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(The FASEB Journal. 2000;14:2303-2314.)
© 2000 FASEB

Uncoupling of ßIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain

DORIT RON*,{dagger}1, ALICIA J. VAGTS*, DOUGLAS P. DOHRMAN*,2, RAMI YAKA*, ZHAN JIANG*, LINA YAO*, JOHN CRABBE{ddagger}, JUDITH E. GRISEL{ddagger},3 and IVAN DIAMOND*,{dagger},§

* Ernest Gallo Research Center,
{dagger} Departments of Neurology,
§ Cellular and Molecular Pharmacology, Pediatrics, and Neuroscience Graduate Program and Center for Neurobiology of Addiction, University of California San Francisco, San Francisco, California 94110-3518, USA; and
{ddagger} Portland Alcohol Research Center, Department of Behavioral Neuroscience, Oregon Health Science University and Veterans Affairs Medical Center, Portland, Oregon 97201, USA

1Correspondence: Ernest Gallo Research Center, 5858 Horton St., Suite 200, Emeryville, CA 94608, USA. E-mail: dorit{at}itsa.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase ßIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of ßIIPKC. Ethanol also inhibits ßIIPKC translocation in response to activation. These results suggest that ethanol inhibition of ßIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of ßIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of ßIIPKC.—Ron, D., Vagts, A. J., Dohrman, D. P., Yaka, R., Jiang, Z., Yao, L., Crabbe, J., Grisel, J. E., Diamond, I. Uncoupling of ßIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.


Key Words: PKC • activated C kinase receptor • cAMP • signal transduction • ethanol • targeting protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROLONGED EXPOSURE TO ethanol leads to neuroadaptive changes characterized by tolerance, physical dependence, and craving (1) . These responses appear to be modulated at least in part by protein kinase C (PKC) (2) . Ethanol alters the normal activities of major neurotransmitter receptors, including glycine (3) , GABAA (4 , 5) , glutamate (6) , and muscarinic receptors (7) , by inhibiting or activating the function of PKC. PKC is also required for ethanol-induced neurite outgrowth (8 , 9) , and up-regulation of Ca2+ channels (10 , 11) . Chronic ethanol intake decreases PKC activity, which is associated with a reduction in phorbol ester binding in selected rat brain areas (12) . In addition, {gamma}PKC knockout mice show low sensitivity to ethanol (13) , whereas {varepsilon}PKC knockout mice are more sensitive in their GABAA responses to ethanol (14) . Although it is clear that ethanol enhances or decreases PKC function, the mechanism by which ethanol alters the activity of PKC is not well understood.

The specificity of protein kinase function is regulated in part by intracellular compartmentalization (15) . This, in turn, is regulated by targeting (anchoring, scaffolding, and adaptor) proteins (15) . PKC is a family of 10 isozymes that translocate to distinct intracellular sites upon activation (16) . Compartmentalization of the translocated PKC isozymes is mediated in part by RACKs (receptors for activated C kinase) (17) . RACK1 has been cloned and identified as a targeting protein for activated ßIIPKC (18) . Among the 10 PKC isozymes, RACK1 is specific for ßIIPKC (19 , 20) . We have discovered recently that RACK1 itself is a translocating protein (20) . Upon PKC activation, RACK1 colocalizes with, binds to and translocates with ßIIPKC (20) . We proposed that RACK1 is a shuttling protein that brings ßIIPKC to its appropriate intracellular site upon activation of PKC (20) .

Recent evidence demonstrates that ethanol causes translocation of specific kinases such as the catalytic subunit of PKA (21) and the {varepsilon} and {delta} isozymes of PKC (22) . Since PKC compartmentalization is mediated by intracellular targeting, such as RACK proteins (23) , one mechanism by which ethanol might affect kinases is by affecting their corresponding targeting proteins. In this study we present evidence that in cultured cells and in vivo, ethanol changes the compartmentalization of RACK1, the targeting protein for ßIIPKC, thereby altering the physiological activity of the kinase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Diacylglycerol (DAG) and phosphatidylserine (PS) were purchased from Avanti (Birmingham, Ala.). Luminol and p-coumaric acid were purchased from Sigma (St. Louis, Mo.). The dopamine D2 agonist tri-hydroxy-N-propyl-noraporphine hydrobromide (NPA) and forskolin were purchased from RBI (Natick, Mass.). RpcAMP was purchased from BioLog Life Science Institute (Bremen, Germany). Recombinant human ßIIPKC was purchased from Panvera (Madison, Wis.). Polyclonal anti-ßIIPKC antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, Calif.). Monoclonal (IgM) anti-RACK1 antibodies were purchased from Transduction Laboratories (Lexington, Ky.). The secondary antibodies FITC-conjugated goat anti-rabbit, Texas red-conjugated goat anti-mouse (IgM), and Cy5-conjugated goat anti-mouse (IgM) antibodies were purchased from Cappel (Cochranville, Pa.). TOTO-3 was purchased from Molecular Probes (Eugene, Oreg.).

