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Dexamethasone-Induced Ras Protein 1 Negatively Regulates Protein Kinase C : Implications for Adenylyl Cyclase 2 Signaling

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana

Received September 20, 2005; accepted February 17, 2006

	Abstract

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We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) , and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1 [EC] ) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D₂ receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated G signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter G_s-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKC autophosphorylation. This effect was selective for the isoform because Dexras1 did not alter autophosphorylation of PKC or PKC. Dexras1 disruption of PKC autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [-³²P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKC coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKC; however, the ability of Dexras1 to interfere with PKC autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKC at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKC activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both G- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

Adenylyl cyclase 2 belongs to the G-stimulated subfamily of adenylyl cyclase isoforms (Hanoune and Defer, 2001). Adenylyl cyclase 2 signaling can be promoted by activators of protein kinase C (PKC) such as diacylglycerol or phorbol esters (Jacobowitz and Iyengar, 1994; Bol et al., 1997). Protein kinase C is a family of serine/threonine kinases composed of at least 12 members that are categorized into three groups based on their regulatory properties: conventional PKCs (cPKCs; , , and ), novel PKCs (nPKCs; , , , and ), and atypical PKCs ( and ) (Newton, 2001). The protein kinase C isoform(s) responsible for phosphorylating AC2 has not been identified, although convention suggests that it is a member of the cPKC or nPKC subfamily because atypical PKCs are insensitive to diacylglycerol stimulation. The ability of Dexras1 to inhibit G_i/o-coupled receptor-stimulated G signaling pathways suggests that Dexras1 may interfere with the conditional activation of AC2 by G dimers (Graham et al., 2002; Takesono et al., 2002; Nguyen and Watts, 2005).

In this report, we reveal that Dexras1 abolishes dopamine D_2L receptor-mediated potentiation of AC2 activity, presumably by interfering with agonist-stimulated G signaling. More significantly, we demonstrate that Dexras1 inhibits phorbol ester stimulation of AC2, thereby implicating a role for Dexras1 in PKC-dependent signaling pathways. To provide insight into the mechanisms for this blockade, we used a series of biochemical, pharmacological, and genetic approaches to demonstrate that Dexras1 inhibits PKC activity. Taken together, our data reveal that Dexras1 negatively modulates AC2 signaling in an indirect fashion by inhibiting both G- and PKC-stimulated AC2 activity.

	Materials and Methods

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Materials. [³H]cAMP was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Fetalclone1 serum and bovine calf serum were purchased from Hyclone (Logan, UT). Rabbit Dexras1 antibody was purchased from Calbiochem (San Diego, CA). Isoform-specific phospho-PKC antibodies (PKC, Thr638; PKC, Ser643; PKC, Thr710) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-PKC and anti-PKC antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-FLAG antibody, isoproterenol, phorbol 12-myristate 13-acetate (PMA), quinpirole, isobutylmethylxanthine, and most other reagents were purchased from Sigma-Aldrich (St. Louis, MO). The cDNA for Dexras1 was obtained from Guthrie cDNA Resource Center (http://www.cdna.org).

Cell Culture. HEK293T cells stably expressing the rat dopamine D_2L receptor were generated as described previously (Neve et al., 2001). HEK-AC2 cells were generated by transfection using Lipofectamine reagent (Invitrogen, Carlsbad, CA) and selection using G418 (900 µg/ml). G418-resistant colonies were screened and selected based on the response to PMA stimulation. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetalclone1 serum, 5% bovine calf serum, 0.05 U/ml penicillin, and 50 mg/ml streptomycin. Cells were grown in a humidified incubator in the presence of 5% CO₂ at 37°C.

Transfections. At 90 to 95% confluence, transient transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. For experiments examining AC2 activity in the presence of only wild-type Dexras1 (Fig. 1), cotransfections were performed using the dual expression vector pBudCE4 (Invitrogen) containing AC2 alone or in combination with Dexras1. All other cotransfections were performed using separate plasmids encoding individual cDNAs and transfections were equalized by mass using pcDNA3.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Dexras1, Gs, G, and PMA modulation of AC2. HEK-D2L cells were cotransfected with AC2 in the absence or presence of Dexras1 using the dual expression vector, pBudCE4, as described under Materials and Methods. At 48 h after transfection, cells were stimulated with isoproterenol (A) or PMA (B) in the absence and presence of 1 µM quinpirole as indicated for 15 min at 37°C in the presence of IBMX. cAMP accumulation was measured as described under Materials and Methods. The data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. cAMP accumulation is presented as picomoles per well above vector-transfected cells. *, p < 0.05 versus matched basal cAMP accumulation. , p < 0.05 versus AC2 transfected cells for each stimulation condition (repeated-measures ANOVA with Bonferroni post-test).

