LOW LEVELS OF ETHANOL STIMULATE AND HIGH LEVELS DECREASE PHOSPHORYLATION IN MICROTUBULE-ASSOCIATED PROTEINS IN RAT BRAIN: AN IN VITRO STUDY

Balwant Ahluwalia*, Syed Ahmad1, Olanrewaju Adeyiga, Barbara Wesley and Shakuntala Rajguru

Laboratory of Endocrine Research, Department of Obstetrics and Gynecology, College of Medicine, Howard University, Washington DC 20060, USA

Received 7 February 2000; in revised form 2 May 2000; accepted 13 May 2000


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphorylation and dephosphorylation of proteins associated with microtubules (MAPs) modulate the functional properties of microtubules (MT). A study was designed to test the hypothesis that ethanol at pharmacologically relevant levels affects phosphorylation of MAPs. Low (6, 12, 24, and 48 mM) and high (96, 384, and 768 mM) levels of ethanol were used in the study. MT prepared from rat brain by successive cycles of assembly–disassembly were found to contain two high molecular weight proteins (MAP2 and MAP1), tubulin, and 70-kDa neurofilament. The kinase activity was determined using [{gamma}32P]ATP as a phosphate donor. The results showed that ethanol primarily stimulated MAP2 phosphorylation. Low levels of ethanol stimulated, whereas high levels decreased, the kinase activity. MAP1 was phosphorylated to a lesser extent. 70-kDa neurofilament and tubulin were phosphorylated, however, the dose-dependent biphasic effect of ethanol on phosphorylation was not found in these cytoskeleton proteins. To determine whether the ethanol-induced kinase activity was cAMP-dependent, the catalytic subunit of cAMP-dependent protein kinase was isolated, purified, and kinase activity was determined with and without ethanol. The results showed that cAMP was not involved in ethanol-induced kinase activity. We conclude that ethanol predominantly stimulates phosphorylation of MAP2 in a dose-dependent manner.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microtubules (MT) are composed of tubulin and a heterogeneous class of proteins collectively known as microtubule-associated proteins (MAPs) (Valee et al., 1984; Tucker, 1990Go; Hirokawa, 1994Go). The roles of MT in various biological functions including mitosis, organization of MT spindle, MT kinetochore, spindle motors, anchoring clusters of neurotransmitter receptors at postsynaptic membranes in the brain, and a variety of cellular functions are clearly defined (Mitchison, 1988Go; Hirokawa, 1994Go; Sakai, 1994Go; Whatley et al., 1994Go; Naoto and Peter, 1998Go). MAPs are an integral part of MT and appear to regulate MT functions via phosphorylation and dephosphorylation. MAP kinases are enriched in brain and reveal prominent staining for MAP kinase in neurons and dendrites (Boulton and Cobb, 1991Go). A number of studies have been performed in vitro and in vivo to explain the ethanol effects in the central nervous system, and it is now accepted that some of the pharmacological effects of this drug can be explained on the basis of phosphorylation (Hoek and Rubin, 1990Go; Ahluwalia and Virmani, 1992Go). In in vitro studies, MAPs and myelin basic proteins have been reported to be excellent substrates for MAP kinase (Ray and Sturgill, 1987Go). MAP kinases are known by a variety of designations, which reflect the protein substrates, such as MAP2, a myelin basic protein, the ribosomal protein S6 kinase (Pelech and Sanghera, 1992Go). Morphological analysis of the cellular cytoskeleton shows that high molecular weight MAPs cross link with MT, neurofilaments, neuronal intermediate filaments, actin, and secretory vesicles, and thereby form an architectural framework (Pedrotti and Islam, 1995Go). In nerve cells, they are part of the cytoskeletal network and are involved in transport of membrane-bound vesicles along MT (Heimann et al., 1985Go).

