Vol. 15, No. 6 November/December I99 I

0145-6008/91/1506-1040$3.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Ethanol Has No Effect on CAMP-Dependent Protein Kinase-, Protein Kinase C-, or Ca*+-CalrnodulinDependent Protein Kinase 11-Stimulated Phosphorylation of Highly Purified Substrates in Vitro Tina K. Machu, Richard W. Olsen, and Michael D. Browning

The actions of ethanol on kinase stimulated phosphorylation were examined using highly purified protein kinases and a variety of purified substrates. Ethanol (25-200 mm) failed to alter the phosphorylationof histone Ila and histone Ills by CAMP-dependentprotein kinase (PKA) and protein kinase C (PKC), respectively. Moreover, ethanol (25-200 mM) did not affect the phosphorylationof synapsin I by Ca’+-celmodulin-dependent protein kinase II (CAM kinase 11). Finally, neither PKA nor PKC stimulated phosphorylation of the GABAA receptor (GABAA-R) was modulated by ethanol at any concentration of ethanol tested. These results suggest that ethanol, in pharmacologicalconcentra~ons,has no direct actions on the ability of these kinases to catalyze the phosphorylationof specific substrate proteins. In particular, ethanol does not appear to directly influence GABAA-R phorphorylation by either PKA or PKC. Key Words: Ethanol, GABAA-R, Phosphorylation.

THANOL HAS NUMEROUS effects at the cellular E level in brain tissue. In particular, ethanol at relatively low concentrations has been shown to modulate ligandgated ion channels, such as the NMDA receptor’s2and the GABAA-R/C1--ionophore ~ o m p l e xThe . ~ stimulatory actions of ethanol on GABAA-R gated C1- flux have been demonstrated both biochemically4*’and electrophysiologi ~ a l l y .However, ~.~ the mechanism through which ethanol produces this response is unknown. One possibility is that ethanol alters the phosphorylation of the GABAA-R. Protein phosphorylation is widely recognized as the primary mechanism for post-translational control of protein activit^,',^ and clear evidence has linked phosphorylation to regulation of receptor function. l o Moreover, recent studies have shown that the purified GABAA-Ris phosphorylated by CAMP-dependent protein kinase (PKA), protein kinase C (PKC), and an as yet uncharacterized kinase.11-14AdFrom the Department ofPharmacology, University of Colorado Health Sciences Center. Denver, Colorado (T.K.M.,M.D.B.) and Department of Pharmacology, University of Calfornia at Los Angeles School of Medicine, Los Angeles, Calijornia (R. W.O.). Received for publication May 13, 1991: accepted July 1. 1991 This work was supported by AA03527 from NIAAA (M.D.B.), NS28772fiom NINDS (National Institute of Neurological Disorders and Stroke) (R. W.O.).and AA05321 from the National Institute on Alcohol Abuse and Alcoholism (T.K.M.). Reprint requests: Tina K. Machu, PhD, Department of Pharmacology, University of Colorado Health Sciences Center, Box C-236, 4200 E. Ninth Avenue, Denver, Colorado 80262. Copyright 0 I991 by The Research Society on Alcoholism. 1040