Immunodetection. Cells were seeded in six-well cluster plates at a concentration of approximately 750,000 cells/well and transfections were performed as described above. At 48 h after transfection (72 h if cells were serum-starved overnight), the plates were placed on ice and lysed with ice-cold lysis buffer [1 mM HEPES, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 1 µg/ml leupeptin, and 1% Nonidet P-40 (NP-40)] for 10 min. The cells were then scraped from the plates, centrifuged at 13,000g for 10 min, and the supernatant was retained for analysis. For subcellular fractionation experiments, cells were lysed with ice-cold lysis buffer (without NP-40) and centrifuged at 100,000g for 30 min at 4°C to generate the pellet (membrane) and supernatant (cytosol) fractions. Membrane pellets were then solubilized in ice-cold lysis buffer containing 1% NP-40. Total protein content was determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL). Protein samples were equalized by dilution and resolved by SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene diflouride membranes (Bio-Rad, Hercules, CA). Nonspecific antibody binding was blocked by incubating membranes overnight in 5% nonfat dried milk at 4°C. Membranes were washed with Tris-buffered saline and incubated with the indicated primary antibody for 3 h. The membranes were washed, and immunodetection was accomplished using an enhanced chemifluorescence Western blotting kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's protocol. After final antibody incubation, membranes were again washed, exposed to enhanced chemifluorescence substrate, and then scanned using the Storm Imaging System (Molecular Dynamics, Sunnyvale, CA). Immunoblots were quantified using ImageQuant software according to manufacturer's instructions. Where indicated, the amount of PKC autophosphorylation was normalized as the ratio of phospho-PKC to total PKC expression (phospho-PKC/total PKC).

Coimmunoprecipitation. Cells in 150-mm tissue culture plates were washed with PBS and then incubated with dithiobis[succinimidylpropionate] (25 mM) at room temperature for 30 min. The reaction was terminated by the addition of 20 mM Tris-HCl, pH 7.5, for 15 min. The cells were then collected in PBS by centrifuging at 200g for 10 min and washed twice with PBS. Cells were then incubated with lysis buffer (PBS, 5 mM EDTA, 0.5% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 25 µg/ml aprotinin) at 4°C for 30 min on a rotating platform. The lysate was cleared by centrifuging at 13,000g for 30 min at 4°C. The supernatant was incubated with protein G Sepharose beads in the absence of antibody for 1 h at 4°C to remove nonspecifically bound proteins. The supernatant was cleared by centrifugation and transferred to a new tube containing Protein G Sepharose beads and 5 µg of primary antibody at 4°C overnight. The beads were pelleted and washed five times with PBS for 2 min and then resuspended in 2x Laemmli sample buffer for immunoblot analysis as described above.

In Vitro PKC Kinase Assay. PKC activity was measured with the SignaTECT PKC assay kit according to the manufacturer's protocol (Promega, Madison, WI) with minor modifications to directly examine PKC activity. In brief, PKC was enriched from cell lysates with DEAE ion exchange chromatography. The eluate was used to measure [-³²P]ATP incorporation into the PKC-specific substrate, neurogranin, under control and PKC-stimulated conditions. The PKC stimulation buffer contained (final concentration) 0.25 mM EGTA, 0.1 mg/ml bovine serum albumin, 0.3 mg/ml phosphatidylserine, 0.03 mg/ml diacylglycerol, 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 nM PMA, 100 µM biotinylated neurogranin peptide substrate, 100 µM ATP, and 0.5 µCi [-³²P]ATP (3000 Ci/mM). The control buffer was essentially identical with the stimulation buffer but did not contain phosphatidylserine, diacylglycerol, or PMA. All reactions were performed in the absence of Ca²⁺ to selectively activate nPKC isoforms. The reaction was incubated in a 30°C water bath for 5 min and terminated by the addition of 7.5 M guanidine hydrochloride. An equal amount of reaction mix was spotted onto a streptavidin-coated membrane (S-adenosylmethionine synthetase-2 biotin capture membrane; Promega), washed three times each with 2 M NaCl, 2 M NaCl, with 1% H₃PO₄, and once with distilled H₂O. The membrane was dried and counted in an LS6500 scintillation counter (Beckman Coulter, Fullerton, CA). Total protein content from the DEAE eluate was determined using a BCA protein assay kit.

Data and Statistical Analysis. Statistical analyses were performed using Prism and InStat software (GraphPad Software Inc., San Diego, CA). A p value of <0.05 defined significance.

