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Activation Loop Phosphorylation Controls Protein Kinase D-Dependent Activation of Nuclear Factor B

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Received for publication March 22, 2004.

Accepted for publication June 29, 2004.

	Abstract

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Activation of the inducible transcription factor nuclear factor B (NF-B) occurs in cells exposed to oxidative stress, and the serine/threonine kinase protein kinase D (PKD) is critical for signal relay to NF-B. We have recently delineated two coordinated events that control PKD activation in response to oxidative stress: phosphorylation at Tyr463 by the tyrosine kinase Abl, and phosphorylation at the activation loop Ser738/Ser742 by the protein kinase C (PKC) isoform PKC. The result is fully active PKD that controls NF-B activation through the IB kinase (IKK) complex. Here, we investigate the mechanism by which PKD controls IKK/NF-B activation. Resveratrol, a potent antioxidant, blocks both PKD activation and NF-B induction. In particular, resveratrol blocked PKD activation loop phosphorylation and activity, and this was caused by a specific inhibition of the Ser738/Ser742 kinase PKC. On the other hand, resveratrol did not affect Abl kinase activity and had no effect on Tyr463 phosphorylation. Moreover, we show that the mechanism by which resveratrol inhibits NF-B is by blocking the translocation of PKD to the IKK complex, specifically by inhibiting Ser738/Ser742 phosphorylation. We therefore propose that rather than acting as an antioxidant, resveratrol specifically blocks oxidative stress-dependent NF-B activation by interfering with PKD phosphorylation and association with the IKK complex.

PKD, formerly known as protein kinase C (PKC)-µ, is a member of a novel subfamily of protein kinases that includes two additional isoforms, PKD2 and PKD3/PKC (Van Lint et al., 2002). PKDs have been shown to be important for G-protein–mediated signaling, Golgi sorting, immune cell signaling, cell growth, and cell survival (Matthews et al., 2000; Rey et al., 2001; Baron and Malhotra, 2002; Storz and Toker, 2003). In addition to interaction with a number of regulatory proteins (Hausser et al., 1999; Johannes et al., 1999; Storz et al., 2000), PKD activity is under the strict control of phosphorylation at two key sites in the catalytic kinase core, the activation loop serines Ser738 and Ser742. Phosphorylation of these two sites is absolutely required for the kinase to achieve catalytic competence, and studies from several laboratories have shown that these residues are directly phosphorylated by several isoforms of the PKC family (Brandlin et al., 2002; Waldron and Rozengurt, 2003; Storz et al., 2004). Our recent studies have focused on the mechanism by which oxidative stress mediates PKD activation, and we have shown that two synergistic signaling pathways are required to promote efficient activation of PKD in response to ROS. First, the tyrosine kinase Src activates Abl, which then directly phosphorylates PKD at Tyr463 in the amino-terminal pleckstrin homology domain (Storz and Toker, 2003). This initial step facilitates the second, rate-limiting step, the phosphorylation of Ser738/Ser742 in the activation loop, mediated by PKC, which itself is also activated by Src (Storz et al., 2004). The net result is a fully active, catalytically competent PKD that relays the signal to IKK/NF-B by an asyet-unknown mechanism.

Numerous chemical inhibitors have been used to investigate the various pathways that converge on the IKK/NF-B complex. Of these, resveratrol (trans-3,4',5-trihydroxystilbene), a known antioxidant, has been shown to block NF-B in response to oxidative stress (Manna et al., 2000). Resveratrol has been shown to function in the prevention of various human pathological processes, including inflammation, atherosclerosis, and carcinogenesis (Tinhofer et al., 2001; Cal et al., 2003). Resveratrol can also promote apoptosis (Dorrie et al., 2001), and although the precise mechanism by which resveratrol exerts its function is not known, the suppression of NF-B has been proposed as one such mechanism (Estrov et al., 2003). For example, resveratrol blocks the phosphorylation and translocation of the p65 subunit of NF-B in tumor necrosis factor (TNF-)-stimulated cells, resulting in reduced transcriptional activity (Manna et al., 2000). However, resveratrol-mediated inhibition of NF-B is not restricted to TNF signaling because it has also been shown to blunt H₂O₂-, PMA-, and lipopolysaccharide-mediated NF-B activation (Manna et al., 2000). More recent studies have suggested that resveratrol is an inhibitor of PKD (Stewart et al., 2000; Haworth and Avkiran, 2001), but again, the mechanism remains undefined.

Here, we used resveratrol in combination with molecular genetic approaches to investigate the mechanism by which PKD promotes NF-B activation in cells exposed to oxidative stress. Our results do not support the notion that resveratrol acts as a general antioxidant to block NF-B activation; rather we show that it specifically blunts PKD activation loop phosphorylation and activation. We further characterize this mechanism and show that resveratrol blocks the association of PKD with the IKK complex. The net effect is an effective inhibition of the PKD/NF-B pathway, which we propose explains the previous findings of inhibition of NF-B by resveratrol in cells exposed to ROS.

	Materials and Methods

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Cell Culture, Antibodies, Reagents, and Purified Proteins. The HeLa cell line was purchased from American Type Culture Collection and maintained in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The -PKD (C-20), -PKC (C-20), -IKK (H-4), -IKK (FL-419), and -Abl (24-11) antibodies were from Santa Cruz Biochemicals (Santa Cruz, CA); -phospho-Ser738/742-PKD (-pSer744/748 in mouse PKD, -pSer738/742 in human PKD) and -phospho-Ser916-PKD (-pSer916 in mouse PKD, -pSer910 in human PKD) were from Cell Signaling Technology Inc. (Beverly, MA); and -Src was from Up-state Biotechnology (Waltham, MA). -HA was purified in house from the 12CA5 hybridoma. The polyclonal -pY463 antibody has been described previously (Storz and Toker, 2003). TNF was a kind gift from H. Wajant (Julius-Maximilians Universität, Würzburg, Germany). Pervanadate was prepared as described previously (Storz et al., 2003). H₂O₂ (30%) was from Fisher Scientific Co. (Pittsburgh, PA). Resveratrol was from BIOMOL Research Laboratories (Ply-mouth Meeting, PA). The PKD-specific substrate peptide used was AALVRQMSVAFFFK. Bovine brain phosphatidylserine and sn-1,2-dioleoylglycerol were from Avanti Polar Lipids (Alabaster, AL). Recombinant PKD or PKD^* (kinase inactive, PKD.K612W) was expressed in insect cells after infection with baculovirus harboring HA-tagged PKD or PKD^* in pFAST-Bac (Invitrogen, Carlsbad, CA) and purified on a nickel- nitrilotriacetic acid affinity column.

Expression Plasmids and Transfections. PKD expression plasmids (amino-terminal HA-tagged PKD in pcDNA3 and the constitutive active HA-PKD.Y463E and HA-PKD.SS738/742EE or inactive HA-PKD.K612W and HA-PKD.SS738/742AA mutants) have been described previously (Storz and Toker, 2003; Storz et al., 2004). PKD.S910A and PKD.S910E expression plasmids are derived from an amino-terminal HA-PKD in pcDNA3. Mutagenesis was carried out by polymerase chain reaction using QuikChange (Stratagene, La Jolla, CA) with the following primer pairs: for PKD.S910A, 5'-CTCGGTGAGCGTGTCGCCATCCTCTGACTCGAG-3' and 5'-CTCGAGTCAGAGGATGGCGACACGCTCACCGAG-3'; for PKD.S910E, 5'-CTCGGTGAGCGTGTCGAAATCCTCTGACTCGAG-3' and 5'-CTCGAGTCAGAGGATTTCGACACGCTCACCGAG-3'. Other expression plasmids were obtained from the following donors: IKK, A. Israel (Institut Pasteur, Paris, France); v-Abl p120, Src.Y527F, B. Schaff-hausen (Tufts University School of Medicine, Boston, MA); PKC.RR144/145AA, S. Ohno (Yokohama City University School of Medicine, Yokohama, Japan); GST-IB amino acids 5-55, T. Maniatis (Harvard University, Cambridge, MA); and GST-Crk amino acids 120-212, R. van Etten (Tufts University School of Medicine and New England Medical Center, Boston, MA). Superfect (QIAGEN, Valencia, CA) was used for transient transfections in luciferase assays, and TransIT HeLa Monster reagent (Mirus, Madison, WI) was used for all other transfections according to the manufacturer's instructions. Cells were stimulated or harvested 24 h after transfection.

Immunoblotting and Immunoprecipitation. Cells were stimulated or harvested 24 h after transfection and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, and 5 mM EDTA, pH 7.4) plus protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysates were used either for immunoblot analysis or proteins were immunoprecipitated by a 1-h incubation with the respective antibody (2 µg), followed by a 30-min incubation with protein G-agarose (Amersham Biosciences Inc., Piscataway, NJ). Immune complexes were washed three times with Tris-buffered saline (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and resolved by SDS-PAGE or subjected to kinase assays.

PKD Kinase Assays. For in vitro kinase assays with immunoprecipitated PKD, after immunoprecipitation (-HA for transfected PKD) and washing, 20 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and 2 mM dithiothreitol) was added to the precipitates, and the kinase reaction was carried out for 20 min after the addition of 10 µl of kinase substrate mix (150 µM PKD-specific substrate peptide, 50 µM ATP, and 10 µCi [-³²P]ATP in kinase buffer). To terminate, the samples were centrifuged, and the supernatants were spotted onto P81 phosphocellulose paper (Whatman, Clifton, NJ). The papers were washed three times with 0.75% phosphoric acid and once with acetone and then dried, and activity was determined by liquid scintillation counting. Protein expression was detected by immunoblotting. For in vitro kinase assays using purified PKD or PKD^*, 1 µg of PKD was incubated with resveratrol, phosphatidylserine/diacylglycerol micelles, and ATP (50 µM ATP and 10 µCi [-³²P]ATP in kinase buffer) for 15 min at room temperature. Mixed phosphatidylserine/diacylglycerol lipid vesicles were prepared as described previously (Storz et al., 2003). The reactions were stopped with sample buffer and subjected to SDS-PAGE. Dried gels were analyzed on a Molecular Imager (Bio-Rad, Hercules, CA).

IKK, Abl, and PKC Kinase Assays. After immunoprecipitation (-PKC, -Abl, and -IKK) and extensive washing, 20 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and 2 mM dithiothreitol) was added to the precipitates, and the kinase reaction was carried out for 30 min by the addition of 10 µl of kinase substrate mix (IKK assays: 2 µg of GST-IB amino acids 5–55, 50 µM ATP, and 10 µCi [-³²P]ATP in kinase buffer; Abl assays: 2 µg GST-Crk amino acids 120–212, 50 µM ATP, and 10 µCi [-³²P]ATP in kinase buffer; PKC assays: 1 µg MBP, 50 µM ATP, and 10 µCi [-³²P]ATP in kinase buffer). To terminate the kinase reaction, 30 µl of 2x SDS sample buffer was added, and the samples were resolved by SDS-PAGE. The gels were dried and analyzed on a Molecular Imager (Bio-Rad). Protein expression was detected by immunoblotting.

Reporter Gene Assays. Cells were transiently cotransfected with an NF-B–reporter construct (NF-B-luc, 5 µg), 1 µg pCS2-(n)-gal, and the protein(s) of interest (1 µg) using Superfect (QIA-GEN). Cells were stimulated 6 h after transfection. Twenty-four hours after transfection, assays for luciferase and -galactosidase activity were performed on total cell lysates using standard assays and measured on a luminometer. Luciferase activity was normalized to the -galactosidase activity. Protein expression was controlled by immunoblot analysis.

	Results

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Resveratrol Inhibits Oxidative Stress-Mediated NF-B Activation. Resveratrol is a naturally occurring phytoalexin produced by certain grapevines, possesses potent antioxidant properties, and has also been shown to block both NF-B and PKD activity in cells (Stewart et al., 2000; Haworth and Avkiran, 2001; Uhle et al., 2003). To investigate the mechanism by which PKD mediates NF-B activation in response to oxidative stress signaling, we first evaluated the NF-B response in HeLa cells exposed to H₂O₂ either in the presence or absence of resveratrol. Consistent with previous reports, resveratrol efficiently blocked oxidative stress-induced NF-B luciferase reporter activity stimulated by either H₂O₂ or pervanadate, a global inhibitor of tyrosine phosphatases (Fig. 1, A and B). As a control, TNF-induced NF-B activation was also blocked by resveratrol. When IKK kinase assays were used as a readout for NF-B activation, resveratrol also completely blocked H₂O₂-induced IKK activation (Fig. 1D), suggesting that the resveratrol block lies upstream of the IKK signalosome. In contrast, resveratrol did not completely abolish TNF-–induced IKK activation, although it was significantly reduced (Fig. 1E). This indicates a difference in the mechanisms by which resveratrol blocks oxidative stress- versus TNF-–induced NF-B activation. Next, we analyzed whether the inhibitory effects of resveratrol are exclusively caused by its antioxidant function or if it can specifically inhibit protein kinases that regulate IKK and NF-B.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Resveratrol inhibits oxidative stress-stimulated NF-B activation. A to C, HeLa cells were transfected with NF-B and -gal reporter genes. 6 hours after transfection, cells were treated with 100 µM resveratrol, and 8 h after transfection, they were stimulated with H2O2 (500 nM), pervanadate (PV, 75 µM), or TNF- (50 ng/ml) for 16 h. Luciferase and -gal reporter gene assays were performed. D and E, HeLa cells were treated with resveratrol (1 h, 100 µM) and stimulated with H2O2 (500 nM, 10 min) or TNF- (50 ng/ml, 5 min). The IKK complex was immunoprecipitated (-IKK), and an IKK substrate phosphorylation kinase assay using GST-IB as a substrate was performed. IKK expression in cells was determined by immunoblot analysis (-IKK/). All results are typical of three independent experiments.

Resveratrol Inhibits NF-B Activation Mediated by Active Alleles of Src, Abl, PKC, and PKD. In HeLa cells, the oxidative stress-induced NF-B activation pathway is mediated by a signaling cascade that involves Src, Abl, PKC, and PKD (Storz et al., 2003, 2004; Storz and Toker, 2003). We therefore used active alleles of Src (Src.Y527F), Abl (v-Abl p120), PKC (PKC.RR144/145AA), or PKD (PKD.Y463E) to specifically activate NF-B in the absence of any exogenous oxidative stress. It is interesting that resveratrol was still able to potently block NF-B induction mediated by active Src, active Abl, active PKC, or active PKD (Fig. 2, A–D). This suggests that resveratrol can directly block one or more steps in the PKD/NF-B pathway even in the absence of exogenous ROS. From these experiments, we conclude that resveratrol can potentially block signaling downstream of either Abl (both Abl and PKD) or downstream of PKC (both PKC and PKD), leading to the inhibition of NF-B induction in all cases. However, because these are activated alleles, this approach does not directly address whether resveratrol blocks the endogenous kinases in response to oxidative stress.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Resveratrol inhibits NF-B activation mediated by active alleles of Src, Abl, PKC, and PKD. A to D, HeLa cells were transfected with NF-B and -gal reporter genes and active Src (Src.Y527F), active Abl (v-Abl p120), active PKC (PKC.RR144/145AA), or active PKD (PKD.Y463E). Six hours after transfection, cells were treated with 100 µM resveratrol for 16 h. Luciferase and -gal reporter gene assays were performed. Protein expression in cells was controlled by immunoblot analysis. All results are typical of three independent experiments. wt, wild type.

Resveratrol Inhibits Oxidative Stress-Mediated Activation of PKC but Not Abl. To determine where in the PKD/NF-B pathway resveratrol inhibits, we evaluated H₂O₂-induced activation of both Abl and PKC, the PKD upstream kinases. It is interesting that resveratrol had no effect on oxidative stress-induced endogenous Abl activation (Fig. 3A), but it significantly (70%) blunted PKC activity as judged by MBP immune-complex kinase assays (Fig. 3B). Thus, again, resveratrol is not acting as a general antioxidant, but it functions to block PKC, which is upstream of PKD and, in turn, upstream of NF-B. Moreover, it is important to note that resveratrol also probably does not block Src activity, because we previously showed that in this pathway, Abl is downstream of Src (Storz and Toker, 2003), and resveratrol does not inhibit Abl (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Resveratrol inhibits PKC but not Abl in response to oxidative stress. HeLa cells were treated with resveratrol (100 µM, 1 h) and stimulated with H2O2 (10 µM, 10 min). Endogenous Abl (A) or PKC (B) were immunoprecipitated, and substrate phosphorylation kinase assays were performed. Abl and PKC expression in cells was controlled by immunoblot analysis. All results are typical of three independent experiments.

Resveratrol Does Not Inhibit PKD Directly. One explanation for the above data is that resveratrol can directly block PKD, which is required for oxidative stress-mediated NF-B activation. To test this question, we first analyzed PKD activity in HeLa cells exposed to H₂O₂. Resveratrol effectively blocked oxidative stress-stimulated PKD activation, as measured in immune-complex substrate kinase assays as well as autophosphorylation (Fig. 4A). Next, we tested whether resveratrol can directly inhibit PKD using recombinant, purified enzyme in in vitro kinase assays. It is interesting that when PKD was incubated in the presence of mixed phosphatidylserine/diacylglycerol vesicles to activate its intrinsic kinase activity, resveratrol did not significantly block this activity at a dose that completely eliminates activity in stimulated cells (100 µM) (Fig. 4B). Finally, a kinase-inactive PKD (PKD^*, PKD.K612W) control (Fig. 4B, left) clearly demonstrated that the phosphorylation of PKD seen in this assay is exclusively caused by autophosphorylation and not by contaminating kinases in the purified PKD preparation. We therefore concluded that resveratrol does not directly inhibit PKD kinase activity but rather that the inhibition of PKD observed in cells must be caused by a block in its activation mechanism.

View larger version (25K): [in this window] [in a new window]   Fig. 4. A, resveratrol blocks oxidative stress-stimulated PKD activation. PKD was overexpressed in HeLa cells. Cells were treated with resveratrol (100 µM, 1 h) and stimulated with H2O2 (10 µM, 10 min). PKD was immunoprecipitated, and substrate-phosphorylation or autophosphorylation assays were performed. PKD expression was controlled by immunoblot analysis (-PKD). B, resveratrol does not directly inhibit PKD. Baculovirus-infected, insect cell-expressed, purified recombinant PKD or PKD* (kinase inactive mutant, PKD.K612W) were incubated with the indicated doses of resveratrol in the presence or absence of phosphatidylserine/dioleoylglycerol (PS/DOG) vesicles. An in vitro autophosphorylation kinase assay was performed. All results are typical of two independent experiments.

Dose Response of PKC, PKD, and NF-B Inhibition by Resveratrol. If resveratrol blocks NF-B activation by blocking the upstream kinases PKC and PKD, then the same concentrations of resveratrol that block NF-B should also block these kinases. To test this, HeLa cells were exposed to increasing doses of resveratrol (0–200 µM) followed by stimulation with H₂O₂. As predicted, the dose-response of inhibition of activation of PKC (Fig. 5A), PKD (Fig. 5B), and NF-B (Fig. 5C) closely correlated, such that 50 to 100 µM resveratrol potently blocked all three activities.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Kinetics of PKC, PKD, and NF-B inhibition. A and B, HeLa cells were treated with resveratrol as indicated (1 h) and stimulated with H2O2 (10 µM, 10 min). Endogenous PKC (A) or overexpressed PKD (B) were immunoprecipitated (IP), and substrate phosphorylation kinase assays were performed. PKC and PKD expression in cells was controlled by immunoblot analysis. C, HeLa cells were transfected with NF-B and -gal reporter genes. Six hours after transfection, cells were treated with resveratrol as indicated, and 8 h after transfection, cells were stimulated with H2O2 (500 nM) for 16 h. Luciferase and -gal reporter gene assays were performed. All results are typical of two independent experiments.

Phosphorylation of PKD at Ser910 Is Not Required for NF-B Activation. We next investigated the mechanism by which resveratrol blocks PKD activation in cells. Ser910 has been described as an in vitro and in vivo auto-phosphorylation site in PKD (Matthews et al., 1999). It has also been shown that PMA stimulates Ser910 phosphorylation and that this is blocked by resveratrol (Haworth and Avkiran, 2001). However, we found that exposure of HeLa cells to oxidative stress, a potent stimulus for PKD, did not result in Ser910 phosphorylation (Fig. 6A). Furthermore, we used a mutational strategy to investigate the functional role of Ser910 in NF-B activation. We found that mutation of Ser910 to either nonphosphorylatable alanine or phosphomimetic glutamic acid had no effect on NF-B induction by H₂O₂ compared with wild-type PKD (Fig. 6B). Finally, because we showed previously that oxidative stress promotes the association of PKD with the IKK complex, we investigated the relevance of Ser910 in this complex formation. Consistent with the lack of an effect on NF-B luciferase activity, neither Ser910 mutant was compromised in association with the IKK complex in coimmunoprecipitation studies, again compared with wild-type PKD (Fig. 6C).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Phosphorylation of PKD at Ser910 is not required for NF-B activation. A, HeLa cells were treated with resveratrol (100 µM, 1 h) and stimulated with H2O2 (10 µM, 10 min). Endogenous PKD was immunoprecipitated (IP) and probed for Ser910 phosphorylation (-pS910). Blots were stripped and reprobed for total PKD. B, HeLa cells were transfected with NF-B and -gal reporter genes and PKD wildtype (wt) or mutants. Six hours after transfection, cells were treated with 500 nM H2O2 for 16 h. Luciferase and -gal reporter gene assays were performed. Protein expression in cells was controlled by immunoblot analysis. C, PKD or PKD mutants and IKK were coexpressed in HeLa cells. Cells were stimulated with H2O2 (10 µM, 10 min). PKD was immunoprecipitated (-HA) and probed for coimmunoprecipitation of IKK. Blots were then stripped and reprobed for total PKD. IKK expression was controlled by immunoblot analysis (-IKK/). The results are typical of three independent experiments.

Resveratrol Inhibits PKD Activation Loop Phosphorylation. Having eliminated Ser910 as the mechanism by which resveratrol blocks PKD activation, we next evaluated Tyr463 and Ser738/Ser742 phosphorylation, both of which are induced in oxidative stress-dependent PKD regulation (Storz and Toker, 2003; Storz et al., 2004). In HeLa cells, resveratrol did not block oxidative stress-stimulated PKD tyrosine phosphorylation, measured with antiphospho-tyrosine (Fig. 7A) or with anti-pY463 (Fig. 7B). On the other hand, resveratrol significantly blocked activation loop phosphorylation of PKD at Ser738/Ser742 (Fig. 7C). These data are in complete agreement with the ability of resveratrol to block PKC (the Ser738/Ser742 kinase) (Fig. 3B) and the lack of inhibition toward Abl (the Tyr463 kinase) (Fig. 3A). Consistent with this model, resveratrol also blocked the association of PKD with PKC, which is observed in cells stimulated with H₂O₂ (Fig. 7D).

View larger version (41K): [in this window] [in a new window]   Fig. 7. Resveratrol inhibits the phosphorylation of PKD at the activation loop but not at Tyr463. A to C, PKD was overexpressed in HeLa cells. HeLa cells were treated with resveratrol (100 µM, 1 h) and stimulated with H2O2 (10 µM, 10 min). PKD was immunoprecipitated (IP) and probed for phosphorylation with -pY (A) or -pY463 (B) or -pS738/742 (C) antibodies. Blots were stripped and reprobed for total PKD (-PKD). C, HeLa cells were treated with resveratrol (100 µM, 1 h) and stimulated with H2O2 (10 µM, 10 min). Endogenous PKD was immunoprecipitated (-PKD), and coimmunoprecipitated PKC was detected by immunoblotting with -PKC. Blots were then stripped and reprobed for total PKD (-PKD). All results are typical of two independent experiments.

Resveratrol Blocks Translocation of PKD to the IKK Complex. To further evaluate the mechanism by which PKD controls IKK/NF-B activation, we determined whether the inhibition of complex formation could account for the inhibitory effects of resveratrol on NF-B activity. We have shown previously that PKD can be coimmunoprecipitated with the IKK complex after exposure of cells to H₂O₂ (Storz and Toker, 2003). We now show that resveratrol completely blocks this association when PKD is immunoprecipitated and IKK is revealed (Fig. 8A) and when IKK is immunoprecipitated and PKD is immunoblotted (data not shown). Second, a PKD.Y463E mutant, which is constitutively phosphorylated at the activation loop Ser738/Ser742 by endogenous PKC (Storz et al., 2004), is also constitutively associated with IKK, but again this is blocked by resveratrol (Fig. 8B), consistent with the ability of resveratrol to block the activation of PKC (Fig. 3B). Next, we investigated the importance of PKD kinase activity and activation loop phosphorylation in translocation to the IKK complex. Translocation of PKD was not dependent on PKD activity, because the both wild-type and kinase-inactive PKD (PKD.K612W) also efficiently coimmunoprecipitated with both IKK and PKC in cells stimulated with H₂O₂ (Fig. 8C). On the other hand, the activation loop PKD mutant (S738A/S742A) was not associated with IKK and also showed impaired (approximately 50%) association with PKC, although this was not complete. Finally, the constitutively active PKD activation loop mutant (S738E/S742E) was constitutively associated with IKK in the absence of stimulation, and a significant proportion was still associated with IKK in cells treated with resveratrol (Fig. 8D). Because PKD activation loop phosphorylation is blocked by resveratrol (Fig. 7C), we conclude from these data that inhibition of translocation of PKD to the IKK complex is the mechanism by which resveratrol blocks the PKC/PKD/NF-B pathway in response to oxidative stress.

View larger version (55K): [in this window] [in a new window]   Fig. 8. Resveratrol blocks oxidative stress-mediated translocation of PKD to the IKK complex. A to D, PKD [wild-type (wt), PKD.K612W (kinase inactive), PKD.Y463E, or PKD.SS738/742EE mutants] and IKK were coexpressed in HeLa cells. HeLa cells were treated with resveratrol (100 µM, 1 h) and/or stimulated with H2O2 (10 µM, 10 min) as indicated. PKD (-HA) was immunoprecipitated (IP) and probed for coimmunoprecipitation of IKK and PKC. Blots were then stripped and reprobed for total PKD. IKK expression was controlled by immunoblot analysis. All results are typical of three independent experiments.

	Discussion

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Resveratrol is a known potent antioxidant and has shown significant efficacy as an anti-inflammatory and antitumorigenic agent in vitro and in vivo (Fremont, 2000). However, not all of the effects of resveratrol can be fully explained by its antioxidant and anticyclooxygenase activities because it can also block cell-cycle progression and cell growth (Joe et al., 2002). Recent studies have shown that in cells, resveratrol also potently blocks NF-B, and separate studies have shown that it can also function as an inhibitor of PKD (Haworth and Avkiran, 2001; Ashikawa et al., 2002). Because we previously linked the activation of PKD to NF-B induction in cells exposed to oxidative stress (Storz and Toker, 2003), we sought to investigate the mechanism by which resveratrol blocks NF-B so as to further understand how PKD promotes NF-B activation. In particular, our goal was to determine whether resveratrol simply functions as a nonspecific antioxidant to block ROS-mediated NF-B activation or whether it can more specifically block one or more steps in the PKD/IKK/NF-B signaling module. Contrary to the notion that resveratrol functions solely as an antioxidant, our data point to this compound as a specific antagonist of two key steps in this pathway: phosphorylation of the PKD activation loop, and interaction of PKD with the IKK complex.

