Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation*

Protein kinase Cζ (PKCζ) 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 PKCζ 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 PKCζ in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCζ activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCζ specific inhibitor (a myristoylated PKCζ pseudosubstrate peptide). Purified, active PKCζ directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCζ mutant results in Bax phosphorylation at serine 184. PKCζ co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCζ by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCζ mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCζ can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCζ 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.

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)(4)(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 Ser 184 through activation of AKT potently suppresses the proapoptotic activity of Bax and prolongs cell survival (6,9). In contrast, c-Jun NH 2 -terminal kinase (JNK)-induced Thr 167 or glycogen synthase kinase-induced Ser 163 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)(13)(14)(15). For example, the classic PKC␣-induced Bcl-2 phosphorylation enhances antiapoptotic function of Bcl2 in association with increased chemoresistance of human leukemia cells (16). Direct interaction between PKC⑀ and Bax results in suppression of the proapoptotic activity of Bax (17). Bad phosphorylation induced by either PKC or PKC 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 is a member of the atypical PKC subfamily and plays a critical role in suppression of mitochondrial-dependent apoptosis (20,21). PKC 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 (Lys 281 ) that is crucial for its kinase activity. A mutation of Lys 3 Trp at Lys 281 site resulted in a kinase-defective dominant negative form of PKC (i.e. K281W) (23). The activation loop and turn motif contain two important phosphorylation sites (i.e. Thr 410 and Thr 560 ). Phosphorylation of Thr 410 and Thr 560 residues is essential for PKC activation (24,25). PKC is insensitive to second messengers such as Ca 2ϩ and diacylglycerol. Thus, its activity is primarily regulated by protein-protein interaction and phosphorylation (11). The signal mechanisms for PKC activation include the PDK1-dependent (i.e. PI3K/PIP3/PDK1/ PKC) and PDK1-independent (i.e. PI3K/PIP3/PKC) 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 and phosphorylates the kinase domain at Thr 410 , which induces Thr 560 phosphorylation. On the other hand, PKC 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 (22). Furthermore, phospholipase D 2 has recently been identified as a novel protein cofactor for activation of PKC that can enhance PKC 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 and Bax in nicotine-induced cancer cell survival has not been established, yet. Here we have demonstrated that PKC functions as a novel nicotine-activated Bax kinase that directly phosphorylates and inactivates Bax in human lung cancer cells.
Cell Culture, cDNA Constructs, and Transfections-WT and Bax Ϫ/Ϫ 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 5Ј-phosphorylated mutagenic primers for various precise deletion mutants were synthesized as follow: S184A, 5Ј-GTC-CTCACCGCCGCGCTCACCATCTGG-3Ј; S184E, 5Ј-GTC-CTCACCGCCGAGCTCACCATCTGG-3Ј. 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: 5Ј-GAGTG-CACCATGGGCGGTGTGAAA-3Ј. 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 Ϫ/Ϫ cells using Lipofectamine TM 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 1ϫ 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 Na 3 VO 4 , 50 mM NaF, and 1 mM ␤-mercaptoethanol) with a mixture of protease inhibitors (Calbiochem). Cells were lysed by sonication and centrifuged at 14,000 ϫ 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 Activity in Vitro-PKC activity was measured using MBP as substrate as previously described (27). Briefly, PKC was immunoprecipitated from cell lysates with an agarose-conjugated PKC antibody. Immunoprecipitated PKC was washed and resuspended in kinase buffer containing 50 mM Hepes (pH 7.5), 100 mM NaCl, 10 mM MgCl 2 , 50 mM NaF, 1 mM NaVO 4 , 1 mM dithiothreitol, and 0.1% Tween 20, 40 g/ml phosphatidylserine, and 2 Ci of [␥-32 P]ATP and MBP (10 g). 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 Ϫ80°C. The activity of PKC was determined by autoradiography. PKC activity was also assessed by measuring the rate of 32 P from [␥-32 P]ATP incorporation into a peptide (ERMRPRKRQGSVRRRV) corresponding to the pseudosubstrate region of PKC⑀ 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 was performed by incubating a polyclonal PKC 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 ␤-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, 20 g/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 NaHCO 3 , 5 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin, 20 g/ml leupeptin, and 40 M modified PKC⑀ pseudosubstrate peptide for 10 min at 30°C. After thermal equilibration, assays were started by the simultaneous addition of 15 M ATP, 0.3 Ci of [␥-32 P]ATP (3000 Ci/mmol), and terminated after 30 min with 100 l of 175 mM phosphoric acid. Then, 100 l 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 [ 32 P]orthophosphoric acid for 120 min. After agonist or inhibitor addition, cells were washed with ice-cold 1ϫ 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 Ϫ80°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 ϫ 10 7 ) were washed with cold 1ϫ 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 ϫ g for 3 min to pellet the nuclei and unbroken cells. The supernatant was centrifuged at 13,000 ϫ g for 10 min to pellet mitochondria as described (9). The second supernatant was further centrifuged at 150,000 ϫ 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 ϫ g for 10 min at 4°C. The resulting supernatant containing mitochondrial proteins was collected. Protein (100 g) 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 agaroseconjugated 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 M ATP, 5 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EGTA, 100 g/ml phosphatidylserine, and 2 Ci of [␥-32 P]ATP and incubated with purified, active PKC 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 M) for 24 h. Mitochondria were isolated by subcellular fractionation and resuspended in freshly prepared 0.1 M Na 2 CO 3 (pH 11.5) and incubated on ice for 30 min. The samples were then centrifuged at 200,000 ϫ 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 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% CO 2 . Cells were washed with 1ϫ 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 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 1ϫ 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 colocalization, 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 colocalization appear yellow.
