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Molecular and Cellular Biology, January 2002, p. 182-195, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.182-195.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Tyrosine Phosphorylation of Protein Kinase C Is Essential for Its Apoptotic Effect in Response to Etoposide

Gonda (Goldschmied) Medical Diagnosis Research Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel,¹ Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892²

Received 28 March 2001/ Returned for modification 23 May 2001/ Accepted 19 September 2001

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 G₁/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 (PKC5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKC. Likewise, activation of caspase 3 and the cleavage of the PKC5 mutant were significantly lower in cells overexpressing PKC5. 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.

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