Nitric Oxide Synthase Regulatory Sites

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 16, Issue of June 5, pp. 10976-10981,1992 Printed in U. S A .

Nitric Oxide Synthase Regulatory Sites PHOSPHORYLATION BY CYCLIC AMP-DEPENDENT PROTEIN KINASE, PROTEIN KINASE C, AND CALCIUM/CALMODULIN PROTEIN KINASE; IDENTIFICATION OF FLAVIN AND CALMODULIN BINDING SITES*

(Received for publication, July 5, 1991)

David S. Bredt, Christopher D. Ferris, and Solomon H. Snyder4 From the Departments of Neuroscience, Pharmacology and Molecular Sciences, and Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by CAMP dependent protein kinase, protein kinase C, and calcium/calmodulin- dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by ~ 7 7 % in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spec- troscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and “cross-talk” between second messenger systems.

Nitric oxide (NO)’ is a messenger molecule involved in diverse processes in many tissues (1, 2). For example, NO may be responsible for the tumoricidal and bactericidal activities of macrophages (3), and in blood vessels it accounts for endothelium-derived relaxing factor activity (4-6). In the brain NO mediates the ability of the excitatory neurotransmitter glutamate to stimulate cyclic GMP levels via N -

* This work was supported by United States Public Health Service Grant MH-18501, Research Scientist Award DA-00074 (to S. H. S.), Training Grant GM-07309 (to D. S. B.), Predoctoral Fellowship MH- 10018 (to C. D. F.), and a gift from Bristol-Myers-Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom all correspondence and reprint requests should be addressed. Tel.: 410-955-3024; Fax: 410-955-3623.

’ The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; PKA, protein kinase A; PKC, protein kinase C; APFX, apoflavodoxin; CaM, calmodulin; CaM-K, calcium/calmodulin-dependent protein kinase; CaM-K-11, calcium/calmodulin protein kinase 11; CPT-CAMP, 8-(4-~hlorophenylthio)adenosine 3’,5’-cyclic monophosphate; HSBA, N-hydroxysuccinimidyl-4-azido benzoate; NMDA, N-methyl-D-aspartate; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide electrophoresis; TPA, 12-O-tetradecanoylphorbol-13- acetate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

methyl-D-aspartate (NMDA) receptors (7, 8). In long term potentiation, a model of memory and learning, where NMDA induces changes in synaptic transmission, NO has been sug- gested as the retrograde messenger (9-11). Similarly, in the cerebellum, NO has been implicated as a messenger in long term depression, a model of motor learning (12). In the peripheral nervous system NO functions as a non-adrenergic non-cholinergic neurotransmitter regulating relaxation of the gastrointestinal tract (13-16), penile erection (17), and blood flow to the adrenal medulla (18).

NO synthase (NOS) (EC 1.14.23) has been purified from brain (19-21) and molecularly cloned (22). Brain NOS is selectively enriched in discrete neuronal populations in the central and peripheral nervous systems (23). The purified enzyme has an absolute requirement for calmodulin and re- quires NADPH for catalytic activity (19). The primary amino acid sequence of NOS contains consensus sequence binding sites for NADPH, FAD, FMN, and calmodulin as well as sites for protein phosphorylation. In the present study we observe that purified brain NOS is phosphorylated by cyclic AMP- dependent PKA, PKC, and calcium/calmodulin protein kinase I1 with all three enzymes acting at different sites and with phosphorylation by PKC dramatically reducing NOS enzymatic activity. We also directly demonstrate the presence of tightly bound FAD and FMN. We show labeling of the purified enzyme with a photoaffinity derivative of calmodulin.

