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(Circulation Research. 1996;78:312-321.)
© 1996 American Heart Association, Inc.

Articles

EJ-Ras Inhibits Phospholipase C₁ but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase

Alan W. Heldman, David E. Kandzari, Robert W. Tucker, Lawrence E. Crawford, Eric R. Fearon, Kenneth S. Koblan, Pascal J. Goldschmidt-Clermont

From the Department of Medicine, Division of Cardiology (A.W.H., D.E.K., L.E.C., P.J.G.-C.), the Department of Oncology (R.W.T.), and the Department of Cell Biology and Anatomy (R.W.T., P.J.G.-C.) of the Johns Hopkins University School of Medicine, Baltimore, Md; the Department of Pathology (E.R.F.), Yale University School of Medicine, New Haven, Conn; and the Department of Cancer Research (K.S.K.), Merck Research Laboratories, West Point, Pa.

Correspondence to Dr Pascal J. Goldschmidt-Clermont, 1023 Ross, Johns Hopkins University, 720 Rutland Ave, Baltimore MD 21287.

Abstract Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21^ras (EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose 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 actin filaments in peripheral ruffling membranes. Polymerization of actin 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 C₁ (PLC1) activity, because inositol tris-phosphate (IP₃) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimulate tyrosine phosphorylation of PLC1, the phospholipase was strongly inhibited by an inhibitory factor present in the cytoplasm of EJ-Ras–transformed cells. In addition, cytoplasmic extracts of EJ-Ras, but not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP₂) hydrolysis catalyzed by a recombinant PLC1 in vitro. Although PIP₂ hydrolysis could not contribute to the reorganization of the actin cytoskeleton induced by PDGF-BB in EJ-Ras–transformed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymerization. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras–transformed cells but actually led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras–transformed cells, cell morphogenic changes in response to PDGF-BB rely importantly on PI3-K and can occur in the complete absence of IP₃ production despite tyrosine phosphorylation of PLC1.

Key Words: actin • platelet-derived growth factor receptor • tyrosine kinase • p21^ras • EJ-Ras

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