Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

Meiguo Xin,Fengqin Gao,W Stratford May,Tammy Flagg,Xingming Deng

doi:10.1074/jbc.m701613200

Abstract

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.

Highlights

(2), oligomerization, and insertion into mitochondrial membranes following stress [3,4,5]
Nicotine Induces Activation of protein kinase C (PKC)␨ and Phosphorylation of Bax in Association with Prolonged Survival of Human Lung Cancer Cells, Which Is Suppressed by a PKC␨-specific Pseudosubstrate Inhibitor—PKC␨ has been implicated in many key cellular functions including cell survival [20, 32]
To ensure that the kinase activity that phosphorylates Myelin basic protein (MBP) is from PKC␨, and not from any other kinase that may be co-immunoprecipitated with PKC␨, we probed the same filter by Western blot using a PKC␫ or PKC␣ antibody, respectively

Summary

Introduction

(2), oligomerization, and insertion into mitochondrial membranes following stress [3,4,5]. To test whether PKC␨ can directly phosphorylate endogenous Bax, endogenous Bax was immunoprecipitated from A549 cells and incubated with purified, active PKC␨ in a kinase assay buffer containing [␥-32P]ATP.

Results

Conclusion