	Results

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Dexras1 Inhibits G- and PMA-Stimulated AC2 Activity. AC2 is conditionally activated by G subunits upon the short-term activation of the G_i/o-coupled dopamine D_2L receptor (Watts and Neve, 1997). Because Dexras1 has been shown to block receptor-mediated G-dependent signaling pathways, we investigated the effects of Dexras1 on dopamine D_2L receptor-mediated potentiation of AC2 activity. The activity of AC2 was examined by stimulating cells with isoproterenol to activate endogenous G_s-coupled -adrenergic receptors. Isoproterenol-stimulated cAMP accumulation was significantly increased above basal levels in AC2 transfected cells (Fig. 1A). The G_s-mediated activation of AC2 was retained in Dexras1-transfected cells, because cAMP accumulation values were comparable with those observed in the absence of Dexras1 (Fig. 1A). In agreement with its short-term regulatory properties, costimulation of cells with isoproterenol and the D₂ receptor agonist quinpirole resulted in greater than 50% potentiation of AC2 activity (Fig. 1A). This effect was completely abolished in cells cotransfected with Dexras1, which is consistent with the ability of Dexras1 to block receptor-mediated activation of G signaling effectors (Fig. 1A).

We examined further the role for Dexras1 in regulating AC2 activity by stimulating cells with the AC2 activator PMA. Cells transfected with only AC2 exhibited robust cAMP accumulation in response to stimulation with PMA compared with basal cAMP levels (Fig. 1B). Similar to the results obtained from G_s stimulation, cAMP accumulation was potentiated greater than 50% after costimulation with PMA and quinpirole compared with the response from PMA stimulation alone (Fig. 1B). It is surprising that Dexras1 cotransfection significantly reduced PMA-stimulated cAMP accumulation by approximately 80% (Fig. 1B). The ability of D_2L receptor activation to potentiate AC2-mediated cAMP accumulation was abolished in Dexras1-cotransfected cells (Fig. 1B). These results suggest that Dexras1 inhibits PKC-mediated activation of AC2 and is the first evidence that Dexras1 may act to obstruct PKC-dependent signaling pathways.

PMA-Stimulated AC2 Activity Is PKC-Dependent. As a first step in identifying the mechanisms by which Dexras1 may be inhibiting PMA stimulation of AC2, we used pharmacological inhibitors of PKC to provide insight into the PKC isoform(s) that may be activating AC2. We used two PKC inhibitors with selectivity for phorbol ester-sensitive PKC isoforms: bisindolylmaleimide is a broad-range PKC inhibitor that targets many members of cPKC and nPKC isoforms, whereas rottlerin has greater specificity for PKC (Gschwendt et al., 1994). HEK-AC2 cells were examined for PMA-stimulated cAMP accumulation in the absence and presence of bisindolylmaleimide or rottlerin. PMA stimulation of AC2 resulted in a greater than 6-fold increase in cAMP accumulation above basal (Fig. 2A). The PKC inhibitors bisindolylmaleimide and rottlerin significantly reduced the PMA-stimulated cAMP response to approximately 2-fold above basal (Fig. 2A). In contrast, neither PKC inhibitor had any effect on PKC-independent activation of AC2 by G_s (Fig. 2A). Isoproterenol-stimulated cAMP accumulation remained at greater than 6-fold above basal in the presence of both inhibitors (Fig. 2A). These data suggest that PKC activity is necessary for phorbol ester stimulation of AC2.

View larger version (21K): [in this window] [in a new window]   Fig. 2. PKC and PMA-dependent activation of AC2. A, HEK-AC2 cells were stimulated with 100 nM PMA or 3 µM isoproterenol in the absence and presence of 25 µM rottlerin or 1 µM bisindolylmaleimide for 15 min at 37°C in the presence of IBMX. cAMP accumulation was measured as described under Materials and Methods. The data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. cAMP accumulation is presented as the -fold increase above basal. Basal cAMP accumulation values were the following: 6.3 ± 0.7; + rottlerin, 4.8 ± 0.8; + bis, 5.9 ± 0.3 pmol/well. *, p < 0.05 versus PMA-stimulated cAMP accumulation alone (repeated-measures ANOVA with Bonferroni posttest). B, HEK-D2L cells were transiently transfected with AC2 in the absence and presence of PKC. At 48 h after transfection cells were stimulated with 100 nM PMA for 15 min at 37°C in the presence of IBMX. cAMP accumulation was subsequently measured as described under Materials and Methods. The data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. *, p < 0.05 versus matched basal cAMP accumulation. , p < 0.05 versus AC2 transfected cells for each stimulation condition (repeated-measures ANOVA with Bonferroni post-test).

