calmodulin-dependent phosphorylation of proteins in the postsynaptic density of adult rat cerebral cortex

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MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 40 (1996) 22 26

Research report

Nitric oxide increases calcium/calmodulin-dependent phosphorylation of proteins in the postsynaptic density of adult rat cerebral cortex Kuo Wu a.*, Jia-ling Xu a, Piin-chau Suen ~', Yung-yu Huang b, Howard T.J. Mount a a Department ofNeuroscience attd Cell Biology, Robert Wood Johnson Medical School, UMDNJ, Piscataway, NJ 08854, USA b Department of Neuroscieme. New York State Psychiatric hlstitute and Department of Psychiato', Columbia UniversiO' Medical College of Physicians and Surgeons, New York, NY 10032, USA Accepted 3 January 1996

Abstract

Nitric oxide (NO) plays important roles in diverse processes, including neurotransmission in the peripheral and central nervous systems. Nitric oxide synthase (NOS), the enzyme that catalyzes formation of NO from t.-arginine, is an intrinsic component of the postsynaptic density (PSD), a specialization of the postsynaptic membrane. This raises the possibility that NO may play a role in postsynaptic function. To begin defining postsynaptic actions of NO, we examined effects of NO on Ca2+/calmodulin-dependent phosphorylation (C/C-DP) of proteins in the cortical PSD of adult rat brain. Treatment of the PSD with sodium nitroprusside, a NO donor, caused a 4-fold increase in C/C-DP of the major PSD protein (mPSDp), relative to C / C treatment alone. Another NO donor, S,S'-dinitrosodithiol, elicited a 2-fold increase in C/C-DP of the mPSDp. Treatment of PSD fractions with L-arginine, a substrate for endogenous NOS, caused a 3-fold increase in C/C-DP activity. The competitive NOS inhibitor, N-L-arginine-methyl ester, decreased basal C/C-DP of cortical mPSDp by 50% and blocked the increase elicited by L-arginine. The inhibitor had no effect on cAMP-dependent phospborylation, suggesting specificity of NO action on C/C-DP. Our observations indicate that NO enhances C/C-DP of PSD proteins. As C/C-DP inactivates NOS, our findings raise the possibility that NO effects on C/C-DP constitute a feedback mechanism for regulation of NOS activity. Keywords: L-Arginine; N-L-Arginine-methyl ester: Calcium/calmodulin-dependent phosphorylation: Major postsynaptic density protein; Nitric oxide; Nitric oxide synthase: Sodium nitroprusside: Synapse

I. Introduction Nitric oxide (NO) is a gaseous transmitter involved in diverse processes [8,9,13,16,32]. Within the CNS, NO may act as a retrograde messenger in long-term potentiation [3,19,31,33] and may also be involved in long-term depression ([12,35]; but see [24]). In addition, NO has been implicated in a variety of disorders of the nervous system, including dementia associated with AIDS [14,25], Alzheimer's and Huntington's diseases [28], and migraine-associated pain [38]. Under physiologic circumstances, NO is synthesized from L-arginine by NO synthase (NOS, EC 1.14.23) [29]. Distinct forms of NOS have been cloned from rat brain [5], endothelium [20,22,34] and macrophages [26,27,44]. Primary amino acid sequence motifs common to all three forms include sites of protein phosphorylation as well as

Corresponding author. Fax: + I (908) 235-4990. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S 0 1 6 9 - 3 2 8 X ( 9 6 ) 0 0 0 2 8 - 9

binding sites for calmodulin, NADPH, FAD and FMN. Calmodulin and NADPH are required for catalytic activity of purified NOS [7]. NOS is phosphorylated by cAMP-dependent protein kinase (PKA) [4,10], protein kinase C (PKC) [4,30] and Ca2+/calmodulin-dependent protein kinase II [4,30]. Phosphorylation by calmodulin kinase II [4,30] dramatically reduces NOS catalyic activity, whereas phosphorylation by PKA apparently does not affect NO biosynthesis [4,10]. The effects of PKC remain unclear. Both increases [30] and decreases [4] in NOS activity have been reported, upon activation of PKC. Recently, NOS was found to be an intrinsic component of the postsynaptic density (PSD) [1,45], a disc-shaped proteinaceous structure attached to the inner surface of the postsynaptic membrane. The PSD also contains several s e r i n e / t h r e o n i n e protein kinases, including P K A [11,36,43], PKC [37] and calmodulin kinases [11,36,41,43], as well as protein tyrosine kinases [18,23]. Because of the localization of NOS with these enzymes, kinases intrinsic

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K. Wu et al. / Molecular Brain Research 40 (1996) 22-26

to the PSD may catalyze phosphorylation of NOS. In the present study, we examined whether NO can in turn modulate phosphorylation of postsynaptic proteins. We report that NO selectively enhances Ca2+/calmodulin-de pendent phosphorylation (C/C-DP) of PSD proteins.

2. Materials and methods 2.1. Materials [~/-33p]ATP (1000-3000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). L-arginine, N-Larginine-methyl ester (N-LAME) and sodium nitroprusside were from Sigma Chemical (St. Louis, MO). S,S'-dinitrosodithiol was a product of Calbiochem (San Diego, CA). Other chemicals were of reagent grade and obtained from regular commercial sources. 2.2. Animals and preparation of the PSD Adult Sprague-Dawley rats were used in all experiments. The animals were killed by exposure to CO 2 vapor and their brains were removed. Cerebral cortices were dissected and stored at - 8 0 ° C until use. Differential centrifugation and Triton X-100 treatment were used to obtain highly purified PSD fractions, as previously described [40,42]. 2.3. Ca2 +/ calmodulin - and cAMP-dependent phosphorylation of PSD proteins Our method for assaying C / C - D P of proteins in the PSD has been described previously [11]. In brief, 50 Ixg samples of the PSD fraction (in a total volume of 100 txl, containing l0 mM MgC12, 1 Ixg leupeptin, 1 txg aprotin, 2 Ixg soybean trypsin inhibitor, 0.5 mM L-microcystin, 1 mM PMSF, 50 mM 1,4-piperazinediethanesulfonic acid/KOH, 1 mM dithiothreitol and 0.2 mM EGTA, pH 7.0) were preincubated at 30°C for 3 min and then incubated with 13 IxM (~/-33p)ATP (5-10 IxCi/sample) in the presence or absence of 0.5 mM Ca 2+ and 30 ~ g / m l calmodulin for 5 or 10 min. To terminate phosphorylation, 30 Izl aliquots of stop solution (0.35 M Tris-HC1, 20% v / v glycerol, 12% mercaptoethanol, 8.7% SDS and 0.1% bromphenol blue, pH 6.7) were added. The reaction mixtures were subjected to SDS-PAGE and autoradiographic analysis. Major postsynaptic density protein (mPSDp), a substrate of C / C - D P within PSD, served as a reference band (M.W. = 51 kDa) for quantitation of NO effects on C / C - D P activity. A similar method was used to assay cAMP-dependent phosphorylation (cAMP-DP). Samples were pre-incubated as above and then incubated with 13 ~M (~/-33p)ATP in the presence of 10 I~M cAMP and 1 mM 3-isobutyl-1methyl-xanthine. Synapsin I (Syn I, 78-81 kDa), a PKA substrate that has been identified both presynaptically

[2,15,39] and within the PSD [2,11,21,36,39], provided a reference band for quantitative assessment of NO effects on cAMP-DP. 2.4. NO donor and NOS inhibitor treatments To examine the effect of NO on C / C - D P of PSD proteins, 1 mM sodium nitroprusside or 0.1 mM S,S'-dinitrosodithiol was included in the reaction mixture. In other experiments, NOS intrinsic to the PSD was used to catalyze the production of NO from exogenous substrate. For the latter experiments, 10-min phosphorylation assays were performed in the presence of L-arginine, 1 p.M FMN, 1 p.M FAD and 1 p.M NADPH. A NOS inhibitor, N-Larginine methyl ester (N-LAME; 0.5 mg/ml), was used to confirm the involvement of NOS in parallel assays. In some experiments, we studied effects of tissue NOS inhibition on subsequent levels of basal C / C - D P and cAMP-DP in isolated PSD. Each cerebral cortex was chopped into eight pieces and pre-incubated for 0, 5 or 10 min (37°C) in 10 vols. of buffer (0.32 M sucrose, 1 mM NaHCO 3, 1 mM MgC12 and 0.5 mM CaC12), with or without N-LAME (0.5 mg/ml). PSD fractions were isolated, pre-incubated for 3 min in the absence of N-LAME, and then subjected to the 3-min phosphorylation assay, described above. As ATP was added after pretreatment with the NOS inhibitor, post-hoc phosphorylation was indeed measured in these experiments. 2.5. Quantitation A scanning densitometer (CliniScan, Helena Laboratories, Beaumont, TX) was used to compare intensity of the mPSDp reference band, under a variety of experimental conditions, to control (absence of phosphorylation activa1

234 4 - 300 kDa 4 - - 240 kDa

4-

mPSDp

4-- 30 kDa

Ca=*/CaM: SNP:

-

-I- + -

-

-

"l''l"

Fig. 1. Effects of sodium nitroprusside on C / C - D P of PSD proteins. PSD samples were incubated with [~-33 P]ATP for 5 min in the presence (lanes 2 and 3) or absence (lanes 1 and 4) of Ca2+/calmodulin, with (lanes 3 and 4) or without (lanes 1 and 2) 1 mM sodium nitroprusside (SNP). Reaction mixtures were subjected to SDS-PAGE and analyzed autoradiographically. SNP treatment enhanced C / C - D P of several proteins with molecular weights between 30 kDa and 300 kDa (compare lanes 2 and 3).

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K. Wu et al. / Molecular Brain Research 40 (1996) 22-26

1 234

mPSDp

Ca2+/CaM: S,S'-DND:

-

- -P -I-I- - "l"

Fig. 2. Effects of S,S'-dinitrosodithiol (S,S'-DND) on C / C - D P of PSD proteins. PSD samples were incubated with (~_3~ P)ATP for 5 min in the presence or absence of S,S'-DND. Treatment with this NO donor increased C / C - D P of mPSDp 2-fold relative to Ca2+/calmodulin alone (compare lanes 3 and 4).

PSD with 1 mM sodium nitroprusside for 5 min increased C / C - D P of several proteins (M.W. = 30-300 kDa). The most prominent of these bands was that of mPSDp, itself a Ca2+/calmodulin-dependent kinase. Sodium nitroprusside induced a 4-fold increase in C / C - D P of mPSDp, relative to treatment with Ca2+/calmodulin alone (Fig. 1, lanes 2 and 3). In the absence of exogenous Ca 2+ and calmodulin, sodium nitroprusside had no effect on basal phosphorylation of PSD proteins (Fig. 1, lane 4). To ascertain whether NO itself was responsible for the increase in C / C - D P , we repeated the experiment with another NO donor, S,S'-dinitrosodithiol. A 5-min treatment with S,S'-dinitrosodithiol (0.1 mM) doubled phosphorylation in the presence of Ca2+/calmodulin (Fig. 2, compare

1

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2

3 4

5

6

7 8

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tors, NO donors or NOS inhibitor). For analysis of NO effects on cAMP-DP, Syn I provided the reference band.

4-- mPSDp

3. Results

3.1. Effects of sodium nitroprusside and S,S'-dinitrosodithiol on C / C-DP of PSD proteins To begin examining the effect of NO on C / C - D P , we used sodium nitroprusside as a NO donor. Treatment of

Inc. time (min): N-LAME: Ca2+/CaM:-

0

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I , I

I

1 2 3 4 5 6 7 8 9 1 0

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I

Caa+/CaM: L-Arginine, lnM: L-Arginlne, I~M: L-Arglnlne, lmM: N-LAME:

- 4- -(- -I- "4" -F --

- -I- - - - - - - - -I- - - - -I- -I-I-

Fig. 3. Effects of endogenous NO on C / C - D P of PSD proteins. PSD samples were incubated for 10 rain with ['y-33P]ATP in the presence (lanes 2-6) or absence (lane 1) of Ca 2 +/calmodulin with (lanes 3-6) or without (lanes 1 and 2) L-arginine, FMN, and FAD, a n d / o r the NOS inhibitor, N-LAME (0.5 m g / m l ; lane 6). Reaction mixtures were then processed for autoradiographic analysis. Treatment with L-arginine and Ca2+/calmodulin (lane 5) caused a 3-fold increase in C / C - D P of mPSDp relative to treatment with Ca 2+/calmodulin alone (lane 2). The effect of L-arginine was blocked by N-LAME (lane 6).

I

I

I

Inc. time (rain): 1~ 5 N-LAME: - cAMP: - "!" -- -I-

I

I

5 + 4"--- -I- -

10

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Fig. 4. Effects of tissue pretreatment with a NOS inhibitor on post-hoe C / C - D P and cAMP-DP of isolated PSD proteins. A: PSD samples, prepared from cerebral cortex that had been pretreated (0, 5 or 10 min) with (lanes 5, 6, 9 and 10) or without (lanes 3, 4, 7 and 8) the NOS inhibitor, N-LAME, were incubated with ['y-33P]ATP in the presence (lanes 2, 4, 6, 8 and 10) or absence (lanes i, 3, 5, 7 and 9) of Ca2+/calmodulin. Five-minute pretreatment with N L A M E caused a 50% decrease in C / C - D P of mPSDp (compare lanes 4 and 6). Ten-minute pretreatment resulted in similar decrease (compare lanes 8 and 10). B: cAMP-DP of PSD proteins was not affected by 5 rain (lanes 4 and 6) or 10 rain (lanes 8 and 10) pretreatment with N-LAME. The position of Syn 1. a PKA substrate, is indicated.

K. Wu et al. / Molecular Brain Research 40 (1996) 22-26

lanes 3 and 4). In the absence of Ca2÷/calmodulin, S,S'dinitrosodithiol had no effect on basal phosphorylation of proteins in the PSD (Fig. 2, lane 2). These results further implicate NO in the enhancement of C/C-DP. 3.2. Effects of endogenously produced NO on C~ C-DP To determine whether NOS endogenous to the PSD might produce NO that would alter C/C-DP activity within the PSD, experiments were repeated with the physiologic NOS substrate, L-arginine. Treatment of PSD fractions with 1 mM L-arginine (10 min) and the NOS cofactors (calmodulin, FMN, FAD and NADPH) increased C/C-DP of mPSDp 3-fold (Fig. 3, lane 5). Lower concentrations of L-arginine (1 nM and 1 ~M) were ineffective (Fig. 3, lanes 3 and 4). The competitive NOS inhibitor, N-LAME, blocked the increase elicited by 1 mM L-arginine (Fig. 3, lane 6). These data suggest that NO produced within the PSD affects C/C-DP of local targets. 3.3. Effects of a NOS inhibitor on C~ C-DP and cAMP-DP To determine whether endogenous production of NO might play a role in the basal phosphorylation of PSD substrates, we assayed PSD proteins isolated from cortical tissue that had been pretreated with N-LAME (0.5 mg/ml, 0, 5 or 10 min). Tissue pretreatment with the inhibitor decreased basal C/C-DP of mPSDp by approximately 50%, but had no effect on cAMP-DP of PSD proteins (Fig. 4A,B, compare lanes 4 and 6, and lanes 8 and 10).

4. Discussion To date, the only well characterized intracellular targets of NO have been guanylyl cyclase and glyceraldehyde dehydrogenase. NO causes the specific activation of guanylate cyclase via NMDA receptors [6,17] and inactivation of glyceraldehyde-3-phosphate dehydrogenase through ADP-ribosylation [46]. Our data establish the enhancement of C/C-DP as a third intracellular action of NO. Two NO donors, sodium nitroprusside and S,S'-dinitrosodithiol, enhanced PSD protein phosphorylation relative to Ca2+/calmodulin alone, indicating that exogenously derived NO increases PSD C/C-DP activity. The observation that pretreatment with L-arginine, a source of NO in the presence of endogenous NOS, also enhanced C/C-DP of PSD proteins indicates that NO production within the PSD increases local C/C-DP. Pretreatment of cortical tissue with the NOS inhibitor, N-LAME, reduced post-hoc C/C-DP, but did not affect PKA activity in the isolated PSD. The apparent specificity of NO action is interesting as only C/C-DP causes inhibition of NO biosynthesis, though NOS is phosphorylated by both kinases [4]. Our findings raise the possibility that NO effects on C/C-DP might play a role in feedback regula-

25

tion of NOS activity. In addition, these results suggest that endogenous production of NO contributes to the maintenance of basal C/C-DP activity in the PSD. It should be noted that NO enhanced C/C-DP of many PSD proteins, rather than a small number of specific substrates. The most prominent affected protein was the mPSDp, a major PSD protein and a Ca2÷/calmodulin-de pendent protein kinase that phosphorylates both itself and other proteins in the PSD. As the mPSDp may have numerous postsynaptic targets, NO might alter the phosporylation status of multiple proteins, including NOS itself. This possibility remains to be explored. The molecular mechanism by which NO enhances C/C-DP also bears investigation. An obvious possibility is that NO binds to the kinase, changing enzyme conformation to a state more favorable for phosphorylation. The present studies clearly indicate that C/C-DP is a target of NO. Purified NOS can be phosphorylated by Ca2+/calmodulin-dependent protein kinase, resulting in down-regulation of NOS activity [30]. In this context, our findings raise the possibility that enhancement of C/C-DP by NO constitutes a mechanism for feedback regulation of NOS activity.

Acknowledgements We thank Ms. Betty Wheeler for excellent technical assistance. This work was supported by NIH Grant HD 23315 and the Medical Research Council of Canada.

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