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Originally published In Press as doi:10.1074/jbc.M406994200 on August 6, 2004

J. Biol. Chem., Vol. 279, Issue 41, 43321-43329, October 8, 2004
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cAMP-dependent Protein Kinase Type I Regulates Ethanol-induced cAMP Response Element-mediated Gene Expression via Activation of CREB-binding Protein and Inhibition of MAPK*

Anastasia Constantinescu{ddagger}§, Meiye Wu{ddagger}, Orna Asher¶, and Ivan Diamond{ddagger}||**

From the {ddagger}Ernest Gallo Clinic and Research Center, Department of Neurology, the ||Department of Cellular and Molecular Pharmacology, and the **Neuroscience Graduate Program and Center for the Neurobiology of Addiction, University of California, San Francisco, Emeryville, California 94608 and the Magen David Adom National Blood Services Center, Tel Hashomer, Ramat-Gan 52621, Israel

Received for publication, June 23, 2004 , and in revised form, July 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that the two types of cAMP-dependent protein kinase (PKA) in NG108-15 cells differentially mediate forskolin- and ethanol-induced cAMP response element (CRE)-binding protein (CREB) phosphorylation and CRE-mediated gene transcription. Activated type II PKA is translocated into the nucleus where it phosphorylates CREB. By contrast, activated type I PKA does not translocate to the nucleus but is required for CRE-mediated gene transcription by inducing the activation of other transcription cofactors such as CREB-binding protein (CBP). We show here that CBP is required for forskolin- and ethanol-induced CRE-mediated gene expression. Forskolin- and ethanol-induced CBP phosphorylation, demonstrable at 10 min, persists up to 24 h. CBP phosphorylation requires type I PKA but not type II PKA. In NG108-15 cells, ethanol and forskolin activation of type I PKA also inhibits several components of the MAPK pathway including B-Raf kinase, ERK1/2, and p90RSK phosphorylation. As a result, unphosphorylated p90RSK no longer binds to nor inhibits CBP. Moreover, MEK inhibition by PD98059 induces a significant increase of CRE-mediated gene activation. Taken together, our findings suggest that inhibition of the MAPK pathway enhances cAMP-dependent gene activation during exposure of NG108-15 cells to ethanol. This mechanism appears to involve type I PKA-dependent phosphorylation of CBP and inhibition of MEK-dependent phosphorylation of p90RSK. Under these conditions p90RSK is no longer bound to CBP, thereby promoting CBP-dependent CREB-mediated gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Repeated exposure to ethanol causes specific changes in several hormone- and neurotransmitter-activated signal transduction pathways, including cAMP and MAPK.1 Both pathways are required for synaptic plasticity related to learning, memory (1, 2), and addictive behaviors (3, 4). Our laboratory has shown that ethanol promotes cAMP signaling by stimulating adenosine A2 receptor activation of adenylyl cyclase (5), leading to activation and translocation of the catalytic subunit (C{alpha}) of PKA to the nucleus (6, 7), phosphorylation of CREB (7), and induction of CRE-mediated gene transcription (8-10).

Several laboratories have determined that MAPK, also known as ERK, is inhibited by acute and chronic ethanol exposure in almost all rat brain regions and in mouse cortex (11-15). In the brain, the MAPK pathway regulates CRE-mediated gene expression via phosphorylation of CREB and CBP (16-18). However, little is known about the interaction of PKA and MAPK in mediating responses to ethanol at a cellular level.

We have shown that ethanol-induced CREB phosphorylation and gene activation in NG108-15 cells are differentially mediated by the two isoforms of PKA (9). Type II PKA is activated by ethanol and translocated to the nucleus, phosphorylating CREB. This step is necessary but not sufficient for gene transcription. By contrast, PKA type I is not translocated into the nucleus and does not phosphorylate CREB. Nevertheless, type I PKA is also required for ethanol-induced gene expression. This suggested that PKA type I regulates a downstream cytoplasmic pathway leading to activation of other transcription cofactors, which interact with phosphorylated CREB to induce gene transcription. The cofactor CBP links the DNA-binding factor CREB to the basal transcription machinery. There is increasing evidence that positive regulation (enhancement of cAMP-dependent gene transcription) is attained through phosphorylation of a factor other than CREB, possibly CBP itself. In this study we demonstrate that ethanol-induced activation of PKA type I causes phosphorylation of CBP as well as inhibition of the MAPK pathway. As a result, 90-KDa ribosomal S6 kinase (p90RSK), the downstream substrate of ERK, is dephosphorylated and no longer binds to nor inhibits CBP, thereby further enhancing CRE-mediated gene transcription.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were purchased from Sigma except where indicated otherwise.

Cell Culture—NG108-15 neuroblastoma x glioma hybrid cells obtained from the cell culture facility at the University of California, San Francisco were grown in 10% Serum Plus (JRH Biosciences) at 37 °C and 10% CO2 as described previously (19). All cells were maintained for 3 days in complete defined medium (20). On day 3, media were changed and the cells maintained in the absence or presence of 100 mM ethanol for various time intervals. Treatment with 1 µM forskolin was carried out for 10 min. 300 µM Rp-Cl-cAMPS (RpI) or Rp-CPT-cAMPS (RpII) (Biolog) was added 2 h prior to ethanol or forskolin exposure and remained throughout the experiments.

Immunocytochemistry—Cells were fixed with 4% paraformaldehyde, blocked in 4% normal goat serum (Jackson ImmunoResearch Laboratories), and incubated overnight with primary antibodies. Monoclonal antibodies for phospho-ERK and polyclonal antibodies for ERK (Cell Signaling Technology) were diluted 1:400. Fluorescein isothiocyanate-conjugated goat anti-mouse and Texas Red-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories) were diluted 1:250. No fluorescence was detected when cells were incubated with fluorophor-conjugated secondary antibody only.

Cell Fractionation—Nuclear fractions were isolated from NG108-15 cells in hypertonic sucrose as described previously (7, 9). The protein content in all fractions was determined by the Bradford Bio-Rad protein assay.

Immunoprecipitation—Nuclear or whole cell extracts (0.5 mg protein) were resuspended in 1 ml of immunoprecipitation buffer (20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, pH 8, 300 mM NaCl, 0.4 mM sodium orthovanadate, 0.4 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2% Triton X-100, 1% Ighepal CA-630) and precleared for 30 min with protein A/G-conjugated agarose. Supernatant from a 10-min centrifugation at 10,000 rpm was incubated overnight with either 2 µg of polyclonal anti-CBP antibodies (Santa Cruz Biotechnology, Inc.) or polyclonal anti-B-Raf antibodies (Upstate Biotechnology) and further incubated with protein A/G-conjugated agarose for 1 h. The immunoprecipitate was centrifuged and washed four times with phosphate-buffered saline, pH 7.0. All incubations were at 4 °C.

B-Raf Kinase Assay—The assay was performed using a kit from Upstate Biotechnology according to the manufacturer protocol. Briefly, B-Raf immunoprecipitates or recombinant B-Raf was incubated in assay buffer with inactive MEK, inactive ERK2, and magnesium-ATP for 30 min at 30 °C. Tubes were then centrifuged, and supernatant was further incubated for 10 min at 30 °C with a mixture containing myelin basic protein as substrate and [{gamma}-32P]ATP. The reaction was stopped by applying the mixture to P81 filter paper. Filters were washed and counted with a Beckman scintillation counter. The kinase activity was then normalized to the amount of B-Raf immunoprecipitated from NG108-15 cells.

Western Blots—Total cell lysates and B-Raf immunoprecipitates were subjected to SDS-PAGE and proteins transferred to polyvinylidene difluoride membranes as described previously (7). For CBP immunoprecipitates, we used 3-8% gradient NuPAGE Novex Tris acetate pre-cast gels (Invitrogen) to ensure the entry of high molecular weight CBP protein. Blots were probed with polyclonal antibodies against ERK, phospho-ERK, phospho-p90RSK (Cell Signaling Technology), and monoclonal anti-phospho-Ser/Thr and anti-AKAP95 antibodies (Transduction Laboratories), diluted 1:1000. Secondary antibodies (Cell Signaling Technology) were horseradish peroxidase-linked goat anti-rabbit (1:1000) for ERK, phospho-ERK, and phospho p90RSK and rabbit anti-mouse (1:2000) for anti-phospho-Ser/Thr and anti-AKAP95. Proteins were detected using a LumiGLO chemiluminescence substrate (PerkinElmer Life Sciences) and exposure to Kodak Biomax film. Scanning densitometry was used to quantitate Western blots using the NIH Image program.