Cell culture
Chinese hamster ovary (CHO) cells stably expressing the long form of the dopamine D2 receptor (D2L) (24) were seeded and grown in Ham’s F12 medium containing 10% fetal bovine serum (FBS) and 2 mM glutamine. After 48 h, media were replaced with Ham’s F12 medium containing 5% serum, 25 mM HEPES (pH 7.4), 2 mM glutamine, 50 µg/ml of human transferrin, 10 µg/ml oleic acid (complexed with 2 mg/ml fatty acid-free bovine serum albumin), 25 µg/ml bovine insulin, and trace elements at the following concentrations: 0.5 nM MnCl2, 0.5 nM [NH4]6Mo7O24, 0.25 nM SnCl4, 25 nM Na3VO4, 5 nM CdSO4, 0.25 nM NiSO4, 15 nM H2SeO3, and 25 nM Na2SiO3. On day 4, the cells were treated with different reagents as described in legends to Figs. 1 and 2 .



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  		Figure 1. Figure 1 .

Ethanol induces translocation of RACK1 to the nucleus. Cells were stained with monoclonal anti-RACK1 antibody (1:100) and polyclonal anti-ßIIPKC antibodies (1:100) and visualized with Texas red-conjugated goat anti-mouse (IgM) (1:500) antibodies and FITC-conjugated goat anti-rabbit (IgG) (1:500). Cells were scanned using a confocal microscope and viewed with a 40x lens. The images are representative of three separate experiments. No staining was observed when anti-RACK1 antibodies and anti-ßIIPKC antibodies were preabsorbed with recombinant RACK1 and ßIIPKC peptide as described in ref 20 . A) CHO/D2L cells treated with 50 mM and; 200 mM of EtOH for 10 min at 37°C. a) ßIIPKC staining in unstimulated cells. b) ßIIPKC staining in cells treated with 50 mM ethanol for 10 min. c) ßIIPKC staining in cells treated with 200 mM ethanol for 10 min. d) RACK1 staining in unstimulated cells. e) RACK1 staining in cells treated with 50 mM ethanol for 10 min. f) RACK1 staining in cells treated with 200 mM ethanol for 10 min. B) CHO/D2L cells treated with 5 mM of EtOH for 60 min at 37°C. a) ßIIPKC staining in unstimulated cells. b) RACK1 staining in unstimulated cells. c) ßIIPKC staining in cells treated with 5 mM ethanol for 60 min. d) RACK1 staining in cells treated with 5 mM ethanol for 60 min.



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  		Figure 2. ßIIPKC translocation is inhibited by ethanol. CHO/D2L cells were treated with 500 nM of the dopamine D2 receptor agonist tri-hydroxy-N-propyl-noraporphine hydrobromide (NPA) (black bars) or 100 nM of ethanol for 30 min at 37°C (hatched bars) or pretreated with ethanol to 30 min before adding NPA (hatched bars). Cells were then stained, visualized, and scored as described in Materials and Methods. The percentage of translocated RACK1 and ßIIPKC was defined as:

Data are the mean ± standard deviation of three separate experiments.

C6 glioma cells were seeded and grown in DMEM containing 5% FBS and Ham’s F12 containing 2 mM glutamine, 20 mM HEPES, 50 µg/ml human transferrin, and trace elements at concentrations described above. Cells were seeded in Nalgene Nunc 4-well chamber slides (Fisher, Pittsburgh, Pa.) and cultures were treated as described in legend of Fig. 4 .



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  		Figure 4. Figure 4 .

