We have studied the signalling of oncogenic Ras proteins in order to more fully understand the transformation process. Many of these studies analyze the effect of overexpression of one specific cellular enzyme, via microinjection and stable transfection of cells. In this project, we have focused on the studies of PLC-gamma. PLC-gamma induced DNA synthesis after microinjection into quiescent NIH 3T3 cells, suggesting a role for PLC-gamma during the G1 restriction point. PLC-gamma1 mutants with defective lipase activity still induced intermediate levels of DNA synthesis. Recently, we have demonstrated that purified PLC-gamma src homology peptides induce DNA synthesis after injection into quiescent NIH 3T3 cells. These results illustrate that in addition to catalytic activity, the structure of a protein also can play an important role in signalling. We are in the process of identifying cellular interacting protein(s) with PLC-gamma To further examine the role of PLC-gamma during the cell cycle and transformation, PLC-gamma was transfected into NIH 3T3 cells and stable transfectants were cloned. The PLC-gamma transfectants (PLC/3T3) expressed elevated levels of PLC and behaved like transformed cells. The PLC/3T3 cells formed foci under G418 selection, grew in an anchorage-independent fashion in soft agar, proliferated in low serum media, have altered cell cycle profiles, and induced tumors after injection into nude mice. This data demonstrates the important role of PLC-gamma in cellular physiology and supports the conclusions drawn from the microinjection studies where PLC-gamma was shown to play a significant role at the G1/S interface.