Luciferase Assay—NG108-15 cells (5 x 104) were transfected in defined media with 150 ng of the pFC-CRE-luciferase plasmid from Stratagene using Effectene (Qiagen) as described by the manufacturer. Where specified, cells were also co-transfected with the expression vector encoding for 12 S E1A and a deletion mutant ({Delta}2-36, a generous gift from Dr. Robert Rooney, Duke University Medical Center). Media were changed 24 h after transfection. To activate CRE-mediated gene expression, the cells were incubated in either 1 µM forskolin or 100 mM ethanol for 10 min, washed, and maintained in fresh media for 5 h before harvesting (10). Cell extracts were prepared and luciferase activity measured as described (8). Each point represents three separate experiments with six replicate samples for each point.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In our previous study we showed that both types of PKA (I and II) are required for ethanol-induced CRE-mediated gene expression (9). We showed that upon activation, type II trans-locates into the nucleus and phosphorylates CREB leading to gene expression. By contrast, type I did not translocate into the nucleus but appeared to activate a cytoplasmic pathway leading to increased gene expression (9). These results suggested that phosphorylation of a factor other than CREB might also be involved.

Ethanol-induced CRE-Luciferase Activity Requires CBP—The cofactor CBP links the DNA-binding factor CREB to the basal transcription machinery. There is increasing evidence that positive regulation (enhancement of cAMP-dependent transcription) is attained through phosphorylation of a factor other than CREB, possibly CBP itself. Therefore, we asked whether CBP and its phosphorylation are essential for ethanol-induced gene expression. NG108-15 cells were transiently cotransfected with pFC-CRE-luciferase plasmid and a vector encoding 12 S E1A, an adenovirus-transforming protein that inhibits the function of CBP (21). Cells were treated with and without ethanol or forskolin for 10 min and luciferase activity measured 5 h later. Fig. 1 shows that treatment with either ethanol or forskolin significantly increases CRE-luciferase activity. Over-expression of full-length CBP inhibitor, E1A, but not a deletion mutant ({Delta}2-36) that lacks the CBP binding site, inhibited ethanol- and forskolin-induced luciferase activity. In untreated cells expression of full-length E1A or its deletion mutant did not change the basal level of luciferase activity. These results suggest that CBP is required for ethanol- and forskolin-induced CRE-mediated gene transcription.



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  		FIG. 1.
Ethanol-induced CRE-luciferase activity requires CBP. NG108-15 cells were transfected with a CRE-luciferase construct (150 ng) and with an expression vector for the CBP inhibitor, E1A, or a deletion mutant, {Delta}2-36, where indicated (mE1A). Media were changed 16 h after transfection as described under "Experimental Procedures." Cells were incubated in either 1 µM forskolin (Fsk) or 100 mM ethanol (EtOH) for 10 min, and luciferase expression was measured 5 h later. The results are expressed as absolute luciferase units (ALU)/mg of protein. Each point represents three separate experiments with 6 replicate samples for each point. Data are presented as means ± S.E. *, significantly different (p < 0.005) from the corresponding untreated cells (Student's t test).

 
Ethanol- and Forskolin-induced CBP Phosphorylation Requires Type I PKA—There is increasing evidence that phosphorylation of CBP enhances CREB-dependent gene transcription (22-25). To determine whether ethanol increases CBP phosphorylation we prepared nuclei of NG108-15 cells incubated in the absence and presence of forskolin or ethanol. CBP, immunoprecipitated from the nuclear extracts, was subjected to SDS electrophoresis, blotted, and probed with antibodies against phosphoserine/threonine. As seen in Fig. 2, A and B, ethanol induces a robust and long-lasting phosphorylation of CBP. At 10 min, the level of CBP phosphorylation induced by 100 mM ethanol is comparable with that induced by 1 µM forskolin.



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  		FIG. 2.
Ethanol- and forskolin-induced CBP phosphorylation requires type I PKA. NG108-15 cells were incubated with 100 mM ethanol for the indicated times, and nuclear extracts were prepared as described under "Experimental Procedures." A, Western blot of CBP immunoprecipitates (IP) from nuclear extracts were probed with monoclonal antibodies that recognize phosphorylated Ser/Thr. The blot shown is representative of three experiments. C, control; F, forskolin. B, densitometric quantitation of phosphorylated CBP in A. C, Western blot of CBP immunoprecipitates from cells treated with 300 µM RpI or RpII for 2 h and 100 mM ethanol for an additional 3 h. Blots were probed with monoclonal antibodies that recognize phosphorylated Ser/Thr. The blot shown is representative of three experiments. D, densitometric quantification of phosphorylated CBP in C.

 
We have shown that type I PKA is required for ethanol-induced gene expression. We proposed that type I PKA activates a cytoplasmic downstream pathway leading to enhanced CREB-dependent gene expression. Therefore, we asked whether type I PKA is responsible for CBP phosphorylation. NG108-15 cells were preincubated with 300 µM RpI or RpII for 2 h followed by incubation with 100 mM ethanol for 3 h. Type I PKA inhibition reduced forskolin- and ethanol-induced CBP phosphorylation, whereas type II inhibition was without effect (Fig. 2, C and D). These results suggest that ethanol- or forskolin-induced CBP phosphorylation requires type I but not type II PKA.

