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Molecular and Cellular Biology, December 1999, p. 8547-8558, Vol. 19, No. 12
0270-7306/99/$04.00+0

Protein Kinase Cdelta Targets Mitochondria, Alters Mitochondrial Membrane Potential, and Induces Apoptosis in Normal and Neoplastic Keratinocytes When Overexpressed by an Adenoviral Vector

Luowei Li, Patricia S. Lorenzo, Krisztina Bogi, Peter M. Blumberg, and Stuart H. Yuspa*

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Science, National Cancer Institute, Bethesda, Maryland 20892

Received 2 April 1999/Returned for modification 18 May 1999/Accepted 19 August 1999

Inactivation of protein kinase Cdelta (PKCdelta ) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCdelta in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCdelta under a cytomegalovirus promoter to overexpress PKCdelta in normal and neoplastic keratinocytes. While PKCdelta overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCalpha , PKCvarepsilon , PKCzeta , and PKCeta , did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCdelta adenovirus. Activation of PKCdelta by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCdelta . Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCdelta translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCdelta -green fluorescent protein fusion protein. Furthermore, activation of PKCdelta in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCdelta -overexpressing keratinocytes. These results indicate that PKCdelta can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

* Corresponding author. Mailing address: Laboratory of Cellular Carcinogenesis and Tumor Promotion, Bldg. 37, Rm. 3B25, National Cancer Institute, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-2612. Fax: (301) 496-8709. E-mail: yuspas{at}mail.nih.gov.

Molecular and Cellular Biology, December 1999, p. 8547-8558, Vol. 19, No. 12
0270-7306/99/$04.00+0


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