Among high molecular weight MAPs, MAP2 (270 kDa) is the major component that has been characterized extensively with regard to both structural and functional properties (Allem and Kreis, 1986Go; Aizawa et al., 1988Go). MAP2 is found almost exclusively in the dendrite/cell body of neurons (Matus et al., 1981Go; Bernhardt and Matus, 1982Go), whereas {tau} MAPs are present in axons (Binder et al., 1985Go). MAP2 is an excellent endogenous substrate for both bound and unbound cytosolic cAMP-dependent and Ca2+/calmodulin-dependent protein kinase (Schulman, 1984aGo; Yamamoto et al., 1985Go). These kinases recognize different sites for phosphorylation in the binding domain of MAP2 (Murthy et al., 1985Go; Tsuyama et al., 1987Go), which interacts with tubulin and diminishes its ability to promote microtubule assembly (Goldenring et al., 1985Go).

Phosphorylation of MAP2 modulates protein–protein interaction and this modification may also determine the association of an interacting domain of MAP2 with other elements of cytoskeletal proteins. In the adult rat, MAP2 appears as a high molecular weight doublet, MAP2a and MAP2b (Schulman, 1984aGo,bGo; Binder et al., 1985Go). MAP2a appears late during the period when MAP2c disappears, whereas MAP2b is present in embryonic and adult brain (Brugg and Matus, 1991Go; Riederer, 1992Go).

Among MAPs, the {tau} protein has been studied extensively and it is reported that phosphorylation changes its biochemical and physical properties (Kosik, 1993Go). Because MAPs are the integral components of neuronal cytoskeletal proteins and phosphorylation modulates their biochemical functions, this study was designed to examine whether ethanol affects the phosphorylation of MAPs.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Radioactive adenosine 5-triphosphate [{gamma}32P] + ATP as the tetra (triethyl ammonium) salt in distilled water with specific activity (20–40 Ci/mmol) was purchased from New England Nuclear (Boston, MA). cAMP, cGMP, ATP, leupeptin, PIPES and 3-isobutyl-methyl-xanthine were purchased from Sigma Chemical Company (St Louis, MO, USA). Molecular weight markers, myosin, ß-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and chemicals for electrophoresis and chromatography such as acrylamide, N,N-methylene-bis-acrylamide, ammonium persulphate, TEMED, DEAE-Sephadex A-50 and Biogel A-15 m were purchased from Bio-Rad (Richmond, CA, USA). Ethanol was obtained from Florida Distiller (Lake Alfred, FL, USA). Film developer (D-196), Rapid Fixer and autoradiographic film (Kodak-x-Omat) were purchased from Kodak Company (Rochester, New York, USA).

Animals
Male Sprague–Dawley rats (150–200 g) were purchased from Charles River (Kingston, NY, USA). Animals were fed commercial feed (Purina Chow) and treated according to guidelines set by the institution. Animals were killed by decapitation and the brains were immediately removed and immersed in ice-cold 0.32 M sucrose prepared in 4 mM Tris–HCl, pH 7.3. The meninges and superficial blood vesicles were removed. Tissues were then minced and were homogenized in 1.5 ml (w/v) of PEEM buffer (0.1 M PIPES, 0.1 mM EDTA, 1.0 mM EGTA, 1.0 mM magnesium sulphate, and 0.1 mM 2-mercaptoethanol, pH 6.6) at 1200 rpm in a Teflon-pestle glass homogenizer for 30 s with four up and four down strokes. The homogenate obtained was centrifuged for 30 min at 20 000 g in a 60 Ti rotor at 4°C in an ultracentrifuge to obtain brain homogenate extract. GTP and leupeptin (1 mM each) were added to the extract to enhance the polymerization of MT and to inhibit proteolysis of MAPs respectively (Tsuyama et al., 1986Go).

Isolation of MAPs
Several methods have been developed for the purification of MAP2. The procedure used in this study was that described by Islam and Burns (1981), as modified by Pedrotti et al. (1993). This results in >97% pure MAPs. Typical recovery of MAPs was approximately 2–3 mg/100 g wet brain tissue.

