QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.042580v1 73/4/1105    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yao, L. Articles by Diamond, I. Search for Related Content PubMed PubMed Citation Articles by Yao, L. Articles by Diamond, I.

Dopamine and Ethanol Cause Translocation of PKC Associated with RACK: Cross-Talk between cAMP-Dependent Protein Kinase A and Protein Kinase C Signaling Pathways

CV Therapeutics, Inc., Palo Alto, California (L.Y., P.F., Z.J., I.D.); Ernest Gallo Clinic and Research Center, Emeryville, California (L.Y., A.G., I.D.); Departments of Neurology (L.Y., I.D.) and Cellular and Molecular Pharmacology (I.D.), and the Neuroscience Graduate Program (I.D.), University of California, San Francisco, California; and Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California (D.M.)

Received October 9, 2007; accepted January 17, 2008

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of PKC and the relationship to PKA activation. In the present study, we used a new PKC antibody, 14E6, that selectively recognized active PKC when not bound to its anchoring protein RACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated PKC and induced translocation of both PKC and its anchoring protein, RACK to a new cytosolic site. The selective PKC agonist, pseudo-RACK, activated PKC but did not cause translocation of the PKC/RACK complex to the cytosol. These data suggest a step-wise activation and translocation of PKC after NPA or ethanol treatment, where PKC first translocates and binds to its RACK and subsequently the PKC/RACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause PKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between PKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.

Stimulation of cells with hormones or neurotransmitters that trigger diacylglycerol (DAG) formation causes activation and translocation of PKC from one subcellular site to another (Mochly-Rosen and Gordon, 1998). Translocation of PKC is associated with anchoring of the activated enzyme to selective receptors for activated C-kinase (RACKs); the functional selectivity of each activated PKC isozyme is determined by its binding to a corresponding RACK (Mochly-Rosen and Gordon, 1998). However, it is not clear how the active enzyme translocates to its functional site where its RACK is located and what other enzymes may be involved in the activation and translocation process.

Alcohol and other addictive drugs seem to converge on specific dopaminergic pathways in the midbrain. In particular, dopamine D2 receptors (D2) have been implicated in the rewarding properties of these drugs (Robbins and Everitt, 1999; Volkow et al., 2004). We previously demonstrated in NG108-15/D2 cells that ethanol and the D2 agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) cause translocation of PKC from the perinuclear region to the cytoplasm (Gordon et al., 1997, 2001). PKC translocation in ethanol-stimulated cells reached maximum at 30 min, whereas NPA-induced PKC translocation was maximal at 10 min (Gordon et al., 1997, 2001). In these cells, ethanol and NPA also activated cAMP-dependent protein kinase A (PKA) (Dohrman et al., 2002; Yao et al., 2002); this activation also occurred within the first minute of stimulation (Dohrman et al., 2002; Yao et al., 2002). PKA is localized at the Golgi apparatus (Dohrman et al., 1996), near the location of PKC in unstimulated cells (Gordon et al., 1997, 2001). In this current study, we found that PKC binding to RACK precedes its translocation and that PKA is required for the translocation of the PKC/RACK complex.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. All reagents were purchased from Sigma (St. Louis, MO) except where indicated. Rp-cAMPS and Sp-cAMPS were purchased from BioLog (La Jolla, CA). Bisindolylmaleimide I (GF 109203X) and Et-18-OCH3 were purchased from Calbiochem (San Diego, CA). 2,10,11-Trihydroxy-N-propylnorapomorphine hydrobromide (NPA) was purchased from Sigma/RBI (Natick, MA). Protease inhibitor tablets (Complete) were purchased from Roche Molecular Biochemicals (Indianapolis, IN).

Cell Culture. NG108-15 cells stably expressing the rat D2L receptor (NG108-15/D2) (Asai et al., 1998) were grown on single-well slides in defined media for 2 days followed by daily replacement until day 4 (Dohrman et al., 1996). The cells were treated as described in the figure legends and fixed as described below (Gordon et al., 1997).