Depletion of PKC Expression by RNA Interference-Human PKC siRNA was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A549 cells were transfected with PKC 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 expression were analyzed by Western blotting using a PKC antibody. Bax phosphorylation or cell viability was assessed following various treatments. Specific silencing of the targeted PKC gene was confirmed by at least three independent experiments.

Nicotine Induces Activation of PKC and Phosphorylation of Bax in Association with Prolonged Survival of Human Lung Cancer Cells, Which Is Suppressed by a PKC-specific
Pseudosubstrate Inhibitor-PKC 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 (Fig. 1A), nicotine may activate PKC to promote cell survival. To test this, A549 cells were treated with increasing concentrations of nicotine for 60 min and PKC was then immunoprecipitated using an agarose-conjugated PKC antibody. Activity of PKC 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 protein. To ensure that the kinase activity that phosphorylates MBP is from PKC, and not from any other kinase that may be co-immunoprecipitated with PKC, we also probed the same filter by Western blot using a PKC or PKC␣ antibody, respectively. Total cell lysate was used as a positive control. Results indicate that the PKC antibody specifically immuno-precipitates PKC and does not co-immunoprecipitates either PKC or PKC␣ (Fig. 1B), indicating its specificity for PKC. Importantly, nicotine significantly enhances PKC 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 substrate for the PKC activity assay as described (28,29). Previous reports have demonstrated that a higher level of phosphorylation by PKC was obtained with the peptide derived from the pseudosubstrate region of PKC⑀ as compared with the peptide derived from the pseudosubstrate region of PKC (28,29), indicating that the PKC⑀ pseudosubstrate peptide is a better substrate for PKC. Therefore, we chose a modified PKC⑀ pseudosubstrate peptide (ERMR-PRKRQGSVRRRV) as a substrate for the PKC activity assay as previously reported (28,29). Results indicate that treatment of A549 cells with nicotine markedly up-regulates PKC 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 is involved in nicotine-induced Bax phosphorylation, A549 cells were exposed to nicotine in the absence or presence of the PKC specific inhibitor (MYR-PKC-PS, a myristoylated PKC pseudosubstrate peptide) or the myristoylated PKC␣ pseudosubstrate peptide (MYR-PKC␣-PS) (20,33,34). Results reveal that MYR-PKC-PS but not MYR-PKC␣-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 in supporting survival of human lung cancer cells.
