Protein kinase C␦ (PKC␦) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKC␦ 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␦ increased the apoptotic effect induced by etoposide, whereas the PKC␦ selective inhibitor rottlerin and the PKC␦ dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKC␦ 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␦ were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKC␦ in the effects of etoposide was examined using cells overexpressing a PKC␦ mutant in which five tyrosine residues were mutated to phenylalanine (PKC␦5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKC␦. Likewise, activation of caspase 3 and the cleavage of the PKC␦5 mutant were significantly lower in cells overexpressing PKC␦5. Using mutants of PKC␦ 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␦ in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKC␦ as an important regulator of this effect.

Protein kinase C␦ (PKC␦) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKC␦ are involved in these two responses. Transfection of cells with PKC␦ mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKC␦ wild-type transfectant. Conversely, transfection with PKC␦ mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKC␦ wild-type transfectant. The tyrosine phosphorylation of PKC␦ and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKC␦ via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKC␦ induced by PDGF. We conclude that the tyrosine phosphorylation of PKC␦ and its association with tyrosine kinases may be an important point of divergence in PKC signaling.

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKC␦ to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKC␦ inhibitor rottlerin and a PKC␦ kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKC␦ in the apoptosis induced by SVNI we used a PKC␦ mutant in which five tyrosine residues were mutated to phenylalanine (PKC␦5). PKC␦5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKC␦. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKC␦5 but did not induce cleavage of PKC␦ or PKC␦5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKC␦ exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKC␦ on specific tyrosines is required for this function.

Nerve growth factor (NGF) is a neurotrophic factor acting on both the peripheral and central nervous systems. In addition, it has been shown to modulate B lymphocyte function through receptorsconsisting of both p75 and TrkA proteins. The low‐affinity NGFR, p75, shares structural homology with the B cell antigen, CD40, tumor necrosis factor (TNF) receptor and Fas antigen (APO‐1), which play a role in cell apoptosis. We studied the effect of NGF on anti‐IgM‐induced apoptosis in human B lymphocytes and the role of protein kinase C (PKC) in this effect. Incubation of Ramos cells with anti‐IgM (10 μg / ml) induced apoptosis which was observed after 6 h and reached plateau levels after 24 h. Addition of NGF to anti‐IgM‐treated cells rescued cells from apoptosis. The NGF effectwas blocked by anti‐NGF antibody and by K252a, a specific inhibitor for the tyrosine kinase activity of TrkA. NGF induced translocation of PKCδ and PKCα from the cytosol to the plasma membrane and translocation of PKCζ to the nucleus. To examine the role of PKC in the inhibitory effect of NGF on anti‐IgM‐induced apoptosis, we used inhibitors of PKCα and PKCδ and found that these treatments did not alter the NGF effect. In contrast, treatment of the cells with oligonucleotide antisense directed against the 5′ coding sequence of PKCζ reduced the expression of PKCζ in the cells and abolished the protective effect of NGF on anti‐IgM‐induced apoptosis. The translocation of PKCζ and the protective effect of NGF were inhibited by the phosphatidylinositol 3 (PI3)‐kinase inhibitors wortmannin and LY294002. The results of this study indicate that NGF is involved in B cell survival and that this effect is mediated by PI3‐kinase‐dependent activation of PKCζ.

In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose‐dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC‐β2 and an increase in that of PKC‐γ. In addition, it induced translocation of PKC‐ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.