Phospholipase D and RalA cooperate with the epidermal growth factor receptor to transform 3Y1 rat fibroblasts

3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) recept or (EGFR cells) become transformed when treated with EGF. A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activ ity. Ra1A, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras -dependent activation of Ra1A. The activation of PLD by EGF in these cells was dependent upon both Ras and Ra1A. In contrast, EGF-induced activation o f Erk1, Erk2, and Jun kinase was dependent on Ras but independent of Ra1A, indicating divergent pathways activated by EGF and mediated by Ras. The tra nsformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and Ra1A. Importantly, overexpression of wild-type Ra1A or an activate d Ra1A mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells , the EGFR cells not only tolerated PLD1 overexpression but also became tra nsformed in the absence of EGF. These data demonstrate that either Ra1A or PLD1 can cooperate with EGF receptor to transform cells.