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. When cells expressing epitope-tagged Pit-2 were infected with A-MuLV the tagged transporters/viral receptors were no longer detected at the plasma membrane, but rather were found internalized as an intracellular pool colocalized with the the A-MuLV gp70 envelope protein. The intracellular pool of Pit-2 observed in A-MuLV infected cells was found to be a lower molecular weight form of Pit-2. This suggests that Pit-2 present in these intracellular complexes may not undergo post translational modifications required for normal Pit-2 processing and trafficking to the plasma membrane. 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. Activation of Ras/PI3 kinase signalling appears to be involved as an initial step in this hydrogen peroxide pathway to stimulate Raf-PK. Further, it was found that exposure of ras-transformed NIH 3T3 cells to hydrogen peroxide, and to PMA, caused significant, and sustained, activation of Raf-PK. These findings support the suggestion that reactive oxygen species may play a role in normal cell signaling pathways, and in the Raf-PK activation process.