©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Brain

L

-Glutamate Decarboxylase

INHIBITION BY PHOSPHORYLATION AND ACTIVATION BY DEPHOSPHORYLATION (*)

(Received for publication, November 17, 1994; and in revised form, January 19, 1995 )

Jun Bao (1)(§) Wai Yiu Cheung (2) Jang-Yen Wu (1)(¶)

From the  (1)Department of Physiology and Cell Biology, University of Kansas, Lawrence, Kansas 66045 and the (2)Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously we showed that the activity of the -aminobutyric acid-synthesizing enzyme L-glutamate decarboxylase (GAD) in crude brain extract is inhibited by ATP and protein phosphatase inhibitors. We suggested that GAD activity is regulated by protein phosphorylation. In this paper we further present evidence to support our hypothesis that protein kinase A and calcineurin may be involved in regulation of GAD activity through phosphorylation and dephosphorylation of GAD, respectively. In addition, the effect of neuronal stimulation on GAD activity in cultured neurons is also included. A model to link neuronal excitation and activation of GAD by Ca-dependent phosphatase is proposed.


INTRODUCTION

-Aminobutyric acid (GABA) (^1)has been established as the major inhibitory neurotransmitter in the vertebrate central nervous system(1, 2) . The rate-limiting step in GABA biosynthesis is the decarboxylation of L-glutamate by L-glutamate decarboxylase (EC 4.1.1.15; GAD)(3) . The level of GABA in the brain is regulated by GAD(4) . Many neurological disorders, such as seizures, Huntington's chorea, Parkinson's disease, stiff-man syndrome, and schizophrenia, have been shown to be related to alterations of the GAD level in the brain(5, 6, 7, 8, 9, 10) . Despite its importance, the precise mechanism underlying the regulation of GAD activity is still not clear.

Reversible phosphorylation is a common mechanism by which the functions of many proteins are regulated in the central nervous system (for review, see (11) ). Regulation of neurotransmitter synthesis by phosphorylation of their synthesizing enzymes has been shown in other neurotransmitter systems(12, 13, 14, 15, 16) . Previously we showed that GAD activity is inhibited by conditions favoring protein phosphorylation, and the inhibition is reversed by phosphatase treatment(17) . This observation suggests that GAD activity is regulated by reversible phosphorylation.

To study further the mechanism of the regulation of GAD activity by protein phosphorylation, we conducted experiments to determine the extent of GAD phosphorylation and GAD activity in crude as well as purified preparations using various protein kinases, phosphatase, and their specific inhibitors. In addition, the effect of neuronal stimulation on GAD activity in cultured neurons is also included. Based on the results presented in this paper and those reported in the literature, a model is proposed to link neuronal excitation to activation of GAD through Ca-dependent dephosphorylation.


EXPERIMENTAL PROCEDURES

Materials

Fresh porcine brains were obtained from a local slaughter house. Triton X-100, 1,2-diolein, L-alpha-phosphatidyl-L-serine, cAMP-dependent protein kinase (protein kinase A; PKA), PKA inhibitory peptide (PKI), PKA catalytic subunit, calmodulin, and calcineurin were from Sigma. Protein kinase C (PKC) and PKC inhibitory peptide were from Upstate Biotechnology, Inc. Okadaic acid was from Moana Bioproducts. Radioisotopes were purchased from Dupont NEN.

Preparation of Synaptosome

Preparation of crude synaptosome was performed as described before(17) . Briefly, fresh porcine brains were homogenized in 0.32 M sucrose (w/v, 15 g/100 ml), and the homogenate was centrifuged at 1,000 times g for 10 min. The supernatant solution was collected and centrifuged at 12,000 times g for 30 min. The resulting supernatant liquid was discarded, and the pellet was gently suspended in Krebs-Ringer phosphate buffer (123 mM NaCl, 3 mM KCl, 0.4 mM MgCl(2), 0.5 mM NaH(2)PO(4), 0.25 mM Na(2)HPO(4), and 1 mg/ml glucose, pH 7.2). The suspension was divided into aliquots for the subsequent experiments.