ditional data suggesting that phosphorylation may underlie the effect of ethanol on GABAA-R function has recently come from studies of the GABAA-R expressed in oocytes.” These authors found ethanol sensitivity required the eight amino acids contained in the yZLsubunit of the receptor complex. Moreover, these eight amino acids contain a consensus sequence site for PKC. The physiological consequences of phosphorylation of the GABAA-R are not yet known. However, the GABAAR bears significant homology with the nicotinic cholinergic receptor,16 in which the rate of desensitization is enhanced by phosphorylation. I’ In addition, two recent reports have provided direct evidence that the catalytic subunit of PKA decreases GABAA-R However, studies are not in agreement whether conditions favoring enhanced phosphorylation of the GABAA-R increase or decrease GABAA-R function.” The mechanisms by which ethanol could influence the in situ phosphorylation of proteins, and the GABAA-R in particular, are not known. Previous studies have suggested that chronic ethanol treatment may influence PKA phosphorylation,’1-26PKC pho~phorylation’~~’’ and Ca’+/calmodulin-dependent protein kinase I1 (CAM kinase 11) pho~phorylation.’~ Ethanol has also been reported to have acute effects on PKA30p3’and PKC phosphorylation system~.~A ’ . ~number ~ of factors could contribute to an ethanol induced change in these studies. For instance, ethanol could directly affect kinase or phosphatase activity or alter the ability of specific proteins to serve as substrates for phosphorylation. Alternatively the levels of kinases, phosphatases, second messengers, and substrates could be changed by ethanol treatment. As a first step in attempting to identify the mechanisms by which ethanol could influence protein phosphorylation, we have examined the ability of ethanol to influence the phosphorylation of purified substrates by purified kinases in vitro. Specifically we assayed the effects of ethanol on the in vitro phosphorylation of substrates such as histone and synapsin I by highly purified forms of PKA, PKC, and CAM kinase 11. Finally we have focussed directly on the GABAA-R and have studied the effects of ethanol on the phosphorylation of this receptor by either PKA or PKC. Alcohol Clin ExpRes, Vol 15. No 6, 1991: pp 1040-1044

LACK OF EFFECT OF ETHANOL ON KINASE ACTIVITY

METHODS Materials The catalytic subunit of PKA was purified from bovine heart as previously described.” PKC was purified as described.35CaM kinase I1 was purified as described,36and was a generous gift of Dr. T. McGuinness. The GABAA-R was purified to near homogeneity as previously deHistone IIa scribed.” Synapsin I was purified as previously desc~ibed.~’ and histone Ills were obtained from US Biochemicals. Calmodulin and calmidazolium were purchased from Sigma. [ y-”P]-ATP was purchased from 1CN. The inhibitor of PKA was prepared as previously described.” The PKC peptide inhibitor was obtained from Gibco. Phosphatidylserine was purchased from Avanti Polar Lipids. All other chemicals were reagent grade or better.

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PAGE, and incorporation of 32Pwas quantified by an AMBIS radioactivity scanner.

Statistics Analysis of variance with repeated measures was used to determine significance of drug treatments, followed by Scheffe’s post hoc test. RESULTS

Phosphorylation of Histone and Synapsin I Our initial studies of PKA and PKC induced phosphorylation used partially purified preparations of histone, designated histone IIa and 111s respectively. As shown in Phosphorylation Conditions Common to all Substrates Fig. 1, PKA stimulated the incorporation of ”P in histone IIa. However, ethanol (25-200 mM) did not alter the ability In all the assays described below, initial velocity of the phosphorylation reaction was measured. of PKA to phosphorylate histone IIa. The PKA inhibitor produced a marked inhibition of PKA phosphorylation of histone IIa in this assay. Similarly, PKC produced phosPhosphorylation of the GABAA-R phorylation of histone 111s. However, ethanol was ineffecThe PKA phosphorylation assay (final volume, 50 pl) consisted of 50 tive at all concentrations tested in producing a change in mM Hepes (pH 7.4), 10 mM MgC12, PKA at 3 &/PI, ethanol 0 to 200 PKC activated phosphorylation (Fig. 2). In contrast, the mM, and 100 p~ ATP (specific activity of lo7dpm/nmol). Samples were incubated at 30’C for 30 min. Prior to PKC stimulated phosphorylation, peptide inhibitor of PKC produced a marked inhibition the GABAA-R was exhaustively dialyzed against 100 mM NaCI, 1 mM of PKC activity towards histone 111s. CAM kinase I1 EDTA, and 10 mM Tris (pH 7.4) to remove factors that inhibit PKC activity.” A modification” of the mixed micelle procedure of Hannum stimulated phosphorylation of synapsin I (Fig. 3) was also et al.“ was used to prepare the receptor in mixed rnicelles of phosphati- examined. The ability of CAM kinase I1 to phosphorylate dylserine, diolein and triton X-100.Briefly, phosphatidylserine and diol- synapsin I was not affected by ethanol concentrations ein, equivalent to 8 mot% and 2.5 mot% of the triton X-100originally ranging from 25 to 200 mM. However, calmidazolium, a in the receptor preparation, respectively, were dried under a stream of potent calmodulin antagonist, produced greater than 90% nitrogen in a glass tube. The dialyzed receptor was added to the tube and inhibition of CAM kinase I1 phosphorylation of synapsin vortexed for 1 min. Receptor was then added to the PKC reaction mixture that was the same as the PKA reaction mixture with the following I. exceptions (in final concentrations): 300 p~ CaCI2 and PKC, 3 pg/ml; PKA was omitted. Samples were incubated for 10 min. The concentration of the receptor in the assay was 8 to 10 pmol/ml. The samples were then subjected to sodiumn dodecyl sulfate-gel electrophoresis (SDS PAGE), and incorporation of 32Pwas quantified using an AMBIS radioactivity scanner.

