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

Abstract

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, PKCepsilon, 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.

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells

“…31,33,50 Likewise, PKCd-induced cell death in HeLa cells and HPVtransformed keratinocytes is presumably p53-independent. 21 In the LNCaP prostate epithelial cell line however, TPA-induced apoptosis was preceded by induction of the cdk inhibitor, p21, and dephosphorylation of the retinoblastoma protein (Rb). 52 An essential role for Rb was confirmed by the demonstration that DU145 prostate epithelial cells, which do not express functional Rb, or LNCaP cells transfected with the Rb inhibitor, E1a, were resistant to TPA-induced apoptosis.…”

“…Our studies in salivary acinar cells are in agreement with data from other epithelial cell models, including breast, prostate and thyroid cells, that demonstrate that activation of PKC with TPA induces apoptosis, 32 ± 34,48 ± 50 and with data from keratinocytes showing that over expression of PKCd induces apoptosis. 21 Whelan and Parker 51 have recently reported that the loss of PKC function is sufficient to induce an apoptotic response in U937 cells and COS-1 cells. Since TPA causes both activation and downregulation of PKC, results obtained using this agent could conceivably reflect either of these processes.…”

The Localization of Protein Kinase Cδ in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

“…Indeed, the role of the nuclear localization of PKCy in its apoptotic effect has been shown in studies showing that PKCy translocated to the nucleus in response to apoptotic stimuli, such as etoposide (9,30), and that the overexpression of a PKCy nuclear localization signal (NLS) mutant abrogates the apoptotic effect of this drug (35). Similarly, PKCy has been shown to translocate to the mitochondria in response to UV radiation (14), phorbol 12-myristate 13-acetate (28), and oxidative stress (29) and to induce cell apoptosis in response to these stimuli. Our results support these studies and provide direct evidence that the localization of PKCy-CA in the cytosol, mitochondria, and nucleus, but not in the ER, promotes cell apoptosis.…”

“…Thus, PKCy translocates to the Golgi in response to ceramide and IFN-g (5,27) to the mitochondria in response to oxidative stress, UV radiation, and phorbol 12-myristate 13-acetate (28,29) and to the nucleus in response to etoposide, g-irradiation, and cytosine arabinoside (9,30). Translocation of PKCy to the Golgi, mitochondria, and nucleus in response to these apoptotic stimuli has been associated with proapoptotic effects of this isoform (5,9,28). In addition, PKCy has been shown to translocate to the endoplasmic reticulum (ER) in cells infected with Sindbis virus and in glioma cells treated with TRAIL, where PKCy exerts antiapoptotic effects (24,25).…”

Protein kinase C as a stress sensor

“…PKCs can interact with the plasma membrane [11], golgi [12], and cytoskeleton [13]. More recently, it has become evident that some PKCs interact with mitochondria [14] and cell nuclei [15]. As a consequence, PKCs are involved in such diverse functions as differentiation, cell growth, apoptosis, gene expression, muscle contraction, metabolism, and endocytosis [16].…”