Phosphorylation of GAD in Synaptosomal Fractions

Aliquots of synaptosomal suspension were subjected to osmotic shock by dilution in water and sonicated. The suspension was adjusted to give a final concentration of 50 mM Tris-Cl, pH 7.3, containing 1 mM 2-aminoethylisothiouronium bromide (AET), 25 mg/ml pepstatin A, 0.1 mg/ml trypsin inhibitor, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine. The mixture was preincubated at 30 °C for 5 min under following conditions: (A) and (D), buffer only; (B), plus 50 µg/ml PKA inhibitory peptide (C), plus 50 µg/ml PKC inhibitory peptide; (E), plus 30 nM okadaic acid; (F), plus 10 µM okadaic acid. The mixtures were incubated further at 37 °C for 1 h in the phosphorylation solution, which included 0.1 mM theophylline, 4 mM EGTA, 5 mM MgCl(2), 0.1 mM cAMP, and 0.1 mM ATP (125 µCi/ml [-P]ATP). In (D)-(F), 4 mM EGTA was replaced by 0.1 mM CaCl(2). The incubation was terminated by centrifugation at 100,000 times g for 45 min, and the supernatants obtained were applied to immunoaffinity anti-GAD IgG columns. After washing with 500 mM potassium phosphate buffer, pH 7.2, containing 4 mM EGTA, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 5 mM benzamidine, GAD was eluted out with 0.2 M acetate buffer, pH 2.0, containing 4 M urea. The eluates were dialyzed against 10 mM potassium phosphate buffer, pH 7.2, containing 1 mM AET, 1 mM aminooxyacetic acid, 4 mM EGTA, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine overnight prior to the separation on a 10% SDS-PAGE. The SDS-PAGE was stained, dried, and subjected to autoradiography. The anti-GAD IgG columns were prepared as described previously(17) .

For GAD assay, the synaptosomes were prepared, lysed, and incubated under various conditions as described above except that no [P]ATP was included. After centrifugation at 100,000 times g, pyridoxal 5`-phosphate was added to the supernatants to give a final concentration of 0.2 mM. Aliquots of the supernatants were used for GAD assays and protein determinations as described previously(17) . No ATP and cAMP were included in the control condition.

Phosphorylation and Dephosphorylation of Purified GAD

A porcine brain was homogenized in 5 mM potassium phosphate, pH 7.2 (w/v, = 1 g/10 ml), as described previously(17) . After centrifugation at 100,000 times g for 45 min, the supernatant was applied to an anti-GAD IgG column. The column was washed extensively with 50 and then 500 mM potassium phosphate, pH 7.2, to remove nonspecifically absorbed proteins. Then the GADbulletanti-GAD IgG complex, which was still attached to the resin, was suspended in 50 mM potassium phosphate, pH 7.2, and divided evenly. Each aliquot containing GADbulletanti-GAD IgG resin was packed into a small column for subsequent experiments. For PKA treatment, the column was first equilibrated with 50 mM Tris-Cl, pH 7.4, 5 mM MgCl(2), and 0.1 mM cAMP. After equilibration, the column was stopped, and the resin was suspended inside the column with 150 µl of equilibration buffer containing 0.4 mM ATP (300 µCi/ml [-P]ATP) and 125 units of catalytic subunit of PKA. After gently mixing, the columns were then kept at 30 °C for 1 h. For PKC treatment, the same procedure was used except that the catalytic subunit of PKA was replaced by 200 ng of PKC, and the equilibration buffer was changed to PKC buffer, which consisted of 50 mM Tris-Cl, pH 7.4, 0.1 mM CaCl(2), 5 mM MgCl(2), 0.03% Triton X-100, 0.31 mg/ml L-alpha-phosphatidyl-L-serine, and 0.06 mg/ml 1,2-diolein. After phosphorylation, the columns were washed with 500 mM potassium phosphate to remove kinases and ATP. In case additional treatment with phosphatase was desirable, the columns were further equilibrated with calcineurin buffer, which included 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.1 mM ZnCl(2), and 1 mM CaCl(2). For phosphatase treatment, the column was stopped, and the resin was suspended inside the column with 150 µl of the above equilibration buffer containing 50 µg of calcineurin and 100 µg of calmodulin or 150 units of calf intestinal alkaline phosphatase. After gentle mixing, columns were kept at 30 °C for 1.5 h, followed by extensive wash with 50 mM and then 500 mM potassium phosphate, containing a mixture of phosphatase inhibitors (2 mM NaF, 200 µM vanadate, 200 µM sodium pyrophosphate, 2 mM EDTA, and 2 mM EGTA). Elution and detection of phosphorylated GAD were performed as described previously(17) .