Dose level of ethanol
The dose level of ethanol used in this, was based on our previous, study (Ahluwalia et al., 1995Go). Low (6, 12, 24, and 48 mM) and high (96, 384, and 768 mM) levels of ethanol were used, the rationale for which was to examine dose-related effects of ethanol.

Phosphorylation procedure
This was performed according to Pant and Veeranna (1995). Varying amounts of ethanol (0–768 mM) were added to 50 µg of protein from the MAPs preparations in 20 mM Tris, pH 7.4, 100 mM NaCl, and 10 mM MgCl2 to a final volume of 100 µl. The phosphorylation reaction was started by adding 10 µCi (5 µl) of [{gamma}32P]ATP (40–80 µCi/mol) to a final concentration of 20 µM at 37°C for 10 min. An aliquot of the reaction mixture was removed and spotted on a 0.5 in2 phosphocellulose pad (Whatman P81). Pads were washed three times with 75 mM phosphoric acid and twice with 95% ethanol. Finally, the dried pads were counted in a Beckman Liquid Scintillation Counter Model LS 5000 CE. The radioactivity (cpm) was the measure of phosphate incorporation. In some experiments, cAMP (2 µM) and 3-isobutyl-1-methyl-xanthine (1 mM) were added to the reaction mixture. In some cases, the phosphorylation reaction was terminated by the addition of 50 µl of SDS-stop solution containing 2% SDS and 2% ß-mercaptoethanol.

SDS–PAGE analysis of the effect of ethanol on phosphorylation of MT preparation
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) was carried out as described by Laemmli (1970), in 1.5-mm thick slabs using a 4–11% gradient of acrylamide. Gels were stained overnight with 0.15% Coomassie blue and destained in destain 1 (R-250 prepared in 7% acetic acid and 35% methanol). Gels were swollen in destain-11 (7% acetic acid and 5% methanol) in the presence of 10% glycerol for 30 min and dried under vacuum. The dried gels were exposed to Kodak x-mat film and autoradiographed for the analysis of [{gamma}32P] labelled protein. The intensity of [32{gamma}P] incorporation (quantitative) into protein was recorded on an image array processor.

Isolation of catalytic subunit of cAMP-dependent protein kinase from MAP2
To isolate the catalytic subunit of cAMP-dependent protein kinase, MAP2 was purified from microtubules by column chromatography on Bio-Gel A-15 mm. Proteins from the column were eluted with PEEM buffer and MAP2-containing fractions were pooled. One ml purified MAP 2 (300 µg of protein) was brought to 10 µM cAMP and incubated for 30 min at 0°C. The sample was applied on a 3 ml column of DEAE-Sephadex A-50 pre-equilibrated with PEEM buffer. The catalytic subunit of cAMP-dependent protein kinase was eluted with PEEM buffer (Theurkauf and Vallee, 1982Go).

Total protein assay
Total protein in the samples (MAPs preparation) was measured by the method of Bradford (1976), using a premixed reagent purchased from Bio-Rad Laboratories. Bovine serum albumin was used as standard in the protein assays.

Statistical analysis
Statistical significance was tested using a two-tailed t-test by means of a statistical package (Statistical Software Inc., Los Angeles, CA) on an IBM computer. Significance between groups was assessed with Kruskal–Wallis analysis of variance. Only P values of <0.05 were considered statistically significant.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of various dose levels of ethanol on MAPs phosphorylation
Figure 1Go shows the phosphorylating activity in MT preparation treated with ethanol (6, 12, 24, 48, 96, 384, and 768 mM). Incorporation of [{gamma}32P] in ethanol-treated samples was calculated as the percentage increase compared to controls (no ethanol). The results show that up to 24 mM ethanol, the incorporation of [{gamma}32P] in MAPs was dose-dependent. For example, at 6 mM ethanol, the increase in [{gamma}32P] incorporation was almost three times that of controls, whereas at 12 and 24 mM, the increase was more than four to five times that of controls. When the level of ethanol was increased to 48, 96, 384, and 768 mM, [{gamma}32P] incorporation decreased in a graded fashion. The differences in [{gamma}32P] incorporation between 24 and 48 mM were not significant (P > 0.05), and at 96, 384, and 768 mM ethanol, [{gamma}32P] incorporation was not significantly different from that of control preparations



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Fig. 1. Biphasic dose level effects of ethanol on incorporation of [{gamma}32P] into microtubule preparations.