Purified PKC Directly Phosphorylates Bax in Vitro and Overexpression of PKC in Cells Results in Bax Phosphorylation at Ser 184 in Vivo-To assess a potential direct role for PKC as a physiological Bax kinase, subcellular distribution of PKC and Bax was examined by immunofluorescent staining. A mouse antibody against human Bax, rabbit polyclonal PKC 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 colocalized with PKC in the cytoplasm of A549 cells. To test whether PKC can directly phosphorylate endogenous Bax, endogenous Bax was immunoprecipitated from A549 cells and incubated with purified, active PKC in a kinase assay buffer containing [␥-32 P]ATP. Results indicate that active PKC directly phosphorylates endogenous Bax in vitro (Fig. 2B). In addition, active PKC can also directly phosphorylate recombinant Bax protein (Fig. 2C). These findings suggest that PKC is a strong candidate for being the direct Bax kinase. To determine whether PKC may be a Bax kinase in vivo, HA-tagged WT, constitutively active A119D, or DN-K281W PKC constructs were transfected into A549 cells that express high levels of endogenous Bax. After transfection for 48 h, cells were metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. Bax phosphorylation was analyzed by autoradiography. Results reveal that overexpression of WT or active but not DN-PKC results in an increased Bax phosphorylation (Fig. 2D). Importantly, the constitutive active PKC has a more potent effect on FIGURE 1. Nicotine stimulates activation of PKC and Bax phosphorylation in association with enhanced survival of human lung cancer cells, which is blocked by a PKC specific inhibitor. A, expression levels of endogenous Bax, Bcl2, and PKC 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 was immunoprecipitated (IP) and incubated with purified MBP in an in vitro kinase assay. PKC activity was analyzed by autoradiography. PKC protein was quantified by Western blot using a PKC antibody. To confirm the specificity of the PKC antibody, the same filter was reprobed using a PKC or PKC␣ antibody, respectively. Total cell lysate was used a positive control for PKC, PKC, or PKC␣ Western blot. C, PKC activity was also measured using a modified PKC⑀ pseudosubstrate peptide with an Ala to Ser mutation as a PKC substrate in A549 cells following nicotine treatment. D, A459 cells expressing high levels of endogenous Bax were metabolically labeled with [ 32 P]orthophosphoric acid and treated with nicotine (1 M) in the absence or presence of the myristoylated PKC pseudosubstrate peptide (MYR-PKC-PS, a PKC specific inhibitor), or the myristoylated PKC␣ pseudosubstrate peptide (MYR-PKC␣-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). Bax phosphorylation compared with WT (Fig. 2D). To identify the site of Bax phosphorylation induced by PKC, WT, S184A, and S184E Bax mutants were co-transfected with the constitutively active A119D PKC mutant into MEF Bax Ϫ/Ϫ cells. After 48 h, cells were then metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. Results reveal that PKC induces Bax phosphorylation exclusively at the Ser 184 site because expression of active PKC 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 downstream substrate. To directly test a role of PKC in regulating the proapoptotic function of Bax, a constitutively active PKC was co-transfected with WT or each of the S184A and S184E Bax mutants into MEF Bax Ϫ/Ϫ 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 enhances survival of MEF Bax Ϫ/Ϫ cells expressing exogenous WT Bax but has no significant effect on survival of MEF Bax Ϫ/Ϫ cells expressing the exogenous S184A or S184E Bax mutant (Fig. 2, F and G). These results indicate that active PKC 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.

Expression of WT or Constitutively Active But Not Dominant Negative PKC 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-induced Bax phosphorylation affects Bax subcellular localization, WT, active, or DN-PKC 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 leads to increased sequestration of Bax in cytosol, whereas expression of DN-PKC 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 After washing with 1ϫ PBS, cells were incubated with a mouse against human Bax and a rabbit against human PKC antibodies. Fluorescein isothiocyanateconjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies were used to visualize Bax (green) and PKC (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 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 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 mutants were transfected into A549 cells that express high levels of endogenous Bax using Lipofectamine 2000. After 48 h, cells were metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. Phosphorylation of Bax was analyzed by autoradiography. Immunoprecipitated Bax protein and HA-PKC 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 mutant into MEF Bax Ϫ/Ϫ cells. After 48 h, cells were then metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. Bax phosphorylation was analyzed by autoradiography. Immunoprecipitated (IP) Bax protein and HA-PKC in total cell lysate were analyzed by Western blot using Bax or HA antibody, respectively. F and G, HA-tagged constitutively active A119D PKC mutant was co-transfected with WT or each of the S184A and S184E Bax mutants into MEF Bax Ϫ/Ϫ cells. After 48 h, expression levels of HA-PKC 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 Ϯ S.D. of three determinations.
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)(36)(37). To test whether expression of PKC affects the stress-induced conformational change of Bax, A549 cells expressing constitutively active or DN-PKC or vector-only control were treated with cisplatin (40 M) 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 reduces the ability of the 6A7 antibody to immunoprecipitate Bax. By contrast, expression of DN-PKC 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 suppresses the cisplatin-induced Bax conformational change in association with prolonged cell survival (Fig. 3,  B-D). Thus, PKC-mediated Bax phosphorylation may prevent a conformational change in Bax and retain Bax in an inactive form.

