EJ-Ras Inhibits Phospholipase Cγ1 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 Goldschmidt-Clermont

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 languageEnglish
Pages (from-to)312-321
Number of pages10
JournalCirculation Research
Volume78
Issue number2
StatePublished - Feb 1 1996
Externally publishedYes

Fingerprint

Phosphatidylinositol 3-Kinase
Polymerization
Actins
NIH 3T3 Cells
Actin Cytoskeleton
Tyrosine
Hydrolysis
Fibroblasts
Phosphatidylinositol 4,5-Diphosphate
Phosphorylation
phospholipase C1
platelet-derived growth factor BB
Stress Fibers
Inositol Phosphates
Phospholipases
Guanosine Triphosphate
Protein Isoforms
Cytoplasm

Keywords

  • Actin
  • EJ-Ras
  • p21
  • Platelet-derived growth factor receptor
  • Tyrosine kinase

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Heldman, A. W., Kandzari, D. E., Tucker, R. W., Crawford, L. E., Fearon, E. R., Koblan, K. S., & Goldschmidt-Clermont, P. (1996). EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase. Circulation Research, 78(2), 312-321.

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 journalArticle

Heldman, AW, Kandzari, DE, Tucker, RW, Crawford, LE, Fearon, ER, Koblan, KS & Goldschmidt-Clermont, P 1996, 'EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase', Circulation Research, vol. 78, no. 2, pp. 312-321.
Heldman, Alan W. ; Kandzari, David E. ; Tucker, Robert W. ; Crawford, Lawrence E. ; Fearon, Eric R. ; Koblan, Kenneth S. ; Goldschmidt-Clermont, Pascal. / EJ-Ras Inhibits Phospholipase Cγ1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase. In: Circulation Research. 1996 ; Vol. 78, No. 2. pp. 312-321.
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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.",
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