Protein kinase C␦ (PKC␦) regulates cell apoptosis in a cell-and stimulus-specific manner. Here, we studied the role of PKC␦ in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKC␦ kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKC␦ mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKC␦ decreased it. PKC␦ acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKC␦ within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKC␦ was essential for its protective effect because overexpression of a caspaseresistant mutant (PKC␦D327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKC␦ to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKC␦ on tyrosine 155. Using a PKC␦Y155F mutant, we found that the phosphorylation of PKC␦ on tyrosine 155 was essential for the cleavage of PKC␦ in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKC␦ on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKC␦ protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKC␦ on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKC␦.

Protein kinase CD (PKCD) regulates cell apoptosis and survival in diverse cellular systems. PKCD translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCD constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCD affects its apoptotic and survival functions. PKCD-Cyto, PKCD-Mito, and PKCD-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCD-ER. PKCD-Cyto and PKCD-Mito underwent cleavage, whereas no cleavage was observed in the PKCD-Nuc and PKCD-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCD-Cyto and PKCD-Mito. In contrast to the apoptotic effects of the PKCD-Cyto, PKCD-Mito, and PKCD-Nuc, the PKCD-ER protected the cells from tumor necrosis factor -related apoptosis-inducing ligand -induced and etoposideinduced apoptosis. Moreover, overexpression of a PKCD kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCD and increased tumor necrosis factor -related apoptosis-inducing ligand -induced apoptosis. The localization of PKCD differentially affected the activation of downstream signaling pathways. PKCD-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCD-Nuc increased c-Jun NH 2 -terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCD-Cyto, whereas c-Jun NH 2 -terminal kinase activation mediated the apoptotic effect of PKCD-Nuc. Our results indicate that the subcellular localization of PKCD plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways.

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495-603 531 tion what happens if the cells with self-renewal capacity are exposed to viral oncogenes.We studied how oncogenes and tumor suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. For this we used neural progenitor cells (NPCs) transduced with modified retroviruses carrying Human Papilloma Virus E6, E7 or E6/E7 oncogenes. HPV 16 E6/E7 is a classical oncogene complex, which degrades tumor suppressors p53 and pRb/p107/p130 and interferes with several other cellular activities. To examine further p53 and pRb involvement in NPC maintenance, we used p53−/− and E7 and E6/E7− expressing NPCs with restored pRb expression, respectively. E6/E7 expressing or p53−/− NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation, and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family and involved MEK-ERK signaling. Low amount of p53 increased self-renewal and proliferation, where as pRb affected only proliferation.This suggests that the oncogenes increase, while p53 and pRb-family tumor suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumor suppressors control tissue homeostasis and highlight their importance in stem cell self-renewal, linked both to cancer and life-long tissue turnover.PKC␦ is a key enzyme in the regulation of cell apoptosis in various cellular systems. PKC␦ can act as a pro or anti-apoptotic kinase depending on the cell type and apoptotic stimuli, however the molecular mechanisms underlying its diverse effects are not understood. Gliomas are characterized by deregulation of cell apoptosis and hence by increased resistance to apoptotic stimuli. Studies in our lab indicate that the expression PKC␦ is inversely correlated with the malignancy of gliomas. Here, we studied the role of PKC␦ in the apoptosis of glioma cells stimulated with etoposide and TRAIL. Using PKC␦WT, PKC␦KD mutant and PKC␦␦siRNAs, we found that PKC␦ played opposite roles in the apoptosis induced by etoposide and TRAIL; PKC␦␦increased the apoptotic effect of etoposide, whereas it reduced cell apoptosis induced by TRAIL. Both etoposide and TRAIL induced tyrosine phosphorylation of PKC␦ albeit on different residues; etoposide on tyrosine 187 and TRAIL on tyrosine 155. Etopo-side and TRAIL also induced differential translocation of PKC␦; etoposide to the nucleus and TRAIL to the ER. Both stimuli induced cleavage of PKC␦ and accumulation of the catalytic fragment. Using PKC␦ single tyrosine mutants, we found that the phosphorylation of PKC␦ on tyrosine 187 was not required for the nuclear translocation of PKC␦, but the phosphorylation on tyrosine 155 was essential for the translocation of PKC␦ to the ER in response to TRAIL. Tyrosine phosphorylation of PKC␦ was also necessary for the act...