Phosphorylation ojHistone The PKA phosphorylation mixture consisted (in final concentration) of histone Ila at 0.66 mg/ml, 50 mM MES (pH 6.0), 10 mM MgC12, PKA at 3 pg/ml, ethanol 0 to 200 mM, and 100 p~ ATP (specific activity, I .4 x lo5 dpm/nmol). The PKC phosphorylation mixture contained (final concentration) 1 mM CaC12, I mM EGTA, 10 mM MgC12, 40 mM MES (pH 6.0), histone Ills at 0.66 mg/ml, diolein 20 pg/ml, and phosphatidylserine 200 pg/ml, PKC at I pg/ml, ethanol 0 to 200 mM, and 100 p~ ATP (specific activity 5 X lo5).Tubes were incubated at 30°C for 5 min and stopped with 1 ml of ice cold 25% trichloroacetic acid (TCA). Bovine serum albumin (BSA), 0.5 mg, was added as a carrier. Tubes were vortexed and centrifuged for 2 min. Tubes were aspirated and 0.5 ml of 25% TCA was added, and tubes were vortexed and spun again. This washing step was repeated twice. Phosphorylation was quantified by Cerenkov counting in a liquid scintillation counter.

Phosphorylation of the GABAAReceptor We next examined the effects of ethanol on the phosphorylation of the purified GABAA-R. The GABAA-R was phosphorylated in vitro by PKA and PKC. PKA phosphorylated a polypeptide with an M,of 58,000. Previous studies’’ have shown that this polypeptide comigrates with one of two muscimol-binding polypeptides found in the 50,000

1

40,000

-

E0

U

Phosphorylation ojsynapsin 1 The CAM kinase I1 phosphorylation mixture (in final concentration) was composed of 50 mM HEPES (pH 7.4), 0.2 mg/ml synapsin I, 5 mM MgCI2, 5 mM CaC12, 30 pg/ml calmodulin, 0.6 pg/ml CAM kinase 11, ethanol 0 to 200 mM, and ATP 50 p~ (specific activity 10’ dpm/nmol). Tubes were incubated for 4 min at 30°C. Samples were subjected to SDS-

0

25

5 0 100 200

Ethanol, mM

0

2 2 110

PKI, ng/ul

Fig. 1. Histone Ila was phosphorylated by PKA in the presence of ethanol (0200 rnM) for 5 min. In Separate experiments, histone Ila was phosphorylated in the presence of PKI (PKA inhibitor), 0 to 110 ng/rl. for 5 min. Ethanol data represent the mean f SEM of 5 experiments. PKA inhibitor data represent the mean f SEM of 3 experiments. ’ p < 0.05.

MACHU ET AL.

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comigrate with a muscimol binding polypeptide." However, ethanol (25-200 mM) had no effect of PKC phosphorylation of this polypeptide (Fig. 5). Phosphorylation of the GABAA-R was blocked under these experimental conditions by specific inhibitors of PKA and PKC.

20

DISCUSSION

Ethanol, mM

'KC Inhibitor, uM

Fig. 2. Histone Ills was phosphotytatedby PKC in the presence of ethanol (0200 mM) for 5 min. In separate assays, the PKC inhibitor, 0 to 1 PM, was present during h i s t m Ills phosphorylati. Ethanol data represent the mean f SEM of four experiments. PKC inhibitor data represent the mean f SEM of three experiments. ' p c 0.05.

