Protein kinase Czeta abrogates the proapoptotic function of Bax through phosphorylation.
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.
Xin
Meiguo
M
University of Florida Shands Cancer Center, Department of Medicine and Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32610-3633, USA.
Gao
Fengqin
F
May
W Stratford
WS
Flagg
Tammy
T
Deng
Xingming
X
D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D009538:Nicotine / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D009538:Nicotine; D011493:Protein Kinase C; D051028:bcl-2-Associated X Protein; D000099221:Protein Kinase C zeta