For the GAD assay, the procedure was the same as described above except that no [P]ATP was included, and GAD was not eluted from the resin. Instead, the resin was suspended in 50 mM potassium phosphate, pH 7.2, containing a mixture of phosphatase inhibitors, 1 mM AET, and 0.2 mM pyridoxal 5`-phosphate. The suspension was used for the GAD assay as described previously(17) . Neither kinases nor phosphatases were added in the control condition.

Effect of Neuronal Stimulation on GAD Activity

Primary neuronal cultures were prepared from brain of 16-day-old rat embryos as described previously(18) . Briefly, cultures of 14-16 days in vitro were divided into six groups with 15 plates/group and subjected to the following treatments.

Group I

Control, cultures were grown in Ca-free EBSS medium (116 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO(4), 1 mM NaH(2)PO(4), 26.3 mM NaHCO(3), 5.5 mM glucose).

Group II

This group was the same as group I except that 2 mM Ca was included.

Group III

This group was the same as group I except that cultures were stimulated by high K stimulation (the same EBSS medium plus 55 mM K) for 5 min.

Group IV

This group was the same as group II except that cultures were stimulated as in group III.

Group V

This group was the same as group IV except that 30 nM okadaic acid was included.

Group VI

This group was the same as group V except that okadaic acid concentration used increased from 30 nM to 10 µM. Cultures were harvested, sonicated in GAD buffer (50 mM potassium phosphate, 1 mM AET, and 0.2 mM pyridoxal 5`-phosphate, pH 7.2) containing protease and phosphatase inhibitors, centrifuged, and assayed for GAD activity.


RESULTS

Effects of Protein Kinase and Phosphatase Inhibitors on Phosphorylation of GAD in Synaptosomal Fractions

In the synaptosomal preparations, GAD was phosphorylated by endogenous kinases with [-P]ATP (Fig. 1, lane A). PKI blocks GAD phosphorylation (Fig. 1, lane B), whereas the inhibitor of PKC had no effect (Fig. 1, lane C). Furthermore, GAD phosphorylation was greatly reduced by replacing EGTA with 0.1 mM Ca (Fig. 1, lane D), presumably through activation of a Ca-dependent phosphatase. This dephosphorylation of GAD was inhibited by okadaic acid at 10 µM but not at 30 nM (Fig. 1, lanes E and F).


Figure 1: Effect of protein kinase inhibitors, phosphatase inhibitors, and Ca on P incorporation into GAD in the synaptosomes. Lanes A-F are autoradiograms of GAD from synaptosomal fractions under various conditions. Phosphorylation was performed in the lysed synaptosomal preparations under the following conditions: lane A, buffer only; lane B, +PKA inhibitor, PKI; lane C, +PKC inhibitor; lane D, +Ca; lane E, +Ca plus 30 nM okadaic acid or lane F, Ca plus 10 µM okadaic acid. At the end of incubation, GAD was isolated from synaptosomal preparations, purified by an anti-GAD immunoaffinity column, and identified by SDS-PAGE autoradiography as detailed under ``Experimental Procedures.'' Arrows indicate both subunits of GAD, 59 kDa and 64 kDa, are phosphorylated. The molecular mass is expressed in kDa.



Effect of Phosphorylation and Dephosphorylation on GAD Activity in Synaptosomal Fractions

The state of phosphorylation of GAD was found to be inversely correlated with its activity (Table 1). GAD activity decreased when the GAD was in the phosphorylated state. Inhibition of GAD phosphorylation or activation of GAD dephosphorylation markedly increased GAD activity.



Phosphorylation and Dephosphorylation of Purified GAD

To ascertain that the above observations are not due to interference from substances present in the crude extract, similar studies were conducted using a purified GAD preparation. Previously we reported one-step purification of GAD using immunoaffinity column chromatography(19) . We used the GAD associated with anti-GAD IgG resin instead of the GAD eluted from the resin as the GAD sample for studies reported here. There are two advantages of using GADbulletanti-GAD IgG resin for studies of GAD activity under various conditions. First, 85% of the GAD was inactivated by elution under acidic condition to dissociate the GADbulletanti-GAD IgG complex(19) , whereas no loss of GAD activity was observed using the resin-bound GAD directly for GAD assays. Second, it was more efficient to remove interfering substances from immobilized GADbulletanti-GAD IgG complex than from GAD in solution. For instance, during the phosphatase experiment, the kinase can be easily removed simply by washing the kinase from the resin.

It was found that the purified GAD was phosphorylated by PKA ( Fig. 2lane A) but not PKC (Fig. 2, lane D). Phosphorylated GAD could be dephosphorylated by treatment with calcineurin (Fig. 2, lane B). Similarly, phosphorylated GAD could be dephosphorylated by calf intestinal alkaline phosphatase, but to a lesser degree (Fig. 2, lane C). It was estimated that there is 0.47 P(i)/subunit of GAD protein from PKA-mediated phosphorylation.


Figure 2: Demonstration of P incorporation in purified GAD preparation by PKA but not PKC and subsequent dephosphorylation by calcineurin. Purified GAD in the form of a GADbulletanti-GAD resin complex was treated under various conditions. Elution of GAD from the anti-GAD immunoaffinity column and detection of phosphorylated GAD by SDS-PAGE were detailed under ``Experimental Procedures.'' In addition, about 0.5 µg of GAD purified by an anti-GAD immunoaffinity column was also analyzed on the same SDS-PAGE and visualized by silver staining as described previously (17) . Lane A, immunoaffinity-purified GAD treated with the catalytic subunit of PKA in the presence of [-P]ATP, cAMP and Mg-ATP. Lane B, P-labeled GAD in lane A treated with calcineurin in the presence of Ca/calmodulin. Lane C, P-labeled GAD in lane A treated with alkaline phosphatase. Lane D, immunoaffinity-purified GAD treated with PKC in the presence of [-P]ATP, Mg-ATP, and PKC activators. Lane E, purified GAD showing both 59- and 64-kDa subunits.



Effects of Phosphorylation and Dephosphorylation on GAD Activity in Purified GAD Preparations

Again, the activity of GAD was found to be inversely correlated with its state of phosphorylation. Inhibition of GAD activity was observed by treatment with PKA, but not PKC. Furthermore, PKA-mediated inhibition of GAD activity was restored by calcineurin or calf intestinal alkaline phosphatase treatment (Table 2).



Effect of Neuronal Stimulation on GAD Activity

As shown in Table 3, a marked increase of GAD activity was observed in high K stimulation in the presence of 2 mM Ca. No activation of GAD was observed by high K stimulation in the absence of Ca. Furthermore, okadaic acid at 30 nM, which is high enough to inhibit protein phosphatase type 1 (IC = 20 nM) and type 2A (IC = 0.2 nM) (20) , had no effect on high K-induced activation of GAD. However, at a high concentration (10 µM) okadaic acid did inhibit high K-induced activation of GAD presumably because of inhibition of protein phosphatase 2B (also called calcineurin; IC = 5 µM(20) ]).




DISCUSSION

In our previous report we showed that GAD activity in crude brain preparations is inhibited by ATP or phosphatase inhibitors(17) . Furthermore, direct phosphorylation of GAD has also been demonstrated in lysed synaptosomal preparations in the presence of [-P]ATP but not [alpha-P]ATP, suggesting that inhibition of GAD activity by ATP is through phosphorylation of the GAD molecule and not by competing with pyridoxal 5`-phosphate for the binding to GAD apoprotein as proposed(21, 22) . In this paper, we have presented evidence to suggest that PKA and calcineurin might be the protein kinase and protein phosphatase responsible for phosphorylation and dephosphorylation of GAD, respectively. Moreover, the activity of GAD appears to be inversely correlated with the extent of GAD phosphorylation; namely, GAD is inhibited when it is phosphorylated and is activated when it is dephosphorylated. Several lines of evidence support our hypothesis that PKA is involved in GAD phosphorylation and regulation of GAD activity. First, phosphorylation of GAD could be demonstrated in both crude as well as purified GAD preparations by catalytic subunit of PKA. Second, GAD phosphorylation by PKA is completely blocked by PKI, a specific inhibitor of PKA. Third, GAD activity is reduced by PKA-mediated phosphorylation, and this inhibition can be blocked by PKI.

