Nicotine Induces Multi-site Phosphorylation of Bad in Association with Suppression of Apoptosis*

Nicotine is an important component in cigarette smoke that can activate the growth-promoting pathways to facilitate the development of lung cancer. How-ever, the intracellular mechanism(s) by which nicotine promotes survival of lung cancer cells remains enig-matic. Bad is a proapoptotic BH3-only member of the Bcl2 family and is expressed in both small cell lung cancer and non-small cell lung cancer cells. Here we report that nicotine potently induces Bad phosphorylation at Ser 112 , Ser 136 , and Ser 155 in a mechanism involv ing activation of MAPKs ERK1/2, PI3K/AKT, and PKA in human lung cancer cells. Nicotine-induced multi-site phosphorylation of Bad results in sequestering Bad from mitochondria and subsequently interacting with 14-3-3 in the cytosol. Treatment of cells with PKC inhibitor (staurosporine), MEK-specific inhibitor (PD98059), PI 3 kinase inhibitor (LY294002), or PKA inhibitor (H89) blocks the nicotine-induced Bad phosphorylation that is associated with enhanced apoptotic cell death. The fact that (cid:1) -adrenergic receptor inhibitor (propranolol) blocks nicotine-induced activation of ERK1/2, AKT, PKA, Bad phosphorylation, and cell survival suggests that nicotine-induced Bad phosphorylation may occur through the upstream (cid:1) -adrenergic receptors. The fact that specific knockdown of Bad expression by RNA interference using short interfering RNA enhances cell survival and that in detergent buffer. Western blot analysis was performed to detect phosphorylated ERK1/2 ( p-ERK1 and p-ERK2 ) or total ERK1/2 ( ERK1 and ERK2 ) by using a phospho-specific ERK antibody or a mixture of ERK1 and ERK2 antibodies. Phosphorylation of AKT was analyzed by West- ern blotting using phosphospecific AKT Thr 308 or Ser 473 antibody, respectively. C A549 cells were treated with various concentrations of nicotine ( i.e. 0.5–10 (cid:3) M for lysed PKA extraction buffer. Kinase assay in vitro described “Experimental Procedures.” activity counting. These implicate

Lung cancer is one of the leading causes of cancer death, with a 5-year relative survival rate of 15% in both men and women worldwide (1)(2). Cigarette smoking is by far the most important risk factor in the development of lung cancer (3). About 90% of male lung cancer deaths and 75-80% female lung cancer deaths in the United States are caused by smoking (1,4). Approximately 25% of the U. S. adult population continue to smoke because of addiction to nicotine, one important compo-nent of cigarette smoking (5)(6).
Nicotine can activate the growth-promoting pathways to facilitate the development of lung cancer and potentially reduce the efficacy of chemotherapeutic agents (7). Although classic nicotine signaling occurs through the nicotinic acetylcholine receptor (nAChR) 1 (8 -9), growing evidence suggests that the adrenergic receptor also plays an important role in nicotine signal transduction pathways, especially in pulmonary adenocarcinoma cells (10 -11).
Bcl-2 family members are key regulators of apoptosis, and their deregulation could be oncogenic (12)(13). Recent observations suggest that responsiveness to therapy and prognosis of lung cancer may be associated with the Bcl-2 family proteins (14).