EXPERIMENTAL PROCEDURES

Materi~ls-[y~~P]ATP, ‘251-calmodulin, and [3H]arginine were purchased from Du Pont-New England Nuclear. N-Hydroxysucci- nimidyl-4-azidobenzoate (HSBA) was purchased from Pharmacia LKB Biotechnology Inc.; all other reagents were obtained from Sigma. CaM-K-I1 was the generous gift of Dr. Mary Kennedy and Dr. Bruce Patton (California Institute of Technology, Pasadena, CA). Flavodoxin protein from M. elsdenni was the generous gift of Dr. Vincent Massey (University of Michigan; Ann Arbor, MI). Apofla- vodoxin (APFX) was prepared by denaturing the purified holoprotein with 5% trichloroacetic acid which releases its bound FMN. Precipi- tated protein was centrifuged, the pellet washed with 5% trichloroacetic acid, and APFX was renatured by neutralization of the pellet with 0.1 M sodium phosphate buffer, pH 7.0. The catalytic subunit of PKA was purified from bovine heart as described (24), and PKC was purified from rat brain as described (25). The specific activities of the purified kinases were 1, 2, and 2 pmol/min/mg for PKA, PKC, and CaM-K-11, respectively, using histone H1 as substrate for PKA and PKC, and synapsin for CaM-K-11.

In Vitro Phosphorylation of NOS-NOS, 20 pg/ml, purified by 2’5”ADP affinity chromatography as described (19), was incubated at 30 ‘C in a final incubation volume of 100 pl. In all experiments, final concentrations were 10 mM MgC12 and 50 p M [32P]ATP. The final concentrations of phosphorylating enzymes were 1,4, and 1 pg/ ml for PKC, CaM-K-11, and catalytic subunit of PKA, respectively. For phosphorylation by both CaM-K-I1 and PKC, 1 mM CaClZ was

10976

Nitric Oxide Synthase 10977

added phosphorylations by PKC also included phosphatidylserine and diacylglycerol, whereas a final calmodulin concentration of 0.05 mg/ml was present for CaM-K-I1 phosphorylation. Reactions were stopped by the addition of loading buffer followed by electrophoresis on 7.5% SDS-PAGE as described by Laemmli (26). Gels were stained with Coomassie Blue, dried, and subjected to autoradiography. Quan- titation of '"P incorporation into NOS was achieved by cutting out the appropriate gel pieces and determining their radioactivity by liquid scintillation counting.

Phosphopeptide Mapping and Phosphorylated Amino Acid Anal- yses-Two-dimensional phosphopeptide maps were exactly produced as described (27). Briefly, the NOS containing gel bands were cut out of the gel and rehydrated by 3 X 10-min incubations with 50% methanol followed by a 30-min incubation in water. Subsequently, these were digested in 1 ml of 50 mM NH,HCOa with 0.3 mg of thermolysin overnight a t 30 "C and lyophilized. The peptides were resuspended, spotted on cellulose thin-layer plates, subjected to electrophoresis along the x axis and ascending chromatography along the .y axis. The plates were then dried and autoradiographed. Phosphoa- mino acid analysis was done as described (27). Briefly, NOS in gel pieces was digested with thermolysin as above followed by acid hydrolysis in 6 M HCI for 1 h at 105 "C. This hydrolysate was concentrated by lyophilization, resuspended in water, and electropho- resed on cellulose plates at pH 1.9 followed by pH 3.5 along with phosphotyrosine, phosphoserine, and phosphothreonine standards. The cellulose plates were stained with ninhydrin to detect the phosphoamino acid standards. The plates were autoradiographed to iden- tify the migration of the "P-labeled amino acids.

Phosphorylation of NOS in 2938 Cells-A stably transfected cell line (293.NOS) was established by co-transfection of pCIS-NOS (22) with pRSV-NE0 into 2938 cells followed by selection for transfec- tants in media containing genticin. Intracellular pools of ATP were labeled by incubation with n2Pi for 3 h in phosphate-free media. Following a 20-min incubation of cells with chlorophenylthio-cyclic AMP (CPT-CAMP) or 12-0-tetradecanoylphorbol-13-acetate (TPA), cells were homogenized in 50 mM Tris.HCI, pH 7.4, containing 50 mM NaF, 10 mM pyrophosphate, 1 mM EDTA, 1 mM EGTA, and 1 mM orthovanadate buffer. Phosphorylation of NOS was quantified by purification of NOS by 2',5'-ADP-agarose and SDS-PAGE and autoradiography. In parallel experiments, NOS activity was measured in homogenates from CPT-CAMP and TPA-treated 293.NOS cells, by measuring the conversion of 10 p~ [:'H]citrulline as described (19). NOS levels from treated cells were quantified by Western blot analysis following separation of protein homogenates by SDS-PAGE exactly as described (28).

Quantitation of NOS Activity in Intact 293.NOS Cells-Maximal NOS activity in intact cells was quantitated by monitoring accumulation of nitrate in the incubation media of cells treated with the calcium ionophore A23187. Cells were grown to near confluence in 1.7-cm wells. Following 15-min treatment with CPT-CAMP or TPA, media was replaced with 0.25 ml of Krebs-Henseleit (7) buffer containing the calcium ionophore A23187 ( 5 pM) and either CPT-CAMP or TPA. Nitrate accumulation in the media was determined following a 2-h incubation by adding 200 pI of media to 200 pl of the Greiss reagent/5% (v/v) H,PO,/l% sulfanilic acid/O.l% N-(1-naph- thy1)ethylenediamine. The reaction of NO, with this reagent produces a pink color, which was quantified at 554 nm against standards in the same buffer.

Fluorescence Measurements-NOS (50 nM, 8 pg/ml), purified to homogeneity as described (19), was denatured by boiling for 3 min in the dark, and protein was pelleted by centrifugation at 12,000 rpm in a microcentrifuge. Fluorescence of the remaining soluble material was analyzed with a Perkin-Elmer Cetus fluorimeter. Phosphodies- terase digestions were performed by the addition of type I1 phosphodiesterase from Crotalus adamanteus venom (0.05 units/ml), followed by overnight incubation at 37 "C in the dark. FMN fluorescence with APFX was quenched by adding an excess of APFX (500 nM) 1 min prior to measuring fluorescence.

Calmodulin Photoaffinity Labeling-N-Hydroxysuccinimidyl-4-a~- idobenzoate-"sI-calmodulin (HSBA-'2sI-calmodulin) was prepared according to the original procedure of Glossman and Streissnig (29) and as modified by McEnery et al. (30). HSBA (stock solution 100 mM in dimethyl sulfoxide) was incubated with 10 pCi of 12sI-calmodulin in 100 pl of 200 mM boric acid, pH 8.5, for 1 h at room temperature in the dark. The final concentration of HSBA was 20 mM. Excess HSBA was quenched with 470 pl of 50 mM Tris. HCI, pH 7.4. Twenty- five pl of the HSBA-1Y511-calmodulin reagent were added to 50-pl aliquots of crude cytosol or purified NOS for 1 h at 4 "C followed by

UV irradiation for 20 min. Proteins were separated by SDS-PAGE, and gels were dried and exposed to x-ray film overnight.

RESULTS

Phosphorylation of NOS-We incubated purified NOS with PKA, PKC, and CaM-K together with ["PIATP for varying durations and, after SDS-PAGE, monitored radioactivity by autoradiography (Fig. lA). All three enzymes phosphorylate NOS, whereas autophosphorylation of PKC and CaM-K is

b 1.0 ~

0.8.

0.6 .

0.4 .

205 -

97-

6 8 -

43 -

0 20 40 60 TlME (min)

FIG. 1. Phosphorylation of NOS by PKC, CaM-K, and PKA. Purified NOS was phosphorylated with each enzyme as described at 30 "C and proteins separated on 7.5% SDS-PAGE. a, following a 30- min incubation with PKC (lane I ) , CaM-K (lane 2), and PKA (lane 3) , autoradiography reveals phosphorylation of the 160-kilodalton NOS band and autophosphorylation of PKC (80 kilodaltons) and CaM-K (50 kilodaltons). b, quantitation of the radioactive "P associated with NOS indicates that within 1 h, phosphorylation of NOS by all three enzymes is nearly stoichiometric with respect to each NOS monomer. Phosphorylation by PKC is most rapid, whereas CaM-K and PKA phosphorylate NOS more slowly. These data are representative of an experiment repeated twice with similar results.

10978 Nitric Oxide Synthase

also apparent. With all three enzymes substantial label is incorporated into NOS in a time-dependent fashion (Fig. 1B) and appears to be nearly stoichiometric with one molecule of :"PP/NOS monomer subunit. PKC provides the most rapid phosphorylation with half-maximal phosphorylation apparent at about 5 min and plateau levels at 15 min. For CaM-K, half-maximal phosphorylation occurs at about 17-20 min with plateau levels at 25-35 min, whereas PKA does not reach plateau levels until 60 min. Phosphorylation for 120 min does not yield any further increase in '*P incorporation into NOS by any of the three kinases (data not shown).

To assess the specificity of phosphorylation, we have observed that phosphorylation is abolished in the absence of magnesium (Table I). For PKC and CaM-K, no phosphorylation can be demonstrated when calcium is omitted. For PKC, phosphorylation is abolished when phosphatidylserine and diacylglycerol are deleted, whereas with CaM-K, phosphorylation is absent when calmodulin is not included in incubation mixtures.

To assess sites of phosphorylation, we cut the phosphorylated bands from SDS gels, digested them with thermolysin, and resolved the resultant phosphopeptides by electrophoresis and thin-layer chromatography in two dimensions (Fig. 2). With PKC the predominant peptide migrates toward the cathode, similar to the mobility of the marker basic fuchsin. In addition, about seven very faint spots are evident at different mobilities. PKA also produces a single predominant phosphopeptide which migrates toward the cathode less than the PKC-derived phosphopeptide. A second minor phosphopeptide is formed with PKA which migrates toward the anode. CaM-K produces a single major phosphopeptide which migrates toward the anode. In addition with CaM-K, we also observe the same phosphopeptides that are produced with PKA and PKC, but at lesser density than the CaM-K predominant phosphopeptide. Several other very faint spots are also evident. Phosphoamino acid analysis reveals that all three kinases phosphorylate NOS on amino acids which co-migrate with phosphoserine (data not shown).

To assess the influence of phosphorylation upon NOS catalytic activity, we incubated the enzyme alone or with the phosphorylating enzymes for 10 and 30 min. When purified NOS is incubated by itself or with calcium at 37 "C, enzyme activity declines 20 and 37%) respectively, at 10 and 30 min. Including calmodulin and calcium with NOS markedly accel- erates the loss of enzyme activity with more than 90% loss of

TABLE I Phosphorylation of purified NOS

Phosphorylation of NOS by PKA, PKC, and CaM-K. Phosphoryl- ation was performed as described for 60 min at 30 "C. Gel bands corresponding to NOS were excised from SDS-PAGE gels and radioactivity quantitated. Phosphorylation of NOS by all three enzymes was specific and near stoichiometric with 1 mol of "P/mol of NOS monomer. Data are means of duplicate observations from an experiment which was replicated with a different NOS preparation with similar results. PS/DAG, phosphatidylserine/diacylglycerol.

Condition M O ~ 3 2 ~ / m ~ 1 NOS

1. PKA (catalytic subunit + MG") 2. PKA - Mg2' 3. PKC ( M F + CaZ+ + PS/DAG) 0.9

5. PKC - Ca2+ 50.01

1.0 50.01

4. PKC - Mg2' 50.01

6. PKC - PS/DAG 0.1 7 . CaM-K (+ M F + Ca2+ + CaM) 0.7 8. CaM-K - Mg?' 50.01 9. CaM-K - Ca2+

10. CaM K - CaM 50.01 50.01

"I- Electrophoresis

b

I "

-+

t cc Electrophoresis

".- - <. ~ . . > .

-+ Electrophoresis

FIG. 2. Two-dimensional phosphopeptide maps of NOS phosphorylated by PKC (a), CaM-KII (b ) , and PKA (c). NOS was phosphorylated as described. After SDS-PAGE, the NOS band was digested with thermolysin and the peptides were separated by electrophoresis and chromatography as described (27). Each kinase predominantly phosphorylates a distinct site, indicated by solid circles. Fainter spots are surrounded by broken circles.


C 1 2 3 4

116-

97-

66-

43-

FIG. 3. Regulation of NOS catalytic activity by activation of PKA and PKC. Stimulation of NOS phosphorylation in 293.NOS cells was done by adding CPT-CAMP or TPA to activate PKA and PKC, respectively. A, CPT-CAMP has no effect on NOS activity, whereas 50 and 500 nM TPA cause a 75% reduction in NOS activity measured as NO; accumulation in response to treatment of intact

catalytic activity a t 30 min. Incubation of purified NOS for 10 or 30 min with PKA, PKC, or CaM-K in the presence or absence of magnesium does not dramatically alter the rate of decline of catalytic activity.

In order to investigate NOS phosphorylation in intact cells, we have established a stably transfected cell line containing the NOS gene. Incubation of 293.NOS cells for 20 min with CPT-CAMP or TPA to activate the endogenous PKA or PKC results in substantial incorporation of added "Pi into NOS purified from these cells (data not shown).

Maximal NOS activity in these CPT-CAMP- and TPA- treated cells was measured by quantitation of NO;, a break- down product of NO, following the addition of the calcium ionophore A23187 which results in production of 2.9 nmol NO;/h/well. Although CPT-CAMP has no apparent effect on NOS catalytic activity, treatment of the cells with 50 or 500 nM TPA results in a 77% reduction in NOS catalytic activity (Fig. 3). This effect of TPA is specifically mediated by protein phosphorylation, as the protein kinase inhibitor H-7 added together with TPA fully reverses the down-regulation of NOS catalytic activity.

In parallel experiments, NOS activity was measured in homogenates from CPT-CAMP and TPA treated 293.NOS cells (Fig. 3B). Again, CPT-CAMP has no effect on NOS activity, whereas 50 and 500 nM TPA reduces enzyme activity 50%, and again H-7 blocks the effect of TPA. The greater magnitude of PKC-mediated down-regulation of NOS in intact cells could be due to a dephosphorylation of NOS, during cell homogenization. Total NOS was measured in cells by Western blotting following treatment with CPT-CAMP and TPA (Fig. 3C). Treatment with these agents does not alter the total quantity of NOS.

Identification of Flavin Binding to NOS-Molecular cloning of NOS reveals substantial homology to cytochrome P-450 reductase, an enzyme with both FAD and FMN recognition sites (31), and the NOS sequence has consensus recognition sites for FAD and FMN (22). To ascertain whether purified NOS possesses tightly bound flavins, we examined the fluorescence spectrum for purified NOS at pH 2.6 and 7.7 when excited at 451 nm (Fig. 4). The native protein displays no fluorescence. When NOS is denatured by boiling for 5 min, substantial fluorescence is apparent with a peak at about 517 nm. Fluorescence at pH 2.6 is greater than at 7.7. I t is well established that the fluorescence of FAD is markedly enhanced at acid pH, whereas FMN fluorescence is somewhat reduced at acid pH (32). In confirmation of these findings we also observe that FAD fluorescence at pH 2.6 is five times greater than at pH 7.7, whereas FMN fluorescence is modestly reduced at the lower pH value (Table 11). Fluorescence derived from purified NOS displays a 25% enhancement of fluorescence at the more acid pH value, suggesting that it may possess a mixture of tightly bound FAD and FMN. To verify the presence of flavins, we utilized APFX which binds tightly and selectively to FMN but not to FAD (33). In the presence of 1 PM APFX, FMN fluorescence is abolished, whereas FAD

cells with 5 pM A23187. The PKC inhibitor H-7 added at 10 pM blocks the effects of TPA on NOS catalytic activity. B, NOS activity quantified in homogenates from the treated cells shows similar results with CPT-CAMP having no effect on enzyme activity and 50 and 500 nM TPA causing a 50% reduction in NOS activity. Again the effects of TPA are blocked by 10 p~ H-7. C, Western blot analysis shows that treatments with kinase activators does not alter NOS levels. Crude protein homogenates (100 pgllane) were separated on SDS- PAGE and blotted with anti-NOS antiserum. Lane I, control; lane 2, 200 p~ CPT-CAMP; lane 3, 500 nM TPA, lane 4, 500 nM TPA and 10 p~ H-7. Data are means of triplicate determinations from a representative experiment repeated five times with similar results.

10980

la ; 80- - ._ C 3

475 510 550 590 Emission(nm)

Nitric Oxide Synthase

I 475 510 550 590

Emission (nm)

FIG. 4. Fluorescenceemission spectraof homogeneous NOS. Fluorescence emission from purified NOS (50 nM) was monitored following excitation at 451 nm. a, the native protein shows no fluorescence, whereas denaturation of the protein releases fluorescent material with an emission maximum at 517 nm, identical to that of flavins. The fluorescence increases 27% upon lowering the pH of the Tris-HC1 buffer system to 2.6, consistent with a 1:l stoichiometry of FMN:FAD. b, phosphodiesterase treatment of the released co-factors increases the fluorescence by 82% again consistent with a 1:l stoichiometry of FMN:FAD. Addition of APFX (500 nM) fully quenches the fluorescence of the phosphodiesterase treated co-factors.

TABLE I1 Comparison of fluorescence for NOS, FAD, and FMN

NOS was purified to homogeneity and denatured by boiling, and fluorescence emission in arbitrary units was monitored a t 517 nm with excitation at 451 nm. Phosphodiesterase digestions and APFX additions were done as described under “Experimental Procedures.” All experiments are consistent with one FAD and one FMN per NOS monomer. Data are from a representative experiment repeated with a seDarate NOS DreDaration.

Before phosphodiesterase After phosphodiesterase (pH 7.7) (pH 7.7)

pH 7.7 +APFX pH 2.6 pH 7.7 +APFX pH 2.6

of FAD to FMN in NOS. After treatment with phosphodiesterase APFX abolishes all fluorescence for NOS as well as FAD and FMN.

Photoaffinity Labeling of NOS with Calmodulin-NOS catalytic activity is absolutely dependent upon calmodulin, which produces half-maximal stimulation of enzyme activity at 10 nM (19). This suggests that NOS possesses a high affinity binding site for calmodulin. To directly test this possibility, we incubated a crude soluble preparation of rat cerebellum or purified NOS with 12sI-calmodulin linked to a photoaffinity ligand (Fig. 5). In the presence of EGTA we do not observe any 12sI-calmodulin labeling of purified or crude NOS. How- ever, in the presence of l p M calcium, a single intense 180- kDa radiolabeled band is observed both with purified NOS and the crude preparation. The radiolabeling of NOS with 12sI-calmodulin is a high affinity saturable process, as incubation with 50 nM unlabeled calmodulin prior to exposure to the photoaffinity calmodulin ligand abolishes labeling of NOS. In crude preparations, even in the presence of EGTA, several bands are labeled by the 12sI-calmodulin photoaffinity ligand.

DISCUSSION

One of the major findings of this study is that NOS is stoichiometrically phosphorylated by PKA, PKC, and CaM- K, with each enzyme predominantly phosphorylating a distinct serine. Phosphorylation of enzymes represents a means of rapidly regulating their enzymatic activity. Purified NOS rapidly loses enzymatic activity (19), and we have not detected changes in NOS activity following in vitro phosphorylation. Very recently, others (34) demonstrated in vitro phosphorylation of brain NOS by PKC and CaM-K-11. These workers found a modest increase in NOS activity following PKC phosphorylation and a dramatic decrease in NOS activity following CaM-K-I1 phosphorylation. However, it was not clear from this study whether pretreatment of the NOS preparation with calcium and/or calmodulin alone caused insta-

- a - b - c P

C P C P - - +

97-

66- 50nM FAD 5 5 25 33 0 27 50nM FMN 35 0 26 36 0 28 50nM NOS 42 6 53 71 0 55

43- fluorescence is unaffected. The fluorescence of 50 nM denatured NOS in the presence of APFX is reduced to levels identical to those provided by 50 nM FAD. The difference between NOS fluorescence at pH 7.7 in the absence and presence of APFX equals the fluorescence of 50 nM FMN. This suggests that each monomer molecule of NOS possesses one bound FAD and one bound FMN molecule. To further establish the identity of the flavins associated with NOS, we have treated NOS with a phosphodiesterase which converts FAD to FMN. After treating 50 nM FAD with the phosphodiesterase, its fluorescence increases 6-7-fold to levels of 50 nM FMN at pH 7.7, whereas no change occurs at pH 2.6. At pH 7.7, phosphodiesterase treatment augments NOS fluorescence by l.&fold, an amount consistent with the conversion

