Nicotine and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are two major components in cigarette smoke. NNK is formed by nitrosation of nicotine and has been identified as the most potent carcinogen. NNK not only can induce DNA damage but also promotes survival of human lung cancer cells. However, the intracellular mechanism(s) by which NNK enhances cell survival remains enigmatic. PKC iota is an atypical PKC isoform and plays an important role in cell survival but the downstream survival substrate(s) has not been identified yet. Bad is a potent proapoptotic BH3-only member of the Bcl2 family and is widely expressed in both small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells. We found that NNK potently induces multisite Bad phosphorylation at ser112, ser136 and ser155 via activation of PKC iota in association with increased survival of human lung cancer A549 cells. Purified active PKC iota can directly phosphorylate Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKC iota in lung cancer H157 cells results in enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src which can function as PKC iota upstream kinase. Treatment of cells with PKC inhibitor (staurosporine) or Src specifc inhibitor (PP2) can block NNK-induced Bad phosphorylation and promotes apoptotic cell death. Since the beta -adrenergic receptor inhibitor (propranolol) blocks NNK-induced activation of PKC iota as well as Bad phosphorylation, this indicates that NNK-induced Bad phosphorylation occurs, at least in part, through the upstream beta-adrenergic receptors. Mechanistically, NNK-induced Bad phosphorylation inhibits its interaction with Bcl-XL and significantly reduces its half-life (i.e. from 24 to 12h). Specific depletion of PKC iota by RNA interference (RNAi) inhibits NNK-induced Bad phosphorylation and survival, suggesting that PKC iota is a required target in NNK/Bad survival signaling. Collectively, these findings reveal a novel role for PKC iota as NNK-activated physiological Bad kinase that can phosphorylate and inactivate the proapoptotic BH3-only protein Bad, which may result in survival and chemoresistance of human lung cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]