title: Tyrosine phosphorylation of protein kinase Cdelta is essential for its apoptotic effect in response to etoposide.
abstract: Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta 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 PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta 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 PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta 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 PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect.
authors.affiliation: Gonda (Goldschmied) Medical Diagnosis Research Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Blass
authors.forename: Ilana
authors.initials: I
authors.lastname: Kronfeld
authors.forename: Gila
authors.initials: G
authors.lastname: Kazimirsky
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Chaya
authors.initials: C
authors.lastname: Brodie
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001578:Benzopyrans / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002469:Cell Separation; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011720:Pyrazoles / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D000098:Acetophenones; D001578:Benzopyrans; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D007527:Isoenzymes; D008164:Luminescent Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D005047:Etoposide; C085746:rottlerin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D053148:Caspase 3; D020169:Caspases