Dexras1 Interferes with PKC Autophosphorylation. The results from our pharmacological experiments prompted us to investigate the effects of Dexras1 on PKC autophosphorylation, because autophosphorylation has been identified as a crucial component in regulating PKC catalytic activity (Newton, 2001). Transfection of PKC into cells resulted in robust immunoreactivity of phospho-serine 643 (Fig. 3A). However, when cells were cotransfected with PKC and Dexras1, PKC autophosphorylation was reduced (Fig. 3A). Because a decrease in PKC expression levels would decrease PKC autophosphorylation levels, the effect of Dexras1 transfection on the expression levels of total PKC was also explored. Immunoblot analysis revealed that the levels of endogenous PKC in the presence of transfected Dexras1 were 98 ± 11% (n = 4) compared with vector-transfected control cells (Fig. 3A, lanes 1 and 2). Likewise, when cells were cotransfected with PKC and Dexras1, the expression of total PKC (endogenous and recombinant) was 100 ± 4% of that from control cells that were transfected with PKC alone (Fig. 3A, lanes 3 and 4). These data provide evidence that Dexras1 interferes with PKC autophosphorylation and not PKC expression. The effect of Dexras1 on PKC autophosphorylation was further analyzed by normalizing the amount of PKC autophosphorylation as a ratio of total PKC expression (phospho-PKC/total PKC) for each transfection condition. This analysis was designed to control directly for any effects that total PKC expression may have on PKC autophosphorylation. The analyses of the normalized data revealed that transfection of Dexras1 reduced PKC autophosphorylation by 27 ± 2% compared with cells transfected with PKC alone (Fig. 3B). The results of this analysis support our initial immunoblot studies and provide stronger evidence that the effects of Dexras1 on PKC autophosphorylation are independent of Dexras1-induced changes in PKC expression.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Dexras1 expression and PKC autophosphorylation. The effect of Dexras1 on PKC autophosphorylation was examined in HEK-D2L cells transiently transfected with PKC, PKC, or PKC in the absence and presence of Dexras1. At 48 h after transfection, cells were serum-starved overnight. Cells were then lysed with ice-cold lysis buffer containing 1% NP-40, and the detergent-soluble cell extracts were examined for autophosphorylation by immunoblot analysis using isoform-specific phospho-PKC antibodies as indicated. The blots were then stripped and reprobed with anti-PKC and/or anti-Dexras1 antibody as indicated. Immunoblots shown are representative of at least three independent experiments. B, the phospho-PKC and PKC blots were examined for pixel intensity for the area under the curve generated for each individual band. The data are presented as phospho-/total PKC and have been normalized to values obtained from PKC transfection alone. The data are presented as mean ± S.E.M. of four independent experiments. *, p < 0.05 compared with PKC transfection alone (one-sample t test).

Dexras1 Inhibits PKC Enzymatic Activity. PKC autophosphorylation at serine 643 has been identified to be a key event in regulating its kinase activity (Li et al., 1997). Therefore, we investigated whether the ability of Dexras1 to disrupt autophosphorylation at this residue translated into an ability to inhibit its enzymatic activity. Cells were transiently transfected with PKC in the absence and presence of Dexras1. DEAE Sepharose ion exchange chromatography was used to enrich PKC before performing an in vitro kinase assay designed to examine nPKC enzymatic activity using neurogranin as a PKC-specific substrate. Transfection of PKC resulted in a greater than 300% increase in PKC-specific enzymatic activity greater than that of endogenous nPKC isoforms (Fig. 4). Cotransfection of Dexras1 reduced PKC kinase activity by approximately 50% (Fig. 4). Immunoblot analysis of eluates after DEAE chromatography revealed that total PKC levels were comparable, whereas there were reduced levels of autophosphorylated PKC in eluates from Dexras1-transfected cells (Fig. 4, inset). These data provide evidence that Dexras1 inhibits PKC kinase activity.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Dexras1 and PKC kinase activity. HEK-D2L cells were transiently transfected with PKC in the absence and presence of Dexras1. At 48 h after transfection, cells were prepared for an in vitro PKC kinase assay as described under Materials and Methods. PKC-specific activity was obtained by subtracting nonspecific activity from total catalytic activity. The data are expressed as mean ± S.E.M. of five independent experiments performed in duplicate. *, p < 0.05 versus untransfected control cells (repeated-measures ANOVA with Bonferroni post-test). Inset, immunoblot analysis of phospho-PKC and total PKC from the eluates after DEAE chromatography. Immunoblots shown are representative of two independent experiments.