Forskolin and ethanol induce RACK1 nuclear compartmentalization in C6 glioma cell line. A) C6 cells were treated with 1 µM forskolin for 30 min at 37°C. The cells were then stained with monoclonal anti-RACK1 antibodies. Cells were scanned using a confocal microscope and viewed with 20x lens. Images shown are individual middle sections of projected Z series. The images are representative of five experiments. B) C6 cells were treated with either 200 mM ethanol (black bars), 1 µM forskolin (gray bars), or media (white bars) for 30 min at 37°C. Cells were stained with anti-RACK1 antibodies and the nuclear marker TOTO-3 (Molecular Probes, 1:10,000). Cells were scanned using a confocal microscope and viewed with 20x lens. Quantitation of compartmentalization of RACK1 with nuclear marker was determined using NIH Image program version 1.61 as described in Materials and Methods. Data are mean ± SD of five experiments. Data were analyzed by Student’s t test, P<0.01. C) C6 cells were treated with 20 µM RpcAMP for 3 h and then with media (gray bars) or 100 mM EtOH (gray bars) for 30 min at 37°C. Cells were also treated with media for 30 min (white bars) or 100 mM ethanol (black bars). Cells were stained with anti-RACK1 antibodies and the nuclear marker TOTO-3 (Molecular Probes, 1:10,000). Cells were scanned using a confocal microscope and viewed with 20x lens. Quantitation of compartmentalization of RACK1 with nuclear marker was determined using NIH Image program version 1.61 as described in Materials and Methods. Data are mean ± SD of three experiments. Data were analyzed by Student’s t test, P<0.01.

Immunostaining and confocal microscopy
CHO/D2L and C6 glioma cells were treated as described in the figure legends. The cells were then washed with cold phosphate-buffered saline (PBS), fixed with ice-cold methanol for 3 min, and washed twice with cold PBS. Cells were incubated for 2 h with 1% normal goat serum in PBS containing 0.1% Triton X-100, followed by overnight incubation at 4°C with the appropriate primary antibody (diluted in PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum albumin). Cells were then washed three times with PBS containing 0.1% Triton and incubated for 1.5 h with FITC-conjugated anti-rabbit (IgG) antibody (1:500, Cappel), Texas red, or Cy5-conjugated anti-mouse (IgM) antibodies together with the nuclear marker TOTO-3 (1:10,000, Molecular Probes). Cells were washed an additional three times with cold PBS containing 0.1% Triton. Slides were mounted using Vectashield and viewed with a Bio-Rad MRC-1024 laser scanning confocal microscope. The confocal images were processed using the computer programs NIH Image version 1.61 (National Institutes of Health) and Adobe PhotoShop (Adobe Systems Inc., San Jose, Calif.). All the images shown are individual middle sections of projected Z series.

Localization of RACK1 and ßIIPKC to the Golgi apparatus
The localization of ßIIPKC and RACK1 to the Golgi apparatus upon PKC activation in CHO/D2L cells was confirmed as described in ref 20 . Briefly, CHO/D2L cells were stained with anti-RACK1 antibodies together with the specific Golgi marker BODIPY-FL C5-ceramide and anti-ßIIPKC antibodies together with the Golgi marker anti-mannosidase antibodies.

Brain sections
Adult male WSC outbred mice (Portland Oregon) were injected intraperitoneally (i.p.) with ethanol (1.5 g/kg) and then exposed to ethanol via vapor chamber for 3 days. Both control mice (exposed to air chambers) and ethanol-treated mice were injected i.p. daily with 1 mmol/kg of the alcohol dehydrogenase inhibitor pyrazole-HCl. After 3 days (chronic) exposure to alcohol, the animals were anesthetized and perfused with 4% paraformaldehyde. Separate mice were injected with acute ethanol (4 g/kg) or saline and perfused 60 min later. Brains were removed, saturated with 30% sucrose and sectioned on a freezing microtome. Slices of 10 µm were blocked in PBS containing 0.3% Triton X-100 and 5% NGS for 1 h at room temperature. Blocking solution was aspirated and sections were then stained with monoclonal anti-RACK antibodies (1:100) and polyclonal anti-ßIIPKC antibodies (1:100) overnight at 4°C. Antibody was aspirated and sections were washed in PBS containing 0.3% Triton and 1% NGS. Sections were incubated for 2 h at RT with secondary antibodies conjugated to Texas red, FITC, and a nuclear marker TOTO-3. Sections were then washed and mounted on Fisher Superfrost glass slides. Three mice were used per treatment. Three slices were mounted and viewed from each mouse brain. Three images were processed from each slice.

Image analysis
Quantification of colocalization with a nuclear marker
Colocalization of RACK1 and/or ßIIPKC with the nuclear marker TOTO-3 was determined using NIH 1.61 Image program as described in ref 20 . Briefly, thresholding was used to separate immunofluorescence pixels from the background and to create binary images. Pairs of binary images of RACK1 and TOTO-3 or ßIIPKC and TOTO-3 were multiplied and divided by 255 to generate a final merged image that could be visualized with an 8 bit gray scale. The percent of RACK1 or ßIIPKC staining merged with the nuclear marker TOTO-3 was calculated using the following equation:

Scoring translocation
Translocation of RACK1 and ßIIPKC in CHO/D2L cells was scored by counting at least three random fields of cells (total of at least 100 cells) per treatment for staining of RACK1 and/or ßIIPKC. The percent of translocated RACK1 and/or ßIIPKC was calculated using the following equation:

Each image contained 20–100 cells.

