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A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase
Biochimica et Biophysica Acta, 1175 (1992) 67-72 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4889/92/$05.00
67
BBAMCR 13285
A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase Hitoshi Nakayama, Hiroyoshi Shikano and Yuichi Kanaoka 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan) (Received 22 April 1992) (Revised manuscript received 10 August 1992)
Key words: Sodium channel; Antipeptide antibody; Protein phosphorylation; Phosphorylation block; Phosphorylated peptide; (Rat " brain)
Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the a subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.
Introduction Phosphorylation is a major mechanism of ion-channel regulation. Although many ion channels are influenced by phosphorylation events, only a few have been biochemically identified and characterized [1,2]. The voltage-gated sodium channel from rat brain is phosphorylated by cAMP-dependent protein kinase (PKA) in purified preparations [3,4] and in situ [5,6]. cAMPdependent phosphorylation of sodium channels in synaptosomes is associated with a reduction in neurotoxin-activated ion flux [5], but the functional significance of phosphorylation under physiological conditions is not known. Noda et al. [7] have deduced the primary structure of rat-brain sodium-channel a sub-
Correspondence to: H. Nakayama, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan. i Present address: Toyama Women's College, Gankaiji 444, Toyama 930-01, Japan. Abbreviations: PKA, cyclic-AMP(cAMP)-dependent protein kinase; ELISA, enzyme-linked immunosorbent assay; STX, saxitoxin; WGA, wheat germ agglutinin; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiotbreitol.
units and proposed five potential phosphorylation sites for PKA. This makes it possible to raise anti-peptide antibodies [16] for the particular regions that may contribute to express the channel function. Raising antipeptide antibodies against the phosphorylation sites may provide useful tools, if they specifically recognize the phosphorylation sites or nearest ones to inhibit the phosphorylation. In this paper, we describe three synthetic peptides that are employed to raise antibodies, characteristics of the elicited antibodies, and analysis of the PKA-catalyzed phosphorylation of rat-brain sodium channels using one of the antibodies.
Experimental Methods
Peptide synthesis and antibody generation. Synthetic peptides corresponding to particular regions of the sodium channel II sequence from rat brain [7] were synthesized by the solid-phase method [8]. Polyclonal antisera were raised in rabbits against peptides corresponding to the following amino-acid residues of the channel sequence: C544-555, 544-555 plus N-terminal cysteine, C561-575, 561-575 plus N-terminal cysteine,
68 and C681-689, 681-689 plus N-terminal cysteine. Peptides were purified by reverse-phase HPLC and their structures were confirmed by amino-acid sequence analysis and amino-acid analysis. For immunization, peptides were coupled to porcine thyroglobulin using N-hydroxysuccinimidyl m-maleimidobenzoate to couple through N-terminal cysteine residues.
Preparation of the purified sodium channel from rat brains. Rat brain lysed P3 was solubilized and purified by successive chromatography of DEAE-Sephadex, hydroxyappatite, and WGA-Sepharose 4B as described in the literature [9]. Specific activity obtained was 680-750 pmol [3H]saxitoxin binding sites/mg protein. Solubilized eel-electroplax sodium channel was partially purified by DEAE-Sephadex as described [17]. Solid-phase ELISA. The binding of the antisera to the synthetic peptides and to the sodium channel was analyzed by indirect solid-phase ELISA [14]. The plates were coated with 50/xl of either the peptide (1/xg/ml) or with the purified sodium channel (2 /zg/ml) in 50 mM sodium carbonate buffer (pH 9.6). Alkaline phosphatase-conjugated goat anti-rabbit IgG was used for the second antibody followed by colorimetry with pnitrophenyl phosphate as substrate. Phosphorylation of the synthetic peptides. Synthetic peptides (0.1 pmol) were incubated in 25 mM HepesTris (pH 7.4), 2 mM MgCI 2 and 30 /xM [y-32p]ATP (3.105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (Sigma) in a final volume of 50 ~1. Various concentrations of anti-(C561-575) antibody (0.1-10 ~M) were added in some series of experiments. The
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Proteolysis of purified 32p-phosphorylated sodium channels. The 32p-phosphorylated sodium channel was subjected to the SDS-PAGE and isolated the phosphorylated a subunit band. The sliced gel was briefly
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reaction was initiated by the addition of [y-32P]ATP and performed at 30°C for 30 min. The reaction was terminated by addition of 0.15 M phosphoric acid (50 ~l). 40 ~I of each sample were loaded on a Whatman P81 phosphoceIlulose paper disk (duplicate) followed by three 2-min washes with 75 mM phosphoric acid (10 /zl) in Petri dishes as described [10]. Radioactivity on the phosphocellulose paper was counted by Cerenkovcounting. In the antibody-blocking experiments, antibody was also phosphorylated and counted by this assay. We carried out the parallel phosphorylation of antibody alone as control and determined the net amount of peptide phosphorylation by subtracting the phosphorylated amount of antibody. Phosphorylation of the sodium channel. Purified sodium channel (0.1 pmol of the STX binding site) was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgC12 and 30/xM [y-aEp]ATP (3" 105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (PKA, Sigma) in a final volume of 50 /xl. Various concentrations of anti-(C561-575) antibody (0.1-10 /~M) were added in some series of experiments. The reaction was initiated by the addition of [T-32p]ATP and performed at 30°C for 30 min. The reaction was stopped by addition of 10 /.d of 5% SDS/25 mM Tris-HCl (pH 6.8)/40% glycerol ( w / v ) / 50 mM dithiothreitol/0.1% bromophenol blue. The samples were then subjected to the SDS-PAGE.
