberg et al., 2003). Tomosyn inhibits priming of large dense-core vesicles in a Ca2+-dependent manner in adrenal chromaffin cells (Yizhar et al., 2004). More recently, we have shown that Rho-associated coiled-coilforming kinase (ROCK) that is activated by Rho small G protein phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable tomosyn complex. This results in inhibition of the formation of the SNARE complex during neurite extension in hippocampal cultured neurons (Sakisaka et al., 2004). The ROCK-catalyzed phosphorylation of syntaxin-1 could act as an "on" switch for tomosyn by enhancing its inhibitory function on the formation of the SNARE complex. Thus, evidence is accumulating that tomosyn acts as a negative regulator for the formation of the SNARE complex, and, thereby, inhibits various vesicle fusion events. However, an "off" switch for tomosyn, which is the molecular regulation that blocks its inhibitory function on the formation of the SNARE complex, remains elusive.
In this study, we show that tomosyn is a target protein for PKA and that the PKA-catalyzed phosphorylation of tomosyn acts as an off switch for tomosyn. It does so by blocking its inhibitory function on the formation of the SNARE complex and, thereby, up-regulating the size of readily releasable synaptic vesicles in neurons.
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Results |
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By using Sp-cAMP and forskolin, we examined whether tomosyn is indeed phosphorylated in intact cells in response to cAMP. Sp-cAMP is a nonhydrolyzable and cell-permeable cAMP analogue that induces activation of PKA in intact cells (Scholubbers et al., 1984), whereas forskolin is a potent activator of adenylate cyclase (Seamon et al., 1981). When 32Pi-labeled NG108 cells expressing HA-tagged wild-type tomosyn (HA-tomosyn) were stimulated by Sp-cAMP or forskolin, the incorporation of 32P into tomosyn was significantly enhanced (Fig. 1 C). The extent of tomosyn phosphorylation that was stimulated by Sp-cAMP and forskolin was reduced by KT5720 and H-89, which are synthetic chemical PKA inhibitors (Kase et al., 1987; Chijiwa et al., 1990). When 32Pi-labeled NG108 cells expressing HA-tomosynS724A were stimulated with Sp-cAMP, the incorporation of 32P into this mutant was not markedly observed. These results indicate that tomosyn is directly phosphorylated by PKA both in vitro and in vivo and that Ser-724 is the major phosphorylation site of tomosyn by PKA.
Reduction by the PKA-catalyzed phosphorylation of tomosyn of its interaction with syntaxin-1 in vitro
We have previously shown that tomosyn interacts with syntaxin-1 in a manner that is competitive with VAMP-2, forms the tomosyn complex, and down-regulates the formation of the SNARE complex (Fujita et al., 1998; Yokoyama et al., 1999; Sakisaka et al., 2004). We examined the effect of the PKA-catalyzed phosphorylation of tomosyn on its interaction with syntaxin-1 in vitro. MBP-tomosyn that was either previously phosphorylated by PKA or left unphosphorylated was incubated with GSTsyntaxin-1bound glutathione beads. Free MBP-tomosyn and MBP-tomosyn that bound to GSTsyntaxin-1 beads were then separated by centrifugation and subjected to SDS-PAGE followed by CBB staining and autoradiography. The amount of phosphorylated MBP-tomosyn that bound to GSTsyntaxin-1 was 20% of the total input, whereas that of nonphosphorylated MBP-tomosyn was
60% of the total input (Fig. 2 A). Kinetic studies revealed that the apparent Kd values of the phosphorylated and nonphosphorylated forms of GSTsyntaxin-1 were 330 and 60 nM, respectively (Fig. 2 B). These results indicate that the PKA-catalyzed phosphorylation of tomosyn decreases its interaction with syntaxin-1 in vitro.
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Enhancement of the formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn in vivo
We have previously shown that the expression of tomosyn increases the formation of the tomosyn complex and, thereby, down-regulates the formation of the SNARE complex in NG108 cells (Sakisaka et al., 2004). We examined whether the PKA-catalyzed phosphorylation of tomosyn affects the formation of the SNARE complex. We cultured NG108 cells expressing myc-tomosyn in the presence and absence of Sp-cAMP and immunoprecipitated syntaxin-1 from the lysate of each NG108 cell. Both myc-tomosyn and VAMP-2 were coimmunoprecipitated with syntaxin-1 in both cells, but the amount of coimmunoprecipitated VAMP-2 in the cells that were pretreated with Sp-cAMP was more than that of control cells (Fig. 3). Conversely, the amount of coimmunoprecipitated myc-tomosyn in cells that were pretreated with Sp-cAMP was less than that of control cells. When either myc-tomosynS724A or S724D was expressed, the amount of coimmunoprecipitated VAMP-2 in the cells expressing myc-tomosynS724D was more than that in the cells expressing myc-tomosynS724A. In addition, the amount of coimmunoprecipitated VAMP-2 in the cells expressing myc-tomosynS724A or S724D was not increased in response to Sp-cAMP. These results are consistent with those obtained by the in vitro experiments shown in Fig. 2 C. They indicate that the tomosyn complex was more predominantly formed in the cells expressing myc-tomosynS724A than in the cells expressing myc-tomosynS724D and that, conversely, the SNARE complex was less predominantly formed in the cells expressing myc-tomosynS724A than in the cells expressing myc-tomosynS724D. These results indicate that the PKA-catalyzed phosphorylation of tomosyn decreases the formation of the tomosyn complex and increases the formation of the SNARE complex in intact cells.
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To further confirm that PKA is the relevant kinase mediating the effect of cAMP in neurotransmitter release from SCG neurons, we applied various reagents that modulate the PKA signaling pathway, including forskolin, KT5720, and H-89 (Fig. 5, EH). Forskolin enhanced neurotransmitter release, which is consistent with the effect of cAMP (Fig. 5, E and H). In contrast, H-89 and a combination of PKA inhibitors (KT5720 and the PKA pseudosubstrate PKI 622 peptide) inhibited the evoked neurotransmitter release (Fig. 5, FH). These results indicate that PKA is the relevant kinase for regulating neurotransmitter release from SCG neurons by increasing the RRP size.
Involvement of the PKA-catalyzed phosphorylation of tomosyn in neurotransmitter release
To investigate the role of the PKA-catalyzed phosphorylation of tomosyn in neurotransmitter release, we analyzed synaptic transmission of SCG neurons by expressing myc-tomosyn, myc-tomosynS724A, myc-tomosynS724D, or tomosyn small interference RNA (siRNA) in the presynaptic neurons. First, we examined electrophysiologically how many neuron pairs are synaptically coupled. For this purpose, we recorded electrical responses in pairs of neurons by generating action potential trains at 2 Hz in the presynaptic neurons. The incidence of synaptic coupling between control neurons (nontransfected pre- and postsynaptic neurons) was 64 ± 4.9% (Fig. 6 A). This value was reduced to 40 ± 13 and 44 ± 6.3% by the expression of myc-tomosyn and myc-tomosynS724D in the presynaptic neurons, respectively, whereas this value was more markedly reduced to 28 ± 11% by the expression of myc-tomosynS724A. Similarly, the incidence of synaptic coupling was reduced to 35 ± 10% by expressing tomosyn siRNA, whereas the incidence of synaptic coupling was 61 ± 14% for control neurons expressing control scramble RNA. Then, we measured the size of EPSPs in synaptically coupled neurons. The mean EPSP amplitude for control synapses of nontransfected neuron pairs was 20 ± 6.8 mV (Fig. 6 B). This value was reduced to 8.2 ± 2.3 and 8.1 ± 2.4 mV by the expression of myc-tomosyn and myc-tomosynS724D in the presynaptic neurons, respectively, whereas this value was more markedly reduced to 4.8 ± 0.7 mV by the expression of myc-tomosynS724A. Similarly, the mean EPSP value was reduced to 6.7 ± 1.7 mV by the expression of tomosyn siRNA, whereas the mean EPSP value was 21 ± 3.2 mV for control neurons expressing control scramble RNA. Disruption of the expression level of tomosyn in either direction compromises neurotransmitter release. Therefore, the expression level of tomosyn seems to be maintained at an optimum level for regulating neurotransmitter release. To confirm that the expression levels of various tomosyn mutants in SCG neurons are similar, we immunostained SCG neurons with a tomosyn pAb and quantified the fluorescence intensities. The immunofluorescence signals for tomosyn mutants were found in the cell body and in discrete puncta surrounding neighboring SCG neurons, suggesting that the expressed tomosyn mutants are translocated into the presynaptic terminals. The expression levels of various tomosyn mutants were apparently similar (Fig. 6 C). The signal for tomosyn in the presynaptic terminals of SCG neurons was fourfold higher than that in the presynaptic terminals expressing the control vector. In addition, the signal for endogenous tomosyn in the presynaptic terminals of SCG neurons expressing tomosyn siRNA were reduced in comparison with that in the presynaptic terminals expressing control scramble RNA. These results indicate that tomosyn and its phosphorylation by PKA are involved in neurotransmitter release from SCG neurons.
