Involvement Of Phospholipase D Research Articles

Phospholipase D, a Novel Therapeutic Target Contributes to the Pathogenesis of Neurodegenerative and Neuroimmune Diseases.

Phospholipase D (PLD) is an enzyme that consists of six isoforms (PLD1-PLD6) and has been discovered in different organisms including bacteria, viruses, plants, and mammals. PLD is involved in regulating a wide range of nerve cells' physiological processes, such as cytoskeleton modulation, proliferation/growth, vesicle trafficking, morphogenesis, and development. Simultaneously, PLD, which also plays an essential role in the pathogenesis of neurodegenerative and neuroimmune diseases. In this review, family members, characterizations, structure, functions and related signaling pathways, and therapeutic values of PLD was summarized, then five representative diseases including Alzheimer disease (AD), Parkinson's disease (PD), etc. were selected as examples to tell the involvement of PLD in these neurological diseases. Notably, recent advances in the development of tools for studying PLD therapy envisaged novel therapeutic interventions. Furthermore, the limitations of PLD based therapy were also analyzed and discussed. The content of this review provided a thorough and reasonable basis for further studies to exploit the potential of PLD in the treatment of neurodegenerative and neuroimmune diseases.

Novel involvement of PLD-PKCδ-CREB axis in regulating FGF-2-mediated pentraxin 3 production in human nasal fibroblast cells.

A higher expression level of mitogenic fibroblast growth factor-2 (FGF-2) has been reported in human nasal mucus of both chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). Meanwhile, we have shown that long pentraxin 3 (PTX3), an essential component of humoral innate immunity that is produced at sites of infection and inflammation, was overproduced in human nasal mucosae and secretions of CRSsNP. Therefore, this study was aimed to investigate how FGF-2 regulates PTX3 expression in human CRSsNP nasal mucosa-derived fibroblast cells (hNMDFs). The FGF-2 treatment caused ptx3 mRNA expression and PTX3 protein induction and secretion. In parallel, a differential expression of FGF receptor (FGFR)-1 to FGFR-4 was observed in hNMDFs and human nasal tissues. While conventionally known PI3K/Akt/mTOR and AP-1 pathways following FGFR activation were shown to be involved, the protein kinase Cδ (PKCδ) and cAMP response element-binding protein (CREB) were also found to be as critical signaling molecules in FGF-2-induced PTX3 induction. The PKCδ and CREB activation could be detected in total cells and in the cell nucleus. Accordingly, a novel CREB binding sequence was detected in the human ptx3 promoter region and could interact with activated CREB in cells challenged with FGF-2. Surprisingly, the phospholipase D (PLD), but not phosphoinositide- and phosphatidylcholine-phospholipase C, was necessarily required for PKCδ and CREB activation. Therefore, we demonstrated here for the first time that FGF-2 mediates PTX3 production not only through PI-3K/Akt/mTOR and AP-1 activation, but also through a novel FGFR-PLD-PKCδ-CREB cellular signaling pathway.

PLD Protein-Protein Interactions With Signaling Molecules and Modulation by PA.

We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein-lipid interactions or as a substrate. The involvement of PLD as an effector of protein-protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2-Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2-PLD2 protein-protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.

Sustained PKCβII activity confers oncogenic properties in a phospholipase D‐ and mTOR‐dependent manner

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCβII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCβII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCβII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCβII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

Phosphatidic acid, phospholipase D and tumorigenesis

Phospholipase D (PLD) is a membrane protein with a double role: maintenance of the structural integrity of cellular or intracellular membranes and involvement in cell signaling through the product of the catalytic reaction, PA, and through protein–protein interaction with a variety of partners. Cross-talk during PLD signaling occurs with other cancer regulators (Ras, PDGF, TGF and kinases). Elevation of either PLD1 or PLD2 (the two mammalian isoforms of PLD) is able to transform fibroblasts and contribute to cancer progression. Elevated total PLD activity, as well as overexpression, is present in a wide variety of cancers such as gastric, colorectal, renal, stomach, esophagus, lung and breast. PLD provides survival signals and is involved in migration, adhesion and invasion of cancer cells, and all are increased during PLD upregulation or, conversely, they are decreased during PLD loss of function. Even-though the end results of PLD action as relates to downstream signaling mechanisms are still currently being elucidated, invasion, a pre-requisite for metastasis, is directly affected by PLD. This review will introduce the classical mammalian PLD’s, PLD1 and PLD2, followed by the mechanisms of intracellular regulation and a status of current investigation in the crucial involvement of PLD in cancer, mostly through its role in cell migration, invasion and metastasis, that has grown exponentially in the last few years.

