Association of phospholipase D activity with the detergent-insoluble cytoskeleton of U937 promonocytic leukocytes

Abstract

Phospholipase D (PLD) regulates cytoskeletal-dependent antimicrobial responses of myeloid leukocytes, including phagocytosis and oxidant generation. However, the mechanisms responsible for this association between PLD activity and the actin cytoskeleton are unknown. We utilized a cell-free system from U937 promonocytes to test the hypothesis that stimulation of PLD results in stable association of the activated lipase with the detergent- insoluble membrane skeleton. Plasma membrane and cytosol were incubated ± guanosine 5'-3-O-(thio)triphosphate (GTPγS), followed by re-isolation and extraction of the washed membranes with octyl glucoside. The detergent- insoluble fraction derived from membranes incubated with GTPγS (DIF(GTPγS)) exhibited 22-fold greater PLD activity than that derived from control membranes (DIF0), when both were assayed in the presence of GTPγS. The DIF contained PLD1, RhoA, and ARF, and the level of each was increased by GTPγS in a dose-dependent manner. The DIF also contained F-actin, vinculin, talin, paxillin, and α-actinin, consistent with its identification as the membrane skeleton. The physiologic relevance of these findings was demonstrated by a similar increase in DIF-associated PLD activity after stimulation of intact U937 cells with opsonized zymosan. These results indicate that stimulation of PLD1 is accompanied by stable association of the activated lipase, RhoA, and ADP-ribosylation factor with the actin-based membrane skeleton.