30,000

20,000

E

0

10,000

Ethanol, mM

Calmldazollum, uM

Fig. 3. Synapsin I was phospfmrylated by CAM kinase II in the presence of ethand (0-200mM) orcalmidazolium. 1 or 10 p ~for, 4 min. Ethanol data represent the mean i SEM of four experiments. CAM kinase I1 data represent the mean i SEM of two expenments. * p < 0.05.

rn 35

s

.E r n

Ethanol, mM

In this study we used highly purified forms of kinases and substrates to examine the direct actions of ethanol on phosphorylation. These assays were camed out under conditions in which specific inhibitors of PKA, PKC, and CAM kinase I1 were demonstrated to block phosphorylation bv these kinases. Under such conditions. we have shown that ethanol, in vitro, has no action on the catalytic activity of PKA, PKC, and CAM kinase 11. These data are consistent with reports indicating that ethanol added to isolated brain membranes had no detectable effect on endogenous phosphorylation by PKA23*2s or PKC.32 We also examined the possibility that some of the effects of ethanol on GABAA-R function might be mediated by direct effects on the ability of the receptor to be phosphorylated. In previous work we and others have demonstrated that both PKA and PKC could phosphorylate the purified GABAA-R in ~ i t r o . " - ' ~In the current experiments we demonstrate that ethanol (25-200 mM) has no effect on the phosphorylation of the GABAA-R by either kinase. Taken together, these data indicate that if acute treatment with pharmacologically relevant concentrations of ethanol does influence phosphorylation by PKA, PKC, or CAM kinase 11, such effects are unlikely to be due to direct actions of ethanol on these kinases. These findings, however, do not Out the possibility that produce changes in the phosphorylation of proteins, and in particular the GABAA-R, by indirect means. For example, acute ethanol could lead to the alteration of second messenger systems known to regulate kinase activity. Indeed, previous work indicates that ethanol can increase intracellular CAMP levels by enhancing G, activation.22

PKI, nglul

Fig. 4. The purified GABAA-R was phosphotylated by PKA in the presence of ethanol (0-200 mM) or PKI (PKA inhibitor), 22 or 110 ng/pl. for 30 min. Values of GABAA-Rphosphorylation are expressed as pmd of =PO, per pmd of muscimol binding. Ethanol data represent the mean f SEM of 5 experiments. PKA inhibitor data represent the mean f SEM of two experiments. p < 0.05.

purified GABAA-R.Ethanol failed to have any effect on PKA stimulated phosphorylation of the receptor at all concentrations tested (Fig. 4). PKC stimulated the phosphorylation Of a polypeptide in the purified receptor preparation with an M, of 56,000 that had also been shown to

0

E Ethanol, mM

PKC inhibitor, uM

Fig. 5. The purified GABAA-R was phosphorylated by PKC in the presence of ethand (0-200mM) or PKC inhibitor, 1 10 pM. for 10 min. Values of GABAA-R phosphorylation are expressed as pmol "PO, per pmd of musamol binding. Ethanol data represent the mean i SEM of 5 experiments. PKC inhibitor data represent the mean i SEM of two experiments. * p c 0.05.