Data show that low dose levels (6, 12, 24, and 48 mM) stimulated, whereas the higher dose levels (96, 384, and 768 mM) decreased phosphorylation relative to 24 mM ethanol. The values at 6, 12, 24, and 48 mM were significantly higher (P < 0.01) compared to controls. Values starting at 96, 384, and 768 mM were similar to controls. No significant differences were found in [{gamma}32P] incorporation between 96, 384, and 768 mM ethanol. Each bar represents the average of at least four experiments (± SD).

 
Gel electrophoretic patterns in MAPs
The electrophoretic patterns of [{gamma}32P] incorporation in MAPs are shown in Fig. 2Go. Results show that ethanol predominantly affected MAP2 phosphorylation. MAP1 was affected to a lesser extent. In MAP2, there was a progressive increase in phosphorylation starting with 6, 12, and 24 mM ethanol. At higher levels of ethanol starting at 48 mM, there was a decrease in phosphorylation relative to 24 mM. At 384 and 768 mM ethanol, the phosphorylation level of MAP2 reached a very low level and was similar to the control (no ethanol) values. MAP1 (350 kDa) was phosphorylated at 12 and 24 mM as shown in Fig. 2Go; at 48 mM ethanol there was a further decrease in phosphorylation activity. At 384 mM and 768 mM, there was no evidence of phosphorylation in MAP1. Tubulin and 70 kDa neurofilament were phosphorylated; however, the biphasic effect of ethanol on phosphorylation as shown in MAP2 was not found in these cytoskeletal proteins. In tubulin, 6 mM ethanol caused a slight increase in phosphorylation and then declined, starting with 12 mM up to 768 mM ethanol. The molecular weights of purified proteins (MAP2 and tubulin) were determined and were found to be identical for high molecular weight and 55 kDa proteins (Fig. 2Go).



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Fig. 2. Dose level biphasic effect of ethanol on phosphorylation of MAPs on SDS gel electrophoresis.

MAP2 was predominantly phosphorylated in the presence of ethanol. Low dose levels (6, 12, 24, and 48 mM) stimulated, whereas high levels (96, 384, and 768 mM) decreased, phosphorylation. MAP1 was phosphorylated to a lesser extent. Tubulin and 70 kDa protein were also phosphorylated, but dose level biphasic effect was observed. The results with 384 and 768 mM ethanol are not shown here, because the phosphorylation at these levels was similar to that in controls (no ethanol added). A minimum of four experiments were conducted with each dose level of ethanol.

 
Effect of ethanol on cAMP- and Ca2+/calmodulin-dependent MAPs phosphorylation
The purpose of this part of the study was to determine whether ethanol-sensitive kinase(s) that phosphorylate MAP2 were cAMP- and/or Ca2+/calmodulin-dependent kinases. The cAMP- and Ca2+/calmodulin-independent kinases are known to phosphorylate MAP2. In order to characterize the ethanol-induced kinase, we used EGTA/EDTA in the homogenization buffer to eliminate the influence of the Ca2+/dependent-kinase. The effect of ethanol on cAMP-dependent kinase was examined in the MT preparation with cAMP added. Figure 3Go (see also inset) shows that there was a minor kinase activity without ethanol and cAMP (see lane 1 and inset). The addition of cAMP significantly stimulated (P < 0.01) the phosphorylation of MAP2 and tubulin (see inset). The kinase activity in MAP2 was higher compared to tubulin (lane 2 and inset). Ethanol significantly stimulated (P < 0.01) kinase activity in both MAP2 and tubulin (lane 3 and inset) and ethanol and cAMP when added together further stimulated phosphorylation of MAP2 only (lane 4 and inset). The phosphorylation activity was significantly higher (P < 0.05) in MAP, compared to tubulin. This suggests that ethanol and cAMP-induced MAP2 phosphorylation occurred at different sites and protein kinase A (cAMP-dependent protein kinase) is associated with MT preparation. In all preparations, phosphorylation of MAPs was significantly higher, compared with tubulin (P < 0.05), suggesting preferential stimulation of phosphorylation of MAPs in the presence of ethanol.



