PLD Activity Research Articles

The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells

Decidualization is a biological and morphological process occurring in hES (human endometrial stromal) cells. Previously, we reported that PLD1 (phospholipase D1) plays an important role in cAMP-induced decidualization of hES cells. In the present study, we focused on how PLD1 expression is up-regulated during decidualization. Treatment with PKA (protein kinase A) inhibitors (Rp-cAMP or H89) or a Ras inhibitor (manumycin) partially inhibited PLD1 expression and decidua formation in response to cAMP treatment. Interestingly, dual inhibition of PKA and Ras completely inhibited PLD1 expression and cAMP-induced decidualization. These results suggest that PLD1 expression during decidualization is controlled additively by PKA and Ras. The use of inhibitors showed that extracellular-signal-regulated kinase, a downstream effector of Ras, was required for PLD activation and the morphological changes during decidualization, but not for the increase in PLD1 protein. Next, to investigate the regulator of the PLD1 gene at the transcriptional level, a promoter assay using deletion mutants of the PLD1 promoter was performed; the result indicated that PR (progesterone receptor) was a possible regulator of the PLD1 gene. In addition, chromatin immunoprecipitation assays on the PLD1 promoter identified PR as a transcription factor for PLD1 expression during 8-Br-cAMP-induced decidualization. Taken together, our findings demonstrate that PKA and Ras are novel regulators of PLD1 expression and also identify PR as a transcription factor for PLD1 expression during the decidualization of hES cells.

Phospholipase D1, but Not D2, Regulates Protein Secretion Via Rho/ROCK in a Ras/Raf-Independent, MEK-Dependent Manner in Rat Lacrimal Gland

A prior study showed that cholinergic agonists activate phospholipase D (PLD). The purpose of this study was to determine whether cholinergic agonists use the PLD pathway to alter protein secretion and to identify the molecular signaling components of this pathway in rat lacrimal gland acini. Rat lacrimal gland acini were isolated by collagenase digestion. Presence and localization of PLD1 and -2 were determined by immunofluorescence and Western blot experiments. Acini were incubated with adenoviruses overnight or the inhibitors 1-butanol, Y-27632, or C3 exotoxin before stimulation with the cholinergic agonist carbachol (Cch, 10(-4) M) for 5 minutes. Western blot analysis was performed for 20 minutes, and protein secretion was measured spectrophotometrically. Activation of ERK, MEK, Pyk2, Ras, and Raf was determined by Western blot analysis. 1-Butanol increased Cch-stimulated protein secretion and decreased ERK activity. Incubation with catalytically inactive PLD1, but not catalytically inactive mutant PLD2 adenovirus, also increased Cch-stimulated protein secretion and decreased ERK activity. Inhibition of Rho with C3 exotoxin and a dominant negative Rho adenovirus and inhibition of ROCK with Y-27632 inhibited Cch-stimulated PLD1 activity, increased protein secretion, and decreased ERK activity. The association of PLD1 and ROCK increased with Cch stimulation, as determined by immunoprecipitation. PMA-stimulated ERK activity was also inhibited by 1-butanol. 1-Butanol had no effect on Cch-stimulated Pyk2, Ras, and Raf activity, but decreased MEK activity. Cholinergic agonists activate PLD1 through Rho and ROCK, which in turn activate MEK and ERK, which attenuate protein secretion in freshly isolated epithelial cells.

Phospholipase D regulates vascular smooth muscle tone in mice

Smooth muscle tone is balanced by a number of intracellular signaling pathways which induce either relaxation or contraction. Pathways that induce contraction include activation of CaV1.2 channels by depolarization, initiation of Ca2+ release from intracellular stores, and inhibition of myosin phosphatase by Rho kinase. Here, we present evidence that phospholipase D (PLD) is involved in alpha-adrenergic-mediated contraction of vascular smooth muscle. Western blot analysis revealed the presence of PLD1 in vascular smooth muscle. In functional studies, the PLD-inhibitor butanol-1 relaxed pre-contracted aorta from wild type mice to a larger extent than the inactive congener butanol-2. The specific effect of butanol-1 was absent in the presence of the CaV1.2 channel blocker isradipine, in pre-contracted aorta from CaV1.2-deficient mice, and in pre-contracted aorta from PLD1/− mice. No specific effect of butanol-1 was observed in aorta from wild type mice being contracted by high extracellular potassium. We suggest that alpha-adrenergic contraction of vascular smooth muscle involves partially activation of PLD1 which probably maintain CaV1.2 channel activity during contraction.

