Protein Kinase C
Abrogates the Proapoptotic Function of Bax through Phosphorylation*
Meiguo Xin,
Fengqin Gao,
W. Stratford May,
Tammy Flagg, , and
Xingming Deng1
University of Florida Shands Cancer Center, Department of Medicine and Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32610-3633
Abstract:
Protein kinase C
(PKC) 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 PKC 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 PKC in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKC activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKC specific inhibitor (a myristoylated PKC pseudosubstrate peptide). Purified, active PKC directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKC mutant results in Bax phosphorylation at serine 184. PKC co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKC by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKC mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKC can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKC 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.
Received for publication February 23, 2007.
Revision received May 22, 2007.
* This was supported by a Flight Attendant Medical Research Institute Clinical Innovator Award, NCI, National Institutes of Health Grant R01 CA112183 (to X. D.), and a National Institutes of Health T32 Training Grant in Cancer Biology (to M. X.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: 1376 Mowry Rd., Cancer/Genetics Research Complex, Rm. 262, P. O. Box 103633, Gainesville, FL 32610-3633. Fax: 352-273-8285; Tel.: 352-273-8170; E-mail: xdeng{at}ufl.edu.
