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Sf9 Insect Cell System
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
219, 638–643 (1996)
0286
Neuronal Nitric Oxide Synthase Specific Autophosphorylation in Baculovirus/Sf9 Insect Cell System Yasuo Watanabe, Keita Terashima, and Hiroyoshi Hidaka1 Department of Pharmacology, Nagoya University School of Medicine, showa-ku, Nagoya 466, Japan Received December 7, 1995 Neuronal Nitric Oxide Synthase (nNOS) is a calmodulin (CaM)-dependent enzyme, which generates the ubiquitous biological mediator nitric oxide. In this study, wild-type or mutant form of rat nNOS was overexpressed in a baculovirus/Sf9 insect cell system and examined for autophosphorylation. Incubation of purified wild-type nNOS in the presence of Mg2+ and ATP result in phosphorylation of nNOS. Deletional mutant of the nNOS amino terminus which contains G-Q-G-A-G-S sequence, however, shows loss of phosphorylation. Furthermore, purified wild-type rat liver cytokine-induced NOS (iNOS), which does not contain G-Q-G-A-G-S sequence, shows no phosphorylation activity. We also found that the ratio of wild-type nNOS phosphorylation was independent of nNOS concentration, consistent with an intramolecular reaction. These data suggest that nNOS-specific autophosphorylation pathway might be involved in the regulation of enzyme activity. © 1996 Academic Press, Inc.
The free radical nitric Oxide (NO) is synthesized from L-Arginine by a member of NO synthase (NOSs) in the presence of various cofactors as well as calmodulin (CaM). Three distinct NOS cDNAs have been confirmed by cloning and characterization. Neuronal NOS (nNOS) (1, 2, 3, 4), which is found in the brain and peripheral nervous system and non-neuronal tissue as well (5), is involved in a non-adrenergic, non-cholinergic neurotransmitter regulation (6), long-term potentiation (7), and long-term depression (8). Cytokine-induced NOS (iNOS) (9, 10) is a Ca2+ insensitive protein containing CaM as a tightly bound subunit (11) and is an important mediator including antimicrobial and antitumor activities. Endothelial NOS (eNOS) (12, 13, 14), which is found predominantly in membrane (15), plays a key role in the regulation of vascular tone and platelet aggregation. Primary structures of each of the enzymes have a number of recognition sites required for enzyme function, containing two consensus binding sequences for NADPH, two for FAD, one for FMN, and one for CaM. In addition to the NH2-terminal catalytic domain (residues 504–608, numbering based on the rat brain nNOS) (16), nNOS contains the PKA phosphorylation consensus sequence (17), K-R-F-G-S (residues 369–374), and a nucleotide binding consensus sequence, G-Q-G-A-G-S (residues 166–171) (18). Although it has been confirmed that not only PKA but also PKC and CaMK can indeed phosphorylate NOS, the physiological significance of these phosphorylation remains unclear (19, 20). To best of our knowledge, there is no study related to phosphorylation activity of NOS itself. In the present study, we found autophosphorylation activity in nNOS but not in inducible NOS (iNOS) using a baculovirus/Sf9 insect cell system. Because the sequence G-Q-G-A-G-S is not present in that of iNOS and endothelial NOS (eNOS), this sequence might have an important role in the regulation of nNOS phosphorylation. To further investigate this hypothesis, we created a deletional mutant of nNOS (designated DnNOS) by PCR-based mutagenesis in which a nucleotide binding consensus sequence, G-Q-G-A-G-S, was removed. MATERIALS AND METHODS Materials. The cDNA rat brain nNOS, rat liver iNOS, and PKA were generous gifts of Dr. Solomon H. Snyder (The Johns 1
To whom correspondence should be addressed. Abbreviations: CaM, calmodulin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PKA, protein kinase A; PKC, protein kinase C; CaMK, calcium/calmodulin dependent protein kinase; PCR, polymerase chain reaction; MOI, multiplicity of infection; PVDF, polyvinylidene; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 638 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Hopkins University School of Medicine, Baltimore, MA), Dr. Hiroyasu Esumi (National Cancer Center Research Institute, Tokyo, Japan), and Dr. Masatoshi Hagiwara (Nagoya University School of Medicine, Nagoya, Japan). [g-32P]ATP (6,000 Ci/mmol) was purchased from Dupont-New England Nuclear. L-[3H] Arginine and ECL detection kit were purchased from Amersham Corp. Restriction enzymes and DNA-modifying enzymes were from Takara Shuzo. Grace’s insect medium was from In Vitrogen. Fetal bovine serum was purchased from Irvine Scientific (Santa Ana, CA). 29,59-ADP-Sepharose was obtained from Pharmacia Biotec Inc. Electrophoresis reagents, and Bradford protein dye reagent were products of Bio-Rad. Anti-nNOS and iNOS antibodies were from Transduction Laboratories (Lexington, KY). Staphylococcus aureus V-8 protease was from Sigma. All other materials and reagents were of the highest quality available from commercial suppliers. cDNA construction and mutagenesis. The cDNA encoding the rat brain nNOS and the rat liver iNOS were introduced into the EcoRI-and BamHI, HindIII-digested pVL1393 transfer vector resulting in pVLnNOS and pVLiNOS. In the DnNOS mutant, the NH2-terminal of CaM-binding domain (residues 725–745) was deleted by PCR-based mutagenesis. PCR clone was isolated, and cDNA sequences were confirmed by dideoxynucleotide sequencing (Sequenase version 2.0, U.S. Biochemical Corp.) Cell culture and selection of recombinant baculovirus. Sf9 cells were cotransfected with linear baculovirus DNA and pVLnNOS, pVLDnNOS, or pVLiNOS. Recombinant baculoviruses were identified by screening occlusion-negative plaques, and the expressed nNOS, DnNOS, or iNOS were detected by Western blots of cell homogenates with anti-nNOS or anti-iNOS antibody. Second-passage recombinant baculovirus was obtained and titered by end-point dilution. Large scale protein expression of nNOS, DnNOS, or iNOS was done in 100–150 ml Corning spinner flasks at an initial cell density of 2 × 106 cells/ml and MOI of 3. Purification of expressed nNOS, DnNOS, and iNOS. Purification procedures were performed as described previously (21), except no hemin chloride was added to the Sf9 cell culture medium. Protein concentration was determined by the method of Bradford (22) using BSA as the standard. In vitro phosphorylation of NOS. NOS (1 mM), purified by 29,59-ADP affinity chromatography, was incubated at 30°C for 30 min in 40 mM Hepes (pH 7.0), 10 mM Mg2+, 1 mM DTT, 1mM EGTA, and 100 mM [g-32P]ATP. Reactions were stopped by the addition of loading buffer followed by SDS-PAGE. Gels were stained with Coomassie Blue and subjected to autoradiography. Quantitation of 32P incorporation into NOS and the specific activity of autophosphorylation or PKAinduced phosphorylation were achieved by TCA precipitation method (23) after the same incubation because only the band of NOS was phosphorylated on SDS-PAGE (Fig. 1). Gel electrophoresis and Western blot analysis. One-dimensional SDS-PAGE was carried out according to the method of Laemmli (24). The electrophoretic transfer of proteins from the SDS-PAGE to the PVDF membrane was performed as described by Towbin et al (25). For immunodetection of the transferred proteins, the procedure of Burnette (26) was used except that the second antibody was linked to horseradish peroxidase. Antigen-antibody complexes were visualized by reacting the bound peroxidase with the chemiluminescence reagent (Du Pont). Peptide mapping. Proteolytic digestion of NOS by Staphylococcus aureus V-8 protease was performed as described previously (27). Phosphopeptides were separated by 15% SDS-PAGE, followed by autoradiography.
RESULT AND DISCUSSION Expression and purification of wild-type and mutant NOS. The wild-type and deletional mutants were expressed using the baculovirus/Sf9 insect cell system and purified on 29,59-ADP9-Sepharose as described under “Materials and methods”. nNOS and DnNOS were purified to >95% homogeneity on SDS-PAGE (Fig. 1A) and Western analyses (Fig. 1B). However, iNOS was partially purified to <20% apparently on SDS-PAGE (Fig. 1A). The specific activity and Km values for L-Arginine of wild-type nNOS at 30°C for 3–20 min were 4–7 nmol of L-Citruline produced/mg/ min and 8–10 mM, respectively, as determined by L-Arginine to L-Citruline conversion (data not shown). The specific activity was 3-fold lower than that reported previously using a baculovirus system (21). Expression of heme-containing enzyme in the baculovirus system often requires hemin supplementation of the culture medium for expression of protein with high specific activity. In the present study, no addition of hemin chloride to the Sf9 cell culture medium might result in the lower specific activity. The specific activity of wild-type iNOS was almost the same as that of wild-type nNOS (data not shown). Since DnNOS does not contain the NH2-terminal catalytic domain of nNOS, it shows no formation of L-Citruline from L-Arginine. However, DnNOS contains a high homology sequence to that of cytochrome P-450 reductase (2) which catalyzes the oxygenation of Nw-hydroxy-L-Arginine (NOHA) with formation of L-Citruline. Indeed, wild-type nNOS activity was maximally activated by a 1:1 molar ratio of DnNOS to wild-type nNOS with 1.2-fold activation (data not shown). The small activation by DnNOS would suggest that the 639
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FIG. 1. SDS–PAGE, Western blot analyses, and autophosphorylation of purified wild-type and mutant NOS. Lane 1, wild-type nNOS; lane 2, mutant nNOS; lane 3, wild-type iNOS. Proteins (2 mg: lane 1 and 2; 10 mg: lane 3) were separated by 10% SDS–PAGE and stained by Coomassie Blue (panel A) or transferred to PVDF membrane and immunoblotted with anti-nNOS or iNOS antibody (panel B). For the analysis of phospho-proteins, proteins were incubated at 30°C for 30 min in 40 mM Hepes (pH 7.0), 10 mM Mg2+, 1 mM DTT, 1 mM EGTA, and 100 mM [g− 32P]ATP, and were subjected to SDS–PAGE, followed by autoradiography (panel C).
formation of NOHA from L-Arginine would be dominant in the reaction in that L-Arginine was catalyzed by nNOS. Phosphorylation of wild-type nNOS. We incubated purified wild-type nNOS, DnNOS, and wild-type iNOS in the presence of Mg2+/[g-32P]ATP and after SDS-PAGE, monitored radioactivity by autoradiography. Wild-type nNOS showed autophosphorylation, whereas DnNOS and wild-type iNOS did very little phosphorylation compared to that of wild-type nNOS (Fig. 1C). nNOS has a consensus sequence (K-R-R-G-S) for phosphorylation by PKA. Wild-type nNOS showed very poor autophosphorylation compared to PKA-induced phosphorylation (Fig. 2A). In the presence and absence of PKA, there were very slow phosphorylations with a molar stoichiometry of approximately 0.15 and 0.6 at 1.5 h, respectively (Fig. 2B). In terms of slow phosphorylation nNOS by PKA was similar to that reported previously (28). Autophosphorylation was not increased in the presence of Ca2+/CaM (data not shown). Both phosphorylations of nNOS did not appear to alter its mobility on SDS-PAGE. We observed that the rate of nNOS autophosphorylation was independent of its concentration (Fig. 3), indicating that this phosphorylation was intramolecular. To further test the role of nNOS on autophosphorylation regulation, the effects of H-89, a specific inhibitor of PKA, and DnNOS on autophosphorylation of nNOS were studied (Fig. 4). H-89 inhibited PKA-
FIG. 2. Phosphorylation of wild-type nNOS. Panel A, wild-type nNOS (1 mM) was incubated without (left lane) or with (right lane) PKA (140 nM) under the same condition as in Fig. 1. Panel B, quantitation of the radioactive 32P incorporation with wild-type nNOS in the indicated time. The inset shows that stoichiometrical analysis of PKA-induced phosphorylation of wild-type nNOS in that autophosphorylation-induced 32P association was omitted. All assays were performed in duplicate, and each point represents the mean ± S.D. from two experiments. 640
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FIG. 3. Concentration dependent of nNOS autophosphorylation. Wild-type nNOS at the indicated concentration was subjected to autophophorylation at 30°C for 20 min under the same condition in Fig. 1. The incorporated 32P (pmol) and its specific activity (mmol/min/mg protein) were determined as described under “Materials and methods”. All assays were performed in duplicate, and each point represents the mean ± S.D. from two experiments.
induced phosphorylation of nNOS completely with a concentration of 1 mM, but did not inhibit autophosphorylation of nNOS, indicating that the autophosphorylation pathway is different from that of cyclic-AMP. The deletional mutant, DnNOS, did not exhibit autophosphorylation itself nor activate the autophosphorylation activity of nNOS. These results exclude that nNOS phosphorylation was due to that by a contaminating Sf9 cell kinase since there would be same kinases in both nNOS and DnNOS fractions. Peptide mapping after limited proteolysis of phosphorylated nNOS. To assess sites of phosphorylation, we cut the phosphorylation bands from SDS-PAGE gels, digested them with Staphylococcus aureus V-8 protease, and peptide mapping analyses were performed (Fig. 5). Limited proteolysis of nNOS yielded two fragments for both autophosphorylation and PKA-induced phosphorylation. These fragments showed difference in their apparent molecular weights in SDS-PAGE indicating that phosphorylation sites of nNOS might be different between auto-and PKA-induced phosphorylation.
FIG. 4. Effects of deletional mutant (DnNOS) and H-89 on the autophosphorylation of nNOS. Wild-type nNOS (1 mM) was subjected to autophosphorylation at 30°C for 20 min in the same condition as described in Fig. 2 with indicated factors. The % changes in phosphorylation of nNOS were determined by TCA precipitation method (23). DnNOS (1 mM) itself has no autophosphorylation activity and no effect on autophosphorylation of wild-type nNOS. PKA (140 nM) increase the 32P incorporation into wild-type nNOS and the effect of PKA was blocked by 1 mM H-89. Data are means of duplicate determinations from two experiments. 641
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FIG. 5. Peptide mapping of the autophosphorylated (lane 1) or PKA-induced phosphorylated (lane 2) wild-type nNOS. The bands of wild-type nNOS (5 mg) were excised from SDS–PAGE gel and subjected to peptide mapping analysis, using 10 mg of Staphylococcus aureus V-8 protease. Phosphopeptides were separated by 15% SDS–PAGE, followed by autoradiography.
Conclusions. This study shows that autophosphorylation of nNOS is observed in which there is a nucleotide binding consensus sequence G-Q-G-A-G-S (residues 166–171) but not substratebinding or enzyme catalytic consensus, R-D-L-Xn-D-F-G-Xn-G-T-P-X3-A/S-P-E. Although some studies have shown that NOS was phosphorylated by PKA, PKC, and CaMK in vitro and in situ (19, 20), to best of our knowledge there is no study related to phosphorylation activity of NOS itself. Our data demonstrate that iNOS, partially purified from the baculovirus/Sf9 insect cell system (the expression level of iNOS was somehow very low compared to that of nNOS in our system), did not undergo autophosphorylation in SDS-PAGE. These results suggest that the nucleotide binding consensus sequence G-Q-G-A-G-S (residues 166–171 might be important for regulation of autophosphorylation of nNOS because iNOS and eNOS do not contain this sequence. The deletional mutant (DnNOS), in which G-Q-G-A-G-S sequence was removed, did not exhibit autophosphorylation itself (See Results and discussions), emphasizing this hypothesis. We did not employ additional steps to further purify iNOS simply to avoid loss of enzyme activity resulting from such steps. Because this autophosphorylation reaction itself was very small, it is not likely to be a physiological phenomenon, and we therefore made no attempt to analyze it in more detail, including the exact site of the autophosphorylation. However nNOS purified from baculovirus/Sf9 insect cell system is post-translationally modified, indicating that nNOS might have been modified by a given amount of phosphorylation before the purification step. The autophosphorylation observed in our study was probably not due to some contaminating Sf9 cell kinase since 1) it was intramolecular (Fig. 3) and 2) addition of DnNOS did not increase the phosphorylation of nNOS (Fig. 4). The physiological significance of the nNOS phosphorylation is still unclear, and the autophosphorylation of nNOS might be sort of basal phosphorylation in a cell because of its Ca2+/CaM, cAMP independence. The specific autophosphorylation of nNOS could have some significance in the regulation of the neuronal system linked to the phosphorylation by PKA, PKC, and CaMK even though no change in nNOS activity was observed upon its autophosphorylation in vitro. REFERENCES 1. 2. 3. 4.
Bredt, D. S., and Snyder, S. H., (1990) Proc. Natl. Acad. Sci. USA 87, 682–685. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder, S. H., (1991) Nature 351, 714–718. Mayer, B., John, M., and Bohme, E., (1990) FEBS Lett. 277, 215–219. Schmidt, H. H., Pollock, J. S., Nakane, M., Gorsky, L. D., Forstermann, U., and Murad, F., (1991) Proc. Natl. Acad. Sci. USA 88, 365–369. 642
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Nakane, M., Schmidt, H. H., Pollock, J. S., Forstermann, U., and Murad, F., (1993) FEBS Lett 316, 175–180. Gillespie, J. S., Liu, X. R., and Martin, W., (1989) Br. J. Pharmacol. 98, 1080–1082. Schuman, E. M., and Madison, D. V., (1991) Science 254, 1503–1506. Shibuki, K., and Okada, D., (1991) Nature 349, 326–328. Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M. F., and Nathan, C. F., (1991) Proc. Natl. Acad. Sci. USA 88, 7773–7777. Xie, Q. W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C., (1992) Science 256, 225–228. Cho, H. J., Xie, Q. W., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., and Nathan, C., (1992) J. Exp. Med. 176, 599–604. Pollock, J. S., Forstermann, U., Mitchell, J. A., Warner, T. D., Schmidt, H. H., Nakane, M., and Murad, F., (1991) Proc. Natl. Acad. Sci. USA 88, 10480–10484. Janssens, S. P., Shimouchi, A., Quertermous, T., Bloch, D. B., and Bloch, K. D., (1992) J. Biol. Chem. 267, 14519– 14522. Marsden, P. A., Schappert, K. T., Chen, H. S., Flowers, M., Sundell, C. L., Wilcox, J. N., Lamas, S., and Michel, T., (1992) FEBS Lett 307, 287–293. Forstermann, U., Pollock, J. S., Schmidt, H. H., Heller, M., and Murad, F., (1991) Proc. Natl. Acad. Sci. USA 88, 1788–1792. Ogura, T., Yokoyama, T., Fujisawa, H., Kurashima, Y., and Esumi, H., (1993) Biochem. Biophys. Res. Commun. 193, 1014–1022. Kemp, B. E., and Pearson, R. B., (1990) Trends Biochem. Sci. 15, 342–346. Hanks, S. K., Quinn, A. M., and Hunter, T., (1988) Science 241, 42–52. Brune, B., and Lapetina, E. G., (1991) Biochem. Biophys. Res. Commun. 181, 921–926. Nakane, M., Mitchell, J., Forstermann, U., and Murad, F., (1991) Biochem. Biophys. Res. Commun. 180, 1396–1402. Nakane, M., Pollock, J. S., Klinghofer, V., Basha, F., Marsden, P. A., Hokari, A., Ogura, T., Esumi, H., and Carter, G. W., (1995) Biochem. Biophys. Res. Commun. 206, 511–517. Bradford, M. M., (1976) Anal. Biochem. 72, 248–254. Wang, Y., and Roach, P. J., (1993) Protein Phosphorylation (Hardie, D. G., Ed.), pp. 121–144, Oxford Univ. Press, New York. Laemmli, U. K., (1970) Nature 227, 680–684. Towbin, H., Staehelin, T., and Gordon, J., (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. Burnette, W. N., (1981) Anal. Biochem. 112, 195–203. Huttner, W. B, DeGennaro, L. J, and Greengard, P., (1981) J. Biol. Chem. 256, 1482–1488. Bredt, D. S., Ferris, C. D., and Snyder, S. H., (1992) J. Biol. Chem. 267, 10976–10981.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
219, 638–643 (1996)
0286
Neuronal Nitric Oxide Synthase Specific Autophosphorylation in Baculovirus/Sf9 Insect Cell System Yasuo Watanabe, Keita Terashima, and Hiroyoshi Hidaka1 Department of Pharmacology, Nagoya University School of Medicine, showa-ku, Nagoya 466, Japan Received December 7, 1995 Neuronal Nitric Oxide Synthase (nNOS) is a calmodulin (CaM)-dependent enzyme, which generates the ubiquitous biological mediator nitric oxide. In this study, wild-type or mutant form of rat nNOS was overexpressed in a baculovirus/Sf9 insect cell system and examined for autophosphorylation. Incubation of purified wild-type nNOS in the presence of Mg2+ and ATP result in phosphorylation of nNOS. Deletional mutant of the nNOS amino terminus which contains G-Q-G-A-G-S sequence, however, shows loss of phosphorylation. Furthermore, purified wild-type rat liver cytokine-induced NOS (iNOS), which does not contain G-Q-G-A-G-S sequence, shows no phosphorylation activity. We also found that the ratio of wild-type nNOS phosphorylation was independent of nNOS concentration, consistent with an intramolecular reaction. These data suggest that nNOS-specific autophosphorylation pathway might be involved in the regulation of enzyme activity. © 1996 Academic Press, Inc.
The free radical nitric Oxide (NO) is synthesized from L-Arginine by a member of NO synthase (NOSs) in the presence of various cofactors as well as calmodulin (CaM). Three distinct NOS cDNAs have been confirmed by cloning and characterization. Neuronal NOS (nNOS) (1, 2, 3, 4), which is found in the brain and peripheral nervous system and non-neuronal tissue as well (5), is involved in a non-adrenergic, non-cholinergic neurotransmitter regulation (6), long-term potentiation (7), and long-term depression (8). Cytokine-induced NOS (iNOS) (9, 10) is a Ca2+ insensitive protein containing CaM as a tightly bound subunit (11) and is an important mediator including antimicrobial and antitumor activities. Endothelial NOS (eNOS) (12, 13, 14), which is found predominantly in membrane (15), plays a key role in the regulation of vascular tone and platelet aggregation. Primary structures of each of the enzymes have a number of recognition sites required for enzyme function, containing two consensus binding sequences for NADPH, two for FAD, one for FMN, and one for CaM. In addition to the NH2-terminal catalytic domain (residues 504–608, numbering based on the rat brain nNOS) (16), nNOS contains the PKA phosphorylation consensus sequence (17), K-R-F-G-S (residues 369–374), and a nucleotide binding consensus sequence, G-Q-G-A-G-S (residues 166–171) (18). Although it has been confirmed that not only PKA but also PKC and CaMK can indeed phosphorylate NOS, the physiological significance of these phosphorylation remains unclear (19, 20). To best of our knowledge, there is no study related to phosphorylation activity of NOS itself. In the present study, we found autophosphorylation activity in nNOS but not in inducible NOS (iNOS) using a baculovirus/Sf9 insect cell system. Because the sequence G-Q-G-A-G-S is not present in that of iNOS and endothelial NOS (eNOS), this sequence might have an important role in the regulation of nNOS phosphorylation. To further investigate this hypothesis, we created a deletional mutant of nNOS (designated DnNOS) by PCR-based mutagenesis in which a nucleotide binding consensus sequence, G-Q-G-A-G-S, was removed. MATERIALS AND METHODS Materials. The cDNA rat brain nNOS, rat liver iNOS, and PKA were generous gifts of Dr. Solomon H. Snyder (The Johns 1
To whom correspondence should be addressed. Abbreviations: CaM, calmodulin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PKA, protein kinase A; PKC, protein kinase C; CaMK, calcium/calmodulin dependent protein kinase; PCR, polymerase chain reaction; MOI, multiplicity of infection; PVDF, polyvinylidene; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 638 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Hopkins University School of Medicine, Baltimore, MA), Dr. Hiroyasu Esumi (National Cancer Center Research Institute, Tokyo, Japan), and Dr. Masatoshi Hagiwara (Nagoya University School of Medicine, Nagoya, Japan). [g-32P]ATP (6,000 Ci/mmol) was purchased from Dupont-New England Nuclear. L-[3H] Arginine and ECL detection kit were purchased from Amersham Corp. Restriction enzymes and DNA-modifying enzymes were from Takara Shuzo. Grace’s insect medium was from In Vitrogen. Fetal bovine serum was purchased from Irvine Scientific (Santa Ana, CA). 29,59-ADP-Sepharose was obtained from Pharmacia Biotec Inc. Electrophoresis reagents, and Bradford protein dye reagent were products of Bio-Rad. Anti-nNOS and iNOS antibodies were from Transduction Laboratories (Lexington, KY). Staphylococcus aureus V-8 protease was from Sigma. All other materials and reagents were of the highest quality available from commercial suppliers. cDNA construction and mutagenesis. The cDNA encoding the rat brain nNOS and the rat liver iNOS were introduced into the EcoRI-and BamHI, HindIII-digested pVL1393 transfer vector resulting in pVLnNOS and pVLiNOS. In the DnNOS mutant, the NH2-terminal of CaM-binding domain (residues 725–745) was deleted by PCR-based mutagenesis. PCR clone was isolated, and cDNA sequences were confirmed by dideoxynucleotide sequencing (Sequenase version 2.0, U.S. Biochemical Corp.) Cell culture and selection of recombinant baculovirus. Sf9 cells were cotransfected with linear baculovirus DNA and pVLnNOS, pVLDnNOS, or pVLiNOS. Recombinant baculoviruses were identified by screening occlusion-negative plaques, and the expressed nNOS, DnNOS, or iNOS were detected by Western blots of cell homogenates with anti-nNOS or anti-iNOS antibody. Second-passage recombinant baculovirus was obtained and titered by end-point dilution. Large scale protein expression of nNOS, DnNOS, or iNOS was done in 100–150 ml Corning spinner flasks at an initial cell density of 2 × 106 cells/ml and MOI of 3. Purification of expressed nNOS, DnNOS, and iNOS. Purification procedures were performed as described previously (21), except no hemin chloride was added to the Sf9 cell culture medium. Protein concentration was determined by the method of Bradford (22) using BSA as the standard. In vitro phosphorylation of NOS. NOS (1 mM), purified by 29,59-ADP affinity chromatography, was incubated at 30°C for 30 min in 40 mM Hepes (pH 7.0), 10 mM Mg2+, 1 mM DTT, 1mM EGTA, and 100 mM [g-32P]ATP. Reactions were stopped by the addition of loading buffer followed by SDS-PAGE. Gels were stained with Coomassie Blue and subjected to autoradiography. Quantitation of 32P incorporation into NOS and the specific activity of autophosphorylation or PKAinduced phosphorylation were achieved by TCA precipitation method (23) after the same incubation because only the band of NOS was phosphorylated on SDS-PAGE (Fig. 1). Gel electrophoresis and Western blot analysis. One-dimensional SDS-PAGE was carried out according to the method of Laemmli (24). The electrophoretic transfer of proteins from the SDS-PAGE to the PVDF membrane was performed as described by Towbin et al (25). For immunodetection of the transferred proteins, the procedure of Burnette (26) was used except that the second antibody was linked to horseradish peroxidase. Antigen-antibody complexes were visualized by reacting the bound peroxidase with the chemiluminescence reagent (Du Pont). Peptide mapping. Proteolytic digestion of NOS by Staphylococcus aureus V-8 protease was performed as described previously (27). Phosphopeptides were separated by 15% SDS-PAGE, followed by autoradiography.
RESULT AND DISCUSSION Expression and purification of wild-type and mutant NOS. The wild-type and deletional mutants were expressed using the baculovirus/Sf9 insect cell system and purified on 29,59-ADP9-Sepharose as described under “Materials and methods”. nNOS and DnNOS were purified to >95% homogeneity on SDS-PAGE (Fig. 1A) and Western analyses (Fig. 1B). However, iNOS was partially purified to <20% apparently on SDS-PAGE (Fig. 1A). The specific activity and Km values for L-Arginine of wild-type nNOS at 30°C for 3–20 min were 4–7 nmol of L-Citruline produced/mg/ min and 8–10 mM, respectively, as determined by L-Arginine to L-Citruline conversion (data not shown). The specific activity was 3-fold lower than that reported previously using a baculovirus system (21). Expression of heme-containing enzyme in the baculovirus system often requires hemin supplementation of the culture medium for expression of protein with high specific activity. In the present study, no addition of hemin chloride to the Sf9 cell culture medium might result in the lower specific activity. The specific activity of wild-type iNOS was almost the same as that of wild-type nNOS (data not shown). Since DnNOS does not contain the NH2-terminal catalytic domain of nNOS, it shows no formation of L-Citruline from L-Arginine. However, DnNOS contains a high homology sequence to that of cytochrome P-450 reductase (2) which catalyzes the oxygenation of Nw-hydroxy-L-Arginine (NOHA) with formation of L-Citruline. Indeed, wild-type nNOS activity was maximally activated by a 1:1 molar ratio of DnNOS to wild-type nNOS with 1.2-fold activation (data not shown). The small activation by DnNOS would suggest that the 639
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FIG. 1. SDS–PAGE, Western blot analyses, and autophosphorylation of purified wild-type and mutant NOS. Lane 1, wild-type nNOS; lane 2, mutant nNOS; lane 3, wild-type iNOS. Proteins (2 mg: lane 1 and 2; 10 mg: lane 3) were separated by 10% SDS–PAGE and stained by Coomassie Blue (panel A) or transferred to PVDF membrane and immunoblotted with anti-nNOS or iNOS antibody (panel B). For the analysis of phospho-proteins, proteins were incubated at 30°C for 30 min in 40 mM Hepes (pH 7.0), 10 mM Mg2+, 1 mM DTT, 1 mM EGTA, and 100 mM [g− 32P]ATP, and were subjected to SDS–PAGE, followed by autoradiography (panel C).
formation of NOHA from L-Arginine would be dominant in the reaction in that L-Arginine was catalyzed by nNOS. Phosphorylation of wild-type nNOS. We incubated purified wild-type nNOS, DnNOS, and wild-type iNOS in the presence of Mg2+/[g-32P]ATP and after SDS-PAGE, monitored radioactivity by autoradiography. Wild-type nNOS showed autophosphorylation, whereas DnNOS and wild-type iNOS did very little phosphorylation compared to that of wild-type nNOS (Fig. 1C). nNOS has a consensus sequence (K-R-R-G-S) for phosphorylation by PKA. Wild-type nNOS showed very poor autophosphorylation compared to PKA-induced phosphorylation (Fig. 2A). In the presence and absence of PKA, there were very slow phosphorylations with a molar stoichiometry of approximately 0.15 and 0.6 at 1.5 h, respectively (Fig. 2B). In terms of slow phosphorylation nNOS by PKA was similar to that reported previously (28). Autophosphorylation was not increased in the presence of Ca2+/CaM (data not shown). Both phosphorylations of nNOS did not appear to alter its mobility on SDS-PAGE. We observed that the rate of nNOS autophosphorylation was independent of its concentration (Fig. 3), indicating that this phosphorylation was intramolecular. To further test the role of nNOS on autophosphorylation regulation, the effects of H-89, a specific inhibitor of PKA, and DnNOS on autophosphorylation of nNOS were studied (Fig. 4). H-89 inhibited PKA-
FIG. 2. Phosphorylation of wild-type nNOS. Panel A, wild-type nNOS (1 mM) was incubated without (left lane) or with (right lane) PKA (140 nM) under the same condition as in Fig. 1. Panel B, quantitation of the radioactive 32P incorporation with wild-type nNOS in the indicated time. The inset shows that stoichiometrical analysis of PKA-induced phosphorylation of wild-type nNOS in that autophosphorylation-induced 32P association was omitted. All assays were performed in duplicate, and each point represents the mean ± S.D. from two experiments. 640
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FIG. 3. Concentration dependent of nNOS autophosphorylation. Wild-type nNOS at the indicated concentration was subjected to autophophorylation at 30°C for 20 min under the same condition in Fig. 1. The incorporated 32P (pmol) and its specific activity (mmol/min/mg protein) were determined as described under “Materials and methods”. All assays were performed in duplicate, and each point represents the mean ± S.D. from two experiments.
induced phosphorylation of nNOS completely with a concentration of 1 mM, but did not inhibit autophosphorylation of nNOS, indicating that the autophosphorylation pathway is different from that of cyclic-AMP. The deletional mutant, DnNOS, did not exhibit autophosphorylation itself nor activate the autophosphorylation activity of nNOS. These results exclude that nNOS phosphorylation was due to that by a contaminating Sf9 cell kinase since there would be same kinases in both nNOS and DnNOS fractions. Peptide mapping after limited proteolysis of phosphorylated nNOS. To assess sites of phosphorylation, we cut the phosphorylation bands from SDS-PAGE gels, digested them with Staphylococcus aureus V-8 protease, and peptide mapping analyses were performed (Fig. 5). Limited proteolysis of nNOS yielded two fragments for both autophosphorylation and PKA-induced phosphorylation. These fragments showed difference in their apparent molecular weights in SDS-PAGE indicating that phosphorylation sites of nNOS might be different between auto-and PKA-induced phosphorylation.
FIG. 4. Effects of deletional mutant (DnNOS) and H-89 on the autophosphorylation of nNOS. Wild-type nNOS (1 mM) was subjected to autophosphorylation at 30°C for 20 min in the same condition as described in Fig. 2 with indicated factors. The % changes in phosphorylation of nNOS were determined by TCA precipitation method (23). DnNOS (1 mM) itself has no autophosphorylation activity and no effect on autophosphorylation of wild-type nNOS. PKA (140 nM) increase the 32P incorporation into wild-type nNOS and the effect of PKA was blocked by 1 mM H-89. Data are means of duplicate determinations from two experiments. 641
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FIG. 5. Peptide mapping of the autophosphorylated (lane 1) or PKA-induced phosphorylated (lane 2) wild-type nNOS. The bands of wild-type nNOS (5 mg) were excised from SDS–PAGE gel and subjected to peptide mapping analysis, using 10 mg of Staphylococcus aureus V-8 protease. Phosphopeptides were separated by 15% SDS–PAGE, followed by autoradiography.
Conclusions. This study shows that autophosphorylation of nNOS is observed in which there is a nucleotide binding consensus sequence G-Q-G-A-G-S (residues 166–171) but not substratebinding or enzyme catalytic consensus, R-D-L-Xn-D-F-G-Xn-G-T-P-X3-A/S-P-E. Although some studies have shown that NOS was phosphorylated by PKA, PKC, and CaMK in vitro and in situ (19, 20), to best of our knowledge there is no study related to phosphorylation activity of NOS itself. Our data demonstrate that iNOS, partially purified from the baculovirus/Sf9 insect cell system (the expression level of iNOS was somehow very low compared to that of nNOS in our system), did not undergo autophosphorylation in SDS-PAGE. These results suggest that the nucleotide binding consensus sequence G-Q-G-A-G-S (residues 166–171 might be important for regulation of autophosphorylation of nNOS because iNOS and eNOS do not contain this sequence. The deletional mutant (DnNOS), in which G-Q-G-A-G-S sequence was removed, did not exhibit autophosphorylation itself (See Results and discussions), emphasizing this hypothesis. We did not employ additional steps to further purify iNOS simply to avoid loss of enzyme activity resulting from such steps. Because this autophosphorylation reaction itself was very small, it is not likely to be a physiological phenomenon, and we therefore made no attempt to analyze it in more detail, including the exact site of the autophosphorylation. However nNOS purified from baculovirus/Sf9 insect cell system is post-translationally modified, indicating that nNOS might have been modified by a given amount of phosphorylation before the purification step. The autophosphorylation observed in our study was probably not due to some contaminating Sf9 cell kinase since 1) it was intramolecular (Fig. 3) and 2) addition of DnNOS did not increase the phosphorylation of nNOS (Fig. 4). The physiological significance of the nNOS phosphorylation is still unclear, and the autophosphorylation of nNOS might be sort of basal phosphorylation in a cell because of its Ca2+/CaM, cAMP independence. The specific autophosphorylation of nNOS could have some significance in the regulation of the neuronal system linked to the phosphorylation by PKA, PKC, and CaMK even though no change in nNOS activity was observed upon its autophosphorylation in vitro. REFERENCES 1. 2. 3. 4.
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