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Originally published In Press as doi:10.1074/jbc.M701613200 on May 24, 2007

J. Biol. Chem., Vol. 282, Issue 29, 21268-21277, July 20, 2007
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Protein Kinase C{zeta} Abrogates the Proapoptotic Function of Bax through Phosphorylation*

Meiguo Xin, Fengqin Gao, W. Stratford May, Tammy Flagg, and Xingming Deng1

From the University of Florida Shands Cancer Center, Department of Medicine and Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32610-3633

Received for publication, February 23, 2007 , and in revised form, May 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C{zeta} (PKC{zeta}) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKC{zeta} in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKC{zeta} in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKC{zeta} activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKC{zeta} specific inhibitor (a myristoylated PKC{zeta} pseudosubstrate peptide). Purified, active PKC{zeta} directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKC{zeta} mutant results in Bax phosphorylation at serine 184. PKC{zeta} co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKC{zeta} by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKC{zeta} mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKC{zeta} can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKC{zeta} may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis through the mitochondrial pathway is mainly controlled and mediated by Bcl2 family proteins. Bax is a major multidomain proapoptotic member of the Bcl2 family that is required for apoptotic cell death (1). However, the signaling mechanism(s) by which Bax is regulated remains enigmatic. It has been proposed that activation of the proapoptotic function of Bax likely occurs through several interdependent mechanisms including translocation from cytosol to mitochondria (2), oligomerization, and insertion into mitochondrial membranes following stress (3–5). Recent reports indicate that the proapoptotic activity of Bax can also be regulated by phosphorylation, a post-translational modification (6–9). Growth factor (i.e. granulocyte macrophate-colony-stimulating factor) or survival agonist (i.e. nicotine)-induced Bax phosphorylation at Ser184 through activation of AKT potently suppresses the proapoptotic activity of Bax and prolongs cell survival (6, 9). In contrast, c-Jun NH2-terminal kinase (JNK)-induced Thr167 or glycogen synthase kinase-induced Ser163 phosphorylation of Bax may enhance the proapoptotic activity of Bax (7–8). Intriguingly, we recently discovered that protein phosphatase 2A functions as a physiological Bax phosphatase that not only dephosphorylates Bax but also potently activates its proapoptotic function (10).

The protein kinase C (PKC)2 family is a multigene family that can be subclassified into three groups including classical, novel, and atypical PKC isoforms according to differences in the lipid activation profile. The mechanisms for activation have been established for sequential phosphorylation, the recruiting of proper localization, and the exchanging of binding proteins (11). Growing evidence indicates that PKC family members play important roles in regulating cell survival and apoptosis (12–15). For example, the classic PKC{alpha}-induced Bcl-2 phosphorylation enhances antiapoptotic function of Bcl2 in association with increased chemoresistance of human leukemia cells (16). Direct interaction between PKC{epsilon} and Bax results in suppression of the proapoptotic activity of Bax (17). Bad phosphorylation induced by either PKC{iota} or PKC{theta} leads to inactivation of Bad (18, 19). All these findings support the notion that Bcl-2 family members potentially function as downstream targets of PKCs in regulating cell survival and cell death.

PKC{zeta} is a member of the atypical PKC subfamily and plays a critical role in suppression of mitochondrial-dependent apoptosis (20, 21). PKC{zeta} consists of four functional domains and motifs, including a PB1 domain in the N terminus, a pseudosubstrate (PS) sequence, a C1 domain of a single Cysrich zinc finger motif, and a kinase domain in the C terminus (22). The kinase domain includes an ATP-binding region, an activation loop, a turn motif, and a hydrophobic motif. The ATP-binding domain contains a Lys residue (Lys281) that is crucial for its kinase activity. A mutation of Lys -> Trp at Lys281 site resulted in a kinase-defective dominant negative form of PKC{zeta} (i.e. K281W) (23). The activation loop and turn motif contain two important phosphorylation sites (i.e. Thr410 and Thr560). Phosphorylation of Thr410 and Thr560 residues is essential for PKC{zeta} activation (24, 25). PKC{zeta} is insensitive to second messengers such as Ca2+ and diacylglycerol. Thus, its activity is primarily regulated by protein-protein interaction and phosphorylation (11). The signal mechanisms for PKC{zeta} activation include the PDK1-dependent (i.e. PI3K/PIP3/PDK1/PKC{zeta}) and PDK1-independent (i.e. PI3K/PIP3/PKC{zeta}) pathways (22). Growth factor or agonist-induced activation of PI3K produces PIP3. On the one hand, PDK1 binds to PIP3 via its PH domain, which results in activation of PDK1. The activated PDK1 interacts with PKC{zeta} and phosphorylates the kinase domain at Thr410, which induces Thr560 phosphorylation. On the other hand, PKC{zeta} directly interacts with PIP3, which releases PS-dependent autoinhibition. Both contributions of PIP3 and PDK1 are necessary for the complete and stable activation of PKC{zeta} (22). Furthermore, phospholipase D2 has recently been identified as a novel protein cofactor for activation of PKC{zeta} that can enhance PKC{zeta} activity through direct interaction in a lipase activity-independent manner (11). Our previous findings reveal that nicotine stimulates Bax phosphorylation in association with increased cell survival (9). However, a direct signal link between PKC{zeta} and Bax in nicotine-induced cancer cell survival has not been established, yet. Here we have demonstrated that PKC{zeta} functions as a novel nicotine-activated Bax kinase that directly phosphorylates and inactivates Bax in human lung cancer cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Nicotine and cisplatin were purchased from Sigma. Purified, active PKC{zeta}, myristoylated PKC{zeta} pseudosubstrate peptide (MYR-PKC{zeta}-PS, Myr-SIYRRGARRWRKL-OH) and the myristoylated PKC{alpha} pseudosubstrate peptide (MYR-PKC{alpha}-PS, Myr-N-FARKGALRQ-NH2) were obtained from Calbiochem. Bax, Bcl2, prohibitin, and PKC{zeta} antibodies, PKC{zeta} siRNA1 (sense strand sequence: CAAGCCAAGCGCUUUAACAtt) and PKC{zeta} siRNA2 (sense strand sequence: CAGAUGGAAUUGCUUACAUtt) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Myelin basic protein (MBP) was obtained from Invitrogen. HA-tagged wild type (WT), constitutively active A119D and dominant negative (DN) K281W PKC{zeta} mutants in pcDNA3 were kindly provided by Dr. Brian Law (University of Florida). Recombinant purified WT, BH1, BH2, and BH3 deletion mutant proteins were purchased from Protein X (San Diego, CA). WT and Bax-/- MEF cells were kindly provided by Dr. Douglas R. Green (St. Jude Children's Research Hospital, Memphis). All reagents used were obtained from commercial sources unless otherwise stated.