Immunocytochemistry and Microscopy. Cells were fixed with ice-cold methanol for 2 to 3 min and rinsed three times with PBS, incubated at room temperature with blocking buffer (1% normal goat serum in PBS and 0.1% Triton X-100) for 3 to 4 h, and then incubated overnight at 4°C in PBS containing 0.1% Triton X-100, 2 mg/ml fatty acid-free bovine serum albumin (Dohrman et al., 1996), primary antibodies specific for PKC (mouse IgG raised against the V5 domain of PKC; Santa Cruz Biotechnology, Santa Cruz, CA), RACK (rat IgG; Stressgen, Victoria, BC, Canada) for RACK, and 14E6 (mouse IgM, raised against the V1 domain of PKC) for active PKC (Souroujon et al., 2004). The cells were then washed three times with PBS, incubated for 1 h at room temperature with goat anti-mouse IgM, anti-mouse IgG, or anti-rat IgG secondary antibodies (Cappel, Aurora, OH) (diluted 1:1000), washed three times with PBS, and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells were imaged using a scanning laser confocal microscope (1024; Bio-Rad Laboratories, Hercules, CA) equipped with a krypton-argon laser attached to a microscope (Optiphot; Nikon, Tokyo, Japan). Images were collected as z-series using Kalman averaging of scans (Gordon et al., 1997). Collected data were processed using NIH Image (http://rsb.info.nih.gov/nih-image/) and Photoshop software (Adobe Systems, Mountain View, CA). All images were obtained under 40x magnification from individual middle sections of the projected z-series.

Quantification of PKC Localization. Fields on each slide were selected at random and cells were scored for perinuclear or cytoplasmic staining by two independent observers who were blind to the experimental conditions. At least four fields were scored for each experiment, for a total number of at least 50 cells per slide.

Cell Fractionation. NG108-15/D2 cells in 100-mm dishes (2 x 10⁶ cells/dish) were incubated with ethanol or NPA for 10 min, washed with ice-cold PBS and lysed on ice in 0.5 ml of lysis buffer containing 50 mM Tris-HCL, pH 7.4, 2.5 mM MgCL₂, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and protease inhibitors (0.1 mM phenylmethyl sulfonyl fluoride, 20 µg/ml soybean trypsin inhibitor, 25 µg/ml aprotinin, 25 µg/ml leupeptin, and 1 mM sodium orthovanadate). Cells were homogenized by 10 passes through a 26-gauge needle and centrifuged at 3000 rpm for 5 min at 4°C. The supernatant was centrifuged for 20 min at 150,000g to separate the membrane pellet from the cytosol (Yao et al., 2002). The supernatant was saved as the cytosolic fraction. The remaining pellets were suspended in 0.5 ml of lysis buffer containing 0.1% Triton X-100, titrated and incubated on ice for 20 min. This suspension was centrifuged as described above, and the Triton-soluble material was collected as the original particulate fraction.

Immunoprecipitation and Western Blot. Five micrograms of PKC monoclonal IgG antibody was incubated with 50 µl of protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Antibody-bound beads were then washed twice with PBS and blocked with 3% bovine serum albumin for 2 h at 4°C. The cytosolic fraction was precleared with protein A/G beads for 30 min at 4°C, incubated with the antibody-bound beads overnight at 4°C and subsequently washed four times with PBS. Bound material was eluted with SDS sample buffer, run on a 10% SDS/PAGE and transferred and probed for PKC (mouse IgG, Santa Cruz, CA) and RACK (rat IgG, Victoria, BC). Secondary antibody was horseradish peroxidase-linked goat anti-mouse or anti-rat (1:1000) (PerkinElmer Life and Analytical Sciences, Waltham, MA). Proteins were detected using chemiluminescence substrate (PerkinElmer Life and Analytical Sciences).