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We then examined whether the PKA-catalyzed phosphorylation of tomosyn is involved in the PACAP-induced facilitation of neurotransmitter release. To investigate the role of PACAP at presynaptic terminals in SCG neurons, we focally applied PACAPs to presynaptic SCG neurons. Focal puff application of PACAP27 or 38 (1 nM in the pipette for 0.5 s) markedly increased EPSP amplitudes and prolonged the time course of EPSP waveforms (Fig. 9 Aa). EPSPs that were elicited every 20 s were increased by PACAP27 and 38 (Fig. 9, Ab and Ac), with the mean of peak amplitudes increased to 75 ± 27 and 66 ± 16% of baseline control responses, respectively (Fig. 9 B). These results indicate that low concentrations of PACAP peptides facilitate neurotransmitter release, which is consistent with a previous study for neuropeptide Y release (May and Braas, 1995). In concordance with cAMP injections into the presynaptic neurons, peptides slightly depolarized the presynaptic membrane (03 mV for 3040 min; not depicted); however, this modest depolarization does not affect the gating kinetics of N-type Ca2+ channels (Regan et al., 1991). Then, we examined whether PKA is involved in the PACAP-induced facilitation of neurotransmitter release by expressing a kinase-dead mutant, PKA-K72H (Iyer et al., 2005), or a mixture of PKA catalytic subunit (PKA C and Cß) siRNAs (Dumaz and Marais, 2003) in the presynaptic neurons. These inhibitory reagents reduced the EPSP amplitude to 8.9 ± 2.1 or 13 ± 0.6 mV in comparison with the value of 20 ± 1.1 mV in nontransfected neuron pairs (Fig. 9 C) and blocked the PACAP27-induced facilitation of neurotransmitter release (Fig. 9, Da and Db). Myc-tomosynS724A also reduced the PACAP27-induced facilitation of neurotransmitter release, confirming the involvement of the PKA-catalyzed phosphorylation of tomosyn in the PACAP-induced facilitation of neurotransmitter release (Fig. 9 Dc). The maximum increase in the mean EPSP amplitude that was caused by PACAP27 (57 ± 30% at 35 min after the application) was significantly reduced by 12 ± 18 (at 31 min; P < 0.05), 30 ± 20 (at 30 min), and 25 ± 8% (at 19 min) with the expression of PKA-K72H, PKA siRNA, and myc-tomosynS724A, respectively (Fig. 9 E). These results suggest that PACAP induces the facilitation of neurotransmitter release through the PKA-catalyzed phosphorylation of tomosyn in SCG neurons and regulates synaptic transmission efficiency in the autonomic nervous system.
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Discussion |
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Second, we have shown that the PKA-catalyzed phosphorylation of tomosyn is involved in neurotransmitter release from SCG neurons. In adrenal chromaffin cells, the PKA-catalyzed phosphorylation of SNAP-25 has been proposed to regulate the priming/depriming of secretory vesicles at a slow rate-limiting step before the final fast priming step and Ca2+-dependent fusion (Nagy et al., 2004). Therefore, another unidentified PKA target has been postulated to be involved in regulation of the priming step that is associated with the quickly releasable pool, which may control the availability of readily releasable vesicles in nerve terminals (Heidelberger and Matthews, 2004). Our results suggest that tomosyn is a candidate PKA target for this penultimate priming step. Upon high, frequent stimulation, the expression of tomosyn S724A (a mutant that is resistant to the phosphorylation of PKA) or S724D (a mutant mimicking the phosphorylated form of tomosyn) increases the EPSP failure rate and causes the appearance of asynchronous EPSP waveforms, suggesting that the mobilization of readily releasable vesicles is strongly reduced when the PKA-catalyzed phosphorylation of tomosyn Ser-724 is impaired. An increase in asynchronous release upon repetitive stimulation has also been observed when the interaction of the synprint site in N-type Ca2+ channels with the SNARE complex is disrupted by synprint peptides (Mochida et al., 1996). It is likely that preventing the formation of the SNARE complex reduces its interaction with N-type Ca2+ channels that are required for synchronous neurotransmitter release. Although further studies are required for our understanding of the molecular mechanism of vesicle priming, we propose that the PKA-catalyzed phosphorylation of tomosyn plays an important role in supplying readily releasable synaptic vesicles to a population of synchronized vesicles, which are able to respond to each action potential during repetitive presynaptic activity. Altogether, our results indicate that tomosyn is a physiologically significant PKA target that controls neurotransmitter release through regulation of the formation of the SNARE complex and vesicle priming.
Third, we have shown that the knockdown of tomosyn decreases neurotransmitter release from SCG neurons, indicating that tomosyn plays both positive and negative roles in neurotransmitter release. The exact reason for these apparently inconsistent data is not known, but one explanation is that the formation of the tomosyn complex may be a prerequisite for the formation of the SNARE complex by analogy with the role of Munc-18 in the formation of the SNARE complex (Gerst, 2003). Because the amount of tomosyn is less than that of syntaxin-1, tomosyn may catalytically facilitate the formation of the SNARE complex. This idea requires the process in which tomosyn is replaced by VAMP-2 to form the SNARE complex. The PKA-catalyzed phosphorylation of tomosyn may be involved in this process for releasing tomosyn form syntaxin-1 and SNAP-25. Another possible explanation is that tomosyn affects another regulatory component besides SNAREs, such as a cytoskeletal network, in the exocytosis of neurotransmitters. Tomosyn is a member of the Lgl family (Katoh and Katoh, 2004). Lgl is part of the cytoskeletal network and is associated with nonmuscle myosin II heavy chain (Strand et al., 1994), which localizes at the presynaptic side of synaptic junctions (Beach et al., 1981) and is involved in neurotransmitter release (Mochida et al., 1994a). Recently, it was found that the aPKC-catalyzed phosphorylation of Lgl inhibits the interaction with myosin II and allows an autoinhibitory intramolecular interaction of the NH2-terminal portion of Lgl with the COOH-terminal portion (Betschinger et al., 2005). Similar to Lgl, tomosyn may interact with myosin II under the control of the PKA-catalyzed phosphorylation of tomosyn, and this interaction may be involved in the exocytosis of neurotransmitters.