Ethanol and Forskolin Inhibition of ERK1/2 Requires Type I PKA—Although type I PKA is required for CBP phosphorylation and CRE-mediated gene expression, type I PKA is not translocated into the nucleus and does not phosphorylate CBP directly. This suggested that type I PKA regulates a cytoplasmic downstream pathway leading to enhanced gene expression. The MAPK pathway, a major signaling pathway in neural cells that can be regulated by PKA, is implicated in the pathophysiology of alcoholism (12, 14, 26-28). Therefore, we asked whether ethanol affects ERK phosphorylation in NG108-15 cells. Our results indicate that exposure to ethanol causes dephosphorylation of pERK (Fig. 3A, left panel). Similar results were obtained after exposure to forskolin (Fig. 3A, left panel). cAMP activates type I and type II PKA. Therefore, we asked whether type I or type II PKA mediates ethanol-induced dephosphorylation of ERK. NG108-15 cells were pretreated with 300 µM RpI or RpII for 2 h followed by additional indicated times with either 1 µM forskolin or 100 mM ethanol. The results in Fig. 3A, left panel, show that RpI but not RpII reverses ethanol- and forskolin-induced ERK dephosphorylation. Control studies (Fig. 3A, right panel) show that the total amount of ERK is unchanged under these conditions. Therefore, the decrease in phosphorylated ERK cannot be explained by a reduction of total ERK protein. These results suggest that PKA type I is required for ethanol-induced inhibition of ERK in NG108-15 cells. ERK dephosphorylation reversed after 4 h despite the continued presence of ethanol up to 24 h (Fig. 3A). Interestingly, when cells were exposed to ethanol for only 10 min and media then replaced with fresh media without ethanol, ERK dephosphorylation persisted for 4 h whether or not ethanol was continuously present, whereas total ERK remained unchanged (Fig. 3B).



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  		FIG. 3.
Ethanol- and forskolin-induced ERK dephosphorylation requires type I PKA. A, Western blot (40 µg of protein) of cell lysates prepared from NG108-15 cells either untreated controls (C) or treated with 1 µM forskolin (F) for 10 min or with 100 mM ethanol for the indicated times in the absence or presence of either RpI or RpII. Blots were probed with antibody against phosphorylated ERK1/2 (pERK, left panel) or total ERK1/2 (ERK, right panel). B, Western blot (40 µg of protein) of cell lysates prepared from NG108-15 cells treated with or without 100 mM ethanol for 10 min and further incubated with fresh media for the indicated times. Blots were probed with antibodies against pERK or ERK. Each blot shown is representative of three experiments. C, NG108-15 cells were either untreated (a-d) or preincubated with 100 mM ethanol for 10 min (e-h), after which the medium was replaced and cells were further incubated for 4 h (b and f), 8 h (c and g), or 24 h (d and h). Cells were fixed, blocked, and probed with antibodies against ERK (red) and pERK (green) as described under "Experimental Procedures." All images are x60 magnification and were obtained with a Bio-Rad 1024 confocal microscope. The data shown are representative of three experiments.

 
It was demonstrated previously that active, phosphorylated ERK resides in the nucleus or cytoplasm depending on its sustained or transient activation, respectively (29). Therefore, we next used cytochemistry to relate the time course of ERK dephosphorylation to ERK intracellular localization. NG108-15 cells were treated with 100 mM ethanol for 10 min, and media then were replaced with fresh media without ethanol for an additional 4, 8, and 12 h, after which cells were fixed and stained as described in the legend for Fig. 3C. The results in Fig. 3C show that in untreated cells, ERK (red) is localized in the perinuclear area and the cytoplasm, whereas pERK (green) is found primarily in the nucleus (Fig. 3C, a). After 10 min with ethanol, pERK is not demonstrable in the nucleus and only unphosphorylated ERK is detected, primarily in the cytoplasm (Fig. 3C, e). With time, ERK phosphorylation is restored and pERK is found again in the nucleus (Fig. 3C, f-h). Untreated cells with only media replacement show no change in ERK activation and localization (Fig. 3C, b-d). These results demonstrate that ethanol induces transient ERK dephosphorylation and translocation from the nucleus in NG108-15 cells.