LACK OF EFFECT OF ETHANOL ON KINASE ACTIVITY

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Moreover, ethanol may also produce increases in intracel- induced 36Cl-influx in spinal cord cultured neurons. Brain Res 17:123lular Ca2+41,42 via the mobilization of intracellular Ca2+ 126, 1986 6. Aguayo LG: Ethanol potentiates the GABA,-activated c1- current ~ t o r e s .During ~ ~ , ~ neuronal stimulation, ethanol may de- in mouse hippocampal and cortical neurons. Eur J Pharmacol 187: 127crease intracellular Ca2+by blockade of voltage sensitive 130, 1990 Ca2+c h a n n e l ~or ~ ~the. ~NMDA ~ receptor c ~ m p l e xIn. ~ ~ ~ 7.~ Nishio M, Narahashi T: Ethanol enhancement of GABA-actiaddition, ethanol could produce changes in the interaction vated chloride current in rat dorsal root ganglion neurons. Brain Res of the kinase/substrate within the cell membrane, as evi- 518~283-286,1990 8. Nestler U,Greengard P: Protein Phosphorylation in the Nervous denced by the enhanced translocation of PKC from cytoSystem. New York, John Wiley & Sons, 1984 sol to membrane in astroglial cells treated acutely with 9. Shenolikar S: Protein phosphorylation: hormones, drugs, and diolein and ethanol.33 bioregulation. FASEB J 2:2753-2764, 1988 Chronic ethanol treatment has been demonstrated to 10. Huganir RL, Greengard P Regulation of neurotransmitter recep alter the phosphorylation of substrate proteins by PKA, tor desensitization by protein phosphorylation. Neuron 5555-567, 1990 1 I . Sweetnam PM, Lloyd J, Gallombardo P, Madison RT, Gallager PKC, and CAM kinase 11. With PKA, alterations have DW, Tallman JF, Nestler U: Phosphorylation of the GABAa/benzodibeen detected in endogenous phosphorylation of brain azepine receptor alpha subunit by a receptor-associated protein kinase. J protein^,^',^^ including the regulatory subunit of PKA.21 Neurochem 51:1274-1284. 1988 PKC activity, on the other hand, has been shown to 12. Kirkness EF, Bovenkerk CF, Ueda T, Turner AJ: Phosphorylation increase in PC12 cells,28but decrease in rat hippocampus of gamma-aminobutyrate (GABA)/benzodiazepine receptors by cyclic and cortex in response to chronic Moreover, AMP dependent protein kinase. Biochem J. 259:613-616, 1989 13. Browning MD, Bureau M, Dudek EM, Olsen R W Protein kinase endogenous CAM kinase I1 activated phosphorylation of C and cyclic AMP-dependent protein kinase phosphorylate the @ subunit synaptic membranes is increased in the brains of rats fed of the purified gamma-aminobutyric acid A receptor. Proc Natl Acad chronically with ethan01.~’Thus, chronic ethanol treat- Sci (USA) 87: 1315- 131 8, 1990 ment has significant effects on protein phosphorylation. 14. Whiting P, McKernan RM, Iverson LL: Another mechanism for Whether these changes occur secondary to an alteration creating diversity in y-aminobutyrate type A receptors: RNA splicing of the catalytic activity of the kinases or the production of directs expression of two forms of 72 subunit, one of which contains a kinase C phosphorylation site. Proc Natl Acad Sci (USA) second messengers, or through a change in the amount of protein 87:9966-9970, 1990 substrate available for phosphorylation has not been de15. Wafford KA, Burnett DM, Leidenheimer NJ, Burt DR, BeiWang termined. J, Kofuji P, Dunwiddie TV, Harris RA, Sikela JM: Ethanol sensitivity In summary, we have demonstrated that in vitro of the GABA-A receptor expressed in Xenopus oocytes requires eight ethanol, in pharmacological concentrations, does not in- amino acids contained in the gamma-21 subunit of the receptor complex. teract with PKA, PKC, or CAM kinase I1 directly to alter Neuron (in press), I99 1 16. Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, the phosphorylation of purified substrates. Thus, previous Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, reports of the influence of ethanol on the protein phos- Seeburg PH, Barnard EA: Sequence and functional expression of the phorylation machinery are likely to be due to indirect GABA-A receptor shows a ligand-gated receptor super-family. Nature effects requiring an intact cellular apparatus. Thus, future (Lond) 328:221-227, 1987 17. Huganir RL, Delcour AH, Greengard P, Hess GP Phosphorylastudies using immunoprecipitation of GABAA-R from intact nerve cell preparations will be required to address tion of the nicotinic acetylcholine receptor regulates its rate of desensitiNature (Lond) 321:774-776, 1986 the question of whether any of the effects of ethanol on zation. 18. Leidenheimer NJ, Machu TK, Endo S, Olsen RW, Harris RA, GABAA-R function could be due to modulation of the Browning M D Cyclic AMP-dependent phosphorylation decreases phosphorylation state of the receptor. GABA-A receptor mediated 36CLuptake by brain microsacs. J NeuroACKNOWLEDGMENTS We thank Ellen M. Dudek and Carolina K. Smith for expert technical assistance.

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