Cell Culture, cDNA Constructs, and Transfections—WT and Bax-/- MEF cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 4 mM L-glutamine as described (26). H23, H69, H82, H157, H358, H460, and H1299 were maintained in RPMI1640 with 10% fetal bovine serum. A549 cells were maintained in F-12K medium with 10% fetal bovine serum and 4 mM L-glutamine. For generation of the phosphomimetic and the nonphosphorylatable Bax mutants, the 5'-phosphorylated mutagenic primers for various precise deletion mutants were synthesized as follow: S184A, 5'-GTCCTCACCGCCGCGCTCACCATCTGG-3'; S184E, 5'-GTCCTCACCGCCGAGCTCACCATCTGG-3'. The T7-tagged WT-Bax/pUC19 construct was used as the target plasmid that contains a unique NdeI restriction site for selection against the unmutated plasmid. The NdeI selection primer is: 5'-GAGTGCACCATGGGCGGTGTGAAA-3'. These Bax mutants were created using a site-directed mutagenesis kit (Clontech) according to the manufacturer's instructions. Each single mutant was confirmed by sequencing of the cDNA and then cloned into the pCIneo (Promega) mammalian expression vector. The pCIneo plasmid containing each Bax mutant cDNA was transfected into MEF Bax-/- cells using LipofectamineTM 2000 according to the manufacturer's instructions (Invitrogen). The expression levels of exogenous Bax were determined by Western blot analysis using a Bax antibody.

Preparation of Total Cell Lysate—Cells were washed with 1x PBS and resuspended in ice-cold 1% CHAPS lysis buffer (1% CHAPS, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, and 1 mM beta-mercaptoethanol) with a mixture of protease inhibitors (Calbiochem). Cells were lysed by sonication and centrifuged at 14,000 x g for 10 min at 4 °C. The resulting supernatant was collected as the total cell lysate and used for protein analysis or co-immunoprecipitation.

Assay of PKC{zeta} Activity in Vitro—PKC{zeta} activity was measured using MBP as substrate as previously described (27). Briefly, PKC{zeta} was immunoprecipitated from cell lysates with an agarose-conjugated PKC{zeta} antibody. Immunoprecipitated PKC{zeta} was washed and resuspended in kinase buffer containing 50 mM Hepes (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 50 mM NaF, 1 mM NaVO4, 1 mM dithiothreitol, and 0.1% Tween 20, 40 µg/ml phosphatidylserine, and 2 µCi of [{gamma}-32P]ATP and MBP (10 µg). The reactions were incubated at 30 °C for 15 min and terminated by addition of SDS sample buffer and boiling the sample for 5 min. The samples were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film at -80 °C. The activity of PKC{zeta} was determined by autoradiography. PKC{zeta} activity was also assessed by measuring the rate of 32P from [{gamma}-32P]ATP incorporation into a peptide (ERMRPRKRQGSVRRRV) corresponding to the pseudosubstrate region of PKC{epsilon} in which an alanine was replaced by a serine as described (28, 29). Briefly, A549 cells were treated with nicotine for 60 min. Specific immunoprecipitation of PKC{zeta} was performed by incubating a polyclonal PKC{zeta} antibody overnight at 0–4 °C with 1 mg of total cell lysate protein in buffer containing 20 mM Tris/HCl (pH 7.5), 0.25 M sucrose, 1.2 mM EGTA, 20 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM sodium vanadate, 1 mM sodium pyrophosphate, 1 mM NaF, Triton X-100 (1%), Nonidet (0.5%), and 150 mM NaCl. Precipitates using non-immune serum were simultaneously prepared to determine blank values. Precipitates were collected on protein A-Sepharose beads, washed, and suspended in 50 mM Tris/HCl (pH 7.5), 1 mM NaHCO3, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 40 µM modified PKC{epsilon} pseudosubstrate peptide for 10 min at 30 °C. After thermal equilibration, assays were started by the simultaneous addition of 15 µM ATP, 0.3 µCi of [{gamma}-32P]ATP (3000 Ci/mmol), and terminated after 30 min with 100 µl of 175 mM phosphoric acid. Then, 100 µl was transferred to P18 filter paper, and washed three times with 75 mM phosphoric acid. The level of peptide phosphorylation was determined by scintillation counting.

Metabolic Labeling, Immunoprecipitation, and Western Blot Analysis—Cells were washed with phosphate-free RPMI medium and metabolically labeled with [32P]orthophosphoric acid for 120 min. After agonist or inhibitor addition, cells were washed with ice-cold 1x PBS and lysed in detergent buffer. Bax was immunoprecipitated using an agarose-conjugated Bax antibody. The samples were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film at -80 °C. Bax phosphorylation was determined by autoradiography. The same filter was then probed by Western blot using a Bax antibody and developed using an ECL kit from Amersham Biosciences as described previously (9).

Subcellular Fractionation—Cells (2 x 107) were washed with cold 1x PBS and resuspended in isotonic mitochondrial buffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM Hepes, pH 7.5) containing protease inhibitor mixture set I, homogenized with a Polytron homogenizer operating for 4 bursts of 10 s each at a setting of 5, then centrifuged at 2000 x g for 3 min to pellet the nuclei and unbroken cells. The supernatant was centrifuged at 13,000 x g for 10 min to pellet mitochondria as described (9). The second supernatant was further centrifuged at 150,000 x g to pellet light membranes. The resulting supernatant is the cytosolic fraction. Mitochondria was washed with mitochondrial buffer twice and resuspended in 1% Nonidet P-40 lysis buffer and rocked for 60 min, then centrifuged at 17,530 x g for 10 min at 4 °C. The resulting supernatant containing mitochondrial proteins was collected. Protein (100 µg) from each fraction was subjected to SDS-PAGE. Bax was analyzed by Western blot using a Bax antibody. The purity of fractions was confirmed by assessing localization of fraction-specific proteins including prohibitin (mitochondria) and caspase 3 (cytosol) (30, 31).

