Abstract
Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (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 Cγ1 (PLCγ1) 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 PLCγ1, 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 PLCγ1 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 IP, production despite tyrosine phosphorylation of PLCγ1.
| Original language | English |
|---|---|
| Pages (from-to) | 312-321 |
| Number of pages | 10 |
| Journal | Circulation Research |
| Volume | 78 |
| Issue number | 2 |
| State | Published - Feb 1 1996 |
| Externally published | Yes |
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Keywords
- Actin
- EJ-Ras
- p21
- Platelet-derived growth factor receptor
- Tyrosine kinase
ASJC Scopus subject areas
- Physiology
- Cardiology and Cardiovascular Medicine
Cite this
EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase. / Heldman, Alan W.; Kandzari, David E.; Tucker, Robert W.; Crawford, Lawrence E.; Fearon, Eric R.; Koblan, Kenneth S.; Goldschmidt-Clermont, Pascal.
In: Circulation Research, Vol. 78, No. 2, 01.02.1996, p. 312-321.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase
AU - Heldman, Alan W.
AU - Kandzari, David E.
AU - Tucker, Robert W.
AU - Crawford, Lawrence E.
AU - Fearon, Eric R.
AU - Koblan, Kenneth S.
AU - Goldschmidt-Clermont, Pascal
PY - 1996/2/1
Y1 - 1996/2/1
N2 - Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (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 Cγ1 (PLCγ1) 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 PLCγ1, 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 PLCγ1 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 IP, production despite tyrosine phosphorylation of PLCγ1.
AB - Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (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 Cγ1 (PLCγ1) 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 PLCγ1, 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 PLCγ1 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 IP, production despite tyrosine phosphorylation of PLCγ1.
KW - Actin
KW - EJ-Ras
KW - p21
KW - Platelet-derived growth factor receptor
KW - Tyrosine kinase
UR - http://www.scopus.com/inward/record.url?scp=0029670961&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029670961&partnerID=8YFLogxK
M3 - Article
C2 - 8575075
AN - SCOPUS:0029670961
VL - 78
SP - 312
EP - 321
JO - Circulation Research
JF - Circulation Research
SN - 0009-7330
IS - 2
ER -