Xin Meiguo, Gao Fengqin, May W Stratford, Flagg Tammy, Deng Xingming
University of Florida Shands Cancer Center, Department of Medicine and Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32610-3633, USA.
J Biol Chem. 2007 Jul 20;282(29):21268-77. doi: 10.1074/jbc.M701613200. Epub 2007 May 24.
Protein kinase Czeta (PKCzeta) 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 PKCzeta 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 PKCzeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCzeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCzeta specific inhibitor (a myristoylated PKCzeta pseudosubstrate peptide). Purified, active PKCzeta directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCzeta mutant results in Bax phosphorylation at serine 184. PKCzeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCzeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCzeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCzeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCzeta 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.
蛋白激酶Cζ（PKCζ）是一种非典型的蛋白激酶C亚型，在维持细胞存活中发挥重要作用，但其具体机制尚未完全明确。Bax是Bcl-2家族的主要成员，是凋亡性细胞死亡所必需的。由于Bax在小细胞肺癌（SCLC）和非小细胞肺癌（NSCLC）细胞中均与PKCζ广泛共表达，因此Bax有可能作为PKCζ的下游靶点，参与调节肺癌细胞的存活和化疗敏感性。在此，我们发现用尼古丁处理细胞不仅能增强PKCζ的活性，还能导致Bax磷酸化并延长细胞存活时间，而PKCζ特异性抑制剂（一种肉豆蔻酰化的PKCζ假底物肽）可抑制这一现象。纯化的活性PKCζ在体外可直接使Bax磷酸化。野生型或组成型激活的A119D而非显性负性的K281W PKCζ突变体的过表达会导致Bax在丝氨酸184处磷酸化。PKCζ与Bax在BH3结构域共定位并相互作用。通过RNA干扰特异性敲低PKCζ可阻断尼古丁刺激的Bax磷酸化，并增强凋亡性细胞死亡。有趣的是，野生型或A119D而非K281W PKCζ突变体的强制表达会导致Bax在细胞质中积累，并阻止Bax发生构象变化，从而延长细胞存活时间。纯化的PKCζ可直接使Bax从经C2-神经酰胺处理的细胞的分离线粒体中解离出来。因此，PKCζ可能作为一种生理性的Bax激酶，直接磷酸化并与Bax相互作用，从而导致Bax在细胞质中被隔离，并消除Bax的促凋亡功能。