Finally, we have shown the physiological significance of the PKA-catalyzed phosphorylation of tomosyn in neurotransmitter release from SCG neurons by using a potent biological ligand to activate the endogenous PKA pathway (PACAP). Consistent with previous studies, focally applied PACAP increases EPSP amplitude along with a slight membrane depolarization that is similar to the introduction of cAMP or forskolin into the presynaptic nerve terminals of SCG neurons (May et al., 1998; Beaudet et al., 2000). However, the effect of PACAP lasts longer than that of cAMP or forskolin. Inhibition of the PKA signaling pathway by the expression of kinase-dead PKA-K72H or a mixture of PKA catalytic subunit (PKA C and Cß) siRNAs significantly reduces the long-lasting PACAP-induced facilitation of neurotransmitter release. The expression of tomosyn-S724A (a mutant that is resistant to the phosphorylation of PKA) also reduces the long-lasting PACAP-induced facilitation of neurotransmitter release. The activity of tomosyn may depend on the level of PKA activation in the presynaptic SCG neurons. In fact, the basal level of PKA activity varies at different synapses (Chavis et al., 1998). In the squid giant synapse, a high resting level of PKA activity tonically enhances neurotransmitter release that is elicited by single action potentials, suggesting that the function of PKA at this synapse is to set the initial efficacy of synaptic transmission (Hilfiker et al., 2001). In addition, PKA has been shown to be involved in the exocytosis of various secretory systems (Evans and Morgan, 2003). Thus, the extent of PKA-catalyzed phosphorylation of tomosyn may modulate exocytosis not only in SCG neurons but also in various secretory systems that under the control of the physiological signaling pathway, such as a trimeric G proteincoupled receptor's signaling pathway (Fig. 9 F).
We have previously shown that ROCK activated by Rho small G protein phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in the inhibition of SNARE complex formation during neurite extension (Sakisaka et al., 2004). The ROCK-catalyzed phosphorylation of syntaxin-1 could act as an on switch for tomosyn by enhancing its inhibitory function on the formation of the SNARE complex. Conversely, the PKA-catalyzed phosphorylation of tomosyn could act as an off switch for tomosyn by blocking its inhibitory function on formation of the SNARE complex. Although the involvement of the ROCK-catalyzed phosphorylation of syntaxin-1 in neurotransmitter release needs to be addressed, tomosyn is an important SNARE regulatory protein in neurotransmitter release, whose activity is regulated via well-known signal transduction pathways.
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Materials and methods |
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Abs
The antitomosyn pAb was prepared as described previously (Fujita et al., 1998). The antisyntaxin-1 mAb, antiVAMP-2 pAb (Synaptic Systems GmbH), antisyntaxin-1 pAb, antiSNAP-25 pAb (Calbiochem), antisynaptophysin mAb (Chemicon), anti-Bassoon mAb (StressGen Biotechnologies), antiPSD-95 family mAb (Upstate Biotechnology), anti-PACAP receptor pAb (Santa Cruz Biotechnology, Inc.), and anti-HA mAb (Babco) were purchased from commercial sources. AlexaFluor-conjugated secondary Abs (Invitrogen) were used to detect primary Abs in immunofluorescence microscopy of SCG neurons.
Phosphorylation of tomosyn by PKA in a cell-free system
Phosphorylation of tomosyn was performed in 50 µl of a reaction mixture containing 50 mM Tris-HCl, pH 7.5, 7 mM MgCl2, 1 mM DTT, 0.06% CHAPS, 1 mM EDTA, 50 µM -[32P]ATP (0.51.2 x 103 cpm/pmol; GE Healthcare), and 25 units of the catalytic subunit of PKA (Promega) at 30°C in the presence or absence of 2 mM pseudosubstrate PKI (Promega). The reaction was stopped by the addition of SDS sample buffer and was boiled for 5 min. Each sample was subjected to SDS-PAGE followed by protein staining with CBB and autoradiography. Relevant gel slices were excised, and scintillation was counted to determine the radioactivity that incorporated into tomosyn.
Phosphorylation of tomosyn in intact cells
NG108 cells were transfected with either pCMV-HA-tomosyn or pCMV-HA-tomosyn-S724A and cultured in DME containing HT (0.10 mM hypoxanthine and 0.16 mM thymidine; Sigma-Aldrich) for 48 h. The cells were washed three times with phosphate-free DME and incubated for 30 min in phosphate-free DME containing HT. Thereafter, the cells were prelabeled with 0.3 mCi/ml [32P]orthophosphate (phosphorus-32; GE Healthcare) for 3 h. The cells were preincubated with or without 1 µM KT5720 (Calbiochem) or 2 µM H-89 (Calbiochem) for 30 min and further stimulated by 1.0 mM Sp-cAMP (Sigma-Aldrich) or 50 µM forskolin (Calbiochem) for 30 min. The cells were lysed with radioimmunoprotein assay buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, and 5 mM EDTA) and immunoprecipitated with anti-HA mAb. The immunoprecipitates were subjected to SDS-PAGE followed by protein staining with CBB and autoradiography.
