Tyrosine phosphorylation of protein kinase Cδ is essential for its apoptotic effect in response to etoposide

Protein kinase C delta (PKC delta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKC delta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKC delta increased the apoptotic effect induced by etoposide, whereas the PKC delta selective inhibitor rottlerin and the PKC delta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKC delta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKC delta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKC delta in the effects of etoposide was examined using cells overexpressing a PKC delta mutant in which five tyrosine residues were mutated to phenylalanine (PKC delta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKC delta. Likewise, activation of caspase 3 and the cleavage of the PKC delta5 mutant were significantly lower in cells overexpressing PKC delta5. Using mutants of PKC delta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKC delta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKC delta as an important regulator of this effect.