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Role of Rho family proteins in phospholipase D activation by growth factors
1Department of Molecular Physiology and Biophysics and Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Abstract:
Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD). In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1. In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor. In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C. Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation. The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1. In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF. We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF. Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA. In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.
Insights
Rac1 mediates epidermal growth factor (EGF)-induced phospholipase D (PLD) activation in fibroblasts. However, Rac1 plays a minimal role in PLD activation by platelet-derived growth factor (PDGF) or protein kinase C, where RhoA is involved.
Area of Science:
- Cell Biology
- Signal Transduction
- Molecular Biology
Background:
- Growth factors activate phospholipase D (PLD) in fibroblasts.
- Rho family GTPases are key regulators of cellular signaling pathways.
Purpose of the Study:
- To elucidate the role of Rac1 and RhoA in growth factor-mediated PLD activation.
- To differentiate the signaling pathways of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in PLD activation.
Main Methods:
- Fibroblast transfection with wild-type and mutant Rac1 constructs (wtRac1, N17Rac1, V12Rac1).
- Measurement of PLD activity in response to EGF, PDGF, and phorbol ester.
- Inhibition of RhoA using C3 transferase from Clostridium botulinum.
- Assessment of phosphoinositide phospholipase C activity.
Main Results:
- Rac1 is essential for EGF-induced PLD activation, as shown by increased activity with wtRac1 and abrogation with N17Rac1.
- Rac1 plays a minimal role in PDGF- or phorbol ester-induced PLD activation.
- RhoA is involved in PDGF-mediated PLD activation.
- Constitutively active V12Rac1 altered cell morphology and basal PLD activity, and attenuated growth factor responses.
Conclusions:
- Rac1 is a major mediator of EGF-induced PLD activation in fibroblasts.
- Rac1 is not significantly involved in PLD activation by PDGF or protein kinase C.
- RhoA plays a role in PDGF-induced PLD activation.