Aw. Heldman et al., EJ-RAS INHIBITS PHOSPHOLIPASE C-GAMMA-1 BUT NOT ACTIN POLYMERIZATION INDUCED BY PLATELET-DERIVED GROWTH FACTOR-BB VIA PHOSPHATIDYLINOSITOL 3-KINASE, Circulation research, 78(2), 1996, pp. 312-321
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.