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Antiserum dilution fold Fig. 1. Binding of anti-peptide antibodies to the corresponding peptides and the purified sodium channel. The plates were coated with 50 ~1 of either the peptide (o, 1 p.g/ml) or the purified sodium channel (e, 2 /.Lg/ml) in 50 mM sodium carbonate buffer (pH 9.6). Alkaline phosphatase-conjugated goat anti-rabbit IgG was used for the second antibody and color development for 10 rain after addition of p-nitrophenyl phosphate was determined by measuring absorbance at 405 nm. (A) Anti-(C544-555) antibody, (B) anti-(C561-575) antibody, and (C) anti-(C681-689) antibody. For control the binding of preimmune serum ([]) is shown.
69 incubated in 50 mM Tris-HCl (pH 9.0), rinsed with the same buffer, and digested with lysyl endoproteinase (Achromobacter proteinase I) (S/E--100, w/w) at 37°C for 24 h. Electrophoresis. SDS-PAGE was carried out using the 4-12% gradient gel of polyacrylamide for the sodium channel [9] or 16.5% polyacrylamide gel for the proteinase-digested samples. After electrophoresis, the gel was dried under vacuum and subjected to the autoradiography. Corresponding radioactive bands were cut out and the radioactivity was determined by Cerenkov-counting. Immunoblot. Electrophoresed polypeptide bands on polyacrylamide gel were electrotransferred [11] onto Immobilon P paper (Millipore) and performed the immunoblot analysis [15]. Alkaline phosphatase-conjugated goat anti rabbit IgG (Capped was used as second antibody and visualized with /3-naphthylphosphate and Fast Blue B.
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Results and Discussion
Specificity of the anti-peptide antibodies In order to elicit antibodies, the synthetic peptides were conjugated to thyroglobulin and injected into rabbits. Although all three anti-peptide antibodies bound to their respective peptides to similar extents in solid-phase ELISA, they differed in their capacity to cross-react with the sodium channel (Fig. 1). Only the anti-peptide (C561-575) antibody can bind to the sodium channel. It probably reflects the exposure a n d / o r conformation of these sequences within the sodium-channel molecule, although five potential phosphorylation sites are considered to be a cluster in the cytoplasmic side [12]. The specificity of the anti-(C561-575) antibody was assessed by immunoblot and the competitive ELISA method. Although the anti-peptide (C561-575) antibody reacted with the sodium channel of 300 kDa as a major component in lysed synaptosome fractions (P3) of rat brains under nonreducing conditions, it also stained 270 kDa (possibly a subunit, see below) and possible aggregates of high molecular weight containing the a subunit (Fig. 2, lane 1). However, it only recognized the a subunit (270 kDa) of the sodium channel which involves the sequence (561-575) and is free from lower-molecular-weight /3 subunits [12] under reducing conditions (lane 2). Preimmune serum did not react with this polypeptide (lane 4). The antipeptide antibody did not recognize the sodium channel from electroplax of electric eels (lane 3), as expected by the fact that the sequences of five potential phosphorylation sites in the rat-brain channel are missing in the electroplax channel. The results suggest that the antibody recognizes the rat-brain sodium channel rather in a sequence-specific fashion. Other evidence of anti-
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Fig. 2. Immunoblot of anti-(C561-575) antibody. SDS-PAGE of lysed P3 fractionsand partiallypurified eel electroplaxsodiumchannel was carried out usingthe 4-12% gradientgel of polyacrylamide. Electrophoresed polypeptidebands were electrotransferredonto Immobilon P paper (Millipore) and the immunoblotanalysiswas performed as described in Experimental Methods. Rat-brain sodium channels not treated with DTT (lane 1); rat-brain sodium channels treated with DTT (lane 2); electroplaxsodiumchannelwithoutDTT (lane 3); DTF-treated rat-brain sodiumchannelimmunoblottedwith preimmuneserum(lane 4). body specificity was obtained by the ELISA assay in the presence of antigen peptides. As shown in Fig. 3, the immunoreaction of the antibody with the purified sodium channel was inhibited by the peptide (C561-575) in a dose-response manner. Two other peptides of residues C544-555 and C681-689 did not inhibit the immunoreaction. Therefore, the anti-peptide (C561-575) antibody is concluded to recognize the a subunit of the rat-brain sodium channel in a sequence-specific manner.
Peptide phosphorylation Three synthetic peptides corresponding to residues 544-555, 561-575, and 681-689 (see sequences in Fig. 6B) of the a subunit of rat-brain sodium channel II include the potential phosphorylation sites of PKA [7]. All three peptides were tested for their ability to undergo phosphorylation in vitro by the catalytic subunit of the PKA. Two peptides, C561-575 and C681689 were phosophorylated efficiently (Fig. 4). However, the peptide C544-555 was not phosphorylated, although it contains the sequence Lys-Arg-Phe-Ser-Ser, possible consensus sequence of RRXS(T) or KRXX-
70 S(T) for potential phosphorylation by PKA [15]. It is not clear why this peptide is not phosphorylated. Phosphorylation of the peptide (561-575) was inhibited in a dose-dependent manner by the addition of the anti(C561-575) antibody, while that of peptide (C681-689) was not inhibited (Fig. 4). The results also suggest that recognition of anti-(C561-575) antibody is sequencespecific.
