Nicotine-induced cell survival is associated with chemoresistance of human lung cancer cells, but our understanding of the intracellular mechanism(s) is fragmentary. Bax is a major proapoptotic member of the Bcl2 family and a molecule required for apoptotic cell death. Growth factor (i.e. granulocyte-macrophage colonystimulating factor)-induced phosphorylation of Bax has been reported to negatively regulate its proapoptotic function. Because Bax is ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells, nicotine may mimic growth factor(s) to regulate the activity of Bax. We found that nicotine potently induces Bax phosphorylation at Ser-184, which results in abrogation of the proapoptotic activity of Bax and increased cell survival. AKT, a known physiological Bax kinase, is activated by nicotine, co

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C 2 -ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C 2 -ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.Apoptosis occurs by activation of an intrinsic or extrinsic pathway and is largely regulated by the Bcl-2 family of apoptotic regulators that is comprised of three subfamilies (1-2). The subfamily including Bcl2, Bcl-XL, and MCL1 members block apoptosis, whereas the Bax subfamily, consisting of Bax and Bak, or the BH3 2 -only subfamily, including Bad, Bid, Bok, Bik, Bim, and PUMA, promote apoptosis (3-8). Bcl2 family members function in a tightly regulated network that protects or induces mitochondrial dysfunction. It is popularly held that antiapoptotic Bcl2 and Bcl-XL heterodimerize with proapoptotic Bax or Bak such that the hydrophobic crevices on their surfaces bind to the exposed BH3 domain of Bax or Bak to block their proapoptotic function. Thus, heterodimerization appears to regulate, at least in part, cell survival or death (5). Furthermore the BH3 domain of the proapoptotic members is required for both their oligomerization and killing activity, although homodimerization does not necessarily correlate with killing activity (9). Genetic studies using Bax and Bak single and double homozygous knock-out mice reveal that either Bax or Bak is essential for inducing mitochondrial dysfunction characterized by the release of potent caspase activators including cytochrome c (Cyt c) and Smac/Diablo that initiate the intrinsic pathway (10).Bax is the major proapoptotic Bcl2 family protein that is widely expressed in v...

Bax, a central death regulator, is required at the decisional stage of apoptosis. We recently identified serine 184 (S184) of Bax as a critical functional switch controlling its proapoptotic activity. Here, we employed the structural pocket around S184 as a docking site to screen the NCI library of small molecules using the UCSF-DOCK program suite. Three compounds, small molecule Bax agonists SMBA1, SMBA2 and SMBA3, induce conformational changes in Bax by blocking S184 phosphorylation, facilitating Bax insertion into mitochondrial membranes and forming Bax oligomers. The latter leads to cytochrome c release and apoptosis in human lung cancer cells, which occurs in a Bax- but not Bak-dependent fashion. SMBA1 potently suppresses lung tumor growth via apoptosis by selectively activating Bax in vivo without significant normal tissue toxicity. Development of Bax agonists as a new class of anti-cancer drugs offers a strategy for the treatment of lung cancer and other Bax-expressing malignancies.

Protein kinase C (PKC) 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 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 in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKC activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKC specific inhibitor (a myristoylated PKC pseudosubstrate peptide). Purified, active PKC directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKC mutant results in Bax phosphorylation at serine 184. PKC co-localizes and interacts with Bax at the BH3 domain. 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 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 leu...

Lung cancer is one leading cause of cancer deaths in both men and women, and the 5-year relative survival rate for all stages combined is only 15% (1). The World Health Organization reported that almost 1 billion men and 250 million women are daily smokers and cigarette smoking causes 90% of lung cancer cases and ϳ1.2 million deaths annually all over the world (1, 2). It is estimated that ϳ90% of male and 75-80% of female lung cancer deaths in the United States each year are caused by smoking (3, 4).One piece of evidence for the connection between cigarette smoking and lung cancer is that lung cancer in women has increased by 600% since 1950 and has reached epidemic levels. This dramatic rise in lung cancer incidence is likely due to the increased prevalence of cigarette smoking, particularly in women over the same time period (5, 6).There are more than 60 known carcinogens in cigarette smoke. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-