Treatment of Cells with Nicotine Results in Increased PKC/ Bax Interaction and Decreased Bcl2/Bax Binding, and Purified PKC Can Directly Disrupt Bcl2/Bax Heterodimeric Complex in
Vitro-Because nicotine can activate PKC to phosphorylate Bax ( Figs. 1 and 2), it is possible that nicotine-induced activation of PKC may facilitate PKC 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, Bax, and Bcl2 were treated with increasing concentrations of nicotine for 60 min. PKC/Bax and Bax/ Bcl2 complexes were immunoprecipitated using a PKC or a Bax antibody, respectively. Results indicate that nicotine stimulates PKC to interact with Bax but not Bcl2 in a dose-dependent manner (Fig. 4A, upper panel). Intriguingly, nicotine-stimulated PKC/Bax association results in decreased Bcl2/Bax binding (Fig. 4A, lower panel). To test whether PKC 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 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 ϫ g for 5 min. Bax-associated PKC (i.e. bound PKC), 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 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 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.
Active But Not DN-PKC Associates with Bax at Its BH3 Domain-To test whether the activity of PKC affects its ability to interact with Bax, HA-tagged WT, active or DN-PKC were transfected into A549 cells using Lipofectamine TM 2000. Coimmunoprecipitation using a monoclonal HA antibody indicates that WT and active but not DN-PKC can interact with Bax (Fig. 5A, upper panel). Reciprocally, Bax associates with both WT and the active PKC but fails to interact with the DN-PKC mutant (Fig. 5A, lower panel). Importantly, the active PKC more efficiently binds to Bax compared with WT (Fig. 5A). Because the DN-PKC mutant resulting from a Lys 3 Trp mutation at Lys 281 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 to associate with Bax. Bax contains the Bcl2 homology (BH) domains including BH1, BH2 and BH3 (38). To directly assess whether PKC binds to Bax at these BH domains, purified PKC was incubated with purified recombinant WT, ⌬BH1, ⌬BH2, or ⌬BH3 Bax deletion mutants in 1% CHAPS lysis buffer at 4°C for 2 h. The PKCassociated Bax was co-immunoprecipitated with a PKC antibody and analyzed by Western blot using a Bax antibody. Results demonstrate that PKC is able to associate with WT, ⌬BH1, and ⌬BH2 but not with the ⌬BH3 Bax mutant (Fig. 5B), indicating that the BH3 domain may be the PKC binding site on Bax.
Purified, Active PKC 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 has a direct effect on mitochondrial integral Bax, A549 cells were treated with C2-ceramide (30 M) for 24 h to induce Bax insertion into mitochondrial membranes. Then, mitochondria were isolated and incubated with purified, active PKC 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 ϫ 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 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

. Treatment of cells with nicotine results in increased PKC/Bax interaction and decreased Bcl2/Bax binding, and purified PKC directly disrupt the Bcl2/Bax heterodimeric complex in vitro.
A, H460 cells expressing high levels of endogenous PKC, Bax, and Bcl2 were treated with increasing concentrations of nicotine for 60 min. A co-immunoprecipitation (IP) experiment was carried out using a PKC or a Bax antibody, respectively. PKC, 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 in a kinase assay buffer at 30°C for 30 min. The samples were centrifuged at 14,000 ϫ g for 5 min. The resulting supernatant and immunocomplex beads were subjected to SDS-PAGE. Bax, bound PKC, bound Bcl2, and free Bcl2 were analyzed by Western blot.

FIGURE 5. PKC directly interacts with Bax at the BH3 domain.
A, the pcDNA3 plasmids bearing HA-tagged WT, constitutively active A119D, and DN-K281W PKC 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-associated Bax or Bax-associated PKC were analyzed by Western blot using Bax or HA antibody, respectively. B, purified PKC (0.1 g) was incubated with purified WT, ⌬BH1, ⌬BH2, or ⌬BH3 Bax deletion mutants (0.1 g each) in 1% CHAPS lysis buffer at 4°C for 1 h. The PKCassociated Bax was co-immunoprecipitated with PKC antibody and analyzed by Western blot.
whether Bax protein slightly associates with or inserts into mitochondrial membranes (2,10). To test whether PKC 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 Na 2 CO 3 (pH 11.5) on ice for 30 min, and centrifuged at 200,000 ϫ 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 in the kinase assay buffer containing [␥-32 P] as described under "Experimental Procedures." Intriguingly, PKC can directly phosphorylate the alkali-resistant, mitochondrial integral Bax (Fig. 6B). This suggests that PKC-induced dissociation of the integral Bax from mitochondria may occur, at least in part, through a mechanism involving phosphorylation of Bax.
