Phospholipase D activity is required for actin stress fiber formation in fibroblasts

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined fun ction. In order to explore its cellular actions, we inactivated the rat PLD 1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). T his was stably expressed in Rat-2 fibroblasts to see if it acted as a domin ant-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 express ing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by similar to 50%, while the PLD a ctivity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in r esponse to LPA or PMA were examined in these clones. All three clones expre ssing rPLD1 V5 failed to form actin stress fibers after treatment,vith 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 cha nge in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. S ince Rho is an activator both of rPLD1 and stress fiber formation, the acti vation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin me diated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also s howed normal vinculin-containing focal adhesions. However, the translocatio n of alpha -actinin to the cytoplasm and to the detergent-insoluble fractio n in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form s tress fibers and that PLD might be involved in the cross-linking of actin f ilaments mediated by alpha -actinin.