BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.

Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis.

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.

Association of Corynebacterium pseudotuberculosis recombinant proteins rCP09720 or rCP01850 with rPLD as immunogens in caseous lymphadenitis immunoprophylaxis.

Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLD + rCP09720 (G3), and rPLD + rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21 days after the last immunization. The animals were evaluated daily for 40 days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (p < .05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (p < .05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLD + rCP09720), and 50% (rPLD + rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function.

Phospholipase D (PLD) proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Previous studies using inhibitors and overexpression of PLD proteins indicate that PLD1 and PLD2 play positive roles in FcεRI-mediated signaling and mast cell function. We used mice deficient in PLD1, PLD2, or both to study the function of these enzymes in mast cells. In contrast to published studies, we found that PLD1 deficiency impaired FcεRI-mediated mast cell degranulation; however, PLD2 deficiency enhanced it. Biochemical analysis showed that PLD deficiency affected activation of the PI3K pathway and RhoA. Furthermore, our data indicated that, although PLD1 deficiency impaired F-actin disassembly, PLD2 deficiency enhanced microtubule formation. Together, our results suggested that PLD1 and PLD2, two proteins that catalyze the same enzymatic reaction, regulate different steps in mast cell degranulation.

Leptin increases TNF-α expression and production through phospholipase D1 in Raw 264.7 cells.

Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.

Protein kinase C-eta and phospholipase D2 pathway regulates foam cell formation via regulator of G protein signaling 2.

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for Galpha(q), which is involved in regulating various vascular functions. To understand how RGS2 regulates foam cell formation, the present study identified signaling pathways controlled by lipopolysaccharide (LPS) and discovered new mechanisms whereby protein kinase C (PKC)-eta and phospholipase D (PLD) 2 regulate RGS2 expression. The toll-like receptor (TLR) 4 agonist LPS caused foam cell formation of Raw264.7 macrophages and dramatically decreased RGS2 mRNA expression. RGS2 down-regulation by LPS was partially recovered by TLR4 small interfering RNA (siRNA). Peritoneal macrophages were separated from wild-type and TLR4 mutant mice, and treatment with LPS showed RGS2 expression decrease in wild-type macrophages but no change in TLR4 mutant macrophages. RGS2 overexpression was suppressed, whereas RGS2 down-regulation accelerated foam cell formation by LPS. Treatment of PKC-eta pseudosubstrate weakened foam cell formation and recovered RGS2 down-regulation by LPS. In addition, LPS or phorbol 12-myristate 13-acetate stimulated PLD activity, and the pretreatment of PLD inhibitor weakened foam cell formation and recovered RGS2 down-regulation. Inhibition of PLD2 expression by siRNA also weakened foam cell formation and partially recovered LPS-mediated RGS2 down-regulation. On the other hand, PLD2 overexpression intensified RGS2 down-regulation and foam cell formation by LPS. These results suggest that LPS causes foam cell formation by increasing PKC-eta and PLD2 activity by down-regulating RGS2 expression via TLR4 dependently.

House dust mite allergen Der f 2-induced phospholipase D1 activation is critical for the production of interleukin-13 through activating transcription factor-2 activation in human bronchial epithelial cells.

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Der f 2-induced interleukin (IL)-13 production. The major house dust mite allergen, Der f 2, increased PLD activity in human bronchial epithelial cells (BEAS-2B), and dominant negative PLD1 or PLD1 siRNA decreased Der f 2-induced IL-13 expression and production. Treatment of Der f 2 activated the phospholipase Cgamma (PLCgamma)/protein kinase Calpha (PKCalpha)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCgamma inhibitors (U73122 and PAO), PKCalpha inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCgamma, PKCalpha, and p38 MAPK act as upstream activators of PLD in Der f 2-treated BEAS-2B cells. Furthermore, expression and production of IL-13 increased by Der f 2 were also blocked by inhibition of PLCgamma, PKCalpha, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCgamma/PKCalpha/p38 MAPK pathway. We found that activating transcription factor-2 (ATF-2) was activated by Der f 2 in BEAS-2B cells and activation of ATF-2 was controlled by PLD1. When ATF-2 activity was blocked with ATF-2 siRNA, Der f 2-induced IL-13 expression and production were decreased. Thus, ATF-2 might be one of the transcriptional factors for the expression of IL-13 in Der f 2-treated BEAS-2B cells. Taken together, PLD1 acts as an important regulator in Der f 2-induced expression and production of IL-13 through activation of ATF-2 in BEAS-2B cells.

Phospholipase D1 plays a key role in TNF-alpha signaling.

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.

Transcription, expression, localization and immunoreactivity of Chlamydophila pneumoniae Phospholipase D protein.

Chlamydophila pneumoniae, a recognized aethiological agent of respiratory infection, is also suspected to play a immuno-pathogenetic role in atherosclerosis by contributing to inflammation and plaque instability. Phospholipase D (PLD) is an enzyme involved in lipid metabolism, in protein transport and signal transduction, all events which can direct or indirect impact on virulence and inflammatory response. To better understand the role of PLD in cell biology and infection by C. pneumoniae, we cloned and expressed the pld gene in Escherichia coli and generated the recombinant PLD (rCpPLD). This product was highly immunogenic in mice, and capable to efficiently detect anti-PLD antibodies in humans. As shown by real-time PCR, PLD gene was expressed in a bi-phasic pattern, with transcriptional peaks corresponding to early and late chlamydial development. Fluorescence microscopy showed that CpPLD localized mostly in the center of inclusion bodies between 8 and 48h from infection and at the periphery of inclusions at 72h. Overall, PLD appears consistently expressed during the developmental cycle of C. pneumoniae and is sensed by the host as an antigen target during infection/exposure to this microorganism. rCpPLD may be a useful tool for future studies concerning the role that this enzyme plays in the pathology of, and immune response to, C. pneumoniae.

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis.

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development.

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.

Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis.

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55-65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.

Up-regulation of phospholipase Cgamma1 and phospholipase D during the differentiation of human monocytes to dendritic cells.

Phospholipase C (PLC)gamma and phospholipase D (PLD) play pivotal roles in the signal transduction required for various cellular responses, including cell proliferation and differentiation. Dendritic cells (DCs), which are professional antigen-presenting cells, can be generated from human monocytes by stimulating the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). We investigated whether PLCgamma and PLD expression levels can be changed during the differentiation of the human monocytes into DCs. The enzymatic activity and protein level of PLC gamma1 were significantly increased in the human monocyte-derived DCs by GM-CSF/IL-4, but the protein levels of PLC gamma2 were unaltered. Moreover, the enzymatic activity and protein level of PLD1b and PLD2 were up-regulated during the differentiation of human monocytes to DCs, but those of PLD1a were not changed. A higher phagocytic activity of DCs was found to be correlated with the up-regulations of PLCgamma1 and PLD, and the phagocytic activity of DCs was inhibited by a PLC-specific inhibitor (U73122) and by a phosphatidic acid acceptor (n-butanol), but to be increased by phosphatidic acid. Thus, suggesting that PLC and PLD participate in the process. This study suggests that the up-regulations of PLCgamma1 and PLD are accompanied by the differentiation of monocytes into DCs, which results in increased phagocytic activity.

A 90-kD phospholipase D from tobacco binds to microtubules and the plasma membrane.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca(2+)-dependent lipid binding domain and is identical to Arabidopsis PLD delta. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

Tissue-specific self-peptides bound by major histocompatibility complex class I molecules of a human pancreatic beta-cell line.

The process of beta-cell destruction in IDDM is mediated, in part, by CD8+ T-cells. Structural characterization of HLA-I-bound self-peptides presented by the human beta-cell line HP-62 was performed to identify possible tissue-specific autoantigens in the context of CD8+ T-cell/HLA-I interactions. The sequences of the beta-cell line HLA-I-bound peptides were compared with sequence databases. Six of the obtained sequences showed homology to known precursor proteins, three of which--GLUT2 receptor, phosphatidylinositol-glycan-specific phospholipase D, and 5-hydroxytryptamine-1F receptor--have a limited, tissue-specific expression. These HLA-bound self-peptides may be part of a pool of autoantigens recognized by beta-cell reactive cytotoxic T-cells.

Immunoprecipitation of a phospholipase D activity with antiphosphotyrosine antibodies.

When granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated human neutrophils were challenged with the chemotactic factor fMet-Leu-Phe, it was possible to detect a time-dependent increase in the hydrolytic (as measured by the production of phosphatidic acid, PA) and the transphosphatidylation (as measured by the production of phosphatidylethanol, PEt) activities of phospholipase D in intact cells prelabeled with a radioactive fatty acid. Both activities were inhibited by preincubation of cells with genistein. Appropriate conditions were developed to test the PLD transphosphatidylation activity against exogenous phosphatidylcholine (PCho) in an in vitro system. As in intact cells, increased PLD activity could be detected in cell lysates obtained from fMet-Leu-Phe-treated cells compared with controls. When lysates were immunoprecipitated with antiphosphotyrosine antibodies, a PLD activity was found only in immune complexes that were prepared from fMet-Leu-Phe-treated cells. Conversely, no activity was found in lysates immunoprecipitated with an irrelevant antibody (GTPase-activating protein, GAP) that nevertheless was able to recognize a tyrosylphosphorylated form of GAP, as demonstrated by western blotting. These data suggest that a PCho-PLD, or a tightly bound protein, is tyrosine phosphorylated during cell activation.