Ethanol or Forskolin Inhibits B-Raf Kinase via Type I PKA—ERK inhibition can be achieved either by down-regulation of kinases up-stream from ERK or by up-regulation of phosphatases. However, because ERK inhibition is PKA-dependent and activation of PKA has been shown to inhibit rather than stimulate several phosphatases implicated in the ERK pathway (30, 31), we favored the first possibility. The kinase immediately up-stream from ERK is MEK. MEK is uniquely activated by the Raf family of kinases (32). Therefore, we examined the amount and activity of B-Raf kinase in NG108-15 cells cultured in the absence and presence of 1 µM forskolin or 100 mM ethanol. We first determined that NG108-15 cells express the 95-KDa isoforms of the B-Raf protein (Fig. 4A). The 68-KDa isoform was not detected. Treatment of cells with either forskolin or ethanol did not change the total amount of B-Raf protein (Fig. 4A). However, the kinase activity of immunoprecipitated B-Raf shows a dramatic decrease during the first 4 h of ethanol exposure (Fig. 4B). Fig. 4C shows that the amount of B-Raf in the immunoprecipitates used in the kinase assay is not significantly changed during the experiment. These results suggest that ethanol inhibits B-Raf kinase activity, resulting in decreased ERK phosphorylation. The results presented in the previous section suggested that type I PKA is required for ethanol inhibition of ERK phosphorylation. Therefore, we next asked whether type I PKA is also required for forskolin and ethanol-induced inhibition of B-Raf kinase activity. NG108-15 cells were pretreated with the 300 µM RpI and/or RpII for 2 h followed either by forskolin for an additional 10 min or by ethanol for 10 min or 4 h. RpI abolished forskolin- and ethanol-induced inhibition of B-Raf kinase activity (Fig. 5), whereas RpII did not. These results suggest that in NG108-15 cells, ethanol activation of type I PKA is required for ethanol-induced inhibition of B-Raf.



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  		FIG. 4.
Ethanol and forskolin inhibit B-Raf kinase activity in NG108-15 cells. A, Western blot (40 µg of protein) of cell lysates prepared from NG108-15 cells, either untreated controls (C) or treated with 1 µM forskolin (F) for 10 min or with 100 mM ethanol for the indicated times. Blots were probed with monoclonal antibodies against B-Raf. The blot shown is representative of three experiments. B, kinase activity of B-Raf immunoprecipitated from NG108-15 cells treated as described in A. The results (CPM/IP) are expressed as counts per minute/density of immunoprecipitated B-Raf measured from the blot shown in C. Data are presented as mean ± S.E., n = 3. *, significantly different (p < 0.02) from the corresponding untreated cells (Student's t test). C, Western blot of the B-Raf immunoprecipitates (IP) obtained in B using polyclonal antibodies against B-Raf. The blot was probed with monoclonal antibodies against B-Raf and is representative of three experiments.

 



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  		FIG. 5.
Ethanol- and forskolin-induced inhibition of B-Raf requires type I PKA. A, kinase activity of B-Raf immunoprecipitated from NG108-15 cells pretreated with 300 µM RpI or RpII for 2 h followed by either 1 µM forskolin for 10 min or 100 mM ethanol for the indicated times. The results (CPM/IP) are expressed as counts per minute/density of immunoprecipitated B-Raf measured from the blot shown in B. Data are presented as mean ± S.E., n = 3. B, Western blot of the B-Raf immunoprecipitates (IP) obtained in A using polyclonal antibodies against B-Raf. The blot was probed with monoclonal antibodies against B-Raf and is representative of three experiments.

 
Ethanol or Forskolin Inhibits Phosphorylation of p90RSK and Its Binding to CBP—The substrate downstream of ERK is the p90RSK (33). Therefore, we first examined the phosphorylation state of p90RSK in NG108-15 cells cultured in the absence or presence of ethanol or forskolin. As expected, treatment of cells with 1 µM forskolin for 10 min significantly decreased phosphorylation of p90RSK (Fig. 6A). Similar results were found with 20 µM PD98059, a MEK inhibitor. Ethanol also induced a transient inhibition of p90RSK phosphorylation, consistent with the results presented in Fig. 3 showing reversible inhibition of ERK phosphorylation by ethanol. In PC12 cells, phosphorylated p90RSK binds to CBP and inhibits CRE-mediated gene transcription (34). Consistent with this observation, we find that dephosphorylation of p90RSK (Fig. 6A) is associated with increased CRE-mediated transcription (Fig. 1). This suggested that the phosphorylated p90RSK-CBP complex blocks CRE-mediated gene transcription, whereas dephosphorylated p90RSK releases CBP and allows initiation of CRE-mediated gene transcription. To test whether phosphorylated p90RSK is bound to CBP, we immunoprecipitated CBP from the nuclei of NG108-15 cells treated with or without ethanol or forskolin for different time intervals. Immunoprecipitates were subjected to SDS electrophoresis, blotted, and probed first with antibodies that recognize phosphorylated p90RSK. The blots were next stripped and probed with antibodies against CBP. Fig. 6B shows that CBP only co-immunoprecipitates with p90RSK when it is phosphorylated but not when p90RSK phosphorylation is reduced by forskolin, ethanol, or the MEK inhibitor PD98059. Fig. 6C shows that there was no change in the recovery of immunoprecipitated CBP throughout the experiment. As an additional control we also probed the same blot with antibodies that recognize another nuclear protein, AKAP95 (35). No band was detected at the 95-KDa position (result not shown).