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Phosphorylation of the sodium channel P K A catalyzed the incorporation of 3.1-3.5 mol of phosphate into the a subunit of the purified sodium channel under the conditions we used. This value is similar to that reported previously [3,6]. The effect of the anti-(C561-575) antibody on phosphorylation was then tested. The sodium channel that was phosphorylated with [32p]ATP in the presence of various concentrations of the antibody or preimmune serum (as control) was separated by SDS-PAGE. As shown in Fig. 5A, phosphorylation of the sodium-channel protein (indicated by an arrow) decreased with increasing concentrations of anti-(C561-575) antibody (lanes 2-6). On the other hand no difference was observed with the addition of preimmune serum (lanes 7-9). The amounts of 32p-phosphorylated sodium channel were quantified by excising the sodium-channel band in the gel and counting its radioactivity. As shown in Fig. 5B, the
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IgG concentration (gM) Fig. 4. Phosphorylation of synthetic peptides and its inhibition by anti-(C561-575) antibody. Synthetic peptide (0.1 pmol), C544-555 ([]), C561-575 (e), and C681-689 (©), was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgCl2 and 30 /~M [y-32p]ATP (3.105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (Sigma) in a final volume of 50/~1. Various concentrations of anti-(C561-575) antibody (0.1-10 /~M) were also added in the series of experiments. The reaction was carried out as described in Experimental Methods. Phosphorylated peptides were trapped on a Whatman P81 phosphocellulose paper disk. Radioactivity on the phosphocellulose paper was counted by Cerenkov-counting. In the antibody-blocking experiments, 32p radioactivity incorporated into the antibody itself was separately counted by the parallel phosphorylation of antibody alone and then subtracted.
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phosphorylation was inhibited in a dose-dependent m a n n e r by the addition of anti-(C561-575) antibody. The inhibition reached 40%, namely 1.2-1.4 tool reduction of the incorporated phosphate at 1 0 / z M of the IgG. These results suggest that Ser-573 in the residues 561-575 is one of the phosphorylation sites of the a subunit, whose phosphorylation is blocked by anti(C561-575) antibody. Therefore, the antibody can be claimed to be sequence-directed or site-directed.
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Peptide concentration, M Fig. 3. Antigenic specificityof anti-(C561-575) antibody. Binding of the antibody to the purified sodium channel was measured in the presence of various concentrations of peptide C544-555 (×), C561575 (e), and C681-689 (•). Experimental conditions were similar to those in Fig. 1 except that the anti-(C561-575) antibody was preincubated with peptides for 1 h at room temperature and color developing was carried out for 30 rain.
Mapping the phosphorylated residues that are blocked by anti-(C561-575) antibody The sodium channel that was phosphorylated in the presence or absence of 5 ~ M I g G was digested with lysyl endoproteinase and further electrophoresed. As shown in Fig. 6A, four phosphorylated peptide fragments were observed in the sample that was phosphorylated without antibody (lane 1). Quantification of each band (3.5, 5, 7, and 8 kDa) by Cerenkov-counting gave incorporated 32p ratios of 0.23, 0.10, 0.55, and
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Fig. 5. Phosphorylation of the sodium channel and its inhibition by anti-(C561-575) antibody. Purified sodium channel (0.1 pmol of the STX binding site) was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgCI2 and 30 g.M [y-32P]ATP (3' 105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (PKA, Sigma) in a final volume of 50 /zl. Various concentrations of anti-(C561-575) antibody (0.1-10/~M) or preimmune serum were also added in some series of experiments. The reaction was initiated by the addition of [y-32P]ATP and performed at 30°C for 30 min. The reaction was stopped by addition of 10/~1 of 5% SDS/25 mM Tris-HCl (pH 6.8)/40% glycerol (w/v)/50 mM dithiothreitol/0.1% bromophenol blue. The samples were then subjected to SDS-PAGE. (A) An autoradiogram of phosphorylated sodium channels with 0/~M (lane 1), 0.1 mM (lane 2), 0.5 mM (lane 3), 1.5 mM (lane 4), 5 mM (lane 5), and 10 mM (lane 6) of anti-(C561-575) antibody, or with 0.1 mM (lane 7), 1.5 mM (lane 8), and 10 mM (lane 9) of preimmune serum. (B) Inhibition curves of the phosphorylation. Radioactivity of the phosphorylated sodium-channel bands was counted and plotted. Anti-(C561-575) antibody (e) and preimmune serum ((3) are shown.