Previous studies have addressed whether resveratrol can directly inhibit PKD activity in vitro. In one study, Stewart et al. (1999) demonstrated a reduced autophosphorylation of purified PKD with increasing doses of resveratrol. An IC₅₀ of 50 µM was reported for inhibition of PKD. A separate group also demonstrated that purified PKD can be inhibited by resveratrol in vitro; however, an IC₅₀ of 200 µM was reported (Haworth and Avkiran, 2001). Using similar in vitro kinase assays, we find that highly purified, recombinant PKD is only modestly inhibited (approximately 20%) by resveratrol at a dose of 100 µM (Fig. 4). It is clear that this cannot account for the complete inhibition of PKD activity observed in cells treated with the same dose of resveratrol (Figs. 4A and 5B). Therefore, a mechanism other than direct inhibition of intrinsic kinase activity must occur in cells to explain the inhibition of PKD, as well as NF-B, by this compound. We therefore turned our attention to phosphorylation of PKD at three distinct sites: Ser910 at the carboxyl terminus, Tyr463 in the pleckstrin homology domain, and Ser738/Ser742 in the activation loop.

Contrary to what has been reported for PMA-stimulated PKD regulation, we were unable to detect an increase in Ser910 phosphorylation induced by oxidative stress (Fig. 6). Moreover, a mutational approach to investigate the potential role of Ser910 in NF-B induction revealed that this site, if it is indeed phosphorylated, is actually dispensable for NF-B activation (Fig. 6). Similar to Ser910, resveratrol also did not block the oxidative stress-stimulated phosphorylation of PKD at Tyr463 (Fig. 7, A and B). We recently showed that Tyr463 is phosphorylated by a Src-Abl signaling pathway in cells exposed to ROS (Storz and Toker, 2003), and consistent with the finding that resveratrol does not inhibit Tyr463 phosphorylation, it also had no effect on both Src and Abl activity stimulated by H₂O₂. Therefore, resveratrol does not function as a nonspecific inhibitor to blunt all pathways leading to the activation of PKD.

In contrast to Ser910 and Tyr463, treatment of cells with resveratrol did significantly inhibit phosphorylation of PKD at the activation loop residues Ser738/Ser742 (Fig. 7C). Because in oxidative-stress signaling these sites are directly phosphorylated by the novel PKC isoform PKC (Storz et al., 2004), we tested whether resveratrol could block PKC activity. As expected, the reduced PKD activation loop phosphorylation seen in cells treated with resveratrol was indeed caused by a block in PKC activity, when oxidative stress was the stimulus (Fig. 3B). Previous studies further support this model, because PKC activity in cells can be blocked with resveratrol concentrations as low as 2 µM (Slater et al., 2003). It has been suggested that one potential mechanism for PKC inhibition is competition with phorbol ester binding by interaction of resveratrol with C1 domains (Slater et al., 2003). Moreover, Yu et al. (2001) recently demonstrated the inhibition of PKC isolated from HeLa cells. The cumulative evidence therefore supports a role for PKC, rather than PKD, as an intracellular target for resveratrol. Moreover, competition of resveratrol for diacylglycerol would also be an explanation for the slight inhibition of purified PKD (which also has two copies of the C1 domain) by resveratrol (Fig. 4B), although it is well-established that the primary mechanism controlling PKD activation is activation loop phosphorylation (Iglesias et al., 1998). Taken together, we propose that resveratrol functions to block the PKC-mediated phosphorylation of PKD at Ser738/Ser742. We also propose that this is a specific inhibitory mechanism toward PKD, because resveratrol also does not block the Abl tyrosine kinase, such that PKD Tyr463 phosphorylation in resveratrol-treated cells is unaffected.

We took advantage of the mechanism by which resveratrol blocks PKD to investigate how PKD signals to NF-B, because resveratrol has been shown to be a potent inhibitor of this transcription factor. Given that resveratrol not only blocks NF-B in response to oxidative stress but also in cells stimulated with the classic NF-B agonists lipopolysaccharide, PMA, and TNF-, it is likely that it functionally blocks at the level of the IKK complex. This is because all of these ligands initiate signaling pathways that ultimately converge at the level of the IKK signalosome, which then relays the signal to IB degradation and ultimately to NF-B activation. Our data are consistent with this notion, because in response to oxidative stress, PKD translocates to the IKK complex, activates IKK, and promotes IB degradation (Storz and Toker, 2003). In the present study, we evaluated the mechanism. First, dose-response experiments showed that resveratrol concentrations that blocked NF-B activity also blocked both PKC and PKD activation by oxidative stress (Fig. 5). Second, resveratrol blocked the oxidative stress-induced translocation of PKD to the IKK complex, measured by coimmunoprecipitation (Fig. 8A). We also show that PKD kinase activity is dispensable for PKD association with both IKK and PKC (Fig. 8C). On the other hand, Ser738/Ser742 phosphorylation is required for PKD translocation to IKK, because a PKD mutant in which these residues cannot be phosphorylated does not associate with IKK after H₂O₂ stimulation, whereas a constitutively active PKD mutant with glutamate residues at the activation loop is constitutively associated with IKK (Fig. 8D). We therefore propose that resveratrol blocks NF-B activation in response to oxidative stress by specifically blocking the activation loop phosphorylation of PKD, mediated by PKC, which in turn blocks the association of PKD with IKK.

Although it is not yet known whether the same mechanism functions in TNF- signaling, it is tempting to speculate that this is indeed the case, because Johannes et al. (1998) have demonstrated the activation of PKD by TNF-. More importantly, PKC has also been linked to TNF-–mediated IB degradation and NF-B activation (Vancurova et al., 2001). It is also worth noting that other studies have also shown that various other PKC isoforms, including PKC, PKCII, PKC, PKC, and PKC have been linked to the activation of NF-B. For example, PMA-stimulated NF-B activation has been shown to be mediated by PKC (Hirano et al., 1995), whereas PKC and PKC seem to participate in TNF-–induced NF-B induction (Leitges et al., 2001; Vancurova et al., 2001). Conventional PKC and novel PKC have been shown to control NF-B induction in T and B cells (Sun et al., 2000; Bauer et al., 2001). Because resveratrol can potentially block some or all of these PKCs, possibly by competing for the natural ligand diacylglycerol, it is likely that PKC is the common target linking resveratrol inhibition of IKK and NF-B in response to all NF-B agonists. This hypothesis is supported by the finding that some of these PKCs have been shown to be inhibited by resveratrol in vivo (Slater et al., 2003), although clearly more work is needed to substantiate this hypothesis.

Although our studies support a specific function for resveratrol in blocking PKC-mediated PKD activation and its subsequent translocation to the IKK complex, we cannot exclude that in certain NF-B activation pathways, resveratrol may also act as a global antioxidant. For example, TNF- has been shown to promote the release of ROS (Shrivastava and Aggarwal, 1999), and thus an additional explanation for the block of NF-B by resveratrol in response to TNF- as well as oxidative stress could indeed be its antioxidant activity. However, we do not favor this model because oxidative stress indirectly activates kinases such as Src or PKC by inhibiting tyrosine and serine/threonine phosphatases. Thus, if resveratrol were merely acting as an antioxidant, one would expect that activation of NF-B by activated alleles of Src, Abl, or PKC would be unaffected by resveratrol, yet it clearly blocks NF-B induction by these upstream kinases (Fig. 2). Moreover, we also evaluated specificity and demonstrated that resveratrol directly inhibits the intrinsic kinase activity of PKC, but not that of Abl (Figs. 3 and 7). This excludes a nonspecific inhibition of general kinase activity by resveratrol, for example, by depleting cellular ATP levels, as has been demonstrated for the compound rottlerin, previously believed to be a specific PKC inhibitor (Soltoff, 2001).

Taken together, our data demonstrate that in oxidative stress signaling, resveratrol can block the activation of NF-B at the level of PKD. This allowed us to investigate the mechanism by which PKD controls NF-B induction at the level of the IKK complex. We found that inhibition of PKD activation loop phosphorylation and loss of translocation to IKK results in a block in NF-B activation. We therefore propose that the concerted action of these two events is responsible for the resveratrol-mediated inhibition of NF-B in oxidative stress signaling. What remains to be determined is how activation loop phosphorylation controls access of PKD to IKK, leading to IKK activation. This is currently under investigation in our laboratory.

	Acknowledgements

 
We thank A. Israel, B. Schaffhaussen, S. Ohno, T. Maniatis, H. Wajant, and R. van Etten for generously providing expression plasmids and reagents. We also thank members of the Toker laboratory for insightful discussions.

	Footnotes

 
This work was supported by National Institutes of Health grant CA75134 (to A.T.) and from the Deutsche Forschungsgemeinschaft grant STO 439/1-1 (to P.S.).

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.000687.

ABBREVIATIONS: ROS, reactive oxygen species; IKK, IB kinase; MBP, myelin basic protein; NF-B, nuclear factor B; PKD, protein kinase D; PKC, protein kinase C; PMA, 12-phorbol 13-myristate acetate; TNF, tumor necrosis factor; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

Address correspondence to: Dr. Alex Toker, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215. E-mail: atoker{at}bidmc.harvard.edu

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