Arabidopsis phospholipase dδ is involved in basal defense and nonhost resistance to powdery mildew fungi.

Plants have evolved a complex array of defensive responses against pathogenic microorganisms. Recognition of microbes initiates signaling cascades that activate plant defenses. The membrane lipid phosphatidic acid, produced by phospholipase D (PLD), has been shown to take part in both abiotic and biotic stress signaling. In this study, the involvement of PLD in the interaction between Arabidopsis (Arabidopsis thaliana) and the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was investigated. This nonadapted pathogen is normally resisted by a cell wall-based defense, which stops the fungal hyphae from penetrating the epidermal cell wall. Chemical inhibition of phosphatidic acid production by PLD increased the penetration rate of Bgh spores on wild-type leaves. The analysis of transfer DNA knockout lines for all Arabidopsis PLD genes revealed that PLDδ is involved in penetration resistance against Bgh, and chemical inhibition of PLDs in plants mutated in PLDδ indicated that this isoform alone is involved in Bgh resistance. In addition, we confirmed the involvement of PLDδ in penetration resistance against another nonadapted pea powdery mildew fungus, Erysiphe pisi. A green fluorescent protein fusion of PLDδ localized to the plasma membrane at the Bgh attack site, where it surrounded the cell wall reinforcement. Furthermore, in the pldδ mutant, transcriptional up-regulation of early microbe-associated molecular pattern response genes was delayed after chitin stimulation. In conclusion, we propose that PLD is involved in defense signaling in nonhost resistance against powdery mildew fungi and put PLDδ forward as the main isoform participating in this process.

Hyper-activated motility in sperm capacitation is mediated by Phospholipase D-dependent actin polymerization

In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm capacitation, however the role of this process in sperm capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human capacitation. Sperm incubated under capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm capacitation.

Phospholipase D and phosphatidic acid signalling in plant response to drought and salinity

The activity of phospholipase D (PLD) in plants increases under different hyperosmotic stresses, such as dehydration, drought, and salinity. Recent results begin to shed light onto the involvement of PLD in response to water deficits and salinity. Different PLDs have unique and overlapping functions in these responses. PLDalpha1 promotes stomatal closure and reduces water loss. PLDalpha1 and PLDdelta are involved in seedling tolerance to salt stress. PLDalpha3 and PLDepsilon enhance plant growth and hyperosmotic tolerance. The different PLDs regulate the production of phosphatidic acid (PA) that is a key class of lipid mediators in plant response to environmental stresses. Further studies on the upstream regulators that activate different PLDs and the downstream effectors of PLDs and PA have the potential to unveil the linkage between the stimulus perception at the cell membrane to intracellular responses to drought and salinity stresses.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Internalization and signal transduction of PrP 106−126 in neuronal cells

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood steps in the pathogenesis of prion diseases. We have thus analyzed the internalization and signal transduction of the neurotoxic fragment of the prion protein PrP 106–126 in the rat neuroblastoma cell line B104 by fluorescence microscopy and quantification by ELISA and in primary neuronal cells from mice. Phospholipase D (PLD) is known to be an enzyme involved in the regulation of secretion, endocytosis and receptor signalling. We determined the PLD activity using a transphosphatidylation assay and could show that PLD is involved in PrP 106–126 internalization. The determination of receptor activity via quantification of ERK1/2 phosphorylation and cAMP level measurement verified the PrP 106–126-induced signal transduction in B104 cells and primary neuronal cells. PrP 106−126-induced a decrease in cAMP level in neuronal cells. These studies indicate the involvement of PLD in PrP 106–126-endocytosis and mediated cellular signalling by an unidentified inhibitory G-protein-coupled receptor and may allow the development of therapeutic agents interfering with prion uptake and/or PLD function using PLD as a possible pharmaceutical target.

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D (PLD) plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression of rice PLD beta 1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLD beta 1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb expression and inhibiting seed germination.

Phospholipase D1 is up-regulated in the mitochondrial fraction from the brains of Alzheimer's disease patients

Mitochondrial dysfunction may play an important role in sporadic Alzheimer's disease (AD) progression. Recently, we have reported that amyloid precursor protein (APP) stimulates phospholipase D (PLD) activity and β-amyloid (Aβ) region of APP is involved in the interaction with PLD1. To elucidate the involvement of PLD in the pathophysiology of AD, we examined the expression of PLD1 and alteration of membrane phospholipid in mitochondrial membranes of control and AD brains using Western blot and phospholipid analysis by thin layer chromatography (TLC). We have found that protein expression of PLD1 was significantly increased in mitochondrial fraction of brains of AD patients compared with that in control brains. Furthermore, the concentration of mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was increased and the content of phosphatidic acid (PA), a product of PLD activity, was up-regulated in the mitochondrial membrane fractions of AD brain compared with that of control brain. These results suggest that up-regulation of PLD1 in the mitochondrial fraction of AD brain might affect the composition of membrane phospholipids and provide a clue to the mechanism underlying the mitochondrial dysfunction associated with AD.

Phospholipase D1 Regulates Cell Migration in a Lipase Activity-independent Manner

Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.

Elicitor-induced Activation of Phospholipases Plays an Important Role for the Induction of Defense Responses in Suspension-cultured Rice Cells

Biphasic generation of reactive oxygen species (ROS) induced by N-acetylchitooligosaccharide elicitor in rice cells was associated with the activation of phopholipase C (PLC) and phospholipase D (PLD). The activation of both enzymes was observed for the first phase of ROS generation, but only the activation of PLD was evident for the second response. Activation of PLD was associated with its recruitment to the membrane. Enzymatic products of these phospholipases, diacylglycerol (DG) and phosphatidic acid (PA), could induce ROS generation by themselves. Moreover, the addition of these lipids compensated the inhibition of the second phase of ROS generation by cycloheximide, indicating the involvement of the synthesis of PLD or related proteins in the second phase of ROS generation. DG and PA also induced the expression of elicitor-responsive genes in the absence of the elicitor. They could not induce phytoalexin biosynthesis by themselves but greatly enhanced the elicitor-induced phytoalexin accumulation. Further, the inhibition of PLD by 1-butanol inhibited the elicitor-induced phytoalexin accumulation, indicating the involvement of PLD and its reaction product, PA, in the induction of phytoalexin biosynthesis. These results indicated the importance of phospholipid signaling, especially by PLD and its product PA, in plant defense responses.

Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.

Down-regulation of Melanogenesis by Phospholipase D2 through Ubiquitin Proteasome-mediated Degradation of Tyrosinase

The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or alpha-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo.

Involvement of Phospholipase D1 in Melanogenesis of Mouse B16 Melanoma Cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.

Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation

Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm capacitation whereas early activation of PKC during capacitation would jeopardize this process.

Participation of phospholipase D and α/β-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c- fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAβC2-1 peptide (Cl8 HAβC2-1 cells), with a sequence (βC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of α-PKC in PLD activation is further underscored by co-immunoprecipitation of α-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c- fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c- fos mRNA levels was observed in Cl8 HAβC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c- fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAβC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a αPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c- fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.

Phospholipase D Is Required in the Signaling Pathway Leading to p38 MAPK Activation in Neutrophil-like HL-60 Cells, Stimulated by N-Formyl-methionyl-leucyl-phenylalanine

Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The differentiation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using phosphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies showed that increasing concentrations of ethanol or 1-butanol but not 2-butanol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p44/p42 activation. These data indicated that activation of phospholipase D (PLD) was required for activation of p38 but not p44/p42. We compared the effect of fMLP with those of tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that ethanol did not inhibit p38 phosphorylation upon stimulation with either GM-CSF or TNF alpha. These results suggested that in cells stimulated with fMLP, PLD was upstream of p38. To further test the involvement of PLD, we used antisense inhibition of human PLD1 expression. Treatment with antisense oligonucleotides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38 activation in neutrophil-like HL-60 cells. In addition, the results obtained with TNF alpha and GM-CSF demonstrated that p38 activation occurred independently of PLD activation.