Protein–protein interaction assays
In vitro binding assay
Binding assays were done according to Ron et al. (19) . Briefly, RACK1-maltose binding fusion protein (1 ml, ~10 µg/ml) was loaded on 0.5 ml amylose resin column (New England Biolabs, Beverly, Mass.) and extensively washed (10 times the column volume) with column buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM sodium azide, and 10 mM ß-mercaptoethanol). ßIIPKC (1 µg purified recombinant SF9 expressed protein) was incubated in overlay buffer [50 mM Tris-HCl pH 7.4, 0.1% bovine serum albumin, 5 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor (SBTI), 0.1% polyethylene glycol, 0.2 M NaCl, 0.1 mM CaCl2, and 12 mM ß-mercaptoethanol] in the presence of PKC activators 50 µg/ml PS, 0.8 µg/ml DAG, and 1 mM CaCl2. The mixture of activated ßIIPKC was incubated with RACK1 on the resin for 15 min at room temperature in the presence and in the absence and presence of 50 mM or 200 mM of ethanol. After washing three times with overlay wash (50 mM Tris-HCl pH 7.4, 0.1% polyethylene glycol, 0.2 M NaCl, 0.1 mM CaCl2, and 12 mM ß-mercaptoethanol) for 10 min each, the complex was eluted with maltose (10 mM), sample buffer was added, and the sample was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Binding of ßIIPKC was detected using anti-ßIIPKC polyclonal antibodies (1:500), followed by chemiluminescent reaction (2.5 mM luminol and 400 µM p-coumaric acid).

Overlay assay
CHO/D2L cells were washed once with cold PBS and lysed in 20 mM Tris-HCl (pH 7.5) containing 10 mM EGTA, 2 mM EDTA, 0.25 M sucrose, 1% Triton X-100, and 10 µg/ml of the following protease inhibitors: SBTI (Sigma), aprotinin, phenylmethylsulfonyl fluoride (Sigma), and leupeptin (Boehringer Mannheim, Mannheim, Germany). Cells were lysed and incubated at 4°C for 30 min. Lysed cells were then centrifuged at 14,000 g at 4°C for 30 min. Protein determination of the triton-soluble material (supernatant) was assessed using Bicinchoninic (BCA) reagent (Pierce, Rockford, Ill.). Approximately 0.5 mg protein was resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was cut into strips and incubated in overlay buffer with activated ßIIPKC (as described above) in the presence and absence of 200 mM ethanol for 15 min at room temperature. The nitrocellulose membrane strips were washed three times with overlay wash for 10 min each. Binding of ßIIPKC was detected using anti-ßIIPKC polyclonal antibodies (1:500), followed by chemiluminescent reaction (2.5 mM luminol and 400 µM p-coumaric acid).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first determined whether the intracellular compartmentalization of RACK1 is altered by exposure to ethanol. Figure 1 confirms our earlier findings (20) that RACK1 is localized to perinuclear structures in unstimulated CHO cells (Fig. 1A-d , 1B-b , and ref 20 ). However, exposure to ethanol caused RACK1 to move to the nucleus (Fig. 1A-e, f , Fig. 1B-d ). Ethanol-induced movement of RACK1 to the nucleus was also observed in C6 glioma cell lines (see Fig. 4B,C ) and NG108–15 neuroblastoma-glioma (data not shown). We concluded that ethanol-induced RACK1 to be compartmentalized in the nucleus since immunofluorescence confocal images are of middle section of projected Z series, and therefore likely to be in the middle of the nucleus. To confirm that RACK1 is indeed compartmentalized in the nucleus in response to ethanol, cells were stained with anti-RACK1 antibodies together with the nuclear marker TOTO-3. Images were quantified as described in Materials and Methods. As shown in Fig. 4B , more than 50% of RACK1 colocalized with the nuclear marker TOTO-3 after exposure to 200 mM ethanol in C6 glioma cells. Ethanol-induced nuclear compartmentalization of RACK1 was observed in all three tested cell lines, but the degree of nuclear compartmentalization varied between cell types as a function of time and concentration. In CHO cells for example, ethanol concentrations as low as 5 mM caused RACK1 to move to the nucleus if cells were exposed for 60 min (Fig. 1B-d ).