Depletion of PKC by RNA Interference Blocks Nicotine-induced Bax Phosphorylation and Enhances Apoptosis-Our data strongly suggest that PKC functions as a physiological Bax kinase to phosphorylate Bax at Ser 184 , which may lead to suppression of the proapoptotic activity of Bax. To test whether PKC is essential for Bax phosphorylation, a RNA interference approach was employed. To avoid potential off-target or nonspecific effects, we tested two different PKC siRNAs (i.e. PKC siRNA1 and PKC siRNA2) at a low concentration (i.e. 10 nM) that generally does not exert nonspecific effects (39). Results indicate that PKC is depleted by either PKC siRNA1 or siRNA2 but not by control siRNA or PKC␣ siRNA (Fig. 7A). Neither PKC siRNA1 nor PKC siRNA2 has any effect on PKC␣ expression. In contrast, PKC␣ siRNA can specifically knock down PKC␣ but has no effect on PKC expression (Fig.  7A). These data reveal that the effect of PKC siRNA on PKC expression is specific and is not a consequence resulting from off-target effects. Functionally, specific disruption of PKC expression by either PKC siRNA1 or siRNA2 but not by control siRNA or PKC␣ 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 may be essential for nicotine-induced Bax phosphorylation to positively regulate lung cancer cell survival.

DISCUSSION
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␣, PKC ␤II, PKC⑀, PKC, and PKC (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 is an atypical PKC isoform that is ubiquitously expressed in both SCLC and NSCLC cells (Fig. 1A). Because nicotine can potently activate  PKC 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. PKC 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 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 specific inhibitor (i.e. MYR-PKC-PS) (Fig. 1), suggesting that Bax may function as a downstream survival substrate of PKC in human lung cancer cells.
Evidence reported here indicates that PKC may be a physiological Bax kinase because PKC 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 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 but not DN-PKC 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 Ser 184 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 cDNA with the WT, S184A, or S184E Bax mutant into MEF Bax Ϫ/Ϫ cells shows that expression of active PKC induces Bax phosphorylation at the Ser 184 site because only WT but not the S184A or S184E Bax mutant can be phosphorylated (Fig. 2E). Because expression of active PKC prolongs survival of MEF Bax Ϫ/Ϫ 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-enhanced cell survival may occur through phosphorylation of Bax at the Ser 184 site. Furthermore, specific depletion of PKC 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 functions as a nicotine-activated Bax kinase that not only can directly phosphorylate but also may inactivate Bax.
Because phosphorylation of Bax at Ser 184 resulted in retention of Bax in cytosol and failure to target mitochondria (9), it is possible that PKC-mediated Bax phosphorylation may also affect the subcellular distribution of Bax. Here, results indicate that overexpression of WT or active PKC leads to Bax accumulation in the cytosol (Fig. 3A), indicating that PKC can prevent Bax from targeting mitochondria. Moreover, overexpression of active PKC 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 enhances Bax mitochondrial localization, conformational change, and apoptotic cell death. These genetic studies reveal that PKC-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/Bax interaction, which leads to dissociation of the Bcl2/Bax heterodimer (Fig. 4A). Intriguingly, the activity of PKC is necessary for its binding to Bax because only active PKC but not DN-PKC has the ability to interact with Bax (Fig.  5). Purified PKC 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 to the BH3 domain of Bax may potentially impede Bax to exert its proapoptotic function. Thus, PKC may provide a double whammy to the proapoptotic function of Bax through interaction with its BH3 domain and phosphorylation at the Ser 184 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-ceramideinduced Bax dephosphorylation promotes Bax targeting and insertion into mitochondrial membrane (10). Here we found that treatment of isolated mitochondria from C2-ceramidetreated cells with purified PKC in vitro results in dissociation of Bax from isolated mitochondrial membranes (Fig. 6A). Further studies indicate that PKC can directly phosphorylate the alkali-resistant, mitochondrial membrane-inserted Bax (Fig.  6B), suggesting that PKC-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-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 as a novel nicotine-activated protein kinase that can directly phosphorylate the multidomain proapoptotic protein, Bax, at Ser 184 . PKC-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 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 that not only phosphorylates but also interacts with Bax to potently inactivate its proapoptotic function.