IL-15 promotes osteoclastogenesis via the PLD pathway in rheumatoid arthritis (148.6)

Abstract IL-15 is a pleiotropic proinflammatory cytokine that appears to help mediate the pathological bone loss. This study was undertaken to explore the signaling molecules essential for osteoclastogenesis mediated by IL-15 in rheumatoid synovial fibroblasts. The levels of RANKL and PLD1 but not PLD2 were upregulated significantly by IL-15, and the RANKL level was significantly upregulated by PA in rheumatoid synovial fibroblasts. Blocking PA production with 1-butanol and siRNA against PLD1 significantly inhibited the IL-15-stimulated expression of RANKL and PLD1. IL-15 levels were significantly higher in serum and synovial fluid from patients with RA than in osteoarthritis patients and healthy controls. IL-15 and PA induced osteoclast formation through the MAPKs and NF-kB signaling pathways. Conclusion. Activation of PLD1 contributes to IL-15-mediated osteoclastogenesis via the MAPKs and NF-kB signaling pathways in rheumatoid synovial fibroblasts. Our data suggest that PLD1 might be an efficient therapeutic strategy for preventing bone destruction in rheumatoid arthritis

The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association

Membrane ruffling is the formation of actin rich membrane protrusions, essential for cell motility. The exact mechanism of ruffling is not fully known. Using YFP and CFP fluorescent chimeras, we show for the first time a co-localization of Phospholipase D2 (PLD2) and Growth factor Receptor Bound protein-2 (Grb2) with actin-rich membrane protrusions of macrophages. Grb2 cooperates with PLD2 in enhancing membrane ruffling, whether in resting cells or in cells stimulated with the growth factor M-CSF, although in the latter an increase in dorsal ruffles was observed, consistent with receptor–ligand internalization. Cells transfected with PLD2 mutated in the PH domain (Y169F) or with Grb2 mutated in the SH2 site (R86K) negate this effect, indicating an association PLD2(Y169)-SH2-Grb2 that was confirmed by immunoprecipitation and Western blotting. The association results in enhanced PLD activity, but the lipase activity can only partially explain the formation of membrane ruffles in vivo. A third component involves the Rho-GTPase Rac2 and it is only when Rac2 is overexpressed along with PLD2 and Rac2 that a full biological effect, including actin polymerization in vivo, is obtained. The mechanism involved is, then, as follows: PLD enzymatic action, after having been increased due to the binding to Grb2-SH2 via Y169, cooperates with Rac2, and the three molecules stimulate actin polymerization and consequently, membrane ruffle formation. Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes.

The TSP motif in AP180 inhibits phospholipase D1 activity resulting in increased efficacy of anticancer drug via its direct binding to carboxyl terminal of phospholipase D1

Phospholipase D (PLD) has two isoforms, PLD1 and PLD2. Both isoforms are possible candidates for the development of anticancer drugs, since PLDs in several cancer cells act as survival factors. The aim of this study was to elucidate the inhibitory mechanism of PLD1 by AP180 in human cancer cells. Transfection of the human AP180 ( hAP180) gene markedly inhibited phobol-12-myristate 13-acetate-induced PLD activity resulting in exacerbation of anticancer drug-induced cell death. Experiments using deletion mutants of hAP180 showed that three amino acids (Thr312–Pro314) are critical for inhibition of PLD1 activity by binding directly to PLD1, and, of these, Ser313 was the most important residue for both binding to and inhibiting PLD1. However, this inhibitory relationship did not exist between hAP180 and PLD2. In addition, the C-terminal region of PLD1 is important for the interaction with hAP180. These results indicated that Thr312–Pro314 (especially Ser313 as a phosphorylation residue) of hAP180 can regulate hPLD1 activity through binding with the C-terminal region of PLD1.

Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase Cα and Extracellular Signal-regulated Kinase Signaling Pathways

Recent studies highlight the existence of a nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PC as a source of second messengers and, particularly, nuclear localization of PC-specific phospholipase D (PLD). In the present study we have identified the nuclear localization sequence (NLS) of PLD1 whose mutation abolished its nuclear import. Recently, we reported that caspase-mediated cleavage of PLD1 generates the N-terminal fragment (NF-PLD1) and C-terminal fragment (CF-PLD1). Here we show that CF-PLD1 but not NF-PLD1, is exclusively imported into the nucleus via its functional NLS, whereas only some portions of intact PLD1 were localized into the nucleus. The NLS of intact PLD1 or CF-PLD1 is required for interaction with importin-β, which is known to mediate nuclear import. The amount of intact PLD1 or CF-PLD1 translocated into nucleus is correlated with its binding affinity with importin-β. Ultimately, nuclear localization of intact PLD1 but not CF-PLD1 mediates the activation of nuclear protein kinase Cα and extracellular signal-regulated kinase signaling pathways. Taken together, we propose that nuclear localization of PLD1 via the NLS and its interaction with importin-β may provide new insights on the functional role of nuclear PLD1 signaling.

Dissecting the Conserved NPxxY Motif of the M3 Muscarinic Acetylcholine Receptor: Critical Role of Asp-7.49 for Receptor Signaling and Multiprotein Complex Formation

Acetylcholine challenge produces M₃ muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M₃ muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα_q/11 after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M₃R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M₃ muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.

LysoPC and PAF Trigger Arachidonic Acid Release by Divergent Signaling Mechanisms in Monocytes

Oxidized low-density lipoproteins (LDLs) play an important role during the development of atherosclerosis characterized by intimal inflammation and macrophage accumulation. A key component of LDL is lysophosphatidylcholine (lysoPC). LysoPC is a strong proinflammatory mediator, and its mechanism is uncertain, but it has been suggested to be mediated via the platelet activating factor (PAF) receptor. Here, we report that PAF triggers a pertussis toxin- (PTX-) sensitive intracellular signaling pathway leading to sequential activation of sPLA2, PLD, cPLA2, and AA release in human-derived monocytes. In contrast, lysoPC initiates two signaling pathways, one sequentially activating PLD and cPLA2, and a second parallel PTX-sensitive pathway activating cPLA2 with concomitant activation of sPLA2, all leading to AA release. In conclusion, lysoPC and PAF stimulate AA release by divergent pathways suggesting involvement of independent receptors. Elucidation of monocyte lysoPC-specific signaling mechanisms will aid in the development of novel strategies for atherosclerosis prevention, diagnosis, and therapy.

The phospholipase D1 pathway modulates macroautophagy

Although macroautophagy is known to be an essential degradative process whereby autophagosomes mediate the engulfment and delivery of cytoplasmic components into lysosomes, the lipid changes underlying autophagosomal membrane dynamics are undetermined. Here, we show that phospholipase D1 (PLD1), which is primarily associated with the endosomal system, partially relocalizes to the outer membrane of autophagosome-like structures upon nutrient starvation. The localization of PLD1, as well as the starvation-induced increase in PLD activity, are altered by wortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting PLD1 may act downstream of Vps34. Pharmacological inhibition of PLD and genetic ablation of PLD1 in mouse cells decreased the starvation-induced expansion of LC3-positive compartments, consistent with a role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of Tau and p62 aggregates in organotypic brain slices. Our in vitro and in vivo findings establish a role for PLD1 in autophagy.

Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.