0.12, respectively; or 0.71-0.81, 0.31-0.35, 1.7-1.9, and 0.37-0.42 mol of 32p was calculated as being incorporated. Experimentally observed molecular masses (3.5, 5, 7, and 8 kDa) of the phosphorylated fragments were inspected by the potential cleavage site by lysyl endoproteinase (lysine residues). They must be derived from cleavage at Lys-550 (N-terminus) and Lys-581 (Cterminus; calculated molecular mass, 3.6 kDa), Lys-683 (N-terminus) and Lys-724 (C-terminus; calculated mass, 4.8 kDa), Lys-585 (N-terminus) and Lys-643 (Cterminus; calculated mass, 7.0 kDa), and Lys-644 (Nterminus) and Lys-724 (C-terminus; calculated mass, 8.3 kDa), respectively (Fig. 6B). In the presence of anti-(C561-575) antibody, however, the phosphorylated band of 3.5 kDa was not observed at all and that of 7 kDa decreased by 46%. On the other hand the phosphorylated amounts in the 5-kDa and 8-kDa fragments were retained (lane 2 in Fig. 6A). The resuits strongly suggest that four of the five potential
P K A phosphorylation sites can be phosphorylated, namely Ser-573, Ser-610, Ser-623, and either Ser-687 or Ser-688; but Ser-553 or Ser-554 is not. This is consistent with the observation that the peptide (C544-555) was not phosphorylated as described above. Anti-(C561-575) antibody completely blocked the phosphorylation at Ser-573 of the 3.5-kDa fragment, as expected for its site-directed characteristics. It is of note that the phosphorylation of the 7-kDa fragment was also blocked by anti-(C561-575) antibody by half. This fragment produced by lysyl endoproteinase digestion is located just after the 3.5-kDa fragment. Therefore, this block is likely to be due to the steric interference of the antibody against the P K A phosphorylation after binding between residues 561 and 575. As the present work clearly shows, the site-directed anti-peptide antibody can block two particular phosphorylation sites of the four in the rat-brain sodium channel. It will provide us with a useful tool to analyze
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Fig. 6. Peptide mapping of 32p-phosphorylated sites in the a subunit of the rat-brain sodium channel. (A) The 32p-phosphorylated sodium channel (0.1 mg) in the absence or presence of anti-(C561-575) antibody (IgG fraction, 5 ~M) was subjected to SDS-PAGE and the phosphorylated a subunit band was isolated. The sliced gel was digested with lysyl endoproteinase (Achromobacter proteinase I) ( S / E = 100, w/w) at 37°C for 24 h. The digest was subjected to SDS-PAGE on a 16.5% polyacrylamide gel. After electrophoresis, the gel was dried under vacuum and subjected to the autoradiography. Lanes 1 and 2 are the samples that were phosphorylated in the absence and presence of the antibody, respectively. (B) Primary structure in the region of the potential phosphorylation sites cluster in rat-brain II sodium-channel a subunit by Noda et al. [7]. Sequences against which antisera were raised are shown by lines and the potential cleavage sites by lysyl endoproteinase are indicated by arrows.
which site(s) of the phosphorylation is actually involved in the sodium-channel modulation, even when applied to crude preparations or cells. Acknowledgment This work was supported in Grant-in Aid for Scientific Research on Priority Area (No.01641501) from the Ministry of Education, Science and Culture of Japan. References 1 Levitan, I.B. (1985) J. Membr. Biol. 87, 177-190. 2 Rossie, S. and Catterall, W.A. (1987) in The Enzyme (Boyer, P.D. and Krebs, E.G., eds), Vol. 18, pp. 335-358, Academic Press, Orlando, FL. 3 Costa, M.R.C., Casanellie, J.E. and Catterall, W.A. (1982) J. Biol. Chem. 257, 7918-7921. 4 Rossie, S., Gordon, D. and Catterall, W.A. (1987) J. Biol. Chem. 262, 17530-17535. 5 Costa, M.R.C. and Catterall, W.A. (1984) J. Biol. Chem. 259, 8210-8218.
6 Rossie, S. and Catterall, W.A. (1987) 262, 12735-12744. 7 Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. and Numa, S. (1986) Nature 320, 189-192. 8 Merrifield, R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154 9 Hartshorn e, R.P. and Catterall, W.A. (1984) J. Biol.Chem. 259, 1667-1675. 10 Flass, D.B., Masaracchia, R.A., Feramisco, J.R. and Kemp, B.E. (1978) Anal. Biochem. 87, 565-575. 11 Towbin, H. and Gordon, J. (1984) J. Immunol. Methods 72, 313-340. 12 Catterall, W.A. (1988) Science 242, 50-61 13 Krebs, E.G. and Beavo, J.A. (1979) Annu. Rev. Biochem. 48, 923-959. 14 Voller, A., Bidwell, D.E. and Bartlett, A. (1976) Bull. World Health Org. 53, 35-46. 15 Blake, M.S., Johnston, H., Russel Jones, G.V. and Gotschlich, E.C. (1984) Anal. Biochem. 136, 175-179. 16 Van Regenmortel, M.H.V., Briand, J.P., Mullers, S. and Plaue, S. (1988) in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (Burdon, R.H. and von Knippenberg, P.H., eds.), Elsevier, Amsterdam. 17 Agnew, W.S., Levinson, S.R., Brabson, J.S. and Raftery, M.A. (1979) Proc. Natl. Acad. Sci. USA 75, 2606-2610.