Dexras1 Interacts with PKC in Intact Cells. We next explored the possibility that Dexras1 may be physically interacting with PKC to disrupt its autophosphorylation and enzymatic activity. We therefore examined the ability of Dexras1 to coimmunoprecipitate with PKC. Cells were cotransfected with PKC and either Dexras1 or vector control. Cell lysates were then subjected to immunoprecipitation with anti-PKC antibody, and the immunoprecipitates were examined for the presence of Dexras1 by immunoblot analysis. The blot revealed an approximately 31-kDa band immunoreactive to anti-Dexras1 antibody after PKC immunoprecipitation (Fig. 5). The band was more reactive from cells transfected with Dexras1 cDNA compared with that of vector-transfected control cells, although Dexras1 immunoreactivity was also observed in the absence of transfected Dexras1 (Fig. 5). These data suggest that PKC can physically associate with endogenous and recombinant Dexras1 in intact cellular systems. Moreover, the combined data implicate this interaction as a mechanism to negatively regulate PKC signaling.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Dexras1 and PKC interactions in intact cells. HEK-D2L cells were cotransfected with PKC and either Dexras1 or pcDNA3 control. At 48 h after transfection, cells were treated with 25 mM dithiobis(succinimidyl)-propionate at room temperature for 30 min. Cell lysates were then prepared for an immunoprecipitation assay as described under Materials and Methods. The lysate samples were incubated with beads alone or in combination with anti-PKC antibody to immunoprecipitate PKC. Samples were then examined for Dexras1 immunoreactivity by immunoblot analysis as described under Materials and Methods. The blot was then stripped and reprobed with anti-PKC antibody. The input lane represents 5 µl of cell lysate samples used for immunoprecipitation. Blot shown is representative of two independent coimmunoprecipitation experiments.

Dexras1 Does Not Inhibit PKC Membrane Translocation. Although Dexras1 possesses a membrane-targeting CAAX motif, there have been conflicting reports as to its localization within the cell (Cismowski et al., 2000; Fang et al., 2000). Furthermore, studies have suggested that not all of Dexras1 effects are isoprenylation-dependent (Graham et al., 2001). One possible mechanism for the ability of Dexras1 to interact and disrupt PKC signaling may be that it is acting in the cytosol to interfere with membrane translocation of this kinase. We investigated this possibility by transfecting cells with PKC in the absence and presence of Dexras1 to examine the ability of PMA to promote membrane translocation of phospho-PKC. Under resting conditions, phosphorylated PKC was found predominantly in the cytoplasmic fractions (Fig. 6, lanes 1 and 3). Stimulating cells with PMA for 30 min resulted in the translocation of PKC to the membrane fractions (Fig. 6, lanes 2 and 4). Although Dexras1 expression decreased the autophosphorylation of PKC, the PMA-induced membrane translocation of PKC was retained in cells cotransfected with Dexras1 (Fig. 6, lanes 3 and 4). These data suggest that Dexras1 does not interfere with the membrane translocation of PKC to inhibit PMA-stimulated AC2 activity.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Dexras1 and PMA-dependent translocation of PKC. HEK-D2L cells were transiently transfected with PKC in the absence and presence of Dexras1. At 48 h after transfection, cells were serum-starved overnight. Thereafter, cells were stimulated with 100 nM PMA for 30 min at 37°C. The reaction was terminated by removing the media and lysing the cells with ice-cold lysis buffer. Cells were then scraped from the plates and centrifuged at 100,000g for 30 min at 4°C to separate the membrane and cytosolic fractions. PKC translocation was examined by immunoblot analysis using a phosphospecific PKC antibody as described under Materials and Methods. Shown is a representative immunoblot from three independent experiments.

Dexras1 Regulation of PKC Autophosphorylation Is Isoprenylation-Dependent. Because Dexras1 did not seem to interfere with cytosolic PKC to inhibit its membrane translocation, we continued our efforts by investigating the requirement for Dexras1 to be membrane-localized to regulate PKC. Our initial experiments used a pharmacological approach to examine the isoprenylation-dependent regulation of PKC by Dexras1. Previous studies have determined that H-Ras is a target for farnesylation at its CAAX box; therefore, we used the farnesyl transferase inhibitor FTI-277 based on the high sequence homology between the CAAX box of Dexras1 (CVIS) and that of H-Ras (CVLS). Consistent with our previous data, transfection of PKC exhibited robust autophosphorylation at serine 643 that was significantly reduced by Dexras1 (Fig. 7). In contrast, treatment of cells with the peptidomimetic FTI-277 abolished the ability of Dexras1 to negatively regulate PKC autophosphorylation (Fig. 7B). PKC autophosphorylation was comparable in the absence and presence of Dexras1 when cells were treated with FTI-277 (Fig. 7). The expression of PKC was not altered by Dexras1 or FTI-277 (data not shown). These data suggest that Dexras1 is targeted to the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKC.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Dexras1 isoprenylation and PKC autophosphorylation. The effect of FTI-277 was examined on Dexras1-mediated disruption of PKC autophosphorylation. A, HEK-D2L cells were transiently transfected with PKC in the absence and presence of Dexras1. At 5 h after transfection, 10 µM FTI-277 was added to the culture media. At 48 h after transfection, cells were serum-starved overnight, lysed with ice-cold lysis buffer containing 1% NP-40, and the detergent-soluble cell extracts were examined for autophosphorylation by immunoblot analysis using a phosphospecific PKC antibody. Immunoblots shown are representative of three independent experiments. B, the phospho-PKC and PKC blots were examined for pixel intensity for the area under the curve generated for each individual band. The data are presented as phospho-/total PKC and have been normalized to values obtained from matched PKC transfection alone. The data are presented as mean ± S.E.M. of three independent experiments. *, p < 0.05 compared with PKC transfection alone (one-sample t test).