Bcl-2 family has at least 20 members in mammals, all of which share at least one Bcl-2 homology (BH) domain (13). Among these domains, the BH3 domain is responsible for interaction with other Bcl2 family proteins and promotes cells undergoing apoptosis (15). Bcl2 family members control cell survival or cell death by regulating the outer mitochondria membrane permeabilization (16). The BH3-only proapoptotic proteins (i.e. Bad, Bid, Bim, Bik, Nix, and Noxa, etc.) can couple death signals to mitochondria and promote apoptosis by quelling the protective action of Bcl-X L (17). Bad is one of the BH3-only proapoptotic members, and phosphorylation of Bad at Ser 112 , Ser 136 , and Ser 155 has been demonstrated to inactivate its proapoptotic function (18 -19) in a mechanism involving binding to 14-3-3 scaffold proteins that results in sequestering Bad from mitochondria and dissociation of Bad from mitochondrial Bcl2 and/or Bcl-X L (20 -22). The active Bad is a dephosphorylated form that localizes at mitochondria and interacts with Bcl-X L to neutralize their anti-apoptotic function. We have recently discovered that nicotine induces Bcl2 phosphorylation at Ser 70 through activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2, and protein kinase c (PKC)␣ that may contribute to nicotine-induced survival and chemoresistance of lung cancer cells (23). Because some lung cancer cells express low or undetectable levels of endogenous Bcl2 (24), it is possible that other Bcl2 family member(s), for example, Bad, may be involved in nicotine-induced survival signal pathway(s). Recent studies indicate that nicotine potently activates both MAPKs ERK1/2 and AKT in association with increased survival of normal lung airway epithelial cells (25). We tested the hypothesis that because AKT and the MAPKs ERK1/2 are reported to function as physiologic Bad kinases (26 -28), nicotine-induced survival and chemoresistance may occur, at least in part, through phosphorylation of Bad.
Protein Isolation-Cells were washed with phosphate buffered saline and resuspended in ice-cold EBC buffer (0.5% Nonidet P-40, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, and 1 mM ␤-mercaptoethanol) with protease inhibitor mixture set I (Calbiochem) and lysed by sonication. The lysates were cleared of insoluble material by centrifugation at 14,000 ϫ g for 10 min at 4°C.
Metabolic Labeling, Immunoprecipitation, and Western Blot Analysis-Cells were washed with phosphate-free RPMI medium 1640 and metabolically labeled with [ 32 P]orthophosphoric acid for 90 min. After treatment, cells were washed with ice-cold phosphate-buffered saline and lysed in detergent buffer. Bad was immunoprecipitated using Bad antibody, as described previously (29 -30). The samples were subjected to SDS-12% PAGE, transferred to a nitrocellulose membrane, and exposed to Kodak X-Omat film at Ϫ80°C. Bad phosphorylation was determined by autoradiography. The same filter was then probed by Western blot analysis with a Bad antibody and developed by using an ECL Kit (Amersham Biosciences), as described previously (30).
Assay of PKA Activity in Vitro-PKA activity was measured using a PKA assay kit according to the manufacturer's instructions (31). A549 cells were treated with nicotine in the absence or presence of inhibitor(s). Cells were then lysed using PKA extraction buffer. Enzyme sample (5 l) was added and incubated at 30°C for 5 mins. Reaction buffer (10 l) for termination was placed onto a pre-numbered membrane square. After washing and rinsing, the membrane was dried, placed into a scintillation vial, and analyzed by scintillation counting.
Cell Viability Assay-The apoptotic and viable cells were detected using an ApoAlert Annexin-V kit (Clontech) according to the manufacturer's instructions. The percentages of Annexin-V low cells (percentage of viable cells) or Annexin-V high cells (percentage of apoptotic cells) were determined by using the data obtained by fluorescence-activated cell sorter analysis as described (32). Cell viability was also confirmed by using the trypan blue dye exclusion method (29).
Subcellular Fractionation-Cells (2 ϫ 10 7 ) were washed with cold 1 ϫ phosphate-buffered saline 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 four 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 the mitochondria as described (33). The second supernatant was further centrifuged at 150,000 ϫ g to pellet light membranes. The resulting supernatant is the cytosolic fraction. Mitochondria were washed with mitochondrial buffer twice, resuspended in 1% Nonidet P-40 lysis buffer, rocked for 60 min, and then centrifuged at 14,000 rpm 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. Bad or phosphorylated Bad was analyzed by Western blot using Bad or phosphospecific Bad antibody, respectively. The purity of fractions was confirmed by assessing localization of mitochondria-specific protein, prohibitin (34).