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Fig. 3. Effect of low levels of ethanol, cAMP or both on MAP phosphorylation.

cAMP (see lane 2 and inset) and ethanol (see lane 3 and inset) singly stimulated MAP2 phosphorylation. When they were added together, there was a further stimulation of phosphorylation (see lane 4 and inset). Tubulin was significantly more phosphorylated (P < 0.05) in the presence of cAMP and ethanol compared to control. Differences between tubulin and MAP2 phosphorylation with ethanol only (see inset third bar) were not significant. Differences between tubulin and MAP2 phosphorylation with ethanol plus cAMP were significant (P < 0.05). A minimum of four experiments were conducted with each dose level of ethanol.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphorylation of key proteins has been implicated as the major intracellular regulatory mechanism and it is clear that phosphorylation and dephosphorylation play a crucial role in stabilizing the microtubular arrays. MT-based motor proteins are involved in anchoring neurotransmitter receptor clusters at postsynaptic densities in brain (Lieuvin et al., 1994Go; Whatley et al., 1994Go). Our results demonstrate that pharmacologically relevant concentrations of ethanol stimulated, whereas higher levels decreased, MAP2 phosphorylation. It appears that phosphorylation inactivates MAPs and phosphorylated MAPs do not promote MT assembly (Ookata et al., 1995Go). Using MT-disrupting (depolymerization) and promoting (polymerization) agents, it has been established that intact MT are required for functional MT network (Whately et al., 1994). That MT depolymerization inhibits GABAergic action and intact MT are necessary for the full activation of GABAaergic responses have been demonstrated (Whately et al., 1994; Macdonald and Olsen, 1994Go). The dose-dependent effect of ethanol on phosphorylation as reported here may explain some of its dose-dependent biphasic effects on neuronal systems, including sedation, anaesthesia, and disruption of neuronal growth and excitability (Tabakoff and Hoffmann, 1987Go). Using PC12 cells, we have shown that ethanol modulates the kinase activity, and phosphorylation and dephosphorylation is the basis of ethanol-induced pharmacological effects on neuronal cells (Virmani and Ahluwalia, 1992Go). In another study, we found that ethanol interrupted cell mitosis (Ahluwalia et al., 1995Go), and that dephosphorylation activates cdc2 kinase with the onset of the M phase of the cell cycle (Gould and Nurse, 1989Go; Ferrell et al., 1991Go). The results of these studies, as mentioned above, suggest that further investigation is needed to confirm the relationship of low dose-stimulated phosphorylation to the biological functional disorders induced by ethanol. The high dose-induced decrease in phosphorylation, as demonstrated in this study, may be explained on the basis of a decrease in the amount of substrate available for phosphorylation or the toxic effect of ethanol on the catalytic activity of kinases. How phosphorylation modulates the ethanol effects on MT is not known. Recently, a protein kinase designated MARK has been cloned that apparently triggers MAPs phosphorylation and causes MT disruption (Drewes et al., 1997Go). MARK preferentially phosphorylates MAP2 and MAP4 and phosphorylates serine/ threonine residues that diminish MT stabilizing activity, resulting in the loss of MT arrays. It will be of interest to examine the relationship of MARK activity in the presence of ethanol. Our study shows that protein kinase A was not involved in the ethanol-induced kinase modulation.