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  		FIG. 6.
Ethanol and forskolin inhibit phosphorylation of p90RSK and binding to CBP. A, Western blot (40 µg of protein) of whole cell lysate from NG108-15 cells treated without (C, control) or with 1 µM forskolin (F) or 20 µM PD98059 (PD) for 10 min or with 100 mM ethanol for the indicated times. Blots were probed with antibodies against p90RSK phosphorylated at Thr-359/Ser-363 (pp90RSK) or antibodies against unphosphorylated p90RSK. The blot shown is representative of three experiments. B and C, NG108-15 cells were treated as described in A. Nuclear extracts were incubated with antibodies against CBP, and the immunoprecipitates were blotted, first probed with antibodies that recognize p90RSK phosphorylated at Thr359/Ser363 (B) and then stripped and probed again with antibodies against CBP (C). Blots are representative of three experiments.

 
Inhibition of MEK Induces CRE-mediated Luciferase Expression in NG108-15 Cells—Ethanol stimulation of CRE-mediated gene expression correlates with ethanol inhibition of B-Raf, ERK, and p90RSK. This suggested the possibility that inhibition of the MAPK pathway could increase CRE-mediated gene expression. To investigate this possibility we inhibited MAPK signaling with PD98059, a specific inhibitor of MEK, and measured downstream CRE-mediated gene transcription. NG108-15 cells require at least 5 h for the reporter luciferase protein to be transcribed and translated after stimulation (10). Therefore, when the incubation with PD98059 was less than 5 h, the media were replaced, and incubation continued up to 5 h. Fig. 7 shows that inhibition of MEK by PD98059 increases the basal level of CRE-dependent transcription at all time points tested to levels that approximate ethanol stimulation of CRE-dependent luciferase activity (Fig. 1). These results suggest that in NG108-15 cells, inhibition of the MAPK pathway enhances CRE-mediated gene expression.



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  		FIG. 7.
MEK inhibition induces CRE-mediated luciferase expression in NG108-15 cells. NG108-15 cells were transfected with a CRE-luciferase construct (150 ng) as described under "Experimental Procedures," and luciferase expression was measured in cells incubated in 20 µM PD98059 for the indicated times. The results are expressed as percent increase over control. Each point represents three separate experiments with 6 replicate samples for each point. Data are presented as means ± S.E. Basal luciferase activity without PD98059 averaged 13.4 ± 2.9 absolute luciferase units/mg of protein/min ± S.E., similar to that shown in Fig. 1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There are several major findings in this study. First, ethanol-induced CRE-mediated gene expression requires CBP. Second, ethanol induces CBP phosphorylation after activation of PKA type I. Third, ethanol-induced activation of PKA type I inhibits the MAPK pathway and reduces p90RSK phosphorylation, thereby leading to enhanced CBP-dependent CREB-mediated gene transcription. A schematic diagram of these pathways and their interaction is shown in Fig. 8.



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  		FIG. 8.
Schematic representation of forskolin (blue arrows) and ethanol (black arrows) activation of CRE-mediated gene expression in NG108-15 cells. A, ethanol (EtOH) inhibits adenosine uptake leading to an increase in extracellular adenosine and activation of the adenosine A2 receptor. This in turn leads to activation of adenylyl cyclase and an increase in cAMP (4). Forskolin bypasses the receptor and directly activates adenylate cyclase leading to increases in cAMP. Increased cAMP activates both types of PKA. Type II PKA translocates to the nucleus, phosphorylating CREB. Phosphorylated CREB is necessary but not sufficient for gene transcription (9). B, type I PKA, activated in cytoplasm, inhibits B-Raf kinase. C, down-regulated B-Raf inhibits the MEK-ERK-p90RSK pathway. D, unphosphorylated p90RSK no longer binds to CBP and releases the restraint on gene expression. In addition, ethanol- and forskolin-induced type I PKA leads to CBP phosphorylation (dashed arrows), enhancing CRE-mediated gene expression.