RNA Interference-A549 cells expressing high levels of endogenous Bad were transfected with Bad siRNA according to the siPORT lipid siRNA transfection protocol from the manufacturer (Ambion Inc). The Bad DNA target sequence for siRNA design was AAGAAGGGACTTC-CTCGCCCG. A control siRNA (non-homologous to any known gene sequence) was used as a negative control. The levels of Bad expression were analyzed by Western blotting using Bad antibody. Specific silencing of targeted Bad gene was confirmed by at least three independent experiments.

Nicotine Induces Bad Phosphorylation in Association with
Increased Survival of Human Lung Cancer Cells-The apoptotic process can be divided into three interdependent phases: induction, decision, and execution. The decision phase is mainly regulated by the Bcl-2 family of apoptotic regulators (35). We have recently demonstrated that nicotine induces Bcl-2 phosphorylation at Ser 70 and potently promotes survival of human small cell lung cancer (SCLC) H69 cells (23). Bad is one of the BH3-only Bcl2 family members, and phosphorylation negatively regulates its proapoptotic activity (22). Interestingly, Bad is more widely expressed in human lung cancer cells, including SCLC and non-small cell lung cancer cells, than Bcl2 (Fig. 1A). This finding suggests that Bad may play a more important role in nicotine-induced survival and chemoresistance of human lung cancer cells, especially in those cells that express low or undetectable levels of endogenous Bcl2. To test whether nicotine can induce Bad phosphorylation and promote cell survival, A549 cells (i.e. human pulmonary adenocarcinoma cell line) expressing high levels of endogenous Bad but no detectable levels of Bcl-2 were tested (Fig. 1A). A549 cells were metabolically labeled with [ 32 P]orthophosphoric acid and treated with nicotine (1 M) for various times as indicated. Results indicate that nicotine potently stimulates Bad phos-  (20,36). Results from metabolic labeling experiments show that nicotine can induce Bad phosphorylation (Fig. 1B), but which are the phosphorylation sites remains unclear. To identify nicotine-induced Bad phosphorylation sites, A549 cells were treated with nicotine (1 ìM), and Bad phosphorylation was assessed using site-phosphospecific Bad antibodies. Results reveal that nicotine induces multi-site Bad phosphorylation at Ser 112 , Ser 136 , and Ser 155 in both a dose-and time-dependent manner (Fig. 2). Interestingly, there is a hierarchical relationship between these three phosphorylation sites, because the peak of nicotineinduced phosphorylation at Ser 112 is observed earlier than phosphorylation at Ser 136 and Ser 155 (i.e. 30 versus 60 min; Fig.  2A). Various lung cancer cell lines, including NCI-H157 and H358 cells that express exogenous Bad, were also tested, and similar results were obtained (data not shown). These results suggest that nicotine-induced survival of human lung cancer cells may occur or be enhanced by a novel mechanism involving multi-site phosphorylation of Bad.
Nicotine Induces Activation of MAPKs ERK1/2, AKT, and PKA in Human Lung Cancer Cells-Nicotine potently stimulates phosphorylation of Bad at multiple sites (i.e. Ser 112 , Ser 136 , and Ser 155 ; Fig. 2) but the upstream protein kinase(s) involved remains unclear. Phosphorylation of Bad at Ser 112 has been reported to depend upon activation of MAPKs ERK1/2, whereas AKT (a downstream serine/threonine kinase of PI3K) or c-AMP-dependent protein kinase A (PKA) is responsible for phosphorylation of Bad at Ser 136 or Ser 155 , respectively (19,22,27). Our results indicate that nicotine simultaneously induces phosphorylation and activation of MAPKs ERK1/2, AKT, and PKA, with a peak observed between 30 and 60 min in a doseresponse manner (Fig. 3). These results suggest that nicotine- . Results indicate that staurosporine or PD98059 potently blocks nicotine-induced MAPKs ERK1/2 activation as well as Ser 112 site phosphorylation of Bad (Fig. 4, A and B). This suggests that nicotine may trigger a PKC/ERK1/2 protein kinase cascade to phosphorylate and inactivate Bad. In addition, LY294002 or H89 also inhibits nicotine-induced Bad phosphorylation at Ser 136 or Ser 155 , respectively (Fig. 4, C and D). These findings provide the pharmacological evidence that AKT and PKA are also involved in nicotine/Bad signaling. Importantly, staurosporine, PD98059, LY294002, and H89 potently block nicotine-induced cell survival after treatment with chemotherapeutic agents (i.e. cisplatin, VP16; Fig. 4E). These results indicate that inhibition of nicotine-induced Bad phosphorylation by blocking these three upstream pathways (i.e. PKC/ERKs, PI3 K/AKT, and PKA) restores the proapoptotic activity of Bad that triggers apoptotic cell death.