An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils

Although phosphatidic acid (PA) regulates a wide variety of physiological processes, its targets remain poorly characterized in human neutrophils. By co-sedimentation with PA-containing vesicles we identified several PA-binding proteins including vesicle amine transport protein-1 (VAT-1), Annexin A3 (ANXA3), Rac2, Cdc42 and RhoG in neutrophil cytosol. Except for ANXA3, protein binding to PA-containing liposomes was calcium-independent. Cdc42 and RhoG preferentially interacted with PA whereas VAT-1 bound to PA or phosphatidylserine with the same affinity. VAT-1 translocated to neutrophil membranes upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation. Inhibition of fMLF-induced PLD activity with the Src kinase inhibitor PP2, the selective inhibitor of PLD FIPI, or of PA formation with primary alcohols reduced VAT-1 translocation. In contrast, inhibition of PA hydrolysis with propranolol enhanced fMLF-mediated VAT-1 recruitment to membranes. PMA also redistributed VAT-1 to membranes in a PKC- and PLD-dependent manner. Though fMLF and PMA increased VAT-1 phosphorylation, different kinases appear to be involved. Cell fractionation revealed that a pool of VAT-1 was co-localized with primary, secondary and tertiary granules and plasma membrane markers in resting neutrophils. Stimulation with fMLF enhanced VAT-1 co-localization with CD32a, a plasma membrane marker. Confocal microscopy revealed that VAT-1 decorates granular structures at the cell periphery and double labeling with VAT-1/lactoferrin antibodies showed a partial co-localization with secondary granules in control and fMLF-stimulated cells. Characterization of these putative PA-binding proteins constitutes another step forward for a better understanding of the role of PLD-derived PA in neutrophil physiology.

An oleate-stimulated, phosphatidylinositol 4,5-bisphosphate-independent phospholipase D in Schizosaccharomyces pombe

Phospholipase D1 (PLD1) is an important enzyme involved in lipid-mediated signal transduction and membrane dynamics in eukaryotes. PLD1 preferentially hydrolyzes phosphatidylcholine to phosphatidic acid. This potent second messenger is involved in cytoskeletal reorganization, secretion, and membrane trafficking in eukaryotic cells. In Saccharomyces cerevisiae, PLD1 is involved in polarized growth and morphogenesis during pheromone response and sporulation. The presence of a PLD activity in Schizosaccharomyces pombe is demonstrated. PLD activity was able to hydrolyze a fluorescently labeled analog of phosphatidylcholine and was capable of performing the transphosphatidylation reaction characteristic of PLDs. Schizosaccharomyces pombe PLD activity was unaffected by phosphatidylinositol 4,5 bisphosphate (PIP(2)), but was slightly stimulated by oleate. PLD activity was shown to increase when the S. pombe cells underwent mating and sporulation. Here, we also report the molecular cloning of the first phospholipase D isoform from an S. pombe genomic DNA library (EMBL accession no. FN547388). Comparisons of three divergent yeasts, S. pombe, S. cerevisiae, and Candida albicans, with respect to the PLD enzymes revealed differences in regulation by oleate and PIP(2). Even with high homology in the protein sequences between the PLD1 enzymes of S. cerevisiae, C. albicans, and S. pombe, there was variation with the effects of the regulators.

Phosphatidic Acid Plays a Regulatory Role in Clathrin-mediated Endocytosis

Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.

Protein Kinase C-η 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.

The sphingosine kinase‐1 survival pathway is a molecular target for the tumor‐suppressive tea and wine polyphenols in prostate cancer

The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway has been associated with cancer promotion and progression and resistance to treatments in a number of cancers, including prostate adenocarcinoma. Here we provide the first evidence that dietary agents, namely, epigallocatechin gallate (EGCg, IC(50)≈75 μM), resveratrol (IC(50)≈40 μM), or a mixture of polyphenols from green tea [polyphenon E (PPE), IC(50)≈70 μM] or grapevine extract (vineatrol, IC(50)≈30 μM), impede prostate cancer cell growth in vitro and in vivo by inhibiting the SphK1/S1P pathway. We establish that SphK1 is a downstream effector of the ERK/phospholipase D (PLD) pathway, which is inhibited by green tea and wine polyphenols. Enforced expression of SphK1 impaired the ability of green tea and wine polyphenols, as well as pharmacological inhibitors of PLD and ERK activities, to induce apoptosis in PC-3 and C4-2B cells. The therapeutic efficacy of these polyphenols on tumor growth and the SphK1/S1P pathway were confirmed in animals using a heterotopic PC-3 tumor in place model. PC-3/SphK1 cells implanted in animals developed larger tumors and resistance to treatment with polyphenols. Furthermore, using an orthotopic PC-3/GFP model, the chemopreventive effect of an EGCg or PPE diet was associated with SphK1 inhibition, a decrease in primary tumor volume, and occurrence and number of metastases. These results provide the first demonstration that the prosurvival, antiapoptotic SphK1/S1P pathway represents a target of dietary green tea and wine polyphenols in cancer.