 
We have previously shown that ethanol inhibits adenosine uptake, leading to increases in extracellular adenosine, activation of adenosine A2 receptors, increased cAMP production, and activation and translocation of PKA into the nucleus. This causes phosphorylation of CREB and stimulation of cAMP-inducible gene expression (7-10). Phosphorylation of CREB at Ser-133 is required but not sufficient for CRE-mediated gene transcription (36). Phosphorylated CREB promotes recruitment of the transcriptional co-activator CBP, which connects the DNA-CREB complex to the basal transcription machinery (37). Although some groups find that binding of CBP to phosphorylated CREB is sufficient to induce CRE-mediated gene transcription (38), others report that CBP itself must be phosphorylated to promote transcription (24, 39-43). However, others suggest that CBP may not even be required for CREB-induced gene transcription (44). We had reported that type I PKA inhibition prevents ethanol-induced CRE-mediated gene expression but does not affect ethanol-induced CREB phosphorylation by type II PKA (9). This suggested that other transcription cofactors, directly or indirectly regulated by PKA type I, may interact with phosphorylated CREB to induce gene transcription (9). Here we demonstrate that CBP appears to be such a required cofactor.

E1A, an adenovirus-transforming protein, inhibits CBP, whereas E1A mutants, which cannot interact with CBP, do not. These viral agents have been used to establish the CBP requirement for CREB-mediated transcription in a variety of cell systems including cortical neurons (40), hippocampal neurons (24, 45), the pituitary cell line AtT20 (46), and the hepatoma cell line HepG2 (47) (for review see (Refs. 21 and 48). In this study we show that E1A abolishes ethanol-induced CRE-luciferase activity, suggesting that CBP is required for ethanol-induced gene transcription in NG108-15 cells.

We also show for the first time that ethanol and forskolin induce a robust and long-lasting phosphorylation of CBP; CBP phosphorylation requires cytoplasmic PKA type I. Several studies indicate that CBP can be phosphorylated by PKA directly (49, 50), but it is not clear which type of PKA was involved. Because ethanol or forskolin did not induce type I PKA translocation into the nucleus, we propose that an intermediate step is required for PKA type I-dependent phosphorylation of CBP in the nucleus (9). There is substantial evidence that CBP is phosphorylated by kinases such as calcium and calmodulin-dependent kinase (CaMK) II and IV (24, 39, 40, 42), cyclinE/Cdk2 (42) and MAPK (41, 43). PKA can activate all of these kinases directly or indirectly. For example, in striatal neurons, PKA-induced Ca2+ release from intracellular stores (51) activates CaMK and conventional PKC in the nucleus. Similarly, Medina et al. (52) demonstrated that Cdk2 is activated by PKA in thyroid cells.

MAPK also can be regulated by PKA (18, 53). Therefore, we asked whether MAPK mediates PKA type I-induced increases in gene expression. Our results show instead that ethanol and forskolin each inhibits MAPK in NG108-15 cells. Moreover, ethanol- and forskolin-induced inhibition of ERK phosphorylation is PKA type I-dependent, because it is abolished by PKA type I inhibition. This is consistent with emerging information that PKA activation or inhibition of MAPK depends on cell context. For example, PKA has been reported to inhibit (54-57) or activate MAPK (56, 58-62). Moreover, PKA has also been reported to inhibit or to activate MAPK in the same Chinese hamster ovary (CHO) cell line, depending on the strain of cells (63-65). Several models have been proposed to explain these paradoxical observations. One model proposes that the 95-KDa isoform of B-Raf kinase is required for PKA activation of MAPK (61). Because 95-KDa B-Raf is expressed in neurons, where PKA activates MAPK, and not in astrocytes, where PKA inhibits MAPK, it was further proposed that B-Raf acts as a molecular switch that permits differential regulation of MAPK by cAMP (56). According to this model, NG108-15 cells should not express B-Raf, because PKA inhibits MAPK in these cells. However, our results clearly demonstrate that type I PKA inhibits MAPK in the NG108-15 cell line despite the fact that these cells, like neuronal cells, express only the 95-KDa isoform of B-Raf. Our findings are consistent with other reports showing that the 68-KDa isoform of B-Raf is undetectable in NG108-15 cells (66). Importantly, we demonstrate that ethanol or forskolin treatment significantly decreases B-Raf kinase activity in NG108-15 cells without changing its amount. Ethanol- and forskolin-induced decreases in B-Raf kinase activity also require type I PKA. cAMP/PKA-dependent inhibition of B-Raf activity has been reported in other systems including PC12 cells, NIH3T3 cells, and neutrophils (67), as well as NG108-15 cells (66). The mechanism of B-Raf inhibition by type I PKA is not yet completely understood but may be related to tissue-specific interaction of B-Raf with 14-3-3 proteins. The mammalian family of 14-3-3 proteins regulates the activity, subcellular localization, and protein-protein interactions of many enzymes. 14-3-3 can bind to proteins that contain a signature recognition motif in their sequence, usually containing a phosphoserine. B-Raf contains three such putative sequences, and 14-3-3 binding protects B-Raf from PKA-mediated inhibition (67). The amount of 14-3-3 protein found in NG108-15 cells (result not shown) may not be sufficient to protect B-Raf from PKA-mediated inhibition.