The ␤-Adrenergic Receptor-specific Inhibitor Propranolol Potently Inhibits Nicotine-induced Bad Phosphorylation and Enhances Apoptosis-It has been reported that nAChRs, espe- Cells were harvested, washed, and lysed in detergent buffer. Western blot analysis was performed to detect phosphorylated ERK1/2 (p-ERK1 and p-ERK2) or total ERK1/2 (ERK1 and ERK2) by using a phospho-specific ERK antibody or a mixture of ERK1 and ERK2 antibodies. Phosphorylation of AKT was analyzed by Western blotting using phosphospecific AKT Thr 308 or Ser 473 antibody, respectively. C, A549 cells were treated with various concentrations of nicotine (i.e. 0.5-10 M) for 30 min. Cells were harvested and lysed using PKA extraction buffer. Kinase assay in vitro was performed as described under "Experimental Procedures." PKA activity was analyzed by scintillation counting.
cially ␣7nAChR and ␤-adrenergic receptor, play important roles in nicotine signaling in lung cancer cells (6,8,10,41). ␣-Bungarotoxin (␣-BTX), a non-competent potent ␣7nAChRspecific inhibitor (42) and propranolol, a ␤-adrenergic receptor inhibitor (43), were selected to test which type of receptor may be involved in nicotine/Bad signaling. A549 cells were treated with nicotine in the presence or absence of various concentrations of propranolol or ␣-BTX. Interestingly, propranolol but not ␣-BTX can completely block nicotine-induced MAPKs ERK1/2, AKT, and PKA activation and Bad phosphorylation at Ser 112 , Ser 136 , and Ser 155 sites (Fig. 5 and data not shown). Functionally, propranolol blocks nicotine-induced cell survival and promotes apoptosis after treatment with cisplatin or VP-16. These results implicate nicotine-induced Bad phosphorylation in a mechanism involving the upstream ␤-adrenergic receptor in pulmonary adenocarcinoma cells.
Nicotine Induces Bad Translocation from Mitochondria into Cytosol, Which Facilitates Association with 14-3-3-Our results indicate that nicotine induces multi-site phosphorylation of Bad in association with increased cell survival. Phosphorylation has been reported to promote Bad translocation from mitochondria into cytosol and interaction with the scaffold protein 14-3-3 (18,21). To assess whether nicotine affects Bad translocation, A549 cells were treated with nicotine for 30 min. Subcellular fractionation experiments were performed to isolate mitochondria and cytosol, as described under "Experimental Procedures." Results reveal that nicotine potently stimulates translocation of Bad from mitochondria into cytosol (Fig.  6A). Phosphorylated Bad is observed only in cytosol, and its accumulation is increased in a dose-dependent manner (Fig.  6A), indicating that nicotine-induced Bad phosphorylation may function to translocate the mitochondrial Bad into cytosol. Coimmunoprecipitation experiments show that nicotine enhances the association between Bad and 14-3-3. These results reveal that nicotine-induced multi-site Bad phosphorylation results in sequestering Bad from mitochondria and functionally blocking its proapoptotic function.
Bad May Be a Required Target for Nicotine-induced Survival of Human Lung Cancer Cells-Bad is ubiquitously expressed in human lung cancer cell lines, whereas Bcl2 is not (Fig. 1A), suggesting that Bad may be a more important potential death target for treatment of patients with lung cancer. To test whether Bad is a necessary target for nicotine-induced survival and chemoresistance of human lung cancer cells, an RNA interference approach was employed. It has recently been dem-onstrated that 21 base pair double-strand RNA (siRNA) is a potent mediator of the RNA interference effect in mammalian cells (44). We used this strategy to target Bad in A549 cells by transfecting cells with Bad siRNA, as described under "Experimental Procedures." Results show that the Bad siRNA can potently and specifically reduce Bad expression by more than 90%, whereas the control siRNA has no effect (Fig. 7A). Depletion of Bad expression was found to prolong cell survival after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16) in the absence or presence of nicotine (Fig. 7B). Nicotine has no additional survival effect in cells expressing Bad siRNA (Fig. 7), indicating that Bad may be a required target for nicotine/survival signaling in human lung cancer cells. DISCUSSION Bcl-2 and related family proteins are major regulators of apoptosis that are critical for development and tumorigenesis in autoimmune and degenerative diseases (13). Bad belongs to the BH3-only Bcl-2 subfamily and shares the conserved BH3 domain (i.e. death domain) with other Bcl2 family members (13,45). All members of this BH3-only subfamily are proapoptotic and have been shown to interact with one or more of the death suppressor family members, such as Bcl-X L (46). Phosphorylation, a post-translational modification, plays a pivotal role in regulating the proapoptotic activity of Bad. Recent studies indicate that the ability of Bad to heterodimerize with Bcl-X L and to promote apoptosis depends upon the phosphorylation status of this proapoptotic protein (27,47). Growth factor (i.e. IL-3 or epidermal growth factor)-induced phosphorylation of Bad at Ser 112 , Ser 136 , and Ser 155 has been reported to disrupt its proapoptotic function (18 -19, 40). Our findings indicate that nicotine mimics growth factors to potently stimulate Bad phosphorylation at these three sites and enhances survival of pulmonary adenocarcinoma A549 cells (Fig. 1). Other lung cancer cell lines (i.e. H157 and H358) were also tested, and similar results were obtained (data not shown), indicating that nicotine can induce Bad phosphorylation and survival in various lung cancer cells. We discovered previously that nicotine induces Bcl2 phosphorylation in association with increased survival of H69 cells expressing high levels of endogenous Bcl2 (23). Unfortunately, most lung cancer cell lines, including A549 cells, do not express detectable levels of Bcl2, but do express high levels of endogenous Bad (Fig. 1A). Therefore, nicotineinduced survival of A549 cells may occur, at least in part, through phosphorylation of Bad at Ser 112 , Ser 136 , and Ser 155 . Phosphorylation at Ser 112 of Bad occurs earlier than Ser 136 or Ser 155 ( Fig. 2A), suggesting that nicotine-induced multi-site Bad phosphorylation may occur in a hierarchical manner. Phosphorylation of Ser 112 may facilitate further phosphorylation of Bad at Ser 136 and Ser 155 sites. However, the signal mechanism and functional significance for this hierarchical phosphorylation remain elusive.
Our results reveal that nicotine-induced multi-site Bad phosphorylation occurs through three signal pathways, including ERKs3 Bad at Ser 112 , PI3K/AKT3 Bad at Ser 136 , and PKA3 Bad at Ser 155 ( Fig. 3 and 4). These results support previous findings that ERKs, AKT, or PKA can function as a Bad Ser 112 , Ser 136 , or Ser 155 kinase, respectively (26 -27, 40, 48). It is known that phosphorylation of Bad on either a single Ser 112 , Ser 136 , or Ser 155 or at multi-site can inactivate the proapoptotic function of Bad (18 -19, 40). PD98059, LY294002, or H89 can potently inhibit nicotine-induced Bad phosphorylation at Ser 112 , Ser 136 , or Ser 155 in association with decreased cell survival, providing further pharmacological evidence that nicotine-induced multi-site Bad phosphorylation and cell survival may occur through multiple signal pathways involving ERK1/2/Bad, PI3K/AKT/Bad, and PKA/Bad (Fig. 8).