We next determined whether ethanol alters the intracellular compartmentalization of ßIIPKC. ßIIPKC was unaffected by ethanol in any of the tested cell lines (Fig. 1 and data not shown). In unstimulated CHO cells ßIIPKC is localized to the cytoplasm (Fig. 1A-a , B-a , and ref 20 ). Exposure to ethanol did not alter the cytoplasmic staining of ßIIPKC even at concentrations as high as 200 mM (Fig. 1A-b, c , Fig. 1B-c ). Furthermore, prolonged incubation with ethanol up to 60 min did not induce ßIIPKC translocation at all tested concentrations (Fig. 1B-c and data not shown). Ethanol, therefore, induced a rapid movement of RACK1 to the nucleus but did not affect the compartmentalization of ßIIPKC.

There is substantial evidence that RACK1 directs the compartmentalization and function of PKC (18 19 20 , 25 26 27 28 29 30 31 32 33 34) . We recently found that RACK1 is a shuttling protein for ßIIPKC (20) . Upon receptor activation, RACK1 binds activated ßIIPKC and translocates it to another site where ßIIPKC is thought to phosphorylate its appropriate substrate(s) (20) . Therefore, we determined whether ethanol-induced nuclear compartmentalization of RACK1 affects the translocation of ßIIPKC in response to receptor activation. We used CHO cells expressing the dopamine D2 receptors (CHO/D2L) as our model system. We previously found that activation of the dopamine D2 receptors with the selective agonist NPA results in the translocation of RACK1 and ßIIPKC (20) . Using specific Golgi markers anti-mannosidase antibodies and BODIPY-FL-C5, the site of activation-induced translocated RACK1 and ßIIPKC in CHO/D2L cells was identified as the Golgi apparatus (20) . Indeed, Fig. 2 shows that in NPA-treated cells, more than 80% of RACK1 and ßIIPKC are localized to the Golgi apparatus (Fig. 2, black bars). We next asked whether exposure to ethanol alters these responses to NPA. Figures 1 and 2 show that exposure to ethanol induced the movement of RACK1 to the nucleus (Fig. 2 , hatched bars), but did not affect the cytosolic compartmentalization of ßIIPKC. On the other hand, when ethanol was added for 30 min prior to NPA, ßIIPKC translocation to the Golgi apparatus was inhibited (Fig. 2) , whereas RACK1 remained targeted to the nucleus by ethanol despite the addition of NPA (Fig. 2 , hatched bar). Therefore, ethanol inhibited NPA-induced translocation of ßIIPKC and changed the direction of RACK1 movement from the Golgi to the nucleus. Since RACK1 remained in the nucleus in the presence of ethanol, it is possible that the inhibition of ßIIPKC translocation by ethanol was due to the miscompartmentalization of RACK1, resulting in uncoupling of the targeting protein RACK1 from its kinase ßIIPKC.

We next determined whether ethanol-induced nuclear compartmentalization of RACK1 and the inhibition of NPA-induced ßIIPKC translocation was due to a direct effect of ethanol on the interaction between ßIIPKC and RACK1 and/or ßIIPKC to other proteins. Recombinant RACK1 was immobilized on an amylose resin column, and ßIIPKC binding was tested in the presence or absence of PKC activators and ethanol. Figure 3A shows that ethanol concentrations as high as 200 mM did not affect the interaction of activated ßIIPKC to RACK1. The inability of ethanol to affect the interaction between RACK1 and ßIIPKC was not due to excess amounts of RACK1 and/or ßIIPKC, since the PKC inhibitors calphostin C and DECA were able to inhibit the binding interaction using same concentration of proteins (20) . We also investigated the possibility that ethanol directly affects the binding of ßIIPKC to other cell proteins. Triton soluble extracts from CHO cells were run on an SDS gel, transferred to nitrocellulose, and overlaid with ßIIPKC in the presence of PKC activators with or without ethanol. Ethanol did not affect the binding of activated ßIIPKC (Fig. 3B ) or inactive ßIIPKC (data not shown) to cell extract proteins. These results suggest that ethanol-induced nuclear compartmentalization of RACK1 and inhibition of ßIIPKC translocation are not due to a direct effect of ethanol on ßIIPKC binding to RACK1 or to other proteins.



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  		Figure 3. Ethanol does not affect ßIIPKC binding to RACK1 (A) or cell extract proteins (B). A) ßIIPKC binding to RACK1. An in vitro binding assay was performed as described in Materials and Methods. ßIIPKC binding to RACK1 in the presence of DAG, PS, and calcium (lanes 1–3) and in the presence of 50 mM and 200 mM ethanol, respectively (lanes 2, 3), and overlay buffer alone (lane 4). Data are representative of three experiments. B) ßIIPKC binding to cell extract proteins. An in vitro overlay assay was performed as described in Materials and Methods. ßIIPKC binding to cell extract proteins in the presence of DAG, PS, and calcium (lanes 1, 2) and in the presence of 200 mM ethanol (lane 2). The 80 kDa band of ßIIPKC in the cell extract is not shown. Data are representative of three experiments.