67
BBAMCR 13285
A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase Hitoshi Nakayama, Hiroyoshi Shikano and Yuichi Kanaoka 1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan) (Received 22 April 1992) (Revised manuscript received 10 August 1992)
Key words: Sodium channel; Antipeptide antibody; Protein phosphorylation; Phosphorylation block; Phosphorylated peptide; (Rat " brain)
Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the a subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.
Introduction Phosphorylation is a major mechanism of ion-channel regulation. Although many ion channels are influenced by phosphorylation events, only a few have been biochemically identified and characterized [1,2]. The voltage-gated sodium channel from rat brain is phosphorylated by cAMP-dependent protein kinase (PKA) in purified preparations [3,4] and in situ [5,6]. cAMPdependent phosphorylation of sodium channels in synaptosomes is associated with a reduction in neurotoxin-activated ion flux [5], but the functional significance of phosphorylation under physiological conditions is not known. Noda et al. [7] have deduced the primary structure of rat-brain sodium-channel a sub-
Correspondence to: H. Nakayama, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan. i Present address: Toyama Women's College, Gankaiji 444, Toyama 930-01, Japan. Abbreviations: PKA, cyclic-AMP(cAMP)-dependent protein kinase; ELISA, enzyme-linked immunosorbent assay; STX, saxitoxin; WGA, wheat germ agglutinin; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiotbreitol.
units and proposed five potential phosphorylation sites for PKA. This makes it possible to raise anti-peptide antibodies [16] for the particular regions that may contribute to express the channel function. Raising antipeptide antibodies against the phosphorylation sites may provide useful tools, if they specifically recognize the phosphorylation sites or nearest ones to inhibit the phosphorylation. In this paper, we describe three synthetic peptides that are employed to raise antibodies, characteristics of the elicited antibodies, and analysis of the PKA-catalyzed phosphorylation of rat-brain sodium channels using one of the antibodies.
Experimental Methods
Peptide synthesis and antibody generation. Synthetic peptides corresponding to particular regions of the sodium channel II sequence from rat brain [7] were synthesized by the solid-phase method [8]. Polyclonal antisera were raised in rabbits against peptides corresponding to the following amino-acid residues of the channel sequence: C544-555, 544-555 plus N-terminal cysteine, C561-575, 561-575 plus N-terminal cysteine,
68 and C681-689, 681-689 plus N-terminal cysteine. Peptides were purified by reverse-phase HPLC and their structures were confirmed by amino-acid sequence analysis and amino-acid analysis. For immunization, peptides were coupled to porcine thyroglobulin using N-hydroxysuccinimidyl m-maleimidobenzoate to couple through N-terminal cysteine residues.
Preparation of the purified sodium channel from rat brains. Rat brain lysed P3 was solubilized and purified by successive chromatography of DEAE-Sephadex, hydroxyappatite, and WGA-Sepharose 4B as described in the literature [9]. Specific activity obtained was 680-750 pmol [3H]saxitoxin binding sites/mg protein. Solubilized eel-electroplax sodium channel was partially purified by DEAE-Sephadex as described [17]. Solid-phase ELISA. The binding of the antisera to the synthetic peptides and to the sodium channel was analyzed by indirect solid-phase ELISA [14]. The plates were coated with 50/xl of either the peptide (1/xg/ml) or with the purified sodium channel (2 /zg/ml) in 50 mM sodium carbonate buffer (pH 9.6). Alkaline phosphatase-conjugated goat anti-rabbit IgG was used for the second antibody followed by colorimetry with pnitrophenyl phosphate as substrate. Phosphorylation of the synthetic peptides. Synthetic peptides (0.1 pmol) were incubated in 25 mM HepesTris (pH 7.4), 2 mM MgCI 2 and 30 /xM [y-32p]ATP (3.105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (Sigma) in a final volume of 50 ~1. Various concentrations of anti-(C561-575) antibody (0.1-10 ~M) were added in some series of experiments. The
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Proteolysis of purified 32p-phosphorylated sodium channels. The 32p-phosphorylated sodium channel was subjected to the SDS-PAGE and isolated the phosphorylated a subunit band. The sliced gel was briefly
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reaction was initiated by the addition of [y-32P]ATP and performed at 30°C for 30 min. The reaction was terminated by addition of 0.15 M phosphoric acid (50 ~l). 40 ~I of each sample were loaded on a Whatman P81 phosphoceIlulose paper disk (duplicate) followed by three 2-min washes with 75 mM phosphoric acid (10 /zl) in Petri dishes as described [10]. Radioactivity on the phosphocellulose paper was counted by Cerenkovcounting. In the antibody-blocking experiments, antibody was also phosphorylated and counted by this assay. We carried out the parallel phosphorylation of antibody alone as control and determined the net amount of peptide phosphorylation by subtracting the phosphorylated amount of antibody. Phosphorylation of the sodium channel. Purified sodium channel (0.1 pmol of the STX binding site) was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgC12 and 30/xM [y-aEp]ATP (3" 105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (PKA, Sigma) in a final volume of 50 /xl. Various concentrations of anti-(C561-575) antibody (0.1-10 /~M) were added in some series of experiments. The reaction was initiated by the addition of [T-32p]ATP and performed at 30°C for 30 min. The reaction was stopped by addition of 10 /.d of 5% SDS/25 mM Tris-HCl (pH 6.8)/40% glycerol ( w / v ) / 50 mM dithiothreitol/0.1% bromophenol blue. The samples were then subjected to the SDS-PAGE.
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Antiserum dilution fold Fig. 1. Binding of anti-peptide antibodies to the corresponding peptides and the purified sodium channel. The plates were coated with 50 ~1 of either the peptide (o, 1 p.g/ml) or the purified sodium channel (e, 2 /.Lg/ml) in 50 mM sodium carbonate buffer (pH 9.6). Alkaline phosphatase-conjugated goat anti-rabbit IgG was used for the second antibody and color development for 10 rain after addition of p-nitrophenyl phosphate was determined by measuring absorbance at 405 nm. (A) Anti-(C544-555) antibody, (B) anti-(C561-575) antibody, and (C) anti-(C681-689) antibody. For control the binding of preimmune serum ([]) is shown.
69 incubated in 50 mM Tris-HCl (pH 9.0), rinsed with the same buffer, and digested with lysyl endoproteinase (Achromobacter proteinase I) (S/E--100, w/w) at 37°C for 24 h. Electrophoresis. SDS-PAGE was carried out using the 4-12% gradient gel of polyacrylamide for the sodium channel [9] or 16.5% polyacrylamide gel for the proteinase-digested samples. After electrophoresis, the gel was dried under vacuum and subjected to the autoradiography. Corresponding radioactive bands were cut out and the radioactivity was determined by Cerenkov-counting. Immunoblot. Electrophoresed polypeptide bands on polyacrylamide gel were electrotransferred [11] onto Immobilon P paper (Millipore) and performed the immunoblot analysis [15]. Alkaline phosphatase-conjugated goat anti rabbit IgG (Capped was used as second antibody and visualized with /3-naphthylphosphate and Fast Blue B.
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Results and Discussion
Specificity of the anti-peptide antibodies In order to elicit antibodies, the synthetic peptides were conjugated to thyroglobulin and injected into rabbits. Although all three anti-peptide antibodies bound to their respective peptides to similar extents in solid-phase ELISA, they differed in their capacity to cross-react with the sodium channel (Fig. 1). Only the anti-peptide (C561-575) antibody can bind to the sodium channel. It probably reflects the exposure a n d / o r conformation of these sequences within the sodium-channel molecule, although five potential phosphorylation sites are considered to be a cluster in the cytoplasmic side [12]. The specificity of the anti-(C561-575) antibody was assessed by immunoblot and the competitive ELISA method. Although the anti-peptide (C561-575) antibody reacted with the sodium channel of 300 kDa as a major component in lysed synaptosome fractions (P3) of rat brains under nonreducing conditions, it also stained 270 kDa (possibly a subunit, see below) and possible aggregates of high molecular weight containing the a subunit (Fig. 2, lane 1). However, it only recognized the a subunit (270 kDa) of the sodium channel which involves the sequence (561-575) and is free from lower-molecular-weight /3 subunits [12] under reducing conditions (lane 2). Preimmune serum did not react with this polypeptide (lane 4). The antipeptide antibody did not recognize the sodium channel from electroplax of electric eels (lane 3), as expected by the fact that the sequences of five potential phosphorylation sites in the rat-brain channel are missing in the electroplax channel. The results suggest that the antibody recognizes the rat-brain sodium channel rather in a sequence-specific fashion. Other evidence of anti-
1
2
3
4
Fig. 2. Immunoblot of anti-(C561-575) antibody. SDS-PAGE of lysed P3 fractionsand partiallypurified eel electroplaxsodiumchannel was carried out usingthe 4-12% gradientgel of polyacrylamide. Electrophoresed polypeptidebands were electrotransferredonto Immobilon P paper (Millipore) and the immunoblotanalysiswas performed as described in Experimental Methods. Rat-brain sodium channels not treated with DTT (lane 1); rat-brain sodium channels treated with DTT (lane 2); electroplaxsodiumchannelwithoutDTT (lane 3); DTF-treated rat-brain sodiumchannelimmunoblottedwith preimmuneserum(lane 4). body specificity was obtained by the ELISA assay in the presence of antigen peptides. As shown in Fig. 3, the immunoreaction of the antibody with the purified sodium channel was inhibited by the peptide (C561-575) in a dose-response manner. Two other peptides of residues C544-555 and C681-689 did not inhibit the immunoreaction. Therefore, the anti-peptide (C561-575) antibody is concluded to recognize the a subunit of the rat-brain sodium channel in a sequence-specific manner.
Peptide phosphorylation Three synthetic peptides corresponding to residues 544-555, 561-575, and 681-689 (see sequences in Fig. 6B) of the a subunit of rat-brain sodium channel II include the potential phosphorylation sites of PKA [7]. All three peptides were tested for their ability to undergo phosphorylation in vitro by the catalytic subunit of the PKA. Two peptides, C561-575 and C681689 were phosophorylated efficiently (Fig. 4). However, the peptide C544-555 was not phosphorylated, although it contains the sequence Lys-Arg-Phe-Ser-Ser, possible consensus sequence of RRXS(T) or KRXX-
70 S(T) for potential phosphorylation by PKA [15]. It is not clear why this peptide is not phosphorylated. Phosphorylation of the peptide (561-575) was inhibited in a dose-dependent manner by the addition of the anti(C561-575) antibody, while that of peptide (C681-689) was not inhibited (Fig. 4). The results also suggest that recognition of anti-(C561-575) antibody is sequencespecific.
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Phosphorylation of the sodium channel P K A catalyzed the incorporation of 3.1-3.5 mol of phosphate into the a subunit of the purified sodium channel under the conditions we used. This value is similar to that reported previously [3,6]. The effect of the anti-(C561-575) antibody on phosphorylation was then tested. The sodium channel that was phosphorylated with [32p]ATP in the presence of various concentrations of the antibody or preimmune serum (as control) was separated by SDS-PAGE. As shown in Fig. 5A, phosphorylation of the sodium-channel protein (indicated by an arrow) decreased with increasing concentrations of anti-(C561-575) antibody (lanes 2-6). On the other hand no difference was observed with the addition of preimmune serum (lanes 7-9). The amounts of 32p-phosphorylated sodium channel were quantified by excising the sodium-channel band in the gel and counting its radioactivity. As shown in Fig. 5B, the
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IgG concentration (gM) Fig. 4. Phosphorylation of synthetic peptides and its inhibition by anti-(C561-575) antibody. Synthetic peptide (0.1 pmol), C544-555 ([]), C561-575 (e), and C681-689 (©), was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgCl2 and 30 /~M [y-32p]ATP (3.105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (Sigma) in a final volume of 50/~1. Various concentrations of anti-(C561-575) antibody (0.1-10 /~M) were also added in the series of experiments. The reaction was carried out as described in Experimental Methods. Phosphorylated peptides were trapped on a Whatman P81 phosphocellulose paper disk. Radioactivity on the phosphocellulose paper was counted by Cerenkov-counting. In the antibody-blocking experiments, 32p radioactivity incorporated into the antibody itself was separately counted by the parallel phosphorylation of antibody alone and then subtracted.
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phosphorylation was inhibited in a dose-dependent m a n n e r by the addition of anti-(C561-575) antibody. The inhibition reached 40%, namely 1.2-1.4 tool reduction of the incorporated phosphate at 1 0 / z M of the IgG. These results suggest that Ser-573 in the residues 561-575 is one of the phosphorylation sites of the a subunit, whose phosphorylation is blocked by anti(C561-575) antibody. Therefore, the antibody can be claimed to be sequence-directed or site-directed.
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Peptide concentration, M Fig. 3. Antigenic specificityof anti-(C561-575) antibody. Binding of the antibody to the purified sodium channel was measured in the presence of various concentrations of peptide C544-555 (×), C561575 (e), and C681-689 (•). Experimental conditions were similar to those in Fig. 1 except that the anti-(C561-575) antibody was preincubated with peptides for 1 h at room temperature and color developing was carried out for 30 rain.
Mapping the phosphorylated residues that are blocked by anti-(C561-575) antibody The sodium channel that was phosphorylated in the presence or absence of 5 ~ M I g G was digested with lysyl endoproteinase and further electrophoresed. As shown in Fig. 6A, four phosphorylated peptide fragments were observed in the sample that was phosphorylated without antibody (lane 1). Quantification of each band (3.5, 5, 7, and 8 kDa) by Cerenkov-counting gave incorporated 32p ratios of 0.23, 0.10, 0.55, and
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Fig. 5. Phosphorylation of the sodium channel and its inhibition by anti-(C561-575) antibody. Purified sodium channel (0.1 pmol of the STX binding site) was incubated in 25 mM Hepes-Tris (pH 7.4), 2 mM MgCI2 and 30 g.M [y-32P]ATP (3' 105 dpm/pmol) with 17 U of purified catalytic subunit of bovine heart cAMP-dependent protein kinase (PKA, Sigma) in a final volume of 50 /zl. Various concentrations of anti-(C561-575) antibody (0.1-10/~M) or preimmune serum were also added in some series of experiments. The reaction was initiated by the addition of [y-32P]ATP and performed at 30°C for 30 min. The reaction was stopped by addition of 10/~1 of 5% SDS/25 mM Tris-HCl (pH 6.8)/40% glycerol (w/v)/50 mM dithiothreitol/0.1% bromophenol blue. The samples were then subjected to SDS-PAGE. (A) An autoradiogram of phosphorylated sodium channels with 0/~M (lane 1), 0.1 mM (lane 2), 0.5 mM (lane 3), 1.5 mM (lane 4), 5 mM (lane 5), and 10 mM (lane 6) of anti-(C561-575) antibody, or with 0.1 mM (lane 7), 1.5 mM (lane 8), and 10 mM (lane 9) of preimmune serum. (B) Inhibition curves of the phosphorylation. Radioactivity of the phosphorylated sodium-channel bands was counted and plotted. Anti-(C561-575) antibody (e) and preimmune serum ((3) are shown.
