title: Phospholipase D activity is required for actin stress fiber formation in fibroblasts.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Kam
authors.forename: J H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016324:Sequence Tagged Sites; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
chemical_list: D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008246:Lysophospholipids; D011994:Recombinant Proteins; D014746:Vimentin; D000185:Actinin; D016596:Vinculin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010739:Phospholipase D; D020741:rho GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate