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
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ABSTRACT |
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INTRODUCTION |
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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, 1986; Aizawa et al., 1988
). MAP2 is found almost exclusively in the dendrite/cell body of neurons (Matus et al., 1981
; Bernhardt and Matus, 1982
), whereas
MAPs are present in axons (Binder et al., 1985
). MAP2 is an excellent endogenous substrate for both bound and unbound cytosolic cAMP-dependent and Ca2+/calmodulin-dependent protein kinase (Schulman, 1984a
; Yamamoto et al., 1985
). These kinases recognize different sites for phosphorylation in the binding domain of MAP2 (Murthy et al., 1985
; Tsuyama et al., 1987
), which interacts with tubulin and diminishes its ability to promote microtubule assembly (Goldenring et al., 1985
).
Phosphorylation of MAP2 modulates proteinprotein 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, 1984a,b
; Binder et al., 1985
). MAP2a appears late during the period when MAP2c disappears, whereas MAP2b is present in embryonic and adult brain (Brugg and Matus, 1991
; Riederer, 1992
).
Among MAPs, the protein has been studied extensively and it is reported that phosphorylation changes its biochemical and physical properties (Kosik, 1993
). 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.
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MATERIALS AND METHODS |
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Animals
Male SpragueDawley rats (150200 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 TrisHCl, 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., 1986).
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 23 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., 1995). 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 (0768 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 [32P]ATP (4080 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.
SDSPAGE analysis of the effect of ethanol on phosphorylation of MT preparation
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was carried out as described by Laemmli (1970), in 1.5-mm thick slabs using a 411% 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 [32P] labelled protein. The intensity of [32
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, 1982).
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 KruskalWallis analysis of variance. Only P values of <0.05 were considered statistically significant.
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RESULTS |
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DISCUSSION |
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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, 1990). 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., 1986
). Another possibility to be considered is that of ethanol causing cell death (Ewald and Shao, 1993
; Cartwright and Smith, 1995
).
A novel protein which has been extensively studied is a protein present in patients with Alzheimer's disease. It has been proposed that phosphorylation can change the biochemical and physical properties of
. When phosphorylated, these structures become long and rigid, but, when dephosphorylated, they become short and elastic (Hagestedt et al., 1989
; Kosik, 1993
). Lindwall and Cole (1984) have shown that
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, 1992). 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. 3
); however, a possibility remains that ethanol can increase intracellular cAMP by enhancing G5 activation (Hoffman and Tabakoff, 1990
). The additive phosphorylation of MAP2 in the presence of cAMP and ethanol in our study (see Fig. 3
) 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 813 mol of phosphate/mol of MAP2 (Burns and Islam, 1984
; Tsuyama et al., 1987
). cAMP-dependent protein kinase can increase this up to a maximum of 2022 mol phosphatase/mol of MAP2 (Matus et al., 1981
). A total of 4046 phosphorylating sites on MAP2/mol have been reported (Tsuyama et al., 1986
).
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.
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FOOTNOTES |
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1 Present address: 30 Ardsley Court, East Brunswick, NJ 08816, USA.
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