Jin Zhaohui, Xin Meiguo, Deng Xingming
University of Florida Shands Cancer Center, Department of Medicine, University of Florida, Gainesville, Florida 32610-0232, USA.
J Biol Chem. 2005 Apr 22;280(16):16045-52. doi: 10.1074/jbc.M413488200. Epub 2005 Feb 10.
Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. NNK cannot only induce DNA damage but also promotes the survival of human lung cancer cells. Protein kinase C (PKC)iota is an atypical PKC isoform and plays an important role in cell survival, but the downstream survival substrate(s) is not yet identified. Bad, a proapoptotic BH3-only member of Bcl2 family, is co-expressed with PKCiota in both small cell lung cancer and non-small cell lung cancer cells. We discovered that NNK potently induces multisite Bad phosphorylation at Ser-112, Ser-136, and Ser-155 via activation of PKCiota in association with increased survival of human lung cancer cells. Purified, active PKCiota can directly phosphorylate both endogenous and recombinant Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKCiota results in an enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the PKC inhibitor (staurosporine) or a Src-specific inhibitor (PP2) can block NNK-induced Bad phosphorylation and promote apoptotic cell death. The beta-adrenergic receptor inhibitor propranolol blocks both NNK-induced activation of PKCiota and Bad phosphorylation, indicating that NNK-induced Bad phosphorylation occurs at least in part through the upstream beta-adrenergic receptor. Mechanistically, NNK-induced Bad phosphorylation prevents its interaction with Bcl-XL. Because the specific depletion of PKCiota by RNA interference inhibits both NNK-induced Bad phosphorylation and survival, this confirms that PKCiota is a necessary component in NNK-mediated survival signaling. Collectively, these findings reveal a novel role for PKCiota as an NNK-activated physiological Bad kinase that can directly phosphorylate and inactivate this proapoptotic BH3-only protein, which leads to enhanced survival and chemoresistance of human lung cancer cells.
亚硝胺4-（甲基亚硝基氨基）-1-（3-吡啶基）-1-丁酮（NNK）由尼古丁亚硝化形成，已被确定为香烟烟雾中最具致癌性的物质。NNK不仅能诱导DNA损伤，还能促进人肺癌细胞的存活。蛋白激酶C（PKC）ι是一种非典型的PKC亚型，在细胞存活中起重要作用，但尚未确定其下游的存活底物。Bad是Bcl2家族中仅含BH3结构域的促凋亡成员，在小细胞肺癌和非小细胞肺癌细胞中均与PKCι共同表达。我们发现，NNK通过激活PKCι，有力地诱导Bad在Ser-112、Ser-136和Ser-155位点发生多位点磷酸化，同时人肺癌细胞的存活率增加。纯化的活性PKCι能在这三个位点直接磷酸化内源性和重组Bad，并在体外破坏Bad/Bcl-XL的结合。PKCι的过表达导致Bad磷酸化增强。NNK还能刺激c-Src的激活，c-Src是一种已知的PKCι上游激酶。用PKC抑制剂（星形孢菌素）或Src特异性抑制剂（PP2）处理细胞，可阻断NNK诱导的Bad磷酸化，并促进凋亡细胞死亡。β-肾上腺素能受体抑制剂普萘洛尔可阻断NNK诱导的PKCι激活和Bad磷酸化，表明NNK诱导的Bad磷酸化至少部分通过上游的β-肾上腺素能受体发生。从机制上讲，NNK诱导的Bad磷酸化阻止了它与Bcl-XL的相互作用。由于RNA干扰特异性消耗PKCι可抑制NNK诱导的Bad磷酸化和细胞存活，这证实PKCι是NNK介导的存活信号通路中的一个必要成分。总的来说，这些发现揭示了PKCι作为一种由NNK激活的生理性Bad激酶的新作用，它能直接磷酸化并使这种仅含BH3结构域的促凋亡蛋白失活，从而导致人肺癌细胞的存活率提高和化疗耐药性增强。
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