Phosphorylation of Bax in Vitro—Purified recombinant Bax protein was obtained from Protein X, Inc. (San Diego, CA). The endogenous Bax was immunoprecipitated using an agarose-conjugated Bax antibody from lysates of A549 cells. Recombinant or endogenous Bax was resuspended in a kinase assay buffer containing 25 mM Tris buffer (pH 7.5), 100 µM ATP, 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 100 µg/ml phosphatidylserine, and 2 µCi of [{gamma}-32P]ATP and incubated with purified, active PKC{zeta} at 30 °C for various times as indicated. The reaction was terminated by addition of SDS sample buffer and boiling prior to SDS-PAGE. Phosphorylation of Bax was determined by autoradiography.

Alkali Extraction of Bax Which Is Peripherally Associated with Mitochondrial Membranes—Cells were treated with C2-ceramide (30 µM) for 24 h. Mitochondria were isolated by subcellular fractionation and resuspended in freshly prepared 0.1 M Na2CO3 (pH 11.5) and incubated on ice for 30 min. The samples were then centrifuged at 200,000 x g for 30 min, and the alkali-extracted membrane pellet was collected as described (2, 10). The alkali-resistant Bax (i.e. nonextractable or integral) in mitochondrial membranes was washed with kinase buffer and used as substrate for PKC{zeta} in vitro kinase assay as described above.

Immunofluorescence—A549 cells were seeded and cultured on a Lab-Tek® chamber slide (Nalge Nunc International) overnight at 37 °C with 5% CO2. Cells were washed with 1x PBS, fixed with ice-cold mixture of methanol/acetone (1:1), and blocked with 10% mouse and rabbit serum. Then, cells were incubated with a mouse against human Bax and a rabbit against human PKC{zeta} primary antibodies for 90 min. After washing, samples were incubated with fluorescein isothiocyanate-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies for 60 min. Cells were washed with 1x PBS and observed under a fluorescent microscope (Zeiss). Pictures were taken and colored with the same exposure setting for each experiment. To determine subcellular regions of protein co-localization, individual red- and green-stained images derived from the same field were merged using Openlab 3.1.5 software from Improvision, Inc. (Lexington, MA). Areas of protein co-localization appear yellow.

Depletion of PKC{zeta} Expression by RNA Interference—Human PKC{zeta} siRNA was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A549 cells were transfected with PKC{zeta} siRNA using Lipofectamine 2000 according to the manufacturer's instructions. A control siRNA (non-homologous to any known gene sequence) was used as a negative control. The levels of PKC{zeta} expression were analyzed by Western blotting using a PKC{zeta} antibody. Bax phosphorylation or cell viability was assessed following various treatments. Specific silencing of the targeted PKC{zeta} gene was confirmed by at least three independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nicotine Induces Activation of PKC{zeta} and Phosphorylation of Bax in Association with Prolonged Survival of Human Lung Cancer Cells, Which Is Suppressed by a PKC{zeta}-specific Pseudosubstrate Inhibitor—PKC{zeta} has been implicated in many key cellular functions including cell survival (20, 32). Because both human small cell (SCLC) cells (i.e. H69 and H82) and non-small cell lung cancer (NSCLC) cells (i.e. H23, H157, H358, H460, H1299, and A549) express high levels of endogenous PKC{zeta} (Fig. 1A), nicotine may activate PKC{zeta} to promote cell survival. To test this, A549 cells were treated with increasing concentrations of nicotine for 60 min and PKC{zeta} was then immunoprecipitated using an agarose-conjugated PKC{zeta} antibody. Activity of PKC{zeta} was measured by an immune complex kinase assay using purified MBP as a substrate as described (27). The same filter was then probed by Western blot to quantify PKC{zeta} protein. To ensure that the kinase activity that phosphorylates MBP is from PKC{zeta}, and not from any other kinase that may be co-immunoprecipitated with PKC{zeta}, we also probed the same filter by Western blot using a PKC{iota} or PKC{alpha} antibody, respectively. Total cell lysate was used as a positive control. Results indicate that the PKC{zeta} antibody specifically immunoprecipitates PKC{zeta} and does not co-immunoprecipitates either PKC{iota} or PKC{alpha} (Fig. 1B), indicating its specificity for PKC{zeta}. Importantly, nicotine significantly enhances PKC{zeta} activity in a dose-dependent manner (Fig. 1B). To further confirm these results, we used a modified pseudosubstrate peptide with an Ala to Ser mutation as a PKC{zeta} substrate for the PKC{zeta} activity assay as described (28, 29). Previous reports have demonstrated that a higher level of phosphorylation by PKC{zeta} was obtained with the peptide derived from the pseudosubstrate region of PKC{epsilon} as compared with the peptide derived from the pseudosubstrate region of PKC{zeta} (28, 29), indicating that the PKC{epsilon} pseudosubstrate peptide is a better substrate for PKC{zeta}. Therefore, we chose a modified PKC{epsilon} pseudosubstrate peptide (ERMRPRKRQGSVRRRV) as a substrate for the PKC{zeta} activity assay as previously reported (28, 29). Results indicate that treatment of A549 cells with nicotine markedly up-regulates PKC{zeta} activity (Fig. 1C), which is consistent with the results obtained by using MBP as a substrate (Fig. 1B). We have previously demonstrated that nicotine can stimulate Bax phosphorylation in association with increased cell survival (9). To assess whether PKC{zeta} is involved in nicotine-induced Bax phosphorylation, A549 cells were exposed to nicotine in the absence or presence of the PKC{zeta} specific inhibitor (MYR-PKC{zeta}-PS, a myristoylated PKC{zeta} pseudosubstrate peptide) or the myristoylated PKC{alpha} pseudosubstrate peptide (MYR-PKC{alpha}-PS) (20, 33, 34). Results reveal that MYR-PKC{zeta}-PS but not MYR-PKC{alpha}-PS potently suppresses nicotine-induced Bax phosphorylation as well as nicotine-prolonged cell survival following cisplatin treatment (Fig. 1, D and E). Other cell lines (i.e. H69, H82, H460, and H1299) were also tested and similar results were obtained (data not shown). These findings suggest that Bax may function as a downstream target of nicotine-activated PKC{zeta} in supporting survival of human lung cancer cells.


Figure 1
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  		FIGURE 1.
Nicotine stimulates activation of PKC{zeta} and Bax phosphorylation in association with enhanced survival of human lung cancer cells, which is blocked by a PKC{zeta} specific inhibitor. A, expression levels of endogenous Bax, Bcl2, and PKC{zeta} in various human lung cancer cells were analyzed by Western blot. B, A549 cells were treated with increasing concentrations of nicotine for 60 min. PKC{zeta} was immunoprecipitated (IP) and incubated with purified MBP in an in vitro kinase assay. PKC{zeta} activity was analyzed by autoradiography. PKC{zeta} protein was quantified by Western blot using a PKC{zeta} antibody. To confirm the specificity of the PKC{zeta} antibody, the same filter was reprobed using a PKC{iota} or PKC{alpha} antibody, respectively. Total cell lysate was used a positive control for PKC{zeta}, PKC{iota}, or PKC{alpha} Western blot. C, PKC{zeta} activity was also measured using a modified PKC{epsilon} pseudosubstrate peptide with an Ala to Ser mutation as a PKC{zeta} substrate in A549 cells following nicotine treatment. D, A459 cells expressing high levels of endogenous Bax were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine (1µM) in the absence or presence of the myristoylated PKC{zeta} pseudosubstrate peptide (MYR-PKC{zeta}-PS, a PKC{zeta} specific inhibitor), or the myristoylated PKC{alpha} pseudosubstrate peptide (MYR-PKC{alpha}-PS). Bax was immunoprecipitated by using an agarose-conjugated Bax antibody. Phosphorylation of Bax was determined by autoradiography (upper). Western blot analysis was performed to confirm and quantify Bax protein (lower). E, A549 cells were treated with cisplatin (40 µM) in the absence or presence of nicotine (1 µM) or MYR-PKC{zeta}-PS or MYR-PKC{alpha}-PS for 48 h. Cells were harvested and analyzed for Annexin-V and PI binding by flow cytometry. The viability was determined by fluorescence-activated cell sorter. Data represent the mean ± S.D. of three determinations.

 
Purified PKC{zeta} Directly Phosphorylates Bax in Vitro and Overexpression of PKC{zeta} in Cells Results in Bax Phosphorylation at Ser184 in Vivo—To assess a potential direct role for PKC{zeta} as a physiological Bax kinase, subcellular distribution of PKC{zeta} and Bax was examined by immunofluorescent staining. A mouse antibody against human Bax, rabbit polyclonal PKC{zeta} antibody, and fluorescein isothiocyanate-conjugated anti-mouse (green) or rhodamine-conjugated anti-rabbit (red) secondary antibodies were used so that cells could be simultaneously stained without cross-reaction. As shown in Fig. 2A, Bax is primarily co-localized with PKC{zeta} in the cytoplasm of A549 cells. To test whether PKC{zeta} can directly phosphorylate endogenous Bax, endogenous Bax was immunoprecipitated from A549 cells and incubated with purified, active PKC{zeta} in a kinase assay buffer containing [{gamma}-32P]ATP. Results indicate that active PKC{zeta} directly phosphorylates endogenous Bax in vitro (Fig. 2B). In addition, active PKC{zeta} can also directly phosphorylate recombinant Bax protein (Fig. 2C). These findings suggest that PKC{zeta} is a strong candidate for being the direct Bax kinase. To determine whether PKC{zeta} may be a Bax kinase in vivo, HA-tagged WT, constitutively active A119D, or DN-K281W PKC{zeta} constructs were transfected into A549 cells that express high levels of endogenous Bax. After transfection for 48 h, cells were metabolically labeled with [32P]orthophosphoric acid for 90 min. Bax phosphorylation was analyzed by autoradiography. Results reveal that overexpression of WT or active but not DN-PKC{zeta} results in an increased Bax phosphorylation (Fig. 2D). Importantly, the constitutive active PKC{zeta} has a more potent effect on Bax phosphorylation compared with WT (Fig. 2D). To identify the site of Bax phosphorylation induced by PKC{zeta}, WT, S184A, and S184E Bax mutants were co-transfected with the constitutively active A119D PKC{zeta} mutant into MEF Bax-/- cells. After 48 h, cells were then metabolically labeled with [32P]orthophosphoric acid for 90 min. Results reveal that PKC{zeta} induces Bax phosphorylation exclusively at the Ser184 site because expression of active PKC{zeta} results in phosphorylation of WT but not S184A or S184E Bax mutants (Fig. 2E). These findings provide both biochemical and genetic evidence that Bax is a physiological PKC{zeta} downstream substrate. To directly test a role of PKC{zeta} in regulating the proapoptotic function of Bax, a constitutively active PKC{zeta} was co-transfected with WT or each of the S184A and S184E Bax mutants into MEF Bax-/- cells. Consistent with our previous findings (9), the nonphosphorylatable S184A Bax mutant has more proapoptotic activity than WT Bax, whereas the phosphomimetic S184E Bax mutant displays no cytotoxity (Fig. 2, F and G). Intriguingly, expression of active PKC{zeta} enhances survival of MEF Bax-/- cells expressing exogenous WT Bax but has no significant effect on survival of MEF Bax-/- cells expressing the exogenous S184A or S184E Bax mutant (Fig. 2, F and G). These results indicate that active PKC{zeta} may abolish the proapoptotic function of WT Bax via phosphorylation but fails to reverse the proapoptotic function of the nonphosphorylatable, constitutively active S184A Bax mutant.


Figure 2
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  		FIGURE 2.
PKC{zeta} co-localizes with Bax and directly phosphorylates Bax at the Ser184 site. A, A549 cells were fixed with methanol and acetone (1:1). After washing with 1x PBS, cells were incubated with a mouse against human Bax and a rabbit against human PKC{zeta} antibodies. Fluorescein isothiocyanate-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies were used to visualize Bax (green) and PKC{zeta} (red) localization patterns under a fluorescent microscope. Red-and green-stained images were merged using Openlab 3.1.5 software. Areas of co-localization appear yellow. B, endogenous Bax was immunoprecipitated from A549 cells and incubated with purified, active PKC{zeta} in an in vitro kinase assay. Phosphorylation of Bax was determined by autoradiography (upper). Western blot analysis was performed to confirm and quantify Bax protein (lower). C, recombinant Bax protein was incubated with purified, active PKC{zeta} in an in vitro kinase assay. Phosphorylation of Bax was determined by autoradiography. D, the pcDNA3 plasmids bearing HA-tagged WT, constitutively active A119D and DN-K281W PKC{zeta} mutants were transfected into A549 cells that express high levels of endogenous Bax using Lipofectamine 2000. After 48 h, cells were metabolically labeled with [32P]orthophosphoric acid for 90 min. Phosphorylation of Bax was analyzed by autoradiography. Immunoprecipitated Bax protein and HA-PKC{zeta} in total cell lysate were analyzed by Western blot using a Bax or HA antibody, respectively. E, WT, S184A, and S184E Bax mutants were co-transfected with the constitutively active A119D PKC{zeta} mutant into MEF Bax-/- cells. After 48 h, cells were then metabolically labeled with [32P]orthophosphoric acid for 90 min. Bax phosphorylation was analyzed by autoradiography. Immunoprecipitated (IP) Bax protein and HA-PKC{zeta} in total cell lysate were analyzed by Western blot using Bax or HA antibody, respectively. F and G, HA-tagged constitutively active A119D PKC{zeta} mutant was co-transfected with WT or each of the S184A and S184E Bax mutants into MEF Bax-/- cells. After 48 h, expression levels of HA-PKC{zeta} and Bax were analyzed by Western blot using HA or Bax antibody, respectively. Cell viability was analyzed as described in the legend for Fig. 1E. Data represent the mean ± S.D. of three determinations.

 
Expression of WT or Constitutively Active But Not Dominant Negative PKC{zeta} Results in Retention of Bax in Cytosol and Prevents Bax from Undergoing a Conformational Change—Bax is located in the cytosol and/or peripherally associated with mitochondrial membranes in unstimulated cells (2). To test whether PKC{zeta}-induced Bax phosphorylation affects Bax subcellular localization, WT, active, or DN-PKC{zeta} were transfected into A549 cells. After 48 h, mitochondria and cytosol were isolated and Bax was analyzed using a Bax antibody. Results reveal that overexpression of WT or active PKC{zeta} leads to increased sequestration of Bax in cytosol, whereas expression of DN-PKC{zeta} results in Bax accumulation in mitochondrial membranes (Fig. 3A). To determine the purity of the subcellular fractions obtained, fraction-specific proteins were assessed by probing the same filters. Prohibitin, an exclusively mitochondrial protein (30), was detected only in the mitochondrial fraction, whereas caspase 3, which is a cytosolic protease in growing cells (31), was detected exclusively in the cytosol (Fig. 3A). This indicates that mitochondrial and cytosolic fractions are highly pure without cross-contamination. The monoclonal antibody 6A7, raised against the peptide amino acids 13–19 in the N terminus of Bax, is not able to bind the soluble form of Bax in healthy cells but can recognize Bax after the conformational change associated with membrane insertion occurs in apoptotic cells (35–37). To test whether expression of PKC{zeta} affects the stress-induced conformational change of Bax, A549 cells expressing constitutively active or DN-PKC{zeta} or vector-only control were treated with cisplatin (40 µM) for 48 h. Antibody binding to Bax was measured by immunofluorescence or immunoprecipitation. Immunoprecipitation of Bax was performed using a 6A7 or pan-Bax antibody and Bax was analyzed by Western blotting using a pan-Bax antibody. Results indicate that treatment of cells with cisplatin potently enhances the ability of the 6A7 antibody to immunoprecipitate Bax compared with the pan-Bax antibody (Fig. 3B, lane 1 versus lane 2). Overexpression of active PKC{zeta} reduces the ability of the 6A7 antibody to immunoprecipitate Bax. By contrast, expression of DN-PKC{zeta} enhances the 6A7 antibody-Bax binding (Fig. 3B). Consistently, Bax immunofluorescence is low or undetectable in untreated vector-only cells, it increases significantly in vector-only cells treated with cisplatin, which is associated with increased apoptotic cell death (Fig. 3, C and D), suggesting that cisplatin is able to induce a conformational change of Bax. Importantly, overexpression of the constitutive active but not DN-PKC{zeta} suppresses the cisplatin-induced Bax conformational change in association with prolonged cell survival (Fig. 3, B–D). Thus, PKC{zeta}-mediated Bax phosphorylation may prevent a conformational change in Bax and retain Bax in an inactive form.


Figure 3
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  		FIGURE 3.
Overexpression of WT or active but not DN-PKC{zeta} facilitates Bax accumulation in cytosol, inhibits cisplatin-induced Bax conformational change, and prolongs cell survival. A, the pcDNA3 plasmids bearing HA-tagged WT, constitutively active A119D and DN-K281W PKC{zeta} mutants were transfected into A549 cells that express high levels of endogenous Bax using Lipofectamine 2000. After 48 h, subcellular fractionation was performed to isolate mitochondrial and cytosolic fractions. Bax protein of the mitochondrial or cytosolic fraction was analyzed by Western blot using a Bax antibody. Fraction-specific proteins (i.e. prohibitin or caspase 3) were assessed by probing the same filters. B, A549 cells expressing active, DN-PKC{zeta}, or vector-only control were treated with cisplatin (40 µM) for 48 h. A co-immunoprecipitation (IP) experiment was carried out using a 6A7 or full-length Bax antibody, respectively. Bax was analyzed by Western blotting using a Bax antibody. C, A549 cells expressing active, DN-PKC{zeta} or vector-only control were treated with cisplatin (40 µM) for 48 h. Cells were incubated with prewarmed (37 °C) growth medium containing MitoTracker (red) for 30 min. Cells were then washed with 1x PBS, fixed, and permeabilized with ice-cold methanol and acetone (1:1), blocked with 10% mouse serum and stained with a mouse monoclonal 6A7 primary and fluorescein isothiocyanate-conjugated anti-mouse secondary (green) antibodies. Images were merged using Openlab 3.1.5 software. Areas of co-localization are yellow. D, A549 cells expressing active, DN-PKC{zeta} or vector-only control were treated with cisplatin (40 µM) for 48 h. Cell viability was assessed as described in the legend for Fig. 1E. Data represent the mean ± S.D. of three separate determinations.