It seemed unlikely that Ca-dependent kinases, such as PKC and calcium calmodulin-dependent kinase, might play a significant role in GAD phosphorylation and regulation of GAD activity. This notion is based on the following observations. First, the conditions used for phosphorylation contained 4 mM EGTA, and hence it is unlikely that Ca-dependent enzymes, e.g. PKC or calmodulin-dependent kinase, are still active under such conditions. However, it must be pointed out that in the presence of activator, e.g. phorbol ester, PKC activity can be increased greatly even in the presence of a very low concentration of Ca(23) . Also, it is known that calmodulin-dependent kinase II can undergo pseudo-irreversible activation by autophosphorylation, which renders calmodulin-dependent kinase II independent of Ca and calmodulin(24) . Hence it is conceivable that even in the presence of EGTA, PKC and calmodulin-dependent kinase II could be still active and participate in GAD phosphorylation. Second, PKC fails to catalyze incorporation of P into GAD protein either in a crude synaptosomal preparation or in a highly purified GAD preparation. Furthermore, PKC or PKC inhibitory peptide has little effect on GAD activity. Third, it has been shown from immunocytochemical and in situ hybridization studies that calmodulin-dependent kinase II and GAD protein or GAD mRNA have different regional and cellular distributions in the brain(25, 26) , suggesting that calmodulin-dependent kinase II is unlikely to be localized in GABA-ergic neurons and thus involved with phosphorylation and regulation of GAD activity.

There are four major types of serine/threonine protein phosphatases: type 1 (protein phosphatase-1) and type 2, which includes three subtypes A, B, and C (protein phosphatases 2A, 2B, and 2C)(27) . These protein phosphatases can be distinguished by their sensitivity to a protein phosphatase inhibitor, okadaic acid(20, 28) . Okadaic acid is a potent inhibitor for protein phosphatases 1 (IC = 20 nM) and 2A (IC = 0.2 nM). Protein phosphatase 2B is inhibited by okadaic acid at a higher concentration (IC = 5 µM), whereas 2C is unaffected by okadaic acid at a concentration as high as 10 µM(20, 28) . Since dephosphorylation of GAD is inhibited by okadaic acid at 10 µM but not at 30 nM (Fig. 1, lanes E and F), it is likely that protein phosphatase 2B (calcineurin) is the phosphatase responsible for GAD dephosphorylation. This is consistent with our previous observation that EGTA or a calcineurin-specific inhibitor, deltamethrin, prevented GAD activation in the crude extract(17) . In addition, it has also been shown that activation of GAD is Ca-facilitated(22) . The fact that calcineurin can dephosphorylate and activate purified GAD in vitro further supports the above notion. Since calcineurin is the major Ca-dependent phosphatase in the brain (29) and has been suggested to be localized in GABA-ergic neurons(30) , it seems reasonable to suggest that calcineurin plays an important role in the GABA synthesis through its activation of GAD activity by dephosphorylation.

In addition to in vitro studies, evidence obtained from in vivo studies as reported in this paper showing that GAD activity in cultured neurons is enhanced by high K stimulation in the presence of Ca, but not in Ca-free media, is also compatible with the above notion. All of the observations discussed above are consistent with our working hypothesis that when GABA-ergic neurons are stimulated, Ca ions enter the nerve terminals through voltage-dependent Ca channels, followed by the release of GABA. The influx of Ca then triggers activation of Ca-dependent phosphatase (one of the candidates is calcineurin) which in turn activates GAD by dephosphorylation, resulting in an increase in GABA synthesis to replenish GABA depletion due to the release.

In summary, phosphorylation by PKA inhibits GAD activity, and dephosphorylation by calcineurin activates it. However, more investigations are required to identify definitely the endogenous kinase and phosphatase responsible for GAD regulation in the brain.


FOOTNOTES

*
This study was supported in part by National Science Foundation Grant BNS-8820581 and National Institutes of Health Grants NS 20978 and NS 20922; by the Marion Merrell Dow Foundation, American Heart Association, Kansas; and by The Office of Naval Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Neurology, Johns Hopkins University, School of Medicine, Baltimore, MD 21287.

To whom correspondence should be addressed: Dept. of Physiology and Cell Biology, University of Kansas, Haworth Hall, Lawrence, KS 66045. Tel.: 913-864-4557; Fax: 913-864-5374.

(^1)
The abbreviations used are: GABA, -aminobutyric acid; GAD, L-glutamate decarboxylase; PKA, cAMP-dependent kinase; PKI, PKA inhibitory peptide; PKC, protein kinase C; AET, 2-aminoethylisothiouronium bromide; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Drs. D. Deupree and M. Yarom for a critical review of the manuscript and Jan Elder and Judy Wiglesworth for typing the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.