The downstream substrate of ERK is p90RSK (Fig. 8). RSK cellular functions include regulation of gene expression by association with and phosphorylation of transcriptional factors such as CREB and CBP. Neurotrophic factors that activate the MAPK/ERK pathway can either decrease or increase CRE-mediated gene transcription. In PC12 cells, for instance, activation of the Ras-ERK pathway by insulin induces phosphorylation of p90RSK. Phosphorylated p90RSK then binds to CBP at the same site that also binds the CBP inhibitor oncoprotein EIA, thereby inhibiting transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene that is regulated by CREB and CBP (34). Other studies show enhancement of CBP and/or CREB transcriptional activity following activation of MAPK pathway (2, 33, 43, 59). Here we demonstrate, however, that under basal conditions p90RSK is phosphorylated and bound to CBP; but PKA activation by ethanol or forskolin inhibits the MAPK pathway, inhibits phosphorylation of p90RSK, and inhibits p90RSK binding to CBP. Similar results are obtained with the MEK inhibitor PD98059. Because ethanol, forskolin, and PD98059 all increase CREB-dependent gene transcription, we propose that in NG108-15 cells, inhibition of MAPK enhances CRE-mediated gene transcription.

Cross-talk between the cAMP and MAPK pathways is involved in many cellular functions. For example, it is well known that both PKA and MAPK can increase transcription of genes containing CRE in their promoter regions (17, 37). In NG108-15 cells, ethanol appears to up-regulate PKA signaling while down-regulating the MAPK pathway. Nevertheless, both of these ethanol-dependent events lead to an increase in CRE-mediated gene expression. This cooperative response is likely to have pathophysiologic significance in those brain regions where ethanol concomitantly increases PKA signaling while inhibiting the MAPK pathway.

Our findings are consistent with several reports showing that in the brain acute and chronic ethanol inhibits ERK in almost all regions (11-15), whereas other reports show that acute ethanol increases PKA signaling, CREB phosphorylation, and CRE-binding activity in vivo and in vitro (6-9, 68). There are, however, no available data about concomitant measurements of both MAPK and CRE-mediated gene expression in the same area of the brain after acute ethanol exposure. Our findings suggest that forskolin- or ethanol-induced inhibition of MAPK signaling appears to increase CRE-mediated gene expression by removing the restraint of CBP on CREB transcriptional activity. Ethanol-induced inhibition of ERK, therefore, might enhance ethanol-induced increases in PKA signaling. It remains to be determined whether ethanol activation of PKA and inhibition of MAPK signaling enhances CRE-dependent gene expression in the nucleus accumbens, an area of the brain involved in ethanol-seeking behavior and voluntary alcohol consumption (10, 69).


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R37 AA10030, funds provided by the State of California for medical research on alcohol and substance abuse through University of California, San Francisco, and Grant DAMD 17-01-1-0061 from the Department of the Army. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Ernest Gallo Clinic and Research Center, 5858 Horton St., Suite 200, Emeryville, CA 94608. Tel.: 510-985-3142; Fax: 510-985-3101; E-mail: anconst{at}itsa.ucsf.edu.

1 The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; pERK, phosphorylated ERK; CREB, cAMP response element-binding protein; CRE, cAMP response element; CBP, CREB-binding protein; p90RSK, 90-KDa ribosomal S6 kinase; PKA, protein kinase A; RpI, Rp-Cl-cAMPS; RpII, Rp-CPT-cAMPS. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Adrienne Gordon for many useful suggestions and Selma Karadottir for help with editing the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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