 
Treatment of Cells with Nicotine Results in Increased PKC{zeta}/Bax Interaction and Decreased Bcl2/Bax Binding, and Purified PKC{zeta} Can Directly Disrupt Bcl2/Bax Heterodimeric Complex in Vitro—Because nicotine can activate PKC{zeta} to phosphorylate Bax (Figs. 1 and 2), it is possible that nicotine-induced activation of PKC{zeta} may facilitate PKC{zeta} to associate with Bax, which may be involved in functionally regulating Bax/Bcl2 heterodimerization. To test this, H460 cells expressing high levels of endogenous PKC{zeta}, Bax, and Bcl2 were treated with increasing concentrations of nicotine for 60 min. PKC{zeta}/Bax and Bax/Bcl2 complexes were immunoprecipitated using a PKC{zeta} or a Bax antibody, respectively. Results indicate that nicotine stimulates PKC{zeta} to interact with Bax but not Bcl2 in a dose-dependent manner (Fig. 4A, upper panel). Intriguingly, nicotine-stimulated PKC{zeta}/Bax association results in decreased Bcl2/Bax binding (Fig. 4A, lower panel). To test whether PKC{zeta} can directly affect Bcl2/Bax herterodimeric complex in vitro, the Bcl2-Bax complex was immunoprecipitated from the lysate of H460 cells disrupted in 1% CHAPS lysis buffer using an agarose-conjugated Bax antibody. The immune complex was incubated with purified, active PKC{zeta} in a kinase buffer at 30 °C for 30 min and proteins released from the complex were identified in the supernatant following centrifugation at 14,000 x g for 5 min. Bax-associated PKC{zeta} (i.e. bound PKC{zeta}), Bax-associated Bcl2 (i.e. bound Bcl2), unbound Bcl2 (i.e. present in the supernatant), and total Bax were analyzed by Western blot. Results reveal that PKC{zeta} can directly disrupt Bcl2/Bax in vitro because decreased levels of Bcl2 were observed on beads and increased levels of Bcl2 were present in the supernatant (Fig. 4B). These findings suggest that PKC{zeta} can directly disrupt the Bax/Bcl2 heterodimer via its binding to Bax, which results in liberation of Bcl2 to benefit the antiapoptotic function of Bcl2.


Figure 4
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  		FIGURE 4.
Treatment of cells with nicotine results in increased PKC{zeta}/Bax interaction and decreased Bcl2/Bax binding, and purified PKC{zeta} directly disrupt the Bcl2/Bax heterodimeric complex in vitro. A, H460 cells expressing high levels of endogenous PKC{zeta}, Bax, and Bcl2 were treated with increasing concentrations of nicotine for 60 min. A co-immunoprecipitation (IP) experiment was carried out using a PKC{zeta} or a Bax antibody, respectively. PKC{zeta}, Bax, and Bcl2 were analyzed by Western blot. Total cell lysate was used as a control. B, the Bax/Bcl2 complex was co-immunoprecipitated from H460 cells using an agarose-conjugated Bax antibody and incubated with increasing concentrations of purified PKC{zeta} in a kinase assay buffer at 30 °C for 30 min. The samples were centrifuged at 14,000 x g for 5 min. The resulting supernatant and immunocomplex beads were subjected to SDS-PAGE. Bax, bound PKC{zeta}, bound Bcl2, and free Bcl2 were analyzed by Western blot.

 
Active But Not DN-PKC{zeta} Associates with Bax at Its BH3 Domain—To test whether the activity of PKC{zeta} affects its ability to interact with Bax, HA-tagged WT, active or DN-PKC{zeta} were transfected into A549 cells using LipofectamineTM 2000. Co-immunoprecipitation using a monoclonal HA antibody indicates that WT and active but not DN-PKC{zeta} can interact with Bax (Fig. 5A, upper panel). Reciprocally, Bax associates with both WT and the active PKC{zeta} but fails to interact with the DN-PKC{zeta} mutant (Fig. 5A, lower panel). Importantly, the active PKC{zeta} more efficiently binds to Bax compared with WT (Fig. 5A). Because the DN-PKC{zeta} mutant resulting from a Lys -> Trp mutation at Lys281 in the ATP-binding domain has no ability to interact with Bax (Fig. 5A), this suggests that a functional ATP-binding domain is critical for PKC{zeta} to associate with Bax. Bax contains the Bcl2 homology (BH) domains including BH1, BH2 and BH3 (38). To directly assess whether PKC{zeta} binds to Bax at these BH domains, purified PKC{zeta} was incubated with purified recombinant WT, {Delta}BH1, {Delta}BH2, or {Delta}BH3 Bax deletion mutants in 1% CHAPS lysis buffer at 4 °C for 2 h. The PKC{zeta}-associated Bax was co-immunoprecipitated with a PKC{zeta} antibody and analyzed by Western blot using a Bax antibody. Results demonstrate that PKC{zeta} is able to associate with WT, {Delta}BH1, and {Delta}BH2 but not with the {Delta}BH3 Bax mutant (Fig. 5B), indicating that the BH3 domain may be the PKC{zeta} binding site on Bax.


Figure 5
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  		FIGURE 5.
PKC{zeta} directly interacts with Bax at the BH3 domain. A, the pcDNA3 plasmids bearing HA-tagged WT, constitutively active A119D, and DN-K281W PKC{zeta} mutants were transfected into A549 cells that express high levels of endogenous Bax using Lipofectamine 2000. After 48 h, a co-immunoprecipitation (IP) experiment was carried out using HA or Bax antibody, respectively. The PKC{zeta}-associated Bax or Bax-associated PKC{zeta} were analyzed by Western blot using Bax or HA antibody, respectively. B, purified PKC{zeta} (0.1 µg) was incubated with purified WT, {Delta}BH1, {Delta}BH2, or {Delta}BH3 Bax deletion mutants (0.1 µg each) in 1% CHAPS lysis buffer at 4 °C for 1 h. The PKC{zeta}-associated Bax was co-immunoprecipitated with PKC{zeta} antibody and analyzed by Western blot.

 
Purified, Active PKC{zeta} Directly Dissociates Bax from the Mitochondria Isolated from C2-ceramide-treated Cells—Our previous findings have demonstrated that treatment of cells with C2-ceramide (a potent protein phosphatase 2A activator) results in Bax dephosphorylation and insertion into mitochondrial membranes (10). To test whether PKC{zeta} has a direct effect on mitochondrial integral Bax, A549 cells were treated with C2-ceramide (30 µM) for 24 h to induce Bax insertion into mitochondrial membranes. Then, mitochondria were isolated and incubated with purified, active PKC{zeta} in a kinase buffer at 30 °C for 30 min. Proteins released from the mitochondria were identified in the supernatant following centrifugation at 14,000 x g for 5 min. Mitochondria-associated Bax in the pellet and the released Bax in the supernatant were analyzed by Western blot. Results reveal that PKC{zeta} directly dissociates Bax from isolated mitochondria in vitro because decreased levels of Bax were observed in the mitochondrial pellet and increased levels of Bax were present in the supernatant (Fig. 6A). It has been demonstrated that an alkali extraction approach can distinguish whether Bax protein slightly associates with or inserts into mitochondrial membranes (2, 10). To test whether PKC{zeta} can induce phosphorylation of the mitochondrial inserted Bax, A549 cells were treated with C2-ceramide for 24 h. Mitochondria were isolated and incubated in 0.1 M Na2CO3 (pH 11.5) on ice for 30 min, and centrifuged at 200,000 x g to yield a mitochondrial pellet. The resulting alkali-extracted mitochondrial membrane pellet was washed three times with a kinase buffer, and incubated with purified, active PKC{zeta} in the kinase assay buffer containing [{gamma}-32P] as described under "Experimental Procedures." Intriguingly, PKC{zeta} can directly phosphorylate the alkali-resistant, mitochondrial integral Bax (Fig. 6B). This suggests that PKC{zeta}-induced dissociation of the integral Bax from mitochondria may occur, at least in part, through a mechanism involving phosphorylation of Bax.


