Role of Rho Family Proteins in Phospholipase D Activation by Growth Factors

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.

Analysis of Platelet-derived Growth Factor-induced Phospholipase D Activation in Mouse Embryo Fibroblasts Lacking Phospholipase C-γ1

“…1). A small but reproducible, increase in PLD activity with EGF and a contrasting robust PLD response to PDGF have been seen in a variety of cell types, including Rat1 and Swiss 3T3 fibroblasts (12,39). The present study thus adds to earlier data reporting differences in the signal transduction pathways for the two growth factors (40 -42).…”

Bacterial protein toxins and lipids: role in toxin targeting and activity

“…Toxins which modify Rho-GTPases, such as C3 from Clostridium botulinum, are able to inhibit PLD activation triggered by various agonists, such as lysophosphatidic acid, growth factors, and PMA in different cell types (Table 2) (Kuribara et al, 1995;Hess et al, 1997;Bacon et al, 1998;Plonk et al, 1998;Meacci et al, 1999). Large clostridial toxins, such as Clostridium difficile toxin B and toxin A, also block PLD activity induced by PMA, or GTP [S] in vitro, probably by reducing PI4-P 5-kinase activity and decreasing PIP 2 level (Schmidt et al, 1996a;Rümenapp et al, 1998).…”

Involvement of Tyrosine Phosphorylation and Protein Kinase C in the Activation of Phospholipase D by H2O2 in Swiss 3T3 Fibroblasts

“…The cells were serum-starved in Dulbecco's modified Eagle's medium supplemented with 0.5% fatty acid-free bovine serum albumin for 24 h before the start of the assay. The procedures for the assay were those of Hess et al (26). For the final 20 h of serum starvation, the cells were labeled with 1 Ci/ml [9,10-3 H]myristic acid.…”

“…For the final 20 h of serum starvation, the cells were labeled with 1 Ci/ml [9,10-3 H]myristic acid. The cells were washed as described (26) and preincubated with 0.3% butan-1-ol for 10 min. The cells were then treated with H 2 O 2 or PDGF for the indicated times, and phosphatidylbutanol formation was measured as described (26).…”

“…The cells were washed as described (26) and preincubated with 0.3% butan-1-ol for 10 min. The cells were then treated with H 2 O 2 or PDGF for the indicated times, and phosphatidylbutanol formation was measured as described (26). Radioactivity incorporated into total phospholipids was measured and used to normalize the results.…”