It is possible that ethanol induces RACK1 to move to the nucleus via a signaling cascade. We found that in C6 glioma cells, the adenylate cyclase activator forskolin induced RACK1 to move to the nucleus (Fig. 4A-b and B (gray bars). Forskolin induced RACK1 nuclear compartmentalization in CHO and NG108–15 cells as well without affecting the compartmentalization of ßIIPKC (data not shown). If ethanol is mediating RACK1 movement to the nucleus via the cAMP signaling cascade, then an inhibitory analog of cAMP should inhibit RACK1 nuclear compartmentalization. Indeed, an inhibitory analog of cAMP, RpcAMP, inhibited forskolin-induced RACK1 nuclear localization (data not shown) and completely prevented ethanol-induced RACK1 nuclear compartmentalization (Fig. 4C , gray). Therefore, it is likely that activation of adenylate cyclase and increase in cAMP levels in the presence of ethanol are mediating the movement of RACK1 to the nucleus in response to ethanol.

Next, we determined whether ethanol-induced nuclear compartmentalization of RACK1 also occurs in vivo. Mice were treated with ethanol acutely (4 g/kg 30 min i.p.) or chronically (3 days vapor in chamber). After acute injection of 4 g/kg ethanol, brain ethanol concentrations were 3.8 ± 0.1 mg/ml. For mice treated for 3 days of ethanol inhalation by vapor chamber, blood alcohol concentrations were initially maintained at ~1.9 mg/ml and declined gradually to an average of 1.1 ± 0.1 mg/ml at the time of removal from the chamber after 72 h. Brain and blood ethanol concentrations were assayed by a previously published chromatographic method (25) . Brain slices were stained for RACK1, and the nuclear marker TOTO-3. RACK1 was widely expressed in the brain (R. Yaka and D. Ron, unpublished results). If ethanol induces nuclear compartmentalization of RACK1 in vivo, then RACK1 should be found in the nuclei and should be localized with the nuclear marker TOTO-3 after exposure to alcohol. Indeed, in the CA2/CA3 region of the hippocampus and granular layer of the cerebellum of ethanol-treated mice, RACK1 was partially localized to nuclei detected after animals were exposed to vapor chambers for 3 days (Fig. 5A, B-b, d and Fig. 5D ). On the other hand, in these regions RACK1 was not detected in nuclei of control animals (Fig. 5A, B-a, c ). Ethanol-induced nuclear compartmentalization of RACK1 was specific to the hippocampus CA2/CA3 region and the cerebellum and was not detected in any other tested brain region (data not shown). After acute exposure to ethanol, however, nuclear compartmentalization of RACK1 was observed only in the cerebellum and not in the hippocampus (Fig. 5D ). It is noteworthy that RACK1 was highly localized to the nuclei in the CA1 regions of hippocampus in naive animals and this was not increased further by alcohol treatment (data not shown). We also tested whether ethanol alters the compartmentalization of ßIIPKC, or induces translocation of the kinase to the nucleus. Ethanol did not induce ßIIPKC translocation to the nuclei in any tested brain region (Fig. 5C, D and data not shown), nor could we detect any other changes in kinase compartmentalization after ethanol treatment (Fig. 5C and data not shown). Therefore, exposure of mice to ethanol induces the nuclear compartmentalization of RACK1 in specific brain regions without changing the compartmentalization of ßIIPKC.



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  		Figure 5. Ethanol induces translocation of RACK1 but not ßIIPKC in brain. Chronic ethanol treatment induces compartmentalization of RACK1 to nuclei in the CA2/CA3 region of the hippocampus (A) and in the granular layer of the cerebellum (B). Adult male WSC outbred mice were exposed to the alcohol dehydrogenase inhibitor pyrazole (a, c) or to pyrazole in an ethanol vapor chamber for 3 days (b, d) as described in Materials and Methods. RACK1 nuclear compartmentalization was assayed by immunostaining with monoclonal anti-RACK1 antibodies (Transduction, 1:100) and visualized with secondary antibodies conjugated to Texas red (1:500) and the nuclear marker TOTO-3. Sections were scanned using a confocal microscope and viewed with a 40x lens. The data were analyzed using the computer program NIH image 1.61 as described in Materials and Methods and processed using Adobe PhotoShop. RACK1 staining is depicted in black and white (a, b) and in green (c, d). TOTO-3 staining is depicted in red (c, d) and merged staining is depicted in yellow/orange (c, d). The figure is representative of three mice that were used per treatment. Three slices were mounted and viewed from each mouse brain. Three images were processed from each slice. a) RACK1 staining in CA2/CA3 region of the hippocampus (A) or cerebellum (B) of pyrazole-treated animal. b) RACK1 staining in CA2/CA3 region of the hippocampus (A) or cerebellum (B) of pyrazole-treated animal in an ethanol vapor chamber for 3 days. c) Merged image of RACK1 and TOTO-3 staining of a pyrazole-treated animal in CA2/CA3 region of the hippocampus (A) or cerebellum (B). d) Merged image of RACK1 and TOTO-3 staining in CA2/CA3 region of the hippocampus (A) or cerebellum (B) pyrazole-treated animal in an ethanol vapor chamber for 3 days. C) Chronic ethanol treatment does not induces translocation of ßIIPKC to nuclei in the CA2/CA3 region of the hippocampus and in the cerebellum. Adult male WSC outbred mice were exposed to the alcohol dehydrogenase inhibitor pyrazole (a, c) or to pyrazole in an ethanol vapor chamber for 3 days (b, d) as described in Materials and Methods. ßIIPKC compartmentalization was assayed by immunostaining with polyclonal anti-ßIIPKC antibodies (Santa Cruz, 1:100) and visualized with secondary antibodies


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here describe a novel mechanism to account, in part, for ethanol-induced changes in PKC activity. We find that ethanol promotes movement of the PKC targeting protein RACK1 to the nucleus in cells in culture and in selected regions in the brain. Compartmentalization of the corresponding kinase ßIIPKC is unaffected by ethanol, but ethanol prevents activation-induced translocation of ßIIPKC. We recently reported that PKC activation causes RACK1 to move to the same site as ßIIPKC and proposed that RACK1 targets ßIIPKC to specific locations by serving as a shuttling protein (20) . Our data here suggest that ethanol uncouples RACK1 from ßIIPKC, thereby interfering with this shuttling function. As depicted in Fig. 6 , our model suggests that when RACK1 is sequestered in the nucleus, it is not available to shuttle ßIIPKC (Fig. 6C ). As a result of uncoupling RACK1 from ßIIPKC, therefore, activation-induced ßIIPKC translocation is inhibited. This model is consistent with the concept that RACK1 modulates ßIIPKC compartmentalization and function (20 , 33) . Furthermore, this model may also account for non-ethanol-related events in which RACK1 levels are reduced. For example, in the cortex of adult rat, a 50% reduction in RACK1 expression is associated with inhibition of activation-induced translocation of PKC (29 , 32) . Thus, activation-induced PKC translocation is inhibited when RACK1 is not available for shuttling. Several studies show that ethanol mediates its effects by activating PKC (reviewed in ref 2 ), and we show a different mechanism in which ethanol inhibits a PKC isozyme. It is therefore likely that ethanol will affect different PKC isozymes differently depending on modes of targeting, receptor, and second messenger systems.



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  		Figure 6. Scheme for ethanol-induced uncoupling of RACK1 from ßIIPKC. A) ßIIPKC (red) and RACK1 (blue) are localized to unique subcellular compartments in unstimulated cells. B) Signaling leads to generation of second messengers and activation of PKC, which results in binding of ßIIPKC to RACK1 and translocation of both proteins to a site where ßIIPKC phosphorylates its substrate. C) Ethanol presumably via activation of adenylate cyclase causes RACK1 to be localized to the nucleus but the compartmentalization of ßIIPKC remains unchanged. As a result ßIIPKC cannot interact with RACK1, thereby preventing PKC activation.

What mechanism might account for the movement of RACK1 to the nucleus? We find that the adenylate cyclase activator forskolin induces RACK1 movement to the nucleus without affecting the compartmentalization of ßIIPKC. This is the first demonstration of a shuttling protein moving to different intracellular sites with different stimuli. RACK1 has recently been shown to bind to different signaling proteins in addition to ßIIPKC (28 , 36 37 38) . Thus, it is possible that activation of adenylate cyclase results in the shuttling of an unidentified protein to the nucleus together with RACK1. Alternatively, it is possible that RACK1 in the nucleus plays a role in events such as cAMP-mediated gene regulation. We are currently investigating these possibilities. It is likely that activation of adenylate cyclase by ethanol induces RACK1 movement to the nucleus, since an inhibitory analog of cAMP (RpcAMP) inhibits ethanol-induced RACK1 nuclear compartmentalization. These results are in line with several reports showing that ethanol increases the activity of adenylate cyclase (39) , increases the production of cAMP (40 41 42) , and activates the cAMP/PKA signal transduction cascade (21 , 43) . It is surprising that ethanol and forskolin have the same magnitude of an effect on RACK1 nuclear compartmentalization. It is possible though that low levels of cAMP are required for RACK1 to move to the nucleus. Another possibility is that C6 glioma cells secret an activator of cAMP signaling that facilitates in ethanol’s effects. We also cannot exclude the possibility that other signaling cascades may be involved in the nuclear compartmentalization of RACK1.