97-

66-

43-

97-

66-

+ Calcium

+ EGTA + Calcium FIG. 5. Calmodulin photoaffinity labeling of NOS. Crude cy-

tosol ( C ; 100 pgllane) or purified NOS (P; 1 pgllane) were incubated with HSBA-linked 12sI-calmodulin, exposed to UV light, and labeled protein bands were autoradiographed following SDS/PAGE. a, in the presence of EGTA (1 mM) several bands are labeled in the crude cytosol with no labeling of purified NOS. b, inclusion of 10 p M free calcium alters the pattern of labeling in the crude cytosol and, with purified NOS, yields an intensely labeled 180-kilodalton band corresponding to 12sI-calmodulin-HSBA linked to NOS. c, the cross-linking interaction is saturable, as inclusion of 50 nM nonradioactive calmodulin (+) fully displaces the labeling of purified NOS in its absence (-1.


bility of NOS enzymatic activity. We find that phosphorylation of NOS following activation of PKC in intact cells leads t o a dramatic decrease in NOS catalytic activity. This finding is consistent with previous physiologic data, demonstrating that phorbol esters inhibit acetylcholine (35) and calcium ionophore (36)-stimulated endothelium-derived relaxing factor release. Thus, PKC phosphorylation similarly regulates NOS activity in neurons and endothelial cells. Stimulation of phosphoinositide turnover in a NOS-containing cell should have two opposing effects on NOS activity. The increased intracellular calcium due to inositol trisphosphate production activates NOS through calmodulin, whereas the increase in diacylglycerol stimulates PKC diminishing NO generation. This down-regulation of NOS activity by PKC represents an important site for "cross-talk'' between the phosphoinositide and NO signaling systems.

We have demonstrated tightly bound FAD and FMN associated with NOS with one molecule each of FAD and FMN/ molecule of NOS. This fits with our observations from molecular cloning that NOS possesses single consensus recognition sites for FAD and FMN, respectively (22). Very recently porcine brain NOS was found to contain 1 mol each of FAD and FMN (37), whereas murine macrophage NOS was found t o contain 1 mol of FAD and 0.5 mol of FMN (38). In the search for homology with other proteins, the only other mammalian enzyme with substantial amino acid sequence homology to NOS is cytochrome P-450 reductase (39). Like NOS, cytochrome P-450 reductase also has recognition sites for NADPH, FAD, and FMN. To our knowledge these two proteins are the only mammalian enzymes with recognition sites for both FAD and FMN. Sulfite reductase, a bacterial enzyme, also has recognition sites for FAD and FMN and also displays substantial amino acid sequence homology to NOS (40). Why do NOS and cytochrome P-450 reductase uniquely bind both FAD and FMN? Both of these enzymes utilize molecular oxygen to hydroxylate substrates (41). However, other enzymes, such as flavin mono-oxygenase, also hydroxylate substrates utilizing molecular oxygen and only possess a single bound flavin (42). NOS and cytochrome P-450 reductase do have in common a necessity to facilitate one-electron trans- fers during catalysis. Electron shuttling between the flavin isoalloxazine rings in cytochrome P-450 reductase is crucial for its radical catalysis (43), and similar electron shuttling is likely involved in the mechanism of NO synthesis.

Utilizing a photoaffinity ligand, we have directly demonstrated the binding of calmodulin to purified NOS. This fits with the requirement of NOS catalytic activity for calmodulin and its apparent high affinity for the enzyme, with half- maximal stimulation of NOS activity of 10 nM calmodulin (19). The amino acid sequence of NOS derived by molecular cloning reveals a recognition site for calmodulin (22). Al- though many enzymes bind calmodulin, to our knowledge NOS is the only oxidative enzyme requiring calmodulin for catalytic activity. Calmodulin serves an important function in regulating NOS activity in response to synaptic transmission. In brain slices the excitatory neurotransmitter glutamate stimulates enhancement of cyclic GMP levels acting via NMDA subtype glutamate receptors (7, 8). This action of

glutamate appears to be mediated by NO, since selective inhibitors of NOS block NMDA stimulation of cyclic GMP levels. At NMDA receptors glutamate selectively opens calcium channels. Calcium entering the neurons binds to calmodulin, which then activates NOS. The NO generated binds to iron at the active site of guanylyl cyclase, increasing its catalytic activity and leading to enhanced formation of cyclic GMP (44).

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