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.

Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites for phorbol esters and the diacylglycerols. X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers. In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme. Our approach was to mutate either the first, the second, or both zinc fingers of PKC␦, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the doseresponse curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the doseresponse curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase C␦ in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains. Protein kinase C (PKC)1 comprises a family of isozymes that mediate signal transduction for the lipophilic second messenger diacylglycerol, regulating a wide array of cellular processes (see Refs. 1 and 2 for review). PKC possesses two functional domains, an N-terminal regulatory domain and a C-terminal catalytic domain. Within the regulatory domain lie two cysteine-rich zinc fingers, responsible for recognition by PKC of diacylglycerol or their ultrapotent analogs, the phorbol esters. In isolation, the individual first and second zinc fingers bind phorbol ester with similar affinities (3, 4), and the molecular details of the interaction between the phorbol esters and the zinc finger domain are beginning to emerge. NMR spectroscopy has yielded the solution structure of the second zinc finger of PKC␣ (5, 6); the structure of the complex between the second zinc finger of PKC␦ and phorbol 13-acetate has been solved by x-ray crystallography (7). Site-directed mutagenesis has further highlighted key residues within the zinc finger structure required for ligand binding (8), and computer modeling is providing insight into how other high affinity ligands, e.g. the indole alkaloids, interact with the zinc finger.An issue that is only beginning to be addressed is the role of the individual zinc fingers within the context of the intact PKC molecule. In pioneering studies, Bell and co-workers (9) had reported that the binding affinity of the second zinc finger of PKC␥ was reduced in constructs containing ...

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of glial cells. In a recent study we found that overexpression of PKC␦ reduced the expression of the astrocytic marker glutamine synthetase (GS). In this study we explored the mechanisms involved in the inhibitory effect of PKC␦ on the expression of glutamine synthetase. Using PKC chimeras we first examined the role of the catalytic and regulatory domains of PKC␦ on the expression of glutamine synthetase. We found that cells stably transfected with chimeras between the regulatory domain of PKC␦ and the catalytic domains of PKC␣ or ⑀ inhibited the expression of GS, similar to the inhibition exerted by overexpression of PKC␦ itself. In contrast, no significant effects were observed in cells transfected with the reciprocal PKC chimeras between the regulatory domains of PKC␣ or ⑀ and the catalytic domain of PKC␦. PKC␦ has been shown to undergo tyrosine phosphorylation in response to various activators. Tyrosine phosphorylation of PKC␦ in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor occurred only in chimeras which contained the PKC␦ regulatory domain. Cells transfected with a PKC␦ mutant (PKC␦5), in which the five putative tyrosine phosphorylation sites were mutated to phenylalanine, showed markedly diminished tyrosine phosphorylation in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor and normal levels of GS. Our results indicate that the regulatory domain of PKC␦ mediates the inhibitory effect of this isoform on the expression of GS. Phosphorylation of PKC␦ on tyrosine residues in the regulatory domain is implicated in this inhibitory effect.

Protein kinase C (PKC) is a complex enzyme system comprised of at least 11 isozymes that serves to mediate numerous extracellular signals which generate lipid second messengers. The discovery of isozyme-selective activators and inhibitors (modulators) of PKC is crucial to ascertaining the role of the individual isozymes in physiological and pathophysiological processes and to manipulating their function. The discovery of such small molecule modulators of PKC is at present a largely unmet pharmacological need. Herein we detail our modeling studies which reveal how the natural product indolactam V (ILV) and its 8-membered ring analogue, the benzolactam 15, bind to the CRD2 activator domain of PKC. These modeling studies reveal that not all PKC ligands possess a common pharmacophore, and further suggest an important role of specific hydrophobic contacts in the PKC-ligand interaction. The modeling studies find strong experimental support from mutagenesis studies on PKC alpha that reveal the crucial role played by the residues proline 11, leucine 20, leucine 24, and glycine 27. Next, we describe the synthesis of two 8-substituted benzolactams starting from L-phenylalanine and characterize their isozyme selectivity; one of the two benzolactams exhibits improved isozyme selectivity relative to the n-octyl-ILV. Lastly, we report inhibition of cellular proliferation of two different breast carcinoma cell lines by the benzolactam 5 and show that the compound preferentially down-regulates PKCbeta in both cell lines.