We found ethanol-induced changes in RACK1 compartmentalization in two brain areas involved in ethanol-induced abnormalities affecting learning, memory, and coordination. We found that mice exposed to ethanol exhibit RACK1 movement to the nuclei in the CA2/CA3 region of the hippocampus and in the cerebellum. On the other hand, the compartmentalization of ßIIPKC was unchanged in these regions. Such miscompartmentalization of RACK1 may contribute to ethanol-induced changes in PKC activity that could lead to ethanol-induced changes in brain function. An ethanol-induced decrease in PKC activity has been previously reported (12 , 44 45) . Since ßIIPKC is a major PKC isozyme in the brain (46) , it is possible that the uncoupling of the kinase from its targeting protein RACK1 is responsible for part of the decrease in PKC activity previously observed with exposure to ethanol. For example, activation of the NMDA receptor in the hippocampus may be involved in processes such as learning and memory (47) . PKC enhances NMDA receptor activity (48) by phosphorylation of specific subunits (49) . Ethanol, on the other hand, inhibits NMDA receptor activation (50) . It is therefore possible that the miscompartmentalization of RACK1 interferes with PKC phosphorylation of the NMDA receptor, thereby limiting normal NMDA receptor activity. Furthermore, a recent report demonstrated that both RACK1 and ßIIPKC associate with ß1 and ß3 subunits of GABAA receptors and that ßIIPKC phosphorylates these subunits (28) . GABAA receptors are important targets for ethanol action (51) . PKC phosphorylation of the GABAA receptor subunits regulates receptor function (52) . It is possible that in response to ethanol, GABAA phosphorylation on ß1 and ß3 subunits will be reduced due to the inhibition of ßIIPKC function due to RACK1 miscompartmentalization. We are currently investigating these possibilities.

In summary, we present here a novel molecular mechanism (Fig. 6) in which ethanol via activation of adenylate cyclase induces uncoupling of the targeting protein RACK1 from its kinase ßIIPKC, thereby preventing PKC translocation and presumably interfering with normal activity of the kinase. This model opens a new avenue of investigation to explain how ethanol modulates signaling cascades. It is therefore of interest to determine whether ethanol alters the function of other targeting proteins.



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  		Figure 5A. (Continued) conjugated to FITC (1:500). Sections were scanned using a confocal microscope and viewed with a 40x lens. The data were analyzed using the computer program NIH image 1.61 as described in Materials and Methods and processed using Adobe PhotoShop. The figure is representative of three mice that were used per treatment. Three slices were mounted and viewed from each mouse brain. Three images were processed from each slice. a) ßIIPKC staining in CA2/CA3 region of the hippocampus of pyrazole-treated animal. b) ßIIPKC staining in CA2/CA3 region of the hippocampus of pyrazole-treated animal in an ethanol vapor chamber for 3 days. c) ßIIPKC staining in cerebellum of pyrazole-treated animal. d) ßIIPKC staining in cerebellum of pyrazole-treated animal in an ethanol vapor chamber for 3 days. D) Nuclear compartmentalization of RACK1 and ßIIPKC in the CA2/CA3 region of the hippocampus and in the cerebellum. The percentage of ßIIPKC (open bars) and RACK1 (solid bars) cocompartmentalization is defined as:


Data are the mean ± SD data obtained from three mice per treatment. Three slices were mounted and viewed from each mouse brain. Three images were processed from each slice.


   ACKNOWLEDGMENTS
 
This work was supported in part by the National Institutes of Health and funds provided by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco. We would like to thank Dr. Adrienne Gordon for helpful discussions and Drs. Robert Messing and Patricia Janak for critical reading of the manuscript.


   FOOTNOTES
 
2 Present Address: Texas A&M University, Health Science Center, Departments of Human Anatomy and Neurobiology, College Station, TX 77843-1114, USA.

3 Present address: Furman University, Department of Psychology, Greenville, SC 29613, USA.

Received for publication March 14, 2000. Revision received May 15, 2000.
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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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