The aim of this project is to better elucidate the role(s) of specific protein kinases in the regulation of cell growth and in malignant transformation. Protein kinase C (PKC) has been shown to play a crucial role in the regulation of a number of physiological processes. Since PKC consists of a family of at least 10 closely related isotypes, it is important to identify cellular targets and biological systems which are selectively modulated by the different PKC isoforms to regulate specific cellular functions. Previous studies established that PKC epsilon is the isoform involved in mediating the phorbol ester tumor promoter (PMA)-induced activation of the Pit-2 phosphate (Pi) transporter/ amphotropic murine leukemia virus (A-MuLV) receptor. Studies utilizing epitope-tagged Pit-2 now have shown that there is a rapid, significant increase in the phosphorylation state of the Pit-2 protein in response to PMA treatment, indicating that activated PKC epsilon may directly phosphorylate Pit-2 to regulate Pi uptake. Additional studies have been carried out to determine if cell signaling is involved in the regulation of Pit-2 and A-MuLV induced cell-cell fusion (synctium formation). It was found that activation of PKC epsilon is required for A-MuLV-induced synctium formation, and that this process is mediated via changes in the Ras-Raf signaling cascade. In placental development, cytotrophoblasts fuse and differentiate into mutinucleated syncytiotrophoblast which is involved in ion exchange and the synthesis of hormones required for fetal growth and development. Recent studies have shown that syncytin, a retroviral envelope protein, is required for trophoblastic cell fusion. Results of experiments carried out with Dr.Daniele Evain-Brion indicate that changes in both PKC activation and in Cu/Zn superoxide dismutase are involved in regulating the fusion of cytotrophoblasts to multinucleated syncytiotrophoblast. These results suggest that PKC mediation of cell signaling pathways may play an important role in the regulation of virus-induced cell-cell fusion. In other studies to determine biological activities specifically regulated by the different isoforms of protein kinase C, it was found that PDGF- and PMA-induced activation of Raf protein kinase (Raf-PK) was selectively mediated through activation of PKC epsilon. Recent interest has focused on the possible role(s) of reactive oxygen species, including hydrogen peroxide, in the regulation of various signal transduction pathways. In addition to PKC epsilon, oxygen free radicals also appear to play an important role in regulating Raf-PK activity. Exposure of NIH 3T3 cells to hydrogen peroxide resulted in stimulation of Raf-PK activity. Importantly, it was found that simultaneous treatment of cells with hydrogen peroxide and PMA resulted in significant and sustained synergistic activation of Raf-PK. These findings support the suggestion that both protein kinase C epsilon and reactive oxygen species play a role in normal cell signaling pathways, and in the Raf-PK activation process.