Figure 6
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  		FIGURE 6.
Purified PKC{zeta} directly dissociates Bax from the mitochondria isolated from C2-ceramide-treated cells. A, A549 cells were treated with C2-ceramide (30 µM) for 24 h. Mitochondria were isolated and incubated with purified, active PKC{zeta} in a kinase buffer at 30 °C for 30 min. Proteins released from the mitochondria were identified in the supernatant following centrifugation at 14,000 x g for 5 min. Mitochondria-associated Bax in the pellet and the released Bax in the supernatant were analyzed by Western blot. B, A549 cells were treated with C2-ceramide for 24 h. Mitochondria were isolated and incubated in 0.1 M Na2CO3 (pH 11.5) on ice for 30 min, and centrifuged at 200,000 x g to yield a mitochondrial pellet. The resulting alkali-extracted mitochondrial membrane pellet was washed three times with a kinase buffer, and incubated with purified, active PKC{zeta} in the kinase assay buffer containing [{gamma}-32P]. Phosphorylation of Bax was determined by autoradiography.

 
Depletion of PKC{zeta} by RNA Interference Blocks Nicotine-induced Bax Phosphorylation and Enhances Apoptosis—Our data strongly suggest that PKC{zeta} functions as a physiological Bax kinase to phosphorylate Bax at Ser184, which may lead to suppression of the proapoptotic activity of Bax. To test whether PKC{zeta} is essential for Bax phosphorylation, a RNA interference approach was employed. To avoid potential off-target or non-specific effects, we tested two different PKC{zeta} siRNAs (i.e. PKC{zeta} siRNA1 and PKC{zeta} siRNA2) at a low concentration (i.e. 10 nM) that generally does not exert nonspecific effects (39). Results indicate that PKC{zeta} is depleted by either PKC{zeta} siRNA1 or siRNA2 but not by control siRNA or PKC{alpha} siRNA (Fig. 7A). Neither PKC{zeta} siRNA1 nor PKC{zeta} siRNA2 has any effect on PKC{alpha} expression. In contrast, PKC{alpha} siRNA can specifically knock down PKC{alpha} but has no effect on PKC{zeta} expression (Fig. 7A). These data reveal that the effect of PKC{zeta} siRNA on PKC{zeta} expression is specific and is not a consequence resulting from off-target effects. Functionally, specific disruption of PKC{zeta} expression by either PKC{zeta} siRNA1 or siRNA2 but not by control siRNA or PKC{alpha} siRNA suppresses nicotine-induced Bax phosphorylation as well as nicotine-stimulated survival of human lung cancer cells (Fig. 7). These findings support the notion that PKC{zeta} may be essential for nicotine-induced Bax phosphorylation to positively regulate lung cancer cell survival.


Figure 7
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  		FIGURE 7.
Depletion of PKC{zeta} by RNA interference suppresses nicotine-induced Bax phosphorylation and enhances apoptosis. A, PKC{zeta} siRNA1, PKC{zeta} siRNA2, PKC{alpha} siRNA, or control siRNA was transfected into A549 using Lipofectamine 2000. The levels of PKC{zeta} or PKC{alpha} expression were analyzed by Western blot. B, A549 cells expressing PKC{zeta} siRNA1, PKC{zeta} siRNA2, PKC{alpha} siRNA, or control siRNA were metabolically labeled with [32P]orthophosphoric acid and treated with nicotine (1 µM) for 60 min. Phosphorylation of Bax was analyzed by autoradiography. C, A549 cells expressing PKC{zeta} siRNA1, PKC{zeta} siRNA2, PKC{alpha} siRNA, or control siRNA were treated with cisplatin (40 µM) in the absence or presence of nicotine (1 µM) for 48 h. Cell viability was assessed as described in the legend for Fig. 1E. Data represent the mean ± S.D. of three determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Nicotine has been found to prolong cell survival, which may be associated with increased chemoresistance of human lung cancer cells, but understanding of the molecular mechanism(s) is fragmentary (9, 40). PKC isoforms that appear to be anti-apoptotic include PKC{alpha}, PKC betaII, PKC{epsilon}, PKC{iota}, and PKC{zeta} (41). Previous studies reveal that the drug-resistant phenotype is associated with expression and/or activity of PKCs in lung cancer cell lines and lung carcinomas (41). PKC{zeta} is an atypical PKC isoform that is ubiquitously expressed in both SCLC and NSCLC cells (Fig. 1A). Because nicotine can potently activate PKC{zeta} and enhance survival of human lung cancer cells (Fig. 1), nicotine-stimulated survival and/or chemoresistance may occur, at least in part, through activation of PKC{zeta}. PKC{zeta} has been reported to be an anti-apoptotic protein kinase (20) but the downstream survival effectors involved have not been fully identified. It is well known that the decision phase for apoptotic cell death is largely regulated by the Bcl-2 family members (42). Thus, Bcl2 family member(s) may be the most attractive candidate for the substrate of PKC{zeta} in nicotine-stimulated survival signaling. Bax, a major multidomain proapoptotic member of the Bcl2 family, is widely expressed in various lung cancer cells. Importantly, nicotine potently induces Bax phosphorylation that can be blocked by a PKC{zeta} specific inhibitor (i.e. MYR-PKC{zeta}-PS) (Fig. 1), suggesting that Bax may function as a downstream survival substrate of PKC{zeta} in human lung cancer cells.

