The aim of this project is to better elucidate the role(s) of specific protein kinases in the regulation of cell growth, malignant transformation, and viral infection. Previous studies established that the Pit2 phosphate (Pi) transporter/amphotropic murine leukemia virus (A-MuLV) receptor is a cellular target of protein kinase C (PKC), and that PKC epsilon is the isoform specifically involved in the up-regulation of Pi uptake by Pit2. Studies now have been carried out to determine if cellular signal transduction pathways and protein kinases also regulate Pit2 virus receptor function and A-MuLV-induced cell-cell fusion (synctium formation). Overexpression of truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide was used to induce viral envelope-mediated cell-cell fusion. This fusion process was dependent on the level of active Pit2 viral receptors present at the cell membrane. Results established that activation of PKC, as well as overexpression of PKC epsilon, enhanced virus-induced cell-cell fusion. Conversely, cell signaling through cyclic AMP to activate protein kinase A blocked this viral fusion process. Other results indicate that modulation of the Ras-Raf signaling cascade also plays an important role in regulating the Pit2 A-MuLV receptor to control virus-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating virus receptor function and virus entry into the cell, and suggest that modulation of these signaling components may be an effective means of altering retroviral infection. In other studies to define the regulatory properties of Pit2, it was noted that an active fraction of Pit2 is found localized to raft lipid microdomains at the plasma membrane. In studies designed to identify cellular proteins that might directly interact with the Pit2 viral receptor, a 24 kD protein was found associated with Pit2 at the membrane. Importantly, activation of protein kinase A was shown to enhance association of this 24 kD protein with the Pit2 receptor. Preliminary evidence suggests that this binding protein may play a role in targeting the active Pit2 viral receptor/phosphate transporter to specific sites on the cell membrane.