It has been found that high levels of ␤-adrenergic receptor are expressed in pulmonary adenocarcinoma cells (10). Mounting evidence now indicates that nicotine can function as a ␤-adrenergic receptor agonist, and its effect is abrogated by FIG. 6. Nicotine induces Bad translocation from mitochondria to cytosol and interaction with 14-3-3. A, A549 cells expressing high levels of endogenous Bad were treated with various concentrations of nicotine (i.e. 0.5-5 M) for 30 min. Cells were harvested and subcellular fractionation was performed as described under "Experimental Procedures." Phosphorylated Bad and total Bad were analyzed by Western blotting as described in the legend to Fig. 2. Prohibitin was used as a mitochondrial marker (34) to verify the purity of each fraction. B, A549 cells were treated with various concentrations of nicotine (0.5-5 M) for 30 min. Cells were harvested, washed, and lysed in detergent buffer. Co-immunoprecipitation experiments were performed using Bad or 14-3-3 antibody, respectively. Total Bad was determined by quantitative immunoprecipitation using a Bad antibody. Bad-associated 14-3-3 (i.e. bound 14-3-3) or 14-3-3-associated Bad (i.e. bound Bad) was analyzed by Western blotting. propranolol (a ␤-adrenergic receptor inhibitor; Refs. 11, 49 -50). Propranolol was found to block nicotine-induced Bad phosphorylation in A549 cells (Fig. 5), but ␣-BTX (␣7 nAChR specific inhibitor) has no effect (data not shown), suggesting that the ␤-adrenergic receptor may be the major upstream receptor for nicotine-stimulated Bad phosphorylation in human lung cancer cells. Importantly, inhibition of nicotine-induced Bad phosphorylation by propranolol restores the proapoptotic function of Bad and results in apoptosis (Fig. 5). Thus, propranolol may have the potential to be developed as a clinically useful drug that specifically targets ␤-adrenergic receptor to block nicotine-stimulated survival signal pathway in patients with lung cancer expressing high levels of ␤-adrenergic receptor and Bad.
Phosphorylation on either Ser 112 or Ser 136 facilitates formation of a complex between Bad and 14-3-3 in the cytosol, blocking its interaction with Bcl-X L at the mitochondrial level (26 -27, 48). Further studies indicate that phosphorylation of Ser 112 , which is located within the center of the Bad BH3 domain, can also directly suppress its proapoptotic function (40). Our findings reveal that nicotine, as a survival agonist, is able to mimic growth factors to functionally inactivate Bad through phosphorylation at these three sites (Figs. 1 and 2). Nicotine-induced multi-site phosphorylation also leads to sequestering Bad from mitochondria in an identical mechanism (Fig. 6). Thus, nicotine may promote survival of human lung cancer cells in a novel mechanism involving multi-site phosphorylation of Bad.
Because Bad is a potent proapoptotic protein that is ubiquitously expressed in both SCLC and non-small cell lung cancer cells (Fig. 1A), targeting Bad phosphorylation may represent a novel therapeutic approach. Our results indicate that specific knockdown of Bad expression by RNA interference enhances both the survival and chemoresistance of A549 cells (Fig. 7). Importantly, this is a specific effect because nicotine has no additional survival effect on cells in which Bad expression is depleted.
In summary, our studies identify a novel nicotine survival signal transduction pathway that depends on phosphorylation of Bad at Ser 112 , Ser 136 , and Ser 155 through activation of MAPKs ERK1/2, AKT, and PKA (Fig. 8). Nicotine-induced multi-site phosphorylation sequesters Bad from mitochondria, where it then interacts with 14-3-3 in the cytosol, which leads to loss of the apoptotic function of Bad and, hence, enhanced cell survival (Fig. 8). Because Bad can function as a target of nicotine in human lung cancer cells, it's use may help to develop novel therapeutic strategies for treatment of patients with lung cancer by blocking the nicotine-activated multiple Bad upstream signal pathways.