Evidence reported here indicates that PKC{zeta} may be a physiological Bax kinase because PKC{zeta} co-localizes with Bax in the cytoplasm and directly phosphorylates either endogenous or recombinant Bax in vitro, indicating its potential, direct role as a Bax kinase (Fig. 2). Confirmation of PKC{zeta} as a physiological Bax kinase was obtained in vivo from results of transfection studies demonstrating that overexpression of HA-tagged WT or constitutively active-PKC{zeta} but not DN-PKC{zeta} in A549 cells, resulted in enhanced phosphorylation of Bax (Fig. 2D), which supports in vitro results. We have previously discovered that nicotine induces Bax phosphorylation at the Ser184 site and prolongs cell survival (9). Genetic studies further demonstrated that the nonphosphorylatable S184A Bax mutant is the active form that has more proapoptotic activity than WT, whereas the phosphomimetic S184E Bax mutant is an inactive form that has no proapoptotic activity (9). In the present study, co-transfection of active PKC{zeta} cDNA with the WT, S184A, or S184E Bax mutant into MEF Bax-/- cells shows that expression of active PKC{zeta} induces Bax phosphorylation at the Ser184 site because only WT but not the S184A or S184E Bax mutant can be phosphorylated (Fig. 2E). Because expression of active PKC{zeta} prolongs survival of MEF Bax-/- cells expressing exogenous WT Bax but has no significant survival effect on cells expressing the S184A Bax mutant (Fig. 2, F and G), this suggests that active PKC{zeta}-enhanced cell survival may occur through phosphorylation of Bax at the Ser184 site. Furthermore, specific depletion of PKC{zeta} expression by RNA interference from lung cancer cells blocks nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death (Fig. 7). These findings provide strong evidence that PKC{zeta} functions as a nicotine-activated Bax kinase that not only can directly phosphorylate but also may inactivate Bax.

Because phosphorylation of Bax at Ser184 resulted in retention of Bax in cytosol and failure to target mitochondria (9), it is possible that PKC{zeta}-mediated Bax phosphorylation may also affect the subcellular distribution of Bax. Here, results indicate that overexpression of WT or active PKC{zeta} leads to Bax accumulation in the cytosol (Fig. 3A), indicating that PKC{zeta} can prevent Bax from targeting mitochondria. Moreover, overexpression of active PKC{zeta} blocks the cisplatin-induced Bax conformational change detected by the 6A7 Bax antibody that only recognizes active, conformationally changed Bax, and leads to prolonged cell survival (Fig. 3). In contrast, expression of DN-PKC{zeta} enhances Bax mitochondrial localization, conformational change, and apoptotic cell death. These genetic studies reveal that PKC{zeta}-mediated Bax phosphorylation may inhibit the Bax conformational change to retain Bax in an inactive form following stress signal.

In addition to Bax phosphorylation, nicotine also stimulates a direct PKC{zeta}/Bax interaction, which leads to dissociation of the Bcl2/Bax heterodimer (Fig. 4A). Intriguingly, the activity of PKC{zeta} is necessary for its binding to Bax because only active PKC{zeta} but not DN-PKC{zeta} has the ability to interact with Bax (Fig. 5). Purified PKC{zeta} can directly disrupt the Bcl2/Bax complex by binding to the BH3 domain of Bax (Figs. 4 and 5). The consequence of this interaction is the release of anti-apoptotic Bcl2 protein from the Bcl2/Bax heterodimer, which may facilitate the formation of the Bcl2 homodimer to enhance the antiapoptotic function of Bcl2. Because the BH3 domain is required for the killing activity of Bax (43, 44), the direct binding of PKC{zeta} to the BH3 domain of Bax may potentially impede Bax to exert its proapoptotic function. Thus, PKC{zeta} may provide a double whammy to the proapoptotic function of Bax through interaction with its BH3 domain and phosphorylation at the Ser184 site.

Mitochondria play a central role in apoptosis that is mainly regulated by Bcl2 family members (45). Bax is a major proapoptotic Bcl2 family member present in the cytosol and/or "peripherally" associates with mitochondrial membranes in an inactive state during normal cell growth. In response to apoptotic stimuli, Bax undergoes specific conformational changes, which allow its targeting/insertion into mitochondrial membranes (46). Our previous findings reveal that C2-ceramide-induced Bax dephosphorylation promotes Bax targeting and insertion into mitochondrial membrane (10). Here we found that treatment of isolated mitochondria from C2-ceramide-treated cells with purified PKC{zeta} in vitro results in dissociation of Bax from isolated mitochondrial membranes (Fig. 6A). Further studies indicate that PKC{zeta} can directly phosphorylate the alkali-resistant, mitochondrial membrane-inserted Bax (Fig. 6B), suggesting that PKC{zeta}-mediated Bax phosphorylation may facilitate extraction of Bax from mitochondrial membranes, which converts Bax from an active form to inactive status. These findings support a notion that PKC{zeta}-mediated suppression of apoptosis may occur through a mechanism by negatively regulating the mitochondrial membrane integration of Bax.

In summary, our findings have identified PKC{zeta} as a novel nicotine-activated protein kinase that can directly phosphorylate the multidomain proapoptotic protein, Bax, at Ser184. PKC{zeta}-induced Bax phosphorylation leads to sequestration of Bax in the cytoplasm, suppression of Bax conformational change, and dissociation of Bax from mitochondrial membranes, which may result in abrogation of the proapoptotic function of Bax. Activated PKC{zeta} can directly interact with Bax at the BH3 domain, and this interaction disrupts the Bcl2/Bax heterodimer to liberate Bcl2, which may potentially impede the proapoptotic function of Bax and benefit the antiapoptotic function of Bcl2. Thus, nicotine-induced survival and chemoresistance of human lung cancer cells may occur in a novel mechanism involving activation of PKC{zeta} that not only phosphorylates but also interacts with Bax to potently inactivate its proapoptotic function.


   FOOTNOTES
 
* This was supported by a Flight Attendant Medical Research Institute Clinical Innovator Award, NCI, National Institutes of Health Grant R01 CA112183 (to X. D.), and a National Institutes of Health T32 Training Grant in Cancer Biology (to M. X.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 1376 Mowry Rd., Cancer/Genetics Research Complex, Rm. 262, P. O. Box 103633, Gainesville, FL 32610-3633. Fax: 352-273-8285; Tel.: 352-273-8170; E-mail: xdeng{at}ufl.edu.

2 The abbreviations used are: PKC, protein kinase C; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; BH, Bcl2 homology domain; PBS, phosphate-buffered saline; DN, dominant negative; WT, wild type; HA, hemagglutinin; PI3K, phosphatidylinositol 3'-OH kinase; siRNA, small interfering RNA; MYR-PKC{zeta}-PS, myristoylated PKC{zeta} pseudosubstrate peptide; MBP, myelin basic protein; PIP3, phosphatidylinositol 1,4,5-trisphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEF, mouse embryonic fibroblast; PDK1, phosphoinositide-dependent kinase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Brian Law (University of Florida) for kindly providing HA-tagged WT, constitutively active A119D, and dominant negative K281W PKC{zeta} DNA constructs.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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