ROLE OF RHO-FAMILY PROTEINS IN PHOSPHOLIPASE-D ACTIVATION BY GROWTH-FACTORS

Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho famil y of small GTPases in growth factor-mediated PLD activation, we used c ells transfected with wild type and mutant Rac1. In response to epider mal growth factor (EGF), PLD activity was greatly increased in Rat1 fi broblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1 consistent with Rac1 me diating the action of this growth factor. In contrast, in cells treate d with platelet-derived growth factor (PDGF) or phorbol ester, the wtR ac1 cells showed little or no enhancement of PLD activity, and the res ponse was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expre ssing constitutively active V12Rac1, but these cells showed other chan ges, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDG F on phosphoinositide phospholipase C activity were not enhanced in ce lls expressing wtRac1 or inhibited in those expressing N17Rac1. In cel ls expressing constitutively active V12Rac1, basal phosphoinositide ph ospholipase C was elevated, but there were no significant effects of E GF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP ribosylates and inactivates RhoA, to investigate the involvement of R hoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17R ac1 showed a decreased PLD in response to PDGF when treated with C3 tr ansferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGP o r protein kinase C.