Phospholipase D1 Drives a Positive Feedback Loop to Reinforce the Wnt/β-Catenin/TCF Signaling Axis

Activation of the Wnt signaling pathway occurs frequently in human cancers, but an understanding of the targets and regulation of this important pathway remains incomplete. In this study, we report that phospholipase D (PLD), a cell survival mediator that is upregulated in cancer, is an important target of the Wnt signaling pathway that functions in a positive feedback loop to reinforce pathway output. PLD1 expression and activity was enhanced by treatment with Wnt3a and glycogen synthase kinase-3 inhibitors, and the Wnt pathway-regulated transcription factors beta-catenin and TCF-4 were required for this effect. Three functional TCF-4-binding sites were identified within the PLD1 promoter. Interestingly, suppressing PLD1 blocked the ability of beta-catenin to transcriptionally activate PLD1 and other Wnt target genes by preventing beta-catenin/TCF-4 complex formation. Conversely, tactics to elevate intracellular levels of phosphatidic acid, the product of PLD1 enzyme activity, enhanced beta-catenin/TCF-4 complex formation as well as beta-catenin-dependent TCF transcriptional activity. In cell-based assays, PLD1 was necessary for the anchorage-independent growth driven by Wnt/beta-catenin signaling, whereas beta-catenin/TCF-4 was necessary for the anchorage-independent growth driven by PLD1 activation. Taken together, our findings define a function for PLD1 in a positive feedback loop of Wnt/beta-catenin/TCF-4 signaling that provides new mechanistic insights into cancer, with implications of novel strategies to disrupt Wnt signaling in cancer.

A Comprehensive Model That Explains the Regulation of Phospholipase D2 Activity by Phosphorylation-Dephosphorylation

We report here that the enzymatic activity of phospholipase D2 (PLD2) is regulated by phosphorylation-dephosphorylation. Phosphatase treatment of PLD2-overexpressing cells showed a biphasic nature of changes in activity that indicated the existence of "activator" and "inhibitory" sites. We identified three kinases capable of phosphorylating PLD2 in vitro-epidermal growth factor receptor (EGFR), JAK3, and Src (with JAK3 reported for the first time in this study)-that phosphorylate an inhibitory, an activator, and an ambivalent (one that can yield either effect) site, respectively. Mass spectrometry analyses indicated the target of each of these kinases as Y(296) for EGFR, Y(415) for JAK3, and Y(511) for Src. The extent to which each site is activated or inhibited depends on the cell type considered. In COS-7, cells that show the highest level of PLD2 activity, the Y(415) is a prominent site, and JAK3 compensates the negative modulation by EGFR on Y(296). In MCF-7, cells that show the lowest level of PLD2 activity, the converse is the case, with Y(296) unable to compensate the positive modulation by Y(415). MTLn3, with medium to low levels of lipase activity, show an intermediate pattern of regulation but closer to MCF-7 than to COS-7 cells. The negative effect of EGFR on the two cancer cell lines MTLn3 and MCF-7 is further proven by RNA silencing experiments that yield COS-7 showing lower PLD2 activity, and MTLn3 and MCF-7 cells showing an elevated activity. MCF-7 is a cancer cell line derived from a low-aggressive/invasive form of breast cancer that has relatively low levels of PLD activity. We propose that PLD2 activity is low in the breast cancer cell line MCF-7 because it is kept downregulated by tyrosyl phosphorylation of Y(296) by EGFR kinase. Thus, phosphorylation of PLD2-Y(296) could be the signal for lowering the level of PLD2 activity in transformed cells with low invasive capabilities.

Dependence of Phospholipase D1 Multi-monoubiquitination on Its Enzymatic Activity and Palmitoylation

Phospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1 H896R, K898R, or D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1 R691G,R695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrantly enlarged vesicles partially co-localized with the Golgi complex but not with early endosomes. The altered localization was reduced by the K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not of PLD1 K898R or PLD2, was blocked by inhibitors of proteasomes but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking between different membrane compartments.

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

BackgroundGlucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.Methodology/Principal FindingsHere, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for 14C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in 14C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of 14C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate 14C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate 14C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.Conclusions/SignificanceThese results suggest that AMPK-mediated PLD1 activation is required for 14C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake.