Vol. 180, No. 3, 1991 November 14, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]396-]402
PHOSPHORYLATION BY CALCIUM CALMODULIN-DEPENDENT PROTEIN KINASE II AND PROTEIN KINASE C MODULATES THE ACTIVITY OF NITRIC OXIDE SYNTHASE Masaki Nakane *#, Jane Mitchell':, Ulrich FOrstermann*:~ and Fetid Murad*:~ *Abbott Laboratories, Abbott Park, Illinois 60064 ~:Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611 Received September 20, 1991
Nitric oxide synthase purified from rat brain, which is Ca 2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylafion. ©1991Aoademic Press, Inc.
Nitric oxide (NO) is a ubiquitous paracrine substance and can also act as an intracellular second messenger in various cells and tissues (1-3).
In brain and neuronal cells, NO is
synthesized from L-arginine by NO synthase (E.C. 1.14.23) in a Ca2+/calmodulin (CAM) dependent manner (4-6). NO mediates the stimulatory actions of excitatory neurotransmitters, such as glutamate, on intracellular concentration of cyclic GMP (3). Brain NO synthase requires NADPH and molecular oxygen, and contains tightly bound tetrahydrobiopterin (H4B), FAD and FMN, indicating a highly regulated enzyme (7, 8). Recently, Bredt et al. obtained cDNA clones of NO synthase from rat brain and found possible phosphorylation sites based on the predicted amino acid sequence (9). However, the regulation of NO synthase activity by phosphorylation has not been described. Here, we investigated the effects of phosphorylation of NO synthase by two kinases that are present in # To whom correspondence should be addressed. ABBREVIATIONS: NO, nitric oxide; CaM, calmodulin; CaM kinase II, calcium calmodulindependent protein kinase II; H4B, tetrahydrobiopterin. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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significant amounts in brain; calcium CaM-dependent protein kinase II (CaM kinase II) and protein kinase C. The data obtained support the hypothesis that NO synthase activity can be regulated by several mechanisms a) direct effects of Ca2+/CaM and co-factors on the enzyme, and b) phosphorylation of the enzyme by one or more protein kinases.
MATERIALS AND METHODS Materials. h,-32p] ATP was purchased from Du Pont/NEN (Boston, MA) and L-[2,3,4,53H]arginine monohydrochloride was from Amersham (Arlington Heights, IL). CaM kinase II and protein kinase C were the generous gifts of T. Yamauchi (Tokyo, Japan) and T. Kuno (Kobe, Japan), respectively. All other chemicals were purchased from Sigma (St. Louis, MO). Purification of NO synthase. Type I NO synthase was purified from rat brain as previously described (10) with some modifications. Rat brains (200g) were homogenized in 5 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 10 ~tg/ml pepstatin, 10 ~g/ml aprotinin, 10 ~tg/ml leupeptin and 100 [tg/ml phenylmethysulfonyl fluoride), and centrifuged at 160,000 x g for 60 min. The supernatant was loaded at 150 ml/hr onto a 2 ml column of 2',5'-ADP Sepharose (Pharmacia) equilibrated with buffer A. The column was washed with 50 ml of buffer A containing 0.5 M NaC1 and then with 10 ml of buffer A. NO synthase was eluted with 10 ml of buffer A containing 20 mM NADPH. The eluted fraction was concentrated to 400 ~tl with Centricon 30 (Amicon) and applied to an FPLC Superose 6 gelpermeation column (HR 10/30, Pharmacia) equilibrated with buffer B (10 mM Tris-HC1, pH 7.5 containing 0.1 M NaC1, 1 mM EDTA, 5 mM 2-mercaptoethanol and 10 % glycerol). The eluted fractions that showed NO synthase activity (see below) and migrated as a single band of 155 kDa on NaDodSO4/polyacrylamide gel electrophoresis were pooled and frozen immediately at -80°C. Under these conditions, the enzyme activity was stable for at least one month. Phosphorylation of purified NO synthase. The phosphorylation was performed by incubating purified NO synthase (0.8 ~tg) in 50 mM HEPES, pH 8.0 containing 10 mM magnesium acetate, 1 mM CaC12, 50 ~tM ATP, 0.4 ~tg of purified kinase. CaM (70 ~tg/ml) was added for CaM kinase II phosphorylation, and phosphatidylserine (20 ~g/ml) and diolein (5 [tg/ml) were added for protein kinase C phosphorylation in final volumes of 20 ~tl (11, 12). The reaction was initiated with ATP and carried out at 30°C for the times indicated. Phosphorylation analysis. The phosphorylation reaction was carried out as described, but using [7-32p]ATP. The reaction was stopped by the addition of 4 ~tl of 8 % NaDodSO 4 in 250 mM Tris-HC1, pH 6.8, 40 % glycerol, 80 mM DTT and 0.05 % bromophenol blue. The samples were subjected to electrophoresis on 7.5 % NaDodSO4/polyacrylamide gels (13). The gel was stained with Coomassie brilliant blue R-250, dried, and subjected to autoradiography on Kodak XOmat film at -80 °C for 4 hr. The incorporation of 32p was quantified by cutting out the appropriate gel pieces and counting the radioactivity by liquid scintillation spectrophotometry. Phosphoamino acid analysis. Appropriate dried gel pieces were washed 3 times with 25 % methanol/10 % acetic acid, followed by 50 % methanol (16). The gel pieces were lyophilized and the phosphorylated protein was digested at 37 °C overnight in 1 ml of 50 mM NH4(X) 3 containing 0.3 mg of trypsin (Sigma). The digest was lyophilized and resuspended in 6 N HCI and hydrolyzed under vacuum for 4 hr at 105 °C. The acid-hydrolyzed peptides were then dried and resuspended in formic acid/acetic acid/water, 1:10:89 (vol/vol), pH 1.9, and spotted on 20 x 10 cm cellulose thin-layer plates (14, 15). Phosphoserine, phosphothreonine, and phosphotyrosine standards (2 mg/ml) with a trace of phenol red were also spotted at the origin of the plate. Electrophoresis was carried out in the same pH 1.9 buffer at 500 V. The plates were dried, developed with 1% ninhydrin in acetone to detect the internal phosphoamino acid standards, and subjected to autoradiography to identify the 32p-labeled phosphoamino acids. The 32p-labeled amino acids were quantified by scraping the phosphoamino acid spots and counting the radioactivity by liquid scintillation spectrophotometry. 1397
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Assay of NO synthase. The phosphorylation reaction was carried out for 10 min and the sample was then diluted 80-fold with 0.1 M HEPES, pH 7.4. NO synthase activity was measured by the conversion of 3H-arginine to 3H-citrulline as described (6, 10) with slight modifications. Diluted samples (50 I.tl) were incubated in 50 mM HEPES, pH 7.4 with 10 I.tM L[2,3,4,5-3H] arginine (9.8 GBq/mmol), 1 mM NADPH, 100 IxM CaC12, 30 nM CaM and 3 ~tM H4B in a final volume of 100 [tl. The reaction was carried out for 15 min at 25 °C and terminated by adding 1 ml of 20 mM HEPES, pH 5.5 containing 2 mM EDTA and 2 mM EGTA. The incubates were applied to 1 ml columns of Dowex AG50WX-8 (Na+form) and eluted twice with 0.5 ml of distilled water. All the liquid was pooled and radioactivity was determined by liquid scintillation counting. Protein determination. Protein was measured using the Bradford reagent (16) with bovine serum albumin as a standard. RESULTS Rate
and
stoichiometry
of p h o s p h o r y l a t e d
NO
synthase.
NO synthase was
phosphorylated by CaM kinase II (Fig. la) or protein kinase C (Fig. lb). Phosphorylation by
A
a
Ca-CaM
i
=
~
o
o
o
o
8
o
-~-
NOS
C 1o
-41(x# CaM Kinase II
~
8
c
o z
6
I
o
b
E '~
~.
+
+
+
+
+
+
Ca/PS/DG
~ ¢L
4
•
CaM Kinase II
I
O
Kinase C
I
O
O
g
.o_ ~-
~,~
NOS
(3
2
m
Kinase C
O~ 0
•
i
,
20
I
I
40
60
Time (min)
Fig. 1. Phosphorylation of type I brain NO synthase by CaM kinase II and protein kinase C. Purified brain NO synthase was phosphorylated by CaM kinase II (a) or protein kinase C (b) for the indicated periods of time as described in Materials and Methods. As a control, NO synthase (NOS) was incubated for 60 min without CaC12/CaM (with 1 mM EGTA) (a) or without CaC12/ phosphatidylserine/diolein (with 1 mM EGTA) (b). (c) The incorporation of 32p was quantified by cutting out the NO synthase bands and counting the radioactivity by liquid scintillation spectrophotometry. 1398
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CaM kinase II was Ca 2+- and CaM-dependent, much faster than the rate of phosphorylation by protein kinase C, and reached a maximum at a final stoichiometry of about 9 moles of phosphate per mole of the subunit of NO synthase (155 kDa) after 60 rain (Fig. lc).
Phosphorylation by
protein kinase C (type I) was Ca 2+-, phosphatidylserine- and diolein-dependent, and reached a maximum at a final stoichiometry of about 3 moles of phosphate per mole of the subunit of NO synthase after 60 min (Fig. lc). Other subtypes of protein kinase C (type II and type III) also phosphorylated NO synthase with the similar stoichiometry (data not shown). Phosphoamino
acid
analysis.
We analyzed the phosphorylated NO synthase for
phosphoamino acid content. NO synthase was phosphorylated by CaM kinase II or protein kinase C on both serine and threonine, but not tyrosine residues (Fig. 2). Effect of p h o s p h o r y l a t i o n on NO synthase activity.
Phosphorylation of NO synthase
by CaM kinase II or protein kinase C altered enzyme activity (Fig. 3). Phosphorylation of the enzyme by CaM kinase II resulted in a marked decrease of the activity to 33 % of control, whereas phosphorylation by protein kinase C increased the activity of the enzyme to 140 % of control. In both cases, the co-factor requirements did not change significantly. Both phosphorylated and nonphosphorylated NO synthase were totally dependent on NADPH, Ca 2+ and CaM for activity. H4B only slightly increased the enzyme activity.
a
b 80
[] Phosphoserine "O
"5
[] PhosphothreoninE
ca
o 60 .c_ E
[] Phosphotyrosine
0 t-cO
o 40 Q.
m
20
n
Phosphorylated by CaM kinase II
Phosphorylated by kinase C
0 Phosphorylated by CaM kinase II
Phosphorylated by kinase C
Fig. 2. Phosphoamino acid analysis of NO synthase. NO synthase was phosphorylated in • the presence of [7-32P] ATP and the 32P-labeled am]"no acids were analyzed as described in Materials and Methods. The cellulose plate was dried, developed with ninhydrin to detect the internal phosphoamino acid standards (arrows), and subjected to autoradiography to identify the 32p-labeled phosphoamino acids (a). The 32p-labeled amino acids were quantified by scraping the phosphoamino acid spots and counting the radioactivity by liquid scintillation spectrophotometry (b). The values are the mean _+S.D. of triplicate determinations by thin layer chromatography. 1399
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10
8 ,
ml
Control
[]
Phosphorylatedby kinase C
[]
Phosphorylated by CaM kinase II
6-
~E
4-
>,E 2-
_
r-
~
E
o~
,~
0
:~
o
~
z,
0
-r
w
+ oJ
o
Fig. 3. Effect of phosphorylation on NO synthase activity. After the phosphorylation reaction with CaM kinase II or protein kinase C, NO synthase activity was measured as described in Materials and Methods. The complete incubation condition included 10 ~tM [3H]arginine, 1 mM NADPH, 100 txM CaC12, 30 nM CaM and 3 I.tM H4B. Also, each component was omitted as indicated. However, the arginine concentration could not be reduced below 50 nM due to the concentration of [3H] arginine. The activity of CaM kinase II-phosphorylated NO synthase could not be determined in the absence of CaM ( * ) because the phosphorylation mixture contained CaM. The values are the means of duplicate determinations.
DISCUSSION The results presented here demonstrate that CaM kinase II phosphorylates brain NO synthase and markedly reduces the enzyme activity, whereas protein kinase C phosphorylates and activates NO synthase. The amino acid sequences surrounding phosphorylation sites for CaM kinase 1I have been determined to be-Arg-X-X-Ser/Thr- (17). In the amino acid sequence of NO synthase predicted from cDNA (9), there are 8 possible phosphorylation sites for serine and 4 possible sites for threonine by CaM kinase II. This is comparable to our stoichiometrical result of about 9 moles phosphate per mole of NO synthase subunit. The number of the phosphorylated threonine sites (about 5 moles phosphate per mole of NO synthase subunit, Fig. 2) was more than the predicted value (4 moles per mole of NO synthase subunit). This may suggest that CaM kinase II also phosphorylated other sequences. It has been shown that CaM kinase II can phosphorylate the sequence of-Lys-X-X-Ser/Thr-, although the apparent K m was 10-fold higher than that for the sequence of-Arg-X-X-Ser/Thr- (18). The consensus amino acid sequences surrounding phosphorylation sites for protein kinase C are not clear, although protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine (19). 1400
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The brain NO synthase is highly dependent on free Ca 2+ at physiologically relevant concentrations (4-6). It is virtually inactive at free Ca 2+ concentrations less than 100 nM, and maximally active at 500 nM Ca 2+. In addition, the enzyme shows an absolute requirement for CaM (5, 6).
