Structural analysis of human phospholipase D1

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been proposed to play roles in numerous cellular pathways including signal transduction and membrane vesicular trafficking. We previously reported the cloning of two mammalian genes, PLD1 and PLD2, that encode PLD activities. We additionally reported that PLD1 is activated in a synergistic manner by protein kinase c-alpha (PKC-alpha), ADP-ribosylation factor 1 (ARF1), and Rho family members. We describe here molecular analysis of PLD1 using a combination of domain deletion and mutagenesis. We show that the amino-terminal 325 amino acids are required for PKC-alpha activation of PLD1 but not for activation by ARF1 and RhoA. This region does not contain the sole PKC-alpha interaction site and additionally functions to inhibit basal PLD activity in vivo. Second, a region of sequence unique to PLD1 (as compared with other PLDs) known as the "loop" region had been proposed to serve as an effector regulatory region but is shown here only to mediate inhibition of PLD1. Finally, we show that modification of the amino terminus, but not of the carboxyl terminus, is compatible with PLD enzymatic function and propose a simple model for PLD activation.     

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

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

Toxicity grading systems. A comparison between the WHO scoring system and the Common Toxicity Criteria when used for nausea and vomiting

Human neutrophil PLD activity stimulated with GTP-gamma-S was reconstituted with recombinant ARF1 in cytosol-depleted cells. PMA-pretreatment of intact cells greatly enhanced the subsequent reconstitution of the ARF1-regulated PLD activity. This enhancement was only observed provided that the intact cells were pretreated with PMA, suggesting the stable recruitment of a cytosolic component, presumably protein kinase C, to the membranes. rARF1-reconstituted PLD activity was not dependent on MgATP, but could be considerably enhanced by MgATP. Maximal effects of MgATP were seen at 1 mM. This enhancement by MgATP could not be attributed to protein kinase C. Neomycin was found to inhibit ARF1-regulated PLD activity suggesting the requirement for polyphosphoinositides. We conclude: (i) that many of the observed effects of PMA may be dependent on the presence of the small GTP-binding protein, ARF, and (ii) polyphosphoinositides are required for ARF1-stimulated PLD activity.     

ADP-ribosylation factor proteins mediate agonist-induced activation of phospholipase D

The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes. Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D. Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells. In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity. However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA. These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.     

ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils

Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads to loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils. PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulated PLD activity. C3 toxin, which inactivates Rho proteins, reduces the ARF-reconstituted PLD activity, illustrating that although Rho alone does not stimulate PLD activity, it synergizes with ARF. To identify the signaling pathway to ARF and Rho activation by fMLP, we used pertussis toxin and wortmannin to examine the requirement for heterotrimeric G proteins of the Gi family and for phosphoinositide 3-kinase, respectively. PLD activity in both intact cells and the ARF-restored response in cytosol-depleted cells is inhibited by pertussis toxin, indicating a requirement for Gi2/Gi3 protein. In contrast, wortmannin inhibited only fMLP-stimulated PLD activity in intact neutrophils, but it has no effect on myr-ARF1-reconstituted activity. fMLP-stimulated translocation of ARF and Rho proteins to membranes is not inhibited by wortmannin. It is concluded that activation of Gi proteins is obligatory for ARF/Rho activation by fMLP, but activation of phosphoinositide 3-kinase is not required.     

Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.     

"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.     

Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation

We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.     

Collagen injection therapy for post-prostatectomy incontinence

Serial deletions of the N-terminal 319 amino acids of rPLD1 expressed in COS-7 cells resulted in increased basal PLD activity. Incubation of the cells with phorbol myristate acetate increased the activity of endogenous and wild-type rPLD1. The mutant rPLD1 with deletion of the first 50 amino acids responded to the phorbol ester, however, rPLD1 with deletions of 115 amino acids or more did not. In cells in which constitutively active V14RhoA was co-expressed with the mutant PLDs, stimulation of PLD activity was observed with all deletion mutants. In membranes from COS-7 cells in which the mutant PLDs were expressed, only the mutant with deletion of 50 N-terminal amino acids responded to added protein kinase C-alpha and phorbol ester, in agreement with the in vivo studies. When myristoylated ADP-ribosylation factor 3 (mARF3) was added together with guanosine 5'-3-O-(thio)triphosphate, all mutants showed stimulation of PLD activity. It is concluded that the site of interaction of protein kinase C with rPLD1 is located in the N-terminal region and that Rho and ARF interact at other sites.     

