Evidence for Rho-mediated Agonist Stimulation of Phospholipase D in Rat1 Fibroblasts

doi:10.1074/jbc.271.22.13135

Evidence for Rho-mediated Agonist Stimulation of Phospholipase D in Rat1 Fibroblasts

EFFECTS OF CLOSTRIDIUM BOTULINUM

From the Howard Hughes Medical Institute and the Departments of Molecular Physiology and Biophysics and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

^‡ Investigator of the Howard Hughes Medical Institute. To whom all correspondence should be addressed.

Abstract

Small GTP-binding proteins of the Rho family are implicated in the in vitro regulation of phosphatidylcholine hydrolysis by phospholipase D (PLD). However, their role in agonist-stimulated PLD activity in whole cells is not clear. The ribosyltransferase C3 from Clostridium botulinum modifies Rho proteins and inhibits their function. When introduced into rat1 fibroblasts by scrape-loading, C3 inhibited PLD activity stimulated by lysophosphatidic acid (LPA), endothelin-1, or phorbol ester. Neither the time course nor agonist dose response for LPA-stimulated PLD activity was altered in C3-treated cells. In contrast to the effects of C3 on PLD activity, agonist-stimulated phosphatidylinositol-phospholipase C activity was not altered in C3-treated cells. Surprisingly, C3 treatment led to a decrease in the amount of RhoA protein, indicating that the loss of PLD activity in response to agonist was partly due to the loss of Rho proteins. As described previously, C3 treatment led to the inhibition of LPA-stimulated actin filament formation. However, disruption of actin filaments with cytochalasin D caused only a minor inhibition of LPA-stimulated PLD activity. Interestingly, stimulation of cells with LPA caused a rapid enrichment of RhoA in the particulate fraction of cell lysates. These data support an in vivo role for RhoA in agonist-stimulated PLD activity that is separate from its role in actin fiber formation.

Footnotes

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↵¹ The abbreviations used are:

PLD

phospholipase D

GTPγS

guanosine 5′-O-(3-thiotriphosphate)

PtdBut

phosphatidylbutanol

C3 transferase

exoenzyme C3 of Clostridium botulinum

LPA

lysophosphatidic acid

ET-1

endothelin-1

PMA

phorbol 12-myristate 13-acetate

G protein

GTP-binding protein

RhoGDI

Rho GDP dissociation inhibitor

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline

IP

inositol phosphate

PAGE

polyacrylamide gel electrophoresis

PI-PLC

phosphatidylinositol-phospholipase C.
↵² A detailed study of the translocation of Rho-family proteins in response to various agonists is being conducted and will be reported elsewhere (I. Fleming, and J. H. Exton, unpublished observations).
- Received February 28, 1996.