Assay for the interaction of tomosyn with syntaxin-1
40 pmol MBP-tomosyn was fully phosphorylated by 25 units of the catalytic subunit of PKA. Phosphorylated tomosyn was incubated with 30 pmol GSTsyntaxin-1 that was bound to 10 µl glutathione beads in the presence of 1% CHAPS at 4°C for 30 min followed by centrifugation and extensive washing. As a control, 40 pmol MBP-tomosyn was incubated under the same conditions as described above except that -[32P]ATP was excluded. The intensity of the bands in SDS-PAGE was determined by densitometry.
Assay for the formation of tomosyn and SNARE complexes
The lysate of NG108 cells was prepared by using a lysis buffer (20 mM Hepes-NaOH, pH 7.4, 90 mM KOAc, 2 mM Mg(OAc)2, 0.5 mM EGTA, 50 mM NaF, 2 mM Na3VO4, and 1% CHAPS; Fujita et al., 1998). The lysates were incubated with an antisyntaxin-1 mAb at 4°C for 2 h. The immune complexes were precipitated with protein GSepharose beads (GE Healthcare) at 4°C for 1 h, washed with the lysis buffer, resolved in 520% gradient SDS-PAGE, and analyzed by immunoblotting with antitomosyn, antiVAMP-2, antiSNAP-25, and antisyntaxin-1 pAbs.
Culture of SCG neurons
Postnatal day 7 Wistar ST rats were decapitated under diethylether anesthesia according to Physiological Society of Japan guidelines. Isolated SCG neurons were maintained in culture for 56 wk as described previously (Mochida et al., 1994b; Mochida, 1995). In brief, SCGs were dissected, desheathed, and incubated with 0.5 mg/ml collagenase (Worthington Biochemical) in L-15 (GIBCO BRL) at 37°C for 10 min. After enzyme digestion, the semidissociated ganglion was triturated gently through a small pore glass pipette until a cloudy suspension was observed. After washing by low speed centrifugation at 1,300 rpm for 3 min, the collected cells were plated onto coverslips in plastic dishes (Corning; 35-mm diameter; approximately one ganglion per dish) containing a growth medium of 84% MEM (GIBCO BRL), 10% FCS (GIBCO BRL), 5% horse serum (GIBCO BRL), 1% penicillin/streptomycin (GIBCO BRL), and 25 ng/ml nerve growth factor (2.5 S; grade II; Alomone Laboratories). The cells were maintained at 37oC in a 95% air/5% CO2 humidified incubator, and the medium was changed twice per week.
Immunocytochemistry
SCG neurons were fixed with 4% PFA for 30 min or acetone for 5 min. After washing with PBS, the cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min. Nonspecific binding was blocked with Block Ace (Dainippon Pharmaceutical) for 2 h. The cells were incubated with primary Abs that were diluted in Block Ace for 1.5 h followed by secondary Abs. The stained cells were observed with a confocal laser microscope (Radiance 2000; Bio-Rad Laboratories) using a 63x oil immersion objective lens (model LSM510; Carl Zeiss MicroImaging, Inc.). Collected data were exported as eight-bit TIFF files and were processed using Adobe Photoshop 7.0.