0.12, respectively; or 0.71-0.81, 0.31-0.35, 1.7-1.9, and 0.37-0.42 mol of 32p was calculated as being incorporated. Experimentally observed molecular masses (3.5, 5, 7, and 8 kDa) of the phosphorylated fragments were inspected by the potential cleavage site by lysyl endoproteinase (lysine residues). They must be derived from cleavage at Lys-550 (N-terminus) and Lys-581 (Cterminus; calculated molecular mass, 3.6 kDa), Lys-683 (N-terminus) and Lys-724 (C-terminus; calculated mass, 4.8 kDa), Lys-585 (N-terminus) and Lys-643 (Cterminus; calculated mass, 7.0 kDa), and Lys-644 (Nterminus) and Lys-724 (C-terminus; calculated mass, 8.3 kDa), respectively (Fig. 6B). In the presence of anti-(C561-575) antibody, however, the phosphorylated band of 3.5 kDa was not observed at all and that of 7 kDa decreased by 46%. On the other hand the phosphorylated amounts in the 5-kDa and 8-kDa fragments were retained (lane 2 in Fig. 6A). The resuits strongly suggest that four of the five potential
P K A phosphorylation sites can be phosphorylated, namely Ser-573, Ser-610, Ser-623, and either Ser-687 or Ser-688; but Ser-553 or Ser-554 is not. This is consistent with the observation that the peptide (C544-555) was not phosphorylated as described above. Anti-(C561-575) antibody completely blocked the phosphorylation at Ser-573 of the 3.5-kDa fragment, as expected for its site-directed characteristics. It is of note that the phosphorylation of the 7-kDa fragment was also blocked by anti-(C561-575) antibody by half. This fragment produced by lysyl endoproteinase digestion is located just after the 3.5-kDa fragment. Therefore, this block is likely to be due to the steric interference of the antibody against the P K A phosphorylation after binding between residues 561 and 575. As the present work clearly shows, the site-directed anti-peptide antibody can block two particular phosphorylation sites of the four in the rat-brain sodium channel. It will provide us with a useful tool to analyze
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Fig. 6. Peptide mapping of 32p-phosphorylated sites in the a subunit of the rat-brain sodium channel. (A) The 32p-phosphorylated sodium channel (0.1 mg) in the absence or presence of anti-(C561-575) antibody (IgG fraction, 5 ~M) was subjected to SDS-PAGE and the phosphorylated a subunit band was isolated. The sliced gel was digested with lysyl endoproteinase (Achromobacter proteinase I) ( S / E = 100, w/w) at 37°C for 24 h. The digest was subjected to SDS-PAGE on a 16.5% polyacrylamide gel. After electrophoresis, the gel was dried under vacuum and subjected to the autoradiography. Lanes 1 and 2 are the samples that were phosphorylated in the absence and presence of the antibody, respectively. (B) Primary structure in the region of the potential phosphorylation sites cluster in rat-brain II sodium-channel a subunit by Noda et al. [7]. Sequences against which antisera were raised are shown by lines and the potential cleavage sites by lysyl endoproteinase are indicated by arrows.
which site(s) of the phosphorylation is actually involved in the sodium-channel modulation, even when applied to crude preparations or cells. Acknowledgment This work was supported in Grant-in Aid for Scientific Research on Priority Area (No.01641501) from the Ministry of Education, Science and Culture of Japan. References 1 Levitan, I.B. (1985) J. Membr. Biol. 87, 177-190. 2 Rossie, S. and Catterall, W.A. (1987) in The Enzyme (Boyer, P.D. and Krebs, E.G., eds), Vol. 18, pp. 335-358, Academic Press, Orlando, FL. 3 Costa, M.R.C., Casanellie, J.E. and Catterall, W.A. (1982) J. Biol. Chem. 257, 7918-7921. 4 Rossie, S., Gordon, D. and Catterall, W.A. (1987) J. Biol. Chem. 262, 17530-17535. 5 Costa, M.R.C. and Catterall, W.A. (1984) J. Biol. Chem. 259, 8210-8218.
6 Rossie, S. and Catterall, W.A. (1987) 262, 12735-12744. 7 Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. and Numa, S. (1986) Nature 320, 189-192. 8 Merrifield, R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154 9 Hartshorn e, R.P. and Catterall, W.A. (1984) J. Biol.Chem. 259, 1667-1675. 10 Flass, D.B., Masaracchia, R.A., Feramisco, J.R. and Kemp, B.E. (1978) Anal. Biochem. 87, 565-575. 11 Towbin, H. and Gordon, J. (1984) J. Immunol. Methods 72, 313-340. 12 Catterall, W.A. (1988) Science 242, 50-61 13 Krebs, E.G. and Beavo, J.A. (1979) Annu. Rev. Biochem. 48, 923-959. 14 Voller, A., Bidwell, D.E. and Bartlett, A. (1976) Bull. World Health Org. 53, 35-46. 15 Blake, M.S., Johnston, H., Russel Jones, G.V. and Gotschlich, E.C. (1984) Anal. Biochem. 136, 175-179. 16 Van Regenmortel, M.H.V., Briand, J.P., Mullers, S. and Plaue, S. (1988) in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (Burdon, R.H. and von Knippenberg, P.H., eds.), Elsevier, Amsterdam. 17 Agnew, W.S., Levinson, S.R., Brabson, J.S. and Raftery, M.A. (1979) Proc. Natl. Acad. Sci. USA 75, 2606-2610.