CaM kinase II is also Ca2+/CaM dependent. It is most highly concentrated in neural
tissues and is thought to be activated by calcium influx into the ceils (17). Thus activation of neuronal cells by neurotransmitters such as glutamate (3) that leads to an increase in intracellular Ca2+ concentrations may not only stimulate NO synthase activity, but at the same time activate CaM kinase II. Phosphorylation of NO synthase by this kinase could represent a negative feedback mechanism for regulation of NO synthase activity. The activity of CaM kinase II can become Ca2+-independent after autophosphorylation of the enzyme (17, 20). So it is conceivable that activated NO synthase may be phosphorylated and inactivated by the autophosphorylated Ca2+-independent CaM kinase II even after cytosolic free Ca 2+ concentrations have returned to basal levels. On the other hand, phosphorylation of NO synthase by protein kinase C resulted in an increase of enzyme activity. Protein kinase C, especially type I, is abundant in brain (21), and the synergistic interactions between protein kinase C and Ca 2+ pathways are involved in the processing and modulation of a variety of cellular responses (22). Thus neurotransmitters that lead to a stimulation of phospholipase C may activate NO synthase in two ways: via the release of Ca 2+ from intracellular stores in response to inositol 1,4,5-trisphosphate, and via the activation of protein kinase C by diacylglycerol. Future experiments will have to determine to what extent these mechanisms contribute to NO synthesis in intact brain cells and other tissues. Our results demonstrate, however, that NO synthesis may be subject to complex regulation not only by Ca2+/CaM and co-factors, but also by phosphorylation of NO synthase. ACKNOWLEDGMENTS: We thank T. Yamauchi (Tokyo Metropolitan Institute for Neurosciences, Tokyo, Japan) and T. Kuno (Kobe University School of Medicine, Kobe, Japan) for their kind gift of protein kinases. We also thank S. Dorwin for her assistance with phosphoamino acid analysis. This work was supported by grants DK 30787 and HL 28474 from the National Institutes of Health, U.S.A. REFERENCES
1.
2. 3.
Murad, F., Ishii, K., Gorsky, L., Ftstermann, U., Kerwin, J. F. and Heller, M. (1990) Nitric Oxide from L-Arginine: A Bioregulatory System (S. Moncada and E. A. Higgs, eds.) pp.301-315, Elsevier Science Publishers B. V., Amsterdam. Ishii, K., Gorsky, L. D., F~Srstermann, U. and Murad, F. (1989) J. Appl. Cardiol. 4, 505-512. Garthwaite, J. (1991) Trends Neurosci. 14, 60-67. 1401
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4.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Knowles, R. G., Palacios, M., Palmer, R. M. J. and Moncada, S. (1989) Proc. Natl. Acad. Sci. USA 86, 5159-5162.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
FOrsterrnann,U., Gorsky, L. D., Pollock, J. S., Ishii, K., Schmidt, H. H. H. W., Heller, M. and Murad, E (1990) Mol. Pharmacol. 38, 7-13. Bredt, D. S. and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. USA 87, 682-685. Mayer, B., John, M., Heizel, B., Werner, E. R., Wachter, H., Schultz, G. and B/Shme, E. (1991) FEBSLett. 288, 187-191. Giovanelli, J., Campos, K. L. and Kaufman, S. (1991) Proc, Natl. Acad. Sci. USA 88, 7091-7095. Bredt, S. D., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R. and Snyder, S. H. (1991) Nature 714-718. Schmidt, H. H. H. W., Pollock, J. S., Nakane, M., Gorsky, L. D., F6rstermann, U. and Murad, E (1991) Proc. Natl. Acad. Sci. USA 88, 365-369. Yamauchi,T. and Fujisawa, H. (1983) Eur. J. Biochem. 132, 15-21. Takai,Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. Laemmli, U. K. (1970)Nature 227, 680-685. Huganir, R. L., Miles, K. and Greengard, P. (1984) Proc. Natl. Acad. Sci. USA 81, 6968-6972. Cooper, J. A., Sefton, B. M. and Hunter, T. (1983) Methods in Enzymology 99, 387402. Bradford, M. M. (1976) Anal, Biochem. 7 2, 248-254. Colbran, R. J., Schworer, C. M., Hashimoto, Y., Fong, Y. L., Rich, D. P., Smith, M. K. and Soderling, T. R. (1989) Biochem. J. 258, 313-325. Pearson, R. B., Woodgett, J. R., Cohen, P. and Kemp, B. E. (1985) J. Biol. Chem. 260, 14471-14476. Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y. and Nishizuka, Y. (1985) J. Biol. Chem. 260, 12492-12499. Hashimoto, ¥., Schworer, C. M., Colbran, R. J. and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055. Nishizuka, Y. (1988) Nature 334, 661-665. Nishizuka, Y. (1984) Nature 308, 693-697.
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PHOSPHORYLATION BY CALCIUM CALMODULIN-DEPENDENT PROTEIN KINASE II AND PROTEIN KINASE C MODULATES THE ACTIVITY OF NITRIC OXIDE SYNTHASE Masaki Nakane *#, Jane Mitchell':, Ulrich FOrstermann*:~ and Fetid Murad*:~ *Abbott Laboratories, Abbott Park, Illinois 60064 ~:Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611 Received September 20, 1991
Nitric oxide synthase purified from rat brain, which is Ca 2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylafion. ©1991Aoademic Press, Inc.
Nitric oxide (NO) is a ubiquitous paracrine substance and can also act as an intracellular second messenger in various cells and tissues (1-3).
In brain and neuronal cells, NO is
synthesized from L-arginine by NO synthase (E.C. 1.14.23) in a Ca2+/calmodulin (CAM) dependent manner (4-6). NO mediates the stimulatory actions of excitatory neurotransmitters, such as glutamate, on intracellular concentration of cyclic GMP (3). Brain NO synthase requires NADPH and molecular oxygen, and contains tightly bound tetrahydrobiopterin (H4B), FAD and FMN, indicating a highly regulated enzyme (7, 8). Recently, Bredt et al. obtained cDNA clones of NO synthase from rat brain and found possible phosphorylation sites based on the predicted amino acid sequence (9). However, the regulation of NO synthase activity by phosphorylation has not been described. Here, we investigated the effects of phosphorylation of NO synthase by two kinases that are present in # To whom correspondence should be addressed. ABBREVIATIONS: NO, nitric oxide; CaM, calmodulin; CaM kinase II, calcium calmodulindependent protein kinase II; H4B, tetrahydrobiopterin. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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significant amounts in brain; calcium CaM-dependent protein kinase II (CaM kinase II) and protein kinase C. The data obtained support the hypothesis that NO synthase activity can be regulated by several mechanisms a) direct effects of Ca2+/CaM and co-factors on the enzyme, and b) phosphorylation of the enzyme by one or more protein kinases.
MATERIALS AND METHODS Materials. h,-32p] ATP was purchased from Du Pont/NEN (Boston, MA) and L-[2,3,4,53H]arginine monohydrochloride was from Amersham (Arlington Heights, IL). CaM kinase II and protein kinase C were the generous gifts of T. Yamauchi (Tokyo, Japan) and T. Kuno (Kobe, Japan), respectively. All other chemicals were purchased from Sigma (St. Louis, MO). Purification of NO synthase. Type I NO synthase was purified from rat brain as previously described (10) with some modifications. Rat brains (200g) were homogenized in 5 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol, 10 ~tg/ml pepstatin, 10 ~g/ml aprotinin, 10 ~tg/ml leupeptin and 100 [tg/ml phenylmethysulfonyl fluoride), and centrifuged at 160,000 x g for 60 min. The supernatant was loaded at 150 ml/hr onto a 2 ml column of 2',5'-ADP Sepharose (Pharmacia) equilibrated with buffer A. The column was washed with 50 ml of buffer A containing 0.5 M NaC1 and then with 10 ml of buffer A. NO synthase was eluted with 10 ml of buffer A containing 20 mM NADPH. The eluted fraction was concentrated to 400 ~tl with Centricon 30 (Amicon) and applied to an FPLC Superose 6 gelpermeation column (HR 10/30, Pharmacia) equilibrated with buffer B (10 mM Tris-HC1, pH 7.5 containing 0.1 M NaC1, 1 mM EDTA, 5 mM 2-mercaptoethanol and 10 % glycerol). The eluted fractions that showed NO synthase activity (see below) and migrated as a single band of 155 kDa on NaDodSO4/polyacrylamide gel electrophoresis were pooled and frozen immediately at -80°C. Under these conditions, the enzyme activity was stable for at least one month. Phosphorylation of purified NO synthase. The phosphorylation was performed by incubating purified NO synthase (0.8 ~tg) in 50 mM HEPES, pH 8.0 containing 10 mM magnesium acetate, 1 mM CaC12, 50 ~tM ATP, 0.4 ~tg of purified kinase. CaM (70 ~tg/ml) was added for CaM kinase II phosphorylation, and phosphatidylserine (20 ~g/ml) and diolein (5 [tg/ml) were added for protein kinase C phosphorylation in final volumes of 20 ~tl (11, 12). The reaction was initiated with ATP and carried out at 30°C for the times indicated. Phosphorylation analysis. The phosphorylation reaction was carried out as described, but using [7-32p]ATP. The reaction was stopped by the addition of 4 ~tl of 8 % NaDodSO 4 in 250 mM Tris-HC1, pH 6.8, 40 % glycerol, 80 mM DTT and 0.05 % bromophenol blue. The samples were subjected to electrophoresis on 7.5 % NaDodSO4/polyacrylamide gels (13). The gel was stained with Coomassie brilliant blue R-250, dried, and subjected to autoradiography on Kodak XOmat film at -80 °C for 4 hr. The incorporation of 32p was quantified by cutting out the appropriate gel pieces and counting the radioactivity by liquid scintillation spectrophotometry. Phosphoamino acid analysis. Appropriate dried gel pieces were washed 3 times with 25 % methanol/10 % acetic acid, followed by 50 % methanol (16). The gel pieces were lyophilized and the phosphorylated protein was digested at 37 °C overnight in 1 ml of 50 mM NH4(X) 3 containing 0.3 mg of trypsin (Sigma). The digest was lyophilized and resuspended in 6 N HCI and hydrolyzed under vacuum for 4 hr at 105 °C. The acid-hydrolyzed peptides were then dried and resuspended in formic acid/acetic acid/water, 1:10:89 (vol/vol), pH 1.9, and spotted on 20 x 10 cm cellulose thin-layer plates (14, 15). Phosphoserine, phosphothreonine, and phosphotyrosine standards (2 mg/ml) with a trace of phenol red were also spotted at the origin of the plate. Electrophoresis was carried out in the same pH 1.9 buffer at 500 V. The plates were dried, developed with 1% ninhydrin in acetone to detect the internal phosphoamino acid standards, and subjected to autoradiography to identify the 32p-labeled phosphoamino acids. The 32p-labeled amino acids were quantified by scraping the phosphoamino acid spots and counting the radioactivity by liquid scintillation spectrophotometry. 1397
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Assay of NO synthase. The phosphorylation reaction was carried out for 10 min and the sample was then diluted 80-fold with 0.1 M HEPES, pH 7.4. NO synthase activity was measured by the conversion of 3H-arginine to 3H-citrulline as described (6, 10) with slight modifications. Diluted samples (50 I.tl) were incubated in 50 mM HEPES, pH 7.4 with 10 I.tM L[2,3,4,5-3H] arginine (9.8 GBq/mmol), 1 mM NADPH, 100 IxM CaC12, 30 nM CaM and 3 ~tM H4B in a final volume of 100 [tl. The reaction was carried out for 15 min at 25 °C and terminated by adding 1 ml of 20 mM HEPES, pH 5.5 containing 2 mM EDTA and 2 mM EGTA. The incubates were applied to 1 ml columns of Dowex AG50WX-8 (Na+form) and eluted twice with 0.5 ml of distilled water. All the liquid was pooled and radioactivity was determined by liquid scintillation counting. Protein determination. Protein was measured using the Bradford reagent (16) with bovine serum albumin as a standard. RESULTS Rate
and
stoichiometry
of p h o s p h o r y l a t e d
NO
synthase.
NO synthase was
phosphorylated by CaM kinase II (Fig. la) or protein kinase C (Fig. lb). Phosphorylation by
A
a
Ca-CaM
i
=
~
o
o
o
o
8
o
-~-
NOS
C 1o
-41(x# CaM Kinase II
~
8
c
o z
6
I
o
b
E '~
~.
+
+
+
+
+
+
Ca/PS/DG
~ ¢L
4
•
CaM Kinase II
I
O
Kinase C
I
O
O
g
.o_ ~-
~,~
NOS
(3
2
m
Kinase C
O~ 0
•
i
,
20
I
I
40
60
Time (min)
Fig. 1. Phosphorylation of type I brain NO synthase by CaM kinase II and protein kinase C. Purified brain NO synthase was phosphorylated by CaM kinase II (a) or protein kinase C (b) for the indicated periods of time as described in Materials and Methods. As a control, NO synthase (NOS) was incubated for 60 min without CaC12/CaM (with 1 mM EGTA) (a) or without CaC12/ phosphatidylserine/diolein (with 1 mM EGTA) (b). (c) The incorporation of 32p was quantified by cutting out the NO synthase bands and counting the radioactivity by liquid scintillation spectrophotometry. 1398
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CaM kinase II was Ca 2+- and CaM-dependent, much faster than the rate of phosphorylation by protein kinase C, and reached a maximum at a final stoichiometry of about 9 moles of phosphate per mole of the subunit of NO synthase (155 kDa) after 60 rain (Fig. lc).
Phosphorylation by
protein kinase C (type I) was Ca 2+-, phosphatidylserine- and diolein-dependent, and reached a maximum at a final stoichiometry of about 3 moles of phosphate per mole of the subunit of NO synthase after 60 min (Fig. lc). Other subtypes of protein kinase C (type II and type III) also phosphorylated NO synthase with the similar stoichiometry (data not shown). Phosphoamino
acid
analysis.
We analyzed the phosphorylated NO synthase for
phosphoamino acid content. NO synthase was phosphorylated by CaM kinase II or protein kinase C on both serine and threonine, but not tyrosine residues (Fig. 2). Effect of p h o s p h o r y l a t i o n on NO synthase activity.
Phosphorylation of NO synthase
by CaM kinase II or protein kinase C altered enzyme activity (Fig. 3). Phosphorylation of the enzyme by CaM kinase II resulted in a marked decrease of the activity to 33 % of control, whereas phosphorylation by protein kinase C increased the activity of the enzyme to 140 % of control. In both cases, the co-factor requirements did not change significantly. Both phosphorylated and nonphosphorylated NO synthase were totally dependent on NADPH, Ca 2+ and CaM for activity. H4B only slightly increased the enzyme activity.
a
b 80
[] Phosphoserine "O
"5
[] PhosphothreoninE
ca
o 60 .c_ E
[] Phosphotyrosine
0 t-cO
o 40 Q.
m
20
n
Phosphorylated by CaM kinase II
Phosphorylated by kinase C
0 Phosphorylated by CaM kinase II
Phosphorylated by kinase C
Fig. 2. Phosphoamino acid analysis of NO synthase. NO synthase was phosphorylated in • the presence of [7-32P] ATP and the 32P-labeled am]"no acids were analyzed as described in Materials and Methods. The cellulose plate was dried, developed with ninhydrin to detect the internal phosphoamino acid standards (arrows), and subjected to autoradiography to identify the 32p-labeled phosphoamino acids (a). The 32p-labeled amino acids were quantified by scraping the phosphoamino acid spots and counting the radioactivity by liquid scintillation spectrophotometry (b). The values are the mean _+S.D. of triplicate determinations by thin layer chromatography. 1399
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10
8 ,
ml
Control
[]
Phosphorylatedby kinase C
[]
Phosphorylated by CaM kinase II
6-
~E
4-
>,E 2-
_
r-
~
E
o~
,~
0
:~
o
~
z,
0
-r
w
+ oJ
o
Fig. 3. Effect of phosphorylation on NO synthase activity. After the phosphorylation reaction with CaM kinase II or protein kinase C, NO synthase activity was measured as described in Materials and Methods. The complete incubation condition included 10 ~tM [3H]arginine, 1 mM NADPH, 100 txM CaC12, 30 nM CaM and 3 I.tM H4B. Also, each component was omitted as indicated. However, the arginine concentration could not be reduced below 50 nM due to the concentration of [3H] arginine. The activity of CaM kinase II-phosphorylated NO synthase could not be determined in the absence of CaM ( * ) because the phosphorylation mixture contained CaM. The values are the means of duplicate determinations.
DISCUSSION The results presented here demonstrate that CaM kinase II phosphorylates brain NO synthase and markedly reduces the enzyme activity, whereas protein kinase C phosphorylates and activates NO synthase. The amino acid sequences surrounding phosphorylation sites for CaM kinase 1I have been determined to be-Arg-X-X-Ser/Thr- (17). In the amino acid sequence of NO synthase predicted from cDNA (9), there are 8 possible phosphorylation sites for serine and 4 possible sites for threonine by CaM kinase II. This is comparable to our stoichiometrical result of about 9 moles phosphate per mole of NO synthase subunit. The number of the phosphorylated threonine sites (about 5 moles phosphate per mole of NO synthase subunit, Fig. 2) was more than the predicted value (4 moles per mole of NO synthase subunit). This may suggest that CaM kinase II also phosphorylated other sequences. It has been shown that CaM kinase II can phosphorylate the sequence of-Lys-X-X-Ser/Thr-, although the apparent K m was 10-fold higher than that for the sequence of-Arg-X-X-Ser/Thr- (18). The consensus amino acid sequences surrounding phosphorylation sites for protein kinase C are not clear, although protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine (19). 1400
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The brain NO synthase is highly dependent on free Ca 2+ at physiologically relevant concentrations (4-6). It is virtually inactive at free Ca 2+ concentrations less than 100 nM, and maximally active at 500 nM Ca 2+. In addition, the enzyme shows an absolute requirement for CaM (5, 6).
CaM kinase II is also Ca2+/CaM dependent. It is most highly concentrated in neural
tissues and is thought to be activated by calcium influx into the ceils (17). Thus activation of neuronal cells by neurotransmitters such as glutamate (3) that leads to an increase in intracellular Ca2+ concentrations may not only stimulate NO synthase activity, but at the same time activate CaM kinase II. Phosphorylation of NO synthase by this kinase could represent a negative feedback mechanism for regulation of NO synthase activity. The activity of CaM kinase II can become Ca2+-independent after autophosphorylation of the enzyme (17, 20). So it is conceivable that activated NO synthase may be phosphorylated and inactivated by the autophosphorylated Ca2+-independent CaM kinase II even after cytosolic free Ca 2+ concentrations have returned to basal levels. On the other hand, phosphorylation of NO synthase by protein kinase C resulted in an increase of enzyme activity. Protein kinase C, especially type I, is abundant in brain (21), and the synergistic interactions between protein kinase C and Ca 2+ pathways are involved in the processing and modulation of a variety of cellular responses (22). Thus neurotransmitters that lead to a stimulation of phospholipase C may activate NO synthase in two ways: via the release of Ca 2+ from intracellular stores in response to inositol 1,4,5-trisphosphate, and via the activation of protein kinase C by diacylglycerol. Future experiments will have to determine to what extent these mechanisms contribute to NO synthesis in intact brain cells and other tissues. Our results demonstrate, however, that NO synthesis may be subject to complex regulation not only by Ca2+/CaM and co-factors, but also by phosphorylation of NO synthase. ACKNOWLEDGMENTS: We thank T. Yamauchi (Tokyo Metropolitan Institute for Neurosciences, Tokyo, Japan) and T. Kuno (Kobe University School of Medicine, Kobe, Japan) for their kind gift of protein kinases. We also thank S. Dorwin for her assistance with phosphoamino acid analysis. This work was supported by grants DK 30787 and HL 28474 from the National Institutes of Health, U.S.A. REFERENCES
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Murad, F., Ishii, K., Gorsky, L., Ftstermann, U., Kerwin, J. F. and Heller, M. (1990) Nitric Oxide from L-Arginine: A Bioregulatory System (S. Moncada and E. A. Higgs, eds.) pp.301-315, Elsevier Science Publishers B. V., Amsterdam. Ishii, K., Gorsky, L. D., F~Srstermann, U. and Murad, F. (1989) J. Appl. Cardiol. 4, 505-512. Garthwaite, J. (1991) Trends Neurosci. 14, 60-67. 1401
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Knowles, R. G., Palacios, M., Palmer, R. M. J. and Moncada, S. (1989) Proc. Natl. Acad. Sci. USA 86, 5159-5162.
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