The dynamic behaviour of the cytoskeleton is provided by MAPs and neurofilaments and phosphorylation appears to be the biochemical process which causes them to bind with the cytoskeleton less tightly, and stabilize them less efficiently (Lewin, 1990Go). Phosphorylation of neurofilament along with MAP2 in our study is interesting, because morphological and biochemical data show that components of neuronal, cytoskeletal, MT, and neurofilaments interact with each other, suggesting that ethanol has multiple sites at which to affect phosphorylation of cytoskeletal proteins. An alcohol-induced increase in phosphorylation can cause disruption of MT, which is known to affect axonal transport, and may thus affect biological function. The disruption of MT, therefore, might interfere with memory formation, one of the major features of ethanol toxicity (Tsuyama et al., 1986Go). Another possibility to be considered is that of ethanol causing cell death (Ewald and Shao, 1993Go; Cartwright and Smith, 1995Go).

A novel protein which has been extensively studied is a {tau} protein present in patients with Alzheimer's disease. It has been proposed that phosphorylation can change the biochemical and physical properties of {tau}. When phosphorylated, these structures become long and rigid, but, when dephosphorylated, they become short and elastic (Hagestedt et al., 1989Go; Kosik, 1993Go). Lindwall and Cole (1984) have shown that {tau} is an effector promoter of tubulin after it has been treated with alkaline phosphatase.

Previous studies have shown that MAP2 is phosphorylated by cAMP-and Ca2+/calmodulin-dependent and -independent protein kinases (Pelech and Sanghera, 1992Go). The biphasic behaviour of MAP2 phosphorylation may be due to structural changes in the proteins or to changes in the activity of the enzymes present in the preparation. However, Machu et al., (1991) reported that ethanol at pharmacological concentrations has no direct effect on cAMP-dependent protein kinase, protein kinase C or Ca2+ calmodulin-dependent protein kinase. Our data also suggest that ethanol does not directly interact with protein kinase A activity (see Fig. 3Go); however, a possibility remains that ethanol can increase intracellular cAMP by enhancing G5 activation (Hoffman and Tabakoff, 1990Go). The additive phosphorylation of MAP2 in the presence of cAMP and ethanol in our study (see Fig. 3Go) suggests that ethanol may stimulate phosphorylation at vacant sites on MAP2 by affecting factors other than cAMP-dependent protein kinase. The role of phosphatases in kinase activity should be considered. It is likely that the decrease in phosphatase activity is responsible for the increased phosphorylation of MAP2 in the presence of ethanol. Studies have reported that bovine MAP2 purified by temperature assembly contains 8–13 mol of phosphate/mol of MAP2 (Burns and Islam, 1984Go; Tsuyama et al., 1987Go). cAMP-dependent protein kinase can increase this up to a maximum of 20–22 mol phosphatase/mol of MAP2 (Matus et al., 1981Go). A total of 40–46 phosphorylating sites on MAP2/mol have been reported (Tsuyama et al., 1986Go).

Although the mechanism responsible for an increased phosphorylation of MAP2 by ethanol observed in this study is not clear, it is apparent that cAMP-, cGMP- and Ca2+-dependent kinases are not involved in the increased incorporation of [32P] into MAP2. Ideal candidates for this effect may be casein kinases, cyclic nucleotide, and Ca2+-independent kinases, or inhibition of phosphatases.

We postulate that increased phosphorylation causes disruption of MT, which may affect axonal transport of material from the cell body into axons. These processes may be important in the maintenance of synaptic viability, and in the maintenance of cytoskeletal structures during the formation and modification of synapses leading to many brain impairments in alcohol users.


    FOOTNOTES
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed at: Department of Obstetrics and Gynecology, College of Medicine, 520 W Street NW Box 23, Washington DC 20059, USA. Back

1 Present address: 30 Ardsley Court, East Brunswick, NJ 08816, USA. Back


    REFERENCES
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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