H-RAS AND RAF-1 COOPERATE IN TRANSFORMATION OF NIH3T3 FIBROBLASTS

We examined the effect of overexpression of growth factor-regulated se cond messenger enzymes, alone and in combination, on transformation of NIH3T3 cells. Signal transducers included phospholipase C-gamma (PLC- gamma), protein kinase C-gamma (PKC-gamma), and two proto-oncogenes, c -H-ras and c-raf-1. Three of these proteins, PLC-gamma PKC-gamma and R af-1, did not transform NIH3T3 cells alone or in combination. c-H-ras, which under its own promoter control has low transforming activity, a lso did not cooperate with PLC-gamma or PKC-gamma. In contrast, the co mbination of normal or oncogenic p21 H-Ras with the Raf-I kinase drama tically increased transformation efficiency. The level of Ras protein required for transformation was reduced in Raf-1 co-transfectants, imp lying that, at low levels of p21 Ras, p74 Raf-I is rate limiting. As t ransformation by Ras depends on jun-mediated transcriptional events, w e also examined H-ras and c-raf-1 cooperation in transcriptional trans activation of TPA-responsive element (TRE)-dependent reporters. Like t he H-ras/c-raf-1 cooperation in transformation, we observed this syner gistic stimulation of TRE-dependent transcription. This pathway for tr ansformation and transcriptional activation by increased levels of nor mal Ras and Raf may be important in tumors that show overexpression bu t lack mutationally activated forms of these two proto-oncogenes.