PKC Activity Assay. Cells grown in 100-mm plates were treated with ethanol or NPA for 10 min, washed with cold PBS, harvested in 1 ml of whole-cell lysis buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 10 mM EGTA, 0.1% Triton X-100, and 1 tablet of protease inhibitor/10 ml), and lysed on ice for 20 min. The lysate was centrifuged at 14,000 rpm for 10 min in an Eppendorf centrifuge. The supernatant was immunoprecipitated for PKC as described above. To assay PKC activity, immunoprecipitates were incubated at 30°C for 20 min with 10 µM ATP, 0.5 µCi of [-³²P]ATP, and a peptide substrate mixture from SignaTECT PKC Assay System (Promega, Madison, WI). PKC activity was detected as described by the manufacturer.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Ethanol and NPA Both Caused Translocation of PKC and RACK to the Same Location. Ethanol and the D2 agonist NPA cause translocation of PKC (Gordon et al., 1997, 2001). Activated PKC associates with the RACK known as β'-coat protein (Csukai et al., 1997). To determine whether RACK translocates together with PKC, NG108-15/D2 cells were treated with either ethanol or the D2 agonist NPA for 10 min and analyzed for translocation of PKC and RACK. Figure 1A shows that ethanol and NPA each induced PKC (green) translocation from the nucleus/perinucleus to the cytoplasm and RACK (red) from the Golgi/perinucleus to the cytoplasm. The merged images (yellow, Fig. 1A) indicate that PKC and RACK are colocalized in the cytoplasm in ethanol- and NPA-treated cells. Cotranslocation and association of the two proteins was confirmed by coimmunoprecipitation. Western blot analysis showed that the amount of PKC in the cytosolic compartment increased concomitantly with the amount of RACK (Fig. 1B), suggesting that PKC and RACK moved together after treatment with either ethanol or NPA.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Ethanol- and NPA-induced translocation of PKC and RACK requires PKA activity in NG108-15/D2 cells. A, NG108/15 cells expressing the D2 were exposed to 100 mM ethanol (EtOH) or the D2 agonist NPA (50 nM) for 10 min. Where indicated, cells were preincubated with or without the PKA inhibitor Rp-cAMPS (Rp, 20 µM) for 20 min before the treatment of ethanol or NPA. Cells were fixed and stained for PKC (green, mouse IgG against the V5 domain of PKC; Santa Cruz Biotechnology, Inc.) and RACK (red), and scanned using a Bio-Rad 1024 confocal microscope. The merged images of PKC with RACK suggest the colocalization (yellow). The data are representative of at least three independent experiments. Preabsorption of the isozyme-specific antibody with the respective peptide antigens were carried out as described in Gordon et al. (Gordon et al., 1997). B, cytosolic fractions were prepared from cells treated with 100 mM ethanol or 50 nM NPA for 10 min, immunoprecipitated, and probed with the mouse PKC or a monoclonal RACK antibody. C, cells were preincubated with or without the PKA inhibitor Rp-cAMPS (Rp, 20 µM) or the adenosine A2A antagonist DMPX (10 µM) for 20 min and then treated with the A2A agonist CGS21680 (CGS, 100 nM) for 10 min. Immunoprecipitation and detection of PKC/RACK translocation were performed as in B.

PKA Activation Is Required and Sufficient to Cause PKC and RACK Translocation. Because ethanol and NPA also activate PKA, and PKA translocation is more rapid than PKC (Dohrman et al., 1996; Gordon et al., 1998, 2001; Yao et al., 2002), we sought to determine whether PKA is required for PKC and RACK translocation and colocalization. Figure 1, A and B, show that the PKA inhibitor Rp-cAMPS prevents the translocation of both PKC and RACK, because the distribution of PKC and RACK seems the same as in control cells. In contrast, NPA- and ethanol-induced translocation of PKC was not affected by Rp-cAMPS (data not shown). To investigate how PKA regulates ethanol- and NPA-induced PKC and RACK translocation, we determined whether activation of PKA is sufficient for PKC and RACK translocation. Figure 2 shows that the PKA activator Sp-cAMPS or activation of the G_s-coupled PGE1 receptor each causes translocation of PKC and RACK to the cytoplasm, similar to ethanol and NPA treatments (Fig. 1A). We demonstrated previously that ethanol activates PKA via adenosine A2A receptors (A2A) (Yao et al., 2002). To determine whether direct activation of the adenosine A2A receptor causes translocation of PKC/RACK, cells were treated with an adenosine A2A agonist CGS21680 for 10 min. We found that CGS21680 mimics ethanol-induced translocation of PKC/RACK (Fig. 1C). This translocation was blocked by the A2A antagonist DMPX or the PKA inhibitor Rp-cAMPS (Fig. 1C). Rp-cAMPS and DMPX had no effect on the localization of PKC and RACK in unstimulated cells (data not shown).

View larger version (72K): [in this window] [in a new window]   Fig. 2. PKA stimulates PLC to induce PKC and RACK translocation. NG108-15/D2 cells were preincubated with or without the PLC inhibitor Et-18-OCH3 (Et-18, 10 µM) or the PKA inhibitor Rp-cAMPS (Rp, 20 µM) for 20 min and then treated with 1 mM Sp-cAMPS (Sp) or 10 µM PGE1 for 10 min. Analyses of PKC and RACK translocation were performed as Fig. 1A.

Ethanol and NPA Caused Translocation of PKC/ RACK Complex via the PLC/PKC System. We have previously shown that ethanol- or NPA-induced translocation of PKC is blocked by the PLC inhibitor Et-18-OCH3 (Gordon et al., 2001). If PLC activation is required for ethanol- and NPA-induced translocation of PKC, then inhibition of PLC activity should also inhibit translocation of PKC/RACK. Indeed, the PLC inhibitor Et-18-OCH3 blocked RACK translocation along with PKC (Fig. 2 and 3). As anticipated, the PKC inhibitor GF 109203X also blocked translocation (data not shown).

View larger version (103K): [in this window] [in a new window]   Fig. 3. Ethanol- and NPA-induced PKC and RACK translocation requires PLC. NG108-15/D2 cells were preincubated with or without the PLC inhibitor Et-18-OCH3 (Et-18, 10 µM), the A2A antagonist DMPX (10 µM), or the D2 antagonist spiperone (SPIP, 10 µM) for 30 min, or PTX (50 ng/ml) overnight, and then treated with 100 mM ethanol or 50 nM NPA for 10 min. Immunostaining for PKC and RACK translocation were carried out as Fig. 1A.

PKC Activation Is Required for Translocation of PKC and RACK. To further investigate whether PKC activation regulates the translocation of PKC and RACK, we used an IgM monoclonal antibody, 14E6, that specifically detects the active conformation of PKC (Souroujon et al., 2004). Figure 4, A and B, show that translocation of PKC (green) together with RACK (blue) began at 1 min and persists for 30 min after the addition of ethanol and NPA. In contrast, PKC staining with 14E6 (red) increased within 1 min, maximized by 10 min, and returned to the basal level by 30 min (Fig. 4, A and B). PKC translocation was observed at the time when 14E6 staining appeared. These data suggest that PKC activation seems to be required for the translocation of PKC and RACK. Consistent with our published observations (Souroujon et al., 2004), PKC activation precedes its binding to RACK and its translocation with RACK to the cytoplasm. Translocation of PKC persisted at 30 min when the activated enzyme was no longer detected by 14E6 (Fig. 4, A and B), suggesting that the 14E6 epitope (V1 domain, the RACK-binding domain) becomes inaccessible when PKC is bound to RACK (Souroujon et al., 2004). We confirmed these findings by directly measuring the catalytic activity of PKC. In accordance with translocation, PKC activity peaked at 10 min, persisted at 30 min and returned to the basal level at 60 min (Fig. 4C).

View larger version (39K): [in this window] [in a new window]   Fig. 4. PKC and RACK translocation as a function of time. NG108-15/D2 cells were incubated with 100 mM ethanol (A) or 50 nM NPA (B) for the indicated times and stained for PKC (green), activated PKC (14E6, red), and RACK (blue). The merged images were produced using anti-mouse IgG against the V5 domain of PKC (Santa Cruz Biotechnology, Inc.), state-specific anti-PKC antibodies, 14E6, and anti-RACK antibodies. C, cells were lysed and immunoprecipitated with anti-mouse PKC antibody (Santa Cruz Biotechnology, Inc.). The immunoprecipitates were assayed for PKC phosphorylation activity as described under Materials and Methods. Data are the mean ± S.E.M. of at least three experiments. *, p < 0.01 compared with control (one-way analysis of variance and Dunnett's test).

View larger version (45K): [in this window] [in a new window]   Fig. 5. PKC activation causes translocation of PKC and RACK. NG108-15/D2 cells were preincubated with or without 1 µM PKC specific peptide inhibitor V1-2, PKC-specific inhibitor V1-1, or conventional PKC-specific inhibitor βC2-4 for 30 min. Cells were then incubated with 100 mM ethanol or 50 nM NPA for 10 min. A, detection of PKC, activated PKC (14E6), and RACK translocation was carried out as in Fig. 4. B, PKC translocation (green) in A was measured as the percentage of cells with cytoplasmic staining of PKC. Data are the mean ± S.E.M. *, p < 0.01 compared with control (one-way analysis of variance and Dunnett's test).

View larger version (64K): [in this window] [in a new window]   Fig. 6. PKC activation and translocation with RACK are separate events. A, NG108-15/D2 cells were pretreated with or without 20 µM Rp-cAMPS and then incubated with the PKC-specific agonist pseudo-RACK (RACK, 1 µM) for 15 min or with 50 nM NPA for 10 min. A, detection of PKC, activated PKC, and RACK translocation was carried out as Fig. 4. B, cells were lysed for fractionation. Twenty micrograms of cytosolic fraction (C) or particulate fraction (P) was loaded on 10% SDS-PAGE and analyzed for PKC by Western blot. The blot shown is representative of three separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The major findings in this study are that ethanol and NPA each can induce translocation of PKC and RACK to a new site and that this process requires PKA activity. After stimulation, PKC translocates from the perinucleus/nucleus to a new perinuclear/Golgi compartment, perhaps where RACK is colocalized in unstimulated cells. Subsequently, PKC and the RACK translocate from the perinucleus/Golgi to the cytosol. Translocation of PKC and RACK to the cytosol occurs only when PKA is activated, a process that is G_i-dependent. Consistent with this observation, the PKC agonist, pseudo-RACK, did not cause the translocation of PKC to the cytosol, although it did activate PKC. Moreover, activation of PKA by Sp-cAMPS, PGE1, or the adenosine A2A receptor alone was sufficient to cause PKC and RACK translocation. Note that PKA-dependent translocation of PKC was inhibited by the PLC inhibitor Et-18-OCH3, suggesting that in addition to the PLC-mediated cross-talk between PKC and PKA signaling, there is a second cross-talk event leading to translocation of PKC/RACK complex that is dependent on PKA activity. Therefore, there is a dual requirement for PKA activity in PKC signaling. A schematic model for ethanol and D2 activation of PKA/PKC cross talk is presented in Fig. 7.

View larger version (22K): [in this window] [in a new window]   Fig. 7. A model of cross-talk between PKA and PKC signaling. Scheme represents dopamine/D2- and ethanol/adenosine A2A-induced PKC and RACK translocation. D2 signaling is indicated by red arrows; ethanol signaling is indicated by blue arrows. D2 activation and ethanol stimulate PLC and increase DAG that activates PKC via Gi proteins. Ethanol inhibits adenosine uptake and increases extracellular adenosine that activates Gs-coupled adenosine A2A receptors. D2 activation releases β that stimulates AC II and IV in the presence of PKC. Both ethanol and D2 activation promote cAMP/PKA signaling. Robust activation of PKA by Sp-cAMPS (black arrow) directly stimulates PLC and activates PKC. Activation of both PKA and PKC leads to the translocation of PKC/RACK complex.

PKC and RACK. Ethanol and NPA also induce translocation of PKC. Ethanol at 100 mM induced maximal translocation at 10 min without affecting cell morphology and viability. Ethanol at 50 mM produced maximal translocation at 48 h (Gordon et al., 1997, 2001). Therefore, we chose 100 mM ethanol and a 10-min incubation time as optimal conditions to define the mechanism and relationship between PKC activation and translocation. Using the antibody 14E6, we show that ethanol and NPA activate PKC and increase the catalytic activity of PKC measured by phosphorylation. We also show that V1-2, an inhibitor of PKC binding to its RACK, prevents ethanol- and NPA-induced translocation of the PKC/RACK complex. In contrast, the peptide inhibitor V1-1 (PKC) or βC2-4 (classic PKC) had no effect. We previously proposed that the site of localization of activated PKC isozymes is determined by the location of isozyme-specific RACKs (Mochly-Rosen and Gordon, 1998). Our data suggest that ethanol and NPA use this mechanism to relocate activated PKC. The activated PKC binds first to its RACK and subsequently translocates from the perinucleus to a new cytoplasmic compartment. Thus, activation of PKC seems to be necessary for PKC and RACK translocation (Fig. 7). However, activation of PKC alone is not sufficient to cause translocation of the PKC/RACK complex because the PKC agonist, pseudo-RACK, does not cause translocation of PKC into the cytoplasm despite activation of PKC. These observations demonstrate that PKA activation induced by ethanol or NPA has a dual role in PKC signaling: first, PKA activates PLC to produce DAG for PKC activation; second, PKA causes relocation of activated PKC/RACK. This is likely to yield different cellular responses, as the protein substrates of PKC should be different in each of these cellular locations.

Cross-Talk between PKA and PKC Signaling. Cross-talk between PKA and PKC signaling pathways is increasingly recognized as a mechanism to regulate signal transduction cascades. However, the molecular events underlying PKA/PKC cross-talk are not clear. Recent work suggests a role for PKA in the activation and translocation of PKC (Yu et al., 1996; Huang et al., 2001). PKA-dependent activation of PKC also occurs in B lymphocytes (Cambier et al., 1987). In addition, activation of dopamine D1 receptors, known to couple to G_s, increases PKC activity and translocation in LTK cells (Yu et al., 1996). In this study, we demonstrate that translocation of PKC and RACK by ethanol and NPA requires PKA activation, but the PKA inhibitor Rp-cAMPS does not inhibit the activation of PKC. These findings suggest that PKA may regulate the location of PKC/RACK complex but does not affect the activation state of the enzyme (Fig. 7). Indeed, prosite analysis reveals a consensus PKA phosphorylation site in RACK (L. Yao, P. Fan, Z. Jiang, and I. Diamond, unpublished observation). Thus, not only do RACKs bind activated PKC isozymes but also RACK phosphorylation may further regulate its translocation to intracellular sites.

One of our most surprising findings is that robust activation of PKA by Sp-cAMPS or PGE1 was sufficient to induce translocation of PKC and RACK. It is noteworthy that direct activation of the adenosine A2A receptor by CGS21680 also caused translocation of PKC/RACK to the same compartment. We propose that activation of PKA stimulates PLCβ, thus increasing DAG levels and causing activation and translocation of PKC (Fig. 7). Indeed, a PLC inhibitor blocks PKC translocation. However, it remains unclear how ethanol, NPA, or PKA activate the correct pool of PKC and how activated PKC translocates with RACK to its functional intracellular sites. One explanation is a "targeting hypothesis": phosphorylation events are controlled in part by the intracellular location of specific kinases in the cell (Hubbard and Cohen, 1993). It has also been suggested that intracellular anchoring proteins regulate cell signaling dynamics in time and space. The Golgi complex is a major subcellular location for PKA in mammalian cells (Nigg et al., 1985; Dohrman et al., 1996; Yao et al., 2002) and is involved in vesicle-mediated protein transport processes (Muñiz et al., 1997). Scott and collaborators and others suggest that some anchoring proteins for PKA, collectively termed AKAPs for A kinase anchoring proteins, also bind inactive PKC in the Golgi (Faux and Scott, 1997; Pawson and Scott, 1997). In addition, RACK, β'-coat protein, is a coatomer protein that moves with vesicles and localizes at the Golgi apparatus (Salama and Schekman, 1995). Thus, AKAPs such as AKAP 350 may act as scaffold proteins that bind PKA, PKC, and RACK (Diviani and Scott, 2001; Shanks et al., 2002) in the Golgi and serve as a platform to organize and regulate PKA and PKC interactions. It remains to be determined which AKAP binds to PKA, PKC, and RACK, and how AKAP targets PKA and PKC to discrete intracellular locations and coordinates multiple components of signal transduction pathways.

Relevance to Alcoholism. Our results provide new insight into some of the cellular events mediated by ethanol and dopamine. Ethanol causes the release of dopamine in the brain (Imperato and Di Chiara, 1986; McBride et al., 1993) and presumably dopamine acts on D2 to mediate rewarding properties of ethanol. We show here that both ethanol and a D2 agonist activate both PKA and PKC signaling pathways via a complex cross-talk between these two signaling cascades. It is tempting to speculate that ethanol and D2 may activate the same signaling pathways because they synergistically activate PKA and PKC signaling (Gordon et al., 2001; Yao et al., 2002). Moreover, ethanol- and dopamine-regulated translocation of PKA and PKC seems to play a role in drinking behaviors; mice lacking PKC show reduced operant ethanol self-administration (Hodge et al., 1999; Olive et al., 2000) and inhibition of the cAMP/PKA signaling pathway generally increases sensitivity to ethanol sedation and reduces ethanol preference and consumption (Moore et al., 1998; Wand et al., 2001; Yao et al., 2002). Taken together with the results in this study, it is possible that drugs which interfere with PKA and PKC cross-talk might be potential therapeutics for alcoholism.

	Footnotes

 
D.M.-R. is a founder of KAI Pharmaceuticals, a company that plans to bring PKC regulators to the clinic. However, this work was carried out in her university laboratory, with the sole support of National Institutes of Health grant AA11147. This research was also supported by National Institutes of Health grant AA010030-12 to I.D. and L.Y.

ABBREVIATIONS: PKC, protein kinase C; DAG, diacylglycerol; D2, dopamine D2 receptor; NPA, 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide; PKA, cAMP-dependent protein kinase A; Rp-cAMPS, adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer; Sp-cAMPS, adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer; GF109203X, Bisindolylmaleimide I; Et-18-OCH3, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine; PBS, phosphate-buffered saline; DMPX, 3,7-dimethyl-1-propargylxanthine; PLC, phospholipase C; PTX, pertussis toxin; PGE1, prostaglandin E1; CGS21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; AKAP, A kinase anchoring protein; A2A, adenosine A2A receptor; IgG, immunoglobulin G.

Address correspondence to: Dr. Lina Yao, CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto, CA 94304. E-mail: lina.yao{at}cvt.com

	References

Top Abstract Materials and Methods Results Discussion References

 
Asai K, Ishii A, Yao L, Diamond I, and Gordon AS (1998) Varying effects of ethanol on transfected cell lines. Alcohol Clin Exp Res 22: 163-166.[CrossRef][Medline]

Baker LP, Nielsen MD, Impey S, Hacker BM, Poser SW, Chan MY, and Storm DR (1999) Regulation and immunohistochemical localization of betagamma-stimulated adenylyl cyclases in mouse hippocampus. J Neurosci 19: 180-192.[Abstract/Free Full Text]

Cambier JC, Newell MK, Justement LB, McGuire JC, Leach KL, and Chen ZZ (1987) Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes. Nature 327: 629-632.[CrossRef][Medline]

Camps M, Carozzi A, Schnabel P, Scheer A, Parker PJ, and Gierschik P (1992) Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature 360: 684-686.[CrossRef][Medline]

Choi DS, Wang D, Dadgar J, Chang WS, and Messing RO (2002) Conditional rescue of protein kinase C epsilon regulates ethanol preference and hypnotic sensitivity in adult mice. J Neurosci 22: 9905-9911.[Abstract/Free Full Text]

Csukai M, Chen CH, De Matteis MA, and Mochly-Rosen D (1997) The coatomer protein β'-COP, a selective binding protein (RACK) for protein kinase C. J Biol Chem 272: 29200-29206.[Abstract/Free Full Text]

Diviani D and Scott JD (2001) AKAP signaling complexes at the cytoskeleton. J Cell Sci 114: 1431-1437.[Abstract]

Dohrman DP, Chen HM, Gordon AS, and Diamond I (2002) Ethanol-induced translocation of protein kinase A occurs in two phases: control by different molecular mechanisms. Alcohol Clin Exp Res 26: 407-415.[Medline]

Dohrman DP, Diamond I, and Gordon AS (1996) Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus. Proc Natl Acad Sci U S A 93: 10217-10221.[Abstract/Free Full Text]

Faux MC and Scott JD (1997) Regulation of the AKAP79-protein kinase C interaction by Ca²⁺/calmodulin. J Biol Chem 272: 17038-17044.[Abstract/Free Full Text]

Federman AD, Conklin BR, Schrader KA, Reed RR, and Bourne HR (1992) Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits. Nature 356: 159-161.[CrossRef][Medline]

Gordon AS, Yao L, Dohrman DP and Diamond I (1998) Ethanol alters the subcellular localization of cAMP-dependent protein kinase and protein kinase C. Alcohol Clin Exp Res 22: 238S-242S.[CrossRef][Medline]

Gordon AS, Yao L, Jiang Z, Fishburn CS, Fuchs S, and Diamond I (2001) Ethanol acts synergistically with a D2 dopamine agonist to cause translocation of protein kinase C. Mol Pharmacol 59: 153-160.[Abstract/Free Full Text]

Gordon AS, Yao L, Wu ZL, Coe IR, and Diamond I (1997) Ethanol alters the subcellular localization of delta- and epsilon protein kinase C in NG108-15 cells. Mol Pharmacol 52: 554-559.[Abstract/Free Full Text]

Hodge CW, Mehmert KK, Kelley SP, McMahon T, Haywood A, Olive MF, Wang D, Sanchez-Perez AM, and Messing RO (1999) Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKCepsilon. Nat Neurosci 2: 997-1002.[CrossRef][Medline]

Huang NK, Lin YW, Huang CL, Messing RO, and Chern Y (2001) Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells. J Biol Chem 276: 13838-13846.[Abstract/Free Full Text]

Hubbard MJ and Cohen P (1993) On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci 18: 172-177.[CrossRef][Medline]

Imperato A and Di Chiara G (1986) Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239: 219-228.[Abstract/Free Full Text]

McBride WJ, Murphy JM, Gatto GJ, Levy AD, Yoshimoto K, Lumeng L, and Li TK (1993) CNS mechanisms of alcohol self-administration. Alcohol Alcohol Suppl 2: 463-467.[Medline]

Mochly-Rosen D, and Gordon AS (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. Faseb J 12: 35-42.[Abstract/Free Full Text]

Moore MS, DeZazzo J, Luk AY, Tully T, Singh CM, and Heberlein U (1998) Ethanol intoxication in Drosophila: genetic and pharmacological evidence for regulation by the cAMP signaling pathway. Cell 93: 997-1007.[CrossRef][Medline]

Muñiz M, Martin ME, Hidalgo J, and Velasco A (1997) Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network. Proc Natl Acad Sci U S A 94: 14461-14466.[Abstract/Free Full Text]

Newton PM and Messing RO (2006) Intracellular signaling pathways that regulate behavioral responses to ethanol. Pharmacol Ther 109: 227-237.[CrossRef][Medline]

Nigg EA, Schafer G, Hilz H, and Eppenberger HM (1985) Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell 41: 1039-1051.[CrossRef][Medline]

Olive MF, Mehmert KK, Messing RO, and Hodge CW (2000) Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol in PKCepsilon-deficient mice. Eur J Neurosci 12: 4131-4140.[CrossRef][Medline]

Olive MF and Messing RO (2004) Protein kinase C isozymes and addiction. Mol Neurobiol 29: 139-154.[CrossRef][Medline]

Park D, Jhon DY, Lee CW, Lee KH, and Rhee SG (1993) Activation of phospholipase C isozymes by G protein beta gamma subunits. J Biol Chem 268: 4573-4576.[Abstract/Free Full Text]

Pawson T and Scott JD (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science 278: 2075-2080.[Abstract/Free Full Text]

Robbins TW and Everitt BJ (1999) Drug addiction: bad habits add up. Nature 398: 567-570.[CrossRef][Medline]

Runnels LW and Scarlata SF (1999) Determination of the affinities between hetero-trimeric G protein subunits and their phospholipase C-beta effectors. Biochemistry 38: 1488-1496.[CrossRef][Medline]

Salama NR and Schekman RW (1995) The role of coat proteins in the biosynthesis of secretory proteins. Curr Opin Cell Biol 7: 536-543.[CrossRef][Medline]

Schechtman D and Mochly-Rosen D (2002) Isozyme-specific inhibitors and activators of protein kinase C. Methods Enzymol 345: 470-489.[Medline]

Shanks RA, Steadman BT, Schmidt PH, and Goldenring JR (2002) AKAP350 at the Golgi apparatus. I. Identification of a distinct Golgi apparatus targeting motif in AKAP350. J Biol Chem 277: 40967-40972.[Abstract/Free Full Text]

Souroujon MC, Yao L, Chen H, Endemann G, Khaner H, Geeraert V, Schechtman D, Gordon AS, Diamond I, and Mochly-Rosen D (2004) State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation. J Biol Chem 279: 17617-17624.[Abstract/Free Full Text]

Tsu RC and Wong YH (1996) Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment. J Neurosci 16: 1317-1323.[Abstract/Free Full Text]

Volkow ND, Fowler JS, Wang GJ, and Swanson JM (2004) Dopamine in drug abuse and addiction: results from imaging studies and treatment implications. Mol Psychiatry 9: 557-569.[CrossRef][Medline]

Wand G, Levine M, Zweifel L, Schwindinger W, and Abel T (2001) The cAMP-protein kinase A signal transduction pathway modulates ethanol consumption and sedative effects of ethanol. J Neurosci 21: 5297-5303.[Abstract/Free Full Text]

Yao L, Arolfo MP, Dohrman DP, Jiang Z, Fan P, Fuchs S, Janak PH, Gordon AS, and Diamond I (2002) betagamma Dimers mediate synergy of dopamine D2 and adenosine A2 receptor-stimulated PKA signaling and regulate ethanol consumption. Cell 109: 733-743.[CrossRef][Medline]

Yao L, Fan P, Jiang Z, Mailliard WS, Gordon AS, and Diamond I (2003) Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-β dimers. Proc Natl Acad Sci U S A 100: 14379-14384.[Abstract/Free Full Text]

Yu PY, Eisner GM, Yamaguchi I, Mouradian MM, Felder RA, and Jose PA (1996) Dopamine D1A receptor regulation of phospholipase C isoform. J Biol Chem 271: 19503-19508.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. K. Kelm, H. E. Criswell, and G. R. Breese The Role of Protein Kinase A in the Ethanol-Induced Increase in Spontaneous GABA Release Onto Cerebellar Purkinje Neurons J Neurophysiol, December 1, 2008; 100(6): 3417 - 3428. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.042580v1 73/4/1105    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yao, L. Articles by Diamond, I. Search for Related Content PubMed PubMed Citation Articles by Yao, L. Articles by Diamond, I.