To provide support for our pharmacological evidence, we used a CAAX box-deficient Dexras1 mutant (C277S) to determine whether Dexras1 must be targeted to the cellular membrane to disrupt PKC autophosphorylation. Initial experiments confirmed the Dexras1 C277S mutant failed to localize to membrane fractions (Fig. 8). In contrast, wild-type Dexras1 was abundantly expressed in both the cytosolic and membrane fractions (Fig. 8). These data demonstrate that in the absence of an intact CAAX box, Dexras1 fails to localize to the cellular membrane. Our subsequent experiments examined the effect of Dexras1 C277S on PKC autophosphorylation. Transient transfection of PKC revealed robust autophosphorylation of this novel PKC isoform that was significantly decreased by cotransfecting wild-type Dexras1 (Fig. 9). In contrast, cotransfection of cells with the Dexras1 C277S mutant failed to alter PKC autophosphorylation (Fig. 9). Phospho-PKC immunoreactivity in the presence of Dexras1 C277S was comparable with that observed when cells were transfected with PKC alone. Immunoblot analysis confirmed the expression of wild-type and mutant Dexras1 proteins in whole-cell lysates (Fig. 9C).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Cellular localization of wild-type and a CAAX box-deficient Dexras1 mutant (C277S). HEK-D2L cells were transiently transfected with pcDNA3, Dexras1, or Dexras1 C277S. At 48 h after transfection, cells were scraped from the plates and centrifuged at 100,000g for 30 min at 4°C to separate the membrane and cytosolic fractions. Dexras1 expression was examined by immunoblot analysis using an anti-Dexras1 antibody as described under Materials and Methods. Shown is a representative blot from three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Dexras1, Dexras1 C277S, and PKC autophosphorylation. A, HEK-D2L cells were cotransfected with PKC, and either wild-type Dexras1 or Dexras1 C277S as indicated. At 48 h after transfection, cells were serum-starved overnight. Cells were then lysed with ice-cold lysis buffer containing 1% NP-40, and the detergent-soluble cell extracts were examined for PKC autophosphorylation by immunoblot analysis as described under Materials and Methods. Immunoblot shown is representative of four independent experiments. B, the phospho-PKC blots were examined for pixel intensity for the area under the curve generated for each individual band. The data have been normalized to values obtained for phospho-PKC immunoreactivity from PKC transfection alone. The data are presented as mean ± S.E.M. of four independent experiments. *, p < 0.05 compared with PKC-transfected conditions (one-sample t test). C, HEK-D2L cells were transiently transfected with pcDNA3, Dexras1, or Dexras1 C277S and examined for Dexras1 expression by immunoblot analysis as described under Materials and Methods using an anti-Dexras1 antibody. Shown is a representative blot from three independent experiments.

Dexras1-Mediated Inhibition of AC2 Signaling Is Isoprenylation-Dependent. We continued our studies with Dexras1 C277S and investigated its effects on PMA-stimulated AC2 activity. Cells were cotransfected with AC2 and either wild-type Dexras1 or Dexras1 C277S and subsequently examined for PMA-stimulated cAMP accumulation. The results of these studies parallel the effects of Dexras1 and Dexras1 C277S on PKC autophosphorylation. Transfection of Dexras1 resulted in a significant decrease in PMA stimulation of AC2 (Fig. 10), which is consistent with our earlier findings. In contrast, Dexras1 C277S failed to inhibit PMA-stimulated AC2 activity; PMA-stimulated cAMP accumulation was comparable in the absence and presence of Dexras1 C277S (Fig. 10). These data demonstrate that an intact CAAX box is also required for Dexras1-mediated inhibition of PMA-stimulated AC2 activity. Taken as a whole, our study suggests that Dexras1 functionally interacts with PKC at the cellular membrane to interfere with PKC-dependent regulation of AC2 signaling.

View larger version (13K): [in this window] [in a new window]   Fig. 10. Dexras1, Dexras1 C277S, and AC2 activity. HEK-D2L cells were cotransfected with PKC and either wild-type Dexras1 or Dexras1 C277S as indicated. At 48 h after transfection, cells were stimulated with 300 nM PMA for 15 min at 37°C in the presence of IBMX. cAMP accumulation was subsequently measured as described under Materials and Methods. The data are presented as mean ± S.E.M. of three independent experiments performed in duplicate (repeated-measures ANOVA with Bonferroni post-test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this report, we provide evidence that Dexras1 may have a dual role in modulating the activation of AC2 signaling by concurrently blocking PKC and G activity—two proteins that function as activators of AC2. Dexras1 seemed to preferentially target G- and PKC-dependent activation of AC2, because G_s-mediated cAMP accumulation was not significantly altered. The ability of Dexras1 to block G_i/o-coupled receptor-mediated potentiation of AC2 activity is consistent with previous reports that Dexras1 may function to negatively regulate G-dependent signaling pathways (Cismowski et al., 2000; Graham et al., 2002; Takesono et al., 2002; Nguyen and Watts, 2005). In contrast, the ability of Dexras1 to interfere with phorbol ester regulation of AC2 activity presents a novel role for Dexras1 in signal transduction.

We provide evidence that Dexras1 acts to negatively regulate PKC signaling in intact cells. Dexras1 significantly reduced PKC autophosphorylation at serine 643 and the functional consequence was a loss of PKC catalytic activity. This is in agreement with a previous study that identified serine 643 of PKC as an important autophosphorylation site for its enzymatic activity (Li et al., 1997). The role for Dexras1 in regulating PKC function seems to be selective for the isoform, as Dexras1 did not interfere with the autoregulation of PKC or PKC. Moreover, Dexras1 regulation of PKC signaling was dependent on isoprenylation-mediated membrane localization, as autophosphorylation of PKC was neither altered by a CAAX box-deficient Dexras1 mutant nor when cells were treated with the farnesyltransferase inhibitor FTI-277. These results are consistent with observations that a constitutively active, but CAAX box-deficient Dexras1 mutant (A178V/C277term) failed to inhibit cAMP-stimulated human growth hormone secretion in AtT-20 corticotroph cells in comparison to the 86% reduction of secretion induced by Dexras1A178V alone (Graham et al., 2001). The data presented in this report support a model in which Dexras1 negatively regulates PKC through an isoprenylation-dependent mechanism: 1) Dexras1 seems to be post-translationally modified by farnesylation of its CAAX box and localizes to the cellular membrane. 2) At the membrane, Dexras1 functionally interacts with PKC and interferes with its autoregulatory mechanisms. 3) The disruption of autophosphorylation results in a decrease in PKC kinase activity. The precise mechanism by which Dexras1 disrupts PKC autophosphorylation (step 2 above) is unclear at this time. Whether Dexras1 blocks PKC-mediated autophosphorylation at serine 643 or if Dexras1 acts to promote the activity of a phosphatase has yet to be determined. It should be noted that Dexras1 regulation of PKC autophosphorylation does not seem to be the sole factor involved in its ability to inhibit phorbol ester-stimulated AC2 activity. The moderate effect of Dexras1 on PKC autoregulation is more likely to be a contributing factor toward its larger effects on AC2 activity. As Dexras1 can also regulate G signaling (Graham et al., 2002; Takesono et al., 2002; Nguyen and Watts, 2005), it may be that Dexras1 interferes with multiple inputs to AC2 that function in an additive or synergistic manner for maximal AC2 activity. Dexras1 may also be involved in other aspects of adenylyl cyclase signaling that have yet to be characterized.

Dexras1 has been proposed to function as a GEF for G_i/o proteins (Cismowski et al., 2000), although there is evidence to suggest that Dexras1 may also regulate pertussis toxininsensitive pathway (Vaidyanathan et al., 2004). In combination with the results of this study, one interpretation for these data may be that Dexras1 can also regulate G_i/o protein signaling through an indirect pathway that involves PKC. For example, the ability of Dexras1 to inhibit receptor-stimulated activation of extracellular signal-regulated kinase (Graham et al., 2002) and heterologous sensitization of AC1 (Nguyen and Watts, 2005) may be partly attributed to the block of PKC activity. Many G_i/o-coupled receptors have been reported to transactivate MAP kinases through a complex signaling pathway that involves G regulation of PKC (Wetzker and Bohmer, 2003). Likewise, pertussis toxin-sensitive sensitization of AC isoforms has also been proposed to occur via an intricate G- and PKC-dependent pathway (Thomas and Hoffman, 1996; Varga et al., 2003; Nguyen and Watts, 2005). Therefore, inhibition of PKC activity may contribute toward the selective blockade of agonist-stimulated G-dependent signaling pathways by Dexras1. Thus, the model for Dexras1 in G protein-coupled receptor signal transduction might be amended to include an indirect regulation of pertussis toxin-sensitive G_i/o protein signaling through a PKC-dependent pathway.

Molecular modeling studies have revealed that serine 643 of PKC is situated at the apex of a "turn motif" that is conserved in ABC kinases (Newton, 2003). Autophosphorylation of PKC isoforms at this serine/threonine residue in the turn motif locks the enzyme in a catalytically competent conformation (Bornancin and Parker, 1996; Edwards et al., 1999). Our discovery that PKC coimmunoprecipitates with endogenous and recombinant Dexras1 from whole-cell lysates suggests that Dexras1 may be interacting with PKC at or near its turn motif to interfere with autophosphorylation of serine 643 and inhibit proper catalytic function. It is currently unclear, however, why Dexras1 specifically targets the isoform. One possible explanation may be that Dexras1 functions as a physiological regulator of PKC activity. In contrast to most PKC isoforms that require "priming" by the upstream phosphoinositide-dependent kinase 1 (PDK1) for catalytic function, PKC has been shown to possess modest kinase activity in the absence of phosphorylation by PDK1 (Stempka et al., 1997; Gschwendt, 1999). This effect has been attributed to a glutamic acid residue situated five positions upstream of the PDK1 phosphorylation site (threonine 505) that may provide the negative charge required for structural integrity and catalytic function (Stempka et al., 1999). Because PKC seems to be processed as a semiactive enzyme, Dexras1 may serve to suppress its basal kinase activity until the proper signal is relayed.

PKC is involved in many cellular processes such as growth, differentiation, and apoptosis (Kikkawa et al., 2002). PKC has been implicated to have a prominent role in oncogenesis. For example, regulation of PKC activity in rat primary tumors using a PKC inhibitory peptide was shown to decrease the metastatic potential of primary mammary tumor as determined by the development of secondary lung metastases (Kiley et al., 1999). In the MDA-MB-231 and MCF-7 human breast cancer cell lines, PKC has been shown to act as a prosurvival and proproliferative factor (McCracken et al., 2003; De Servi et al., 2005). Furthermore, PKC has been identified to be the predominant isoform expressed in MCF-7 cells, and antiestrogen resistance of these cells is associated with the up-regulation of PKC expression (Shanmugam et al., 1999; Nabha et al., 2005). Together, these studies suggest factors that act to impair PKC signaling may have a role in regulating oncogenesis. Dexras1 expression has been shown to inhibit clonogenic growth of MCF-7 and A549 cells (Vaidyanathan et al., 2004), which suggests that Dexras1 may have a regulatory role in oncogenesis by inhibiting PKC activity. Although protein kinase C has historically been believed to be pro-oncogenic by activating MAP kinase pathways (Hofmann, 2004), PKC isoforms might also regulate cellular proliferation by promoting adenylyl cyclase signaling, because studies support a positive role for cAMP in cell growth and proliferation (Stork and Schmitt, 2002). Thus, the antiproliferative effects of Dexras1 might be associated with its ability to inhibit PKC activity to negatively regulate two distinct oncogenic pathways: cAMP signaling, as we demonstrate in this report, and MAP kinase activation (Graham et al., 2002; Nguyen and Watts, 2005).

In summary, the current study identifies a novel role for Dexras1 in cellular communication. We provide evidence that Dexras1 acts to negatively modulate AC2 signaling by interfering with PKC activity through an isoprenylation-dependent mechanism. These results are consistent with reports of a pertussis toxin-independent mechanism for Dexras1 in intact cells (Vaidyanathan et al., 2004). The role for Dexras1 in regulating PKC activity may provide novel therapeutic targets for drug therapy, because many physiological and pathophysiological processes are associated with altered PKC signaling.

	Acknowledgements

 
We thank Jamie L. Doran for critical reading of the manuscript. We also thank Drs. Alex Toker, Peter Parker, and Shigeo Ohno for providing cDNA for the , , and PKC isoforms. The Dexras1 C277S mutant was a gift from Dr. Stephen Lanier and Govindan Vaidyanathan.

	Footnotes

 
This work was supported by National Institutes of Health grants MH60397 and DA13680 and a Purdue Research Foundation grant.

ABBREVIATIONS: Dexras1, dexamethasone-induced Ras protein 1; GEF, guanine nucleotide exchange factor; AC, adenylyl cyclase; HEK, human embryonic kidney; PDK1, phosphoinositide-dependent kinase 1; PMA, phorbol 12-myristate 13-acetate; FTI-277, methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate; PKC, protein kinase C; MAP, mitogen-activated protein; IBMX, 3-isobutyl-1-methylxanthine; NP-40, Nonidet P-40; PBS, phosphate-buffered saline; ANOVA, analysis of variance; cPKC, conventional protein kinase C; nPKC, novel protein kinase C.

Address correspondence to: Dr. Val J. Watts, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, RHPH 210, West Lafayette, IN 47907. E-mail: wattsv{at}pharmacy.purdue.edu

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