EJ-RAS INHIBITS PHOSPHOLIPASE C-GAMMA-1 BUT NOT ACTIN POLYMERIZATION INDUCED BY PLATELET-DERIVED GROWTH FACTOR-BB VIA PHOSPHATIDYLINOSITOL 3-KINASE

Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21(ras) (EJ-Ras) produces morphogenic changes characterized by de creased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts los e their ''stressed'' conformation and adopt a ''relaxed'' morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of ac tin filaments in peripheral ruffling membranes. Polymerization of acti n could be induced in EJ-Ras-transformed cells by exposure to platelet -derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase C-gamma 1 (PLC gamma 1) activity, becaus e inositol tris-phosphate (IP3) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimula te tyrosine phosphorylation of PLC gamma 1, the phospholipase was stro ngly inhibited by an inhibitory factor present in the cytoplasm of EJ- Ras-transformed cells. In addition, cytoplasmic extracts of EJ-Ras, bu t not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis catalyzed by a recombinant PLC gamma 1 in vitro. Al though PIP2 hydrolysis could not contribute to the reorganization of t he actin cytoskeleton induced by PDGF-BB in EJ-Ros-transEormed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymeri zation. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras-transformed cells but actua lly led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras-transformed cells, cell morphogenic change s in response to PDGF-BB rely importantly on PI3-K and can occur in th e complete absence of IP3 production despite tyrosine phosphorylation of PLC gamma 1.