Expression and knockdown of PKA or tomosyn in SCG neurons
The mammalian expression vector pBS-H1 (Yamada et al., 2005) was used for the expression of siRNA. The following inserts were used: the PKA C genespecific insert was a 21-nucleotide sequence corresponding to nt 885905 (5'-AAGTGGTTTGCCACAACTGAC-3') of PKA C
cDNA, and the PKA Cß genespecific insert was nt 2545 (5'-AAGAGTTCCTAGCCAAAGCCA-3') of PKA Cß cDNA, which was separated by a 10-nucleotide noncomplementary spacer (5'-TTGATATCCG-3') from the reverse complement of the same 21-nucleotide sequence (Dumaz and Marais, 2003). An expression vector for tomosyn siRNA was constructed (Sakisaka et al., 2004). Various tomosyn or PKA expression vectors and the tomosyn or PKA siRNA expression vector were microinjected into the nuclei of SCG neurons through a microglass pipette along with 10 kD fluorescein dextran (Invitrogen) as described previously (Mochida et al., 2003). Entry of the constructs into the cell was monitored by fluorescence intensity in the nucleus or that of the dye in the cell body. The cells were maintained at 37°C in a 95% air/5% CO2 humified incubator for 4070 h, and the injected neurons were identified with an inverted microscope (Diaphot 300; Nikon) equipped with an epifluorescence unit and a fluorescence microscope system (Arugas/HiSCA; Hamamatsu Photonics). The injected neurons were examined as the presynaptic neurons in paired neuronal recordings.
Synaptic transmission between SCG neurons
EPSPs were recorded as described previously (Mochida et al., 1994a). Conventional intracellular recordings were made from two neighboring neurons by using microelectrodes filled with 1 M KAc (7090 M). EPSPs were recorded from a nontransfected neuron, whereas action potentials were generated in the transfected neuron with various tomosyn expression vectors or the tomosyn siRNA expression vector by the passage of current through an intracellular recording electrode. Synaptic couples with subthreshold EPSPs that did not produce postsynaptic action potentials were selected for further study. Neurons were superfused with a modified Krebs' solution consisting of 136 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 11 mM glucose, and 3 mM Na-Hepes, pH 7.4, except for recordings of EPSPs with 10 stimuli, which were superfused in a Krebs' solution containing 0.2, 0.5, and 1 mM CaCl2. Electrophysiological data were collected and analyzed by using software written by the late L. Tauc (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France; Mochida et al., 1996) and analyzed with Origin 7.0 software (Microcal Software). The peak amplitudes of EPSPs were averaged, and the resulting values were smoothed by an eight-point moving average algorithm and plotted against recording time with time = 0 indicating the start of the presynaptic injection of cAMP (Sigma-Aldrich), forskolin, H-89, a mixture of PKI 622 (Calbiochem) and KT5720, or the start of puff application of PACAP27 and 38 (Sigma-Aldrich). Reagents for injection were dissolved in 150 mM KAc, 5 mM Mg2+-ATP, and 10 mM Hepes, pH 7.3, and were introduced into the presynaptic cell body by diffusion from a suction glass pipette (1520-M
tip resistance). 5% Fast Green FCF (Sigma-Aldrich) was included via the peptide injection solution to confirm entry into the presynaptic cell body. The injection pipette was removed 5 or 10 min after starting injection. To measure RRP size, 0.5 M sucrose was puff applied to presynaptic neurons every 3 min (Mochida et al., 1998). Each sucrose response was recorded with Clampex, and the integral values that were measured by Fetchan (pClamp 8.1; Axon Instruments, Inc.) were averaged and normalized. Each experiment was performed in a minimum of four synaptically coupled SCG neuron pairs. Data values with associated error are shown in the text, and figures represent means ± SEM. A two-tailed t test was applied to compare effects among presynaptic neurons that were transfected with various tomosyn expression vectors or between the presynaptic neurons that were transfected with tomosyn siRNA and those transfected with scramble RNA.
Online supplemental material
Fig. S1 shows the localization of PACAP receptor in SCG neurons. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200504055/DC1.
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Acknowledgments |
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This work was supported by grants-in-aid for scientific research (to S. Mochida) and cancer research (to Y. Takai) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2004). T. Sakisaka is a recipient of a Human Frontier Science Program Career Development Award (2003).
Submitted: 11 April 2005
Accepted: 22 August 2005
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References |
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