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Molecular and Cellular Biology, June 2001, p. 4055-4066, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.4055-4066.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Phospholipase D Activity Is Required for Actin Stress Fiber Formation in Fibroblasts

Yoonseok Kam and John H. Exton*

Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 2 November 2000/Returned for modification 30 November 2000/Accepted 26 March 2001

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha -actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha -actinin.

* Corresponding author. Mailing address: Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 322-6494. Fax: (615) 322-4381. E-mail: john.exton{at}mcmail.vanderbilt.edu.

Molecular and Cellular Biology, June 2001, p. 4055-4066, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.4055-4066.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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