Effects of brefeldin A on phosphatidylcholine phospholipase D and inositolphospholipid metabolism in HL-60 cells

The involvement of the small GTP-binding protein ADP-ribosylation factor (ARF) in guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent activation of phospholipase D (PLD) in HL-60 cells has been well established in vitro. In this study, we tested the effect of brefeldin A, which prevents ARF activation by inhibiting guanine-nucleotide-exchange activity, on PLD stimulation by receptor agonists (formyl-Met-Leu-Phe and ATP) and by the phorbol ester phorbol 12-myristate 13-acetate (PMA) in differentiated HL-60 cells. However, brefeldin A did not affect the activation of PLD at a time (1 h) when it eliminated the activity of the trans-Golgi enzyme galactosyltransferase. It also did not inhibit PLD activity in Golgi-enriched membranes treated with GTP[S] with or without ARF in vitro. However, longer times of brefeldin A treatment (>6 h), progressively and completely inhibited the activation of PLD by formyl-Met-Leu-Phe and partly inhibited (approximately 50%) the activation by PMA. In contrast, long-term brefeldin A treatment did not inhibit the effect of GTP[S] on PLD in permeabilized HL-60 cells. Long-term brefeldin A treatment completely inhibited inositol phosphate production in response to formyl-Met-Leu-Phe and ATP, indicating that it affected inositolphospholipid-specific phospholipase C activity. These data indicate that the rapid inhibitory effect of brefeldin A on Golgi function is not associated with inhibition of receptor-mediated or PMA-mediated PLD activation in HL-60 cells. However, longer-term effects, presumably arising from the disruption of the Golgi, lead to a total inhibition of agonist activation of PLD and inositolphospholipid-specific phospholipase C. In summary, these results do not support a role for brefeldin-A-sensitive ARF in agonist regulation of PLD in HL-60 cells.     

Possible target components for the inhibitory effect of N-ethylmaleimide on the activation of neutrophil NADPH oxidase

Potential target components for the inhibitory effect of covalent sulfhydryl-modifying reagent N-ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell-free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O2-generation in the cell-free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47phox was as effective as untreated p47phox both in the kinase-dependent and in the amphiphile-dependent cell-free activation systems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47phox was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G-protein rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation of NADPH oxidase.     

Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells

A series of radiolabelled ceramides (D-erythro and L-threo) and dihydroceramides (DL-erythro and DL-threo) with 2, 4 or 6 carbon N-acyl groups were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to synthesis of short-chain sphingomyelin and glucosylceramide, radiolabelled sphingosine and sphinganine were released from short-chain ceramides and dihydroceramides and subsequently utilized for synthesis of long-chain ceramide and sphingolipids. Substrate preference for short-chain sphingomyelin synthesis in cells was D-erythro-ceramides > L-threo-ceramides > DL-erythro-dihydroceramides > DL-threo-dihydroceramides, and C4- and C6-analogues were preferred over the C2-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-chain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. D-erythro-C6-Ceramide was the preferred substrate for short-chain glucosylceramide synthesis. D-erythro-C2-ceramide inhibited incorporation of [3H]serine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) and in a dose-dependent manner. Over a similar time period, [3H]choline-labelling of sphingomyelin was not affected. Inhibition of [3H]serine-labelling of sphingolipids appeared to correlate with release of [3H]long-chain bases from short-chain ceramides and dihydroceramides and synthesis of long-chain sphingolipids. However, some discrepancies between DL-erythro-C4- and C6-dihydroceramides, and D-erythro-C2-ceramide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [3H]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de novo sphingolipid synthesis by short-chain ceramides and dihydroceramides could not be related to inhibition of serine palmitoyltransferase activity in vitro.     

Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coli, causes a dominant-negative phenotype

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups-d18:1-C2:0 (C2-ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1-C6:0 (C6-ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3, synthetic d18:1-C18:0 GM3 (C18-Cer GM3, where C18:0 is a stearoyl group), or even d18:1-C2:0 GM3 (C2-Cer GM3), which itself contains a PCD-causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen-induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.     

Human phospholipase D1 can be tyrosine-phosphorylated in HL-60 granulocytes

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.     

Biochemistry and cell biology of phospholipase D in human neutrophils

Neutrophils play a major role host defense against invading microbes. Recent studies have emphasized the importance of the phospholipase D (PLD) in the signalling cascade leading to neutrophil activation. Phospholipase D catalyzes the hydrolysis of phospholipids to generate phosphatidic acid with secondarily generation of diradylglycerol; both of these products have been implicated as second messengers. Herein, we discuss the regulation and the biochemistry of the receptor-regulated PLD in human neutrophils. In vivo and in vitro studies suggest an activation mode in which initial receptor-linked activation of phospholipase C generates diacylglycerol and inositol trisphosphate. The resulting calcium flux along with the diacylglycerol activate a conventional isoform of protein kinase C (PKC), probably PKC beta 1. This PKC, in turn phosphorylates a plasma membrane component resulting in PLD activation and a second outpouring of diradylglycerol. The small GTP-binding proteins, RhoA and ARF, also participate in this process, and synergize with a 50 kDa cytosolic regulatory factor.     

Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2

Phospholipase D (PLD) has been implicated in a variety of cellular processes including vesicular transport, the respiratory burst, and mitogenesis. PLD1, first cloned from human, is activated by small GTPases such as ADP-ribosylation factor (ARF) and RhoA. Rodent PLD2, which is approximately 50% identical to PLD1 has recently been cloned from mouse embryo (Colley, W., Sung, T., Roll, R., Jenco, J., Hammond, S., Altshuller, Y., Bar-Sagi, D., Morris, A., and Frohman, M. (1997) Curr. Biol. 7, 191-201) and rat brain (Kodaki, T., and Yamashita, S. (1997) J. Biol. Chem. 272, 11408-11413). We describe herein the cloning from a B cell library and expression of human PLD2 (hPLD2). The open reading frame is predicted to encode a 933-amino acid protein (Mr of 105,995); this corresponds to the size of the protein expressed in insect cells using recombinant baculovirus. The deduced amino acid sequence shows 53 and 90% identity to hPLD1 and rodent PLD2, respectively. The mRNA for PLD2 was widely distributed in various tissues including peripheral blood leukocytes, and the distribution was distinctly different from that of hPLD1. hPLD1 and hPLD2 both showed a requirement for phosphatidylinositol 4,5-bisphosphate. Both isoforms showed optimal activity at 10-20 mol % phosphatidylcholine in a mixed lipid vesicle system and showed comparable basal activities in the presence of phosphatidylinositol 4,5-bisphosphate. Unexpectedly, ARF-1 stimulated the activity of hPLD2 expressed in insect cells about 2-fold, compared with a 20-fold stimulation of hPLD1 activity. Thus, not only PLD1 but also hPLD2 activity can be positively regulated by both phosphatidylinositol 4,5-bisphosphate and ARF.     

Rho prevents apoptosis through Bcl-2 expression: implications for interleukin-2 receptor signal transduction

Here we describe a Rho-mediated apoptosis suppression pathway driven by Bcl-2 expression in the interleukin (IL)-4- or IL-2-dependent murine T cell line TS1 alpha beta. IL-2, but not IL-4, induces Bcl-2 expression through RhoA activation which is inhibited by the specific Rho family inhibitor, Clostridium difficile Toxin B, as well as by a dominant negative RhoA mutant. Using transient transfections of RhoA mutants tagged with the vesicular stomatitis virus glycoprotein, we show that a constitutively active RhoA mutant induces Bcl-2 expression and prevents apoptosis upon IL-4 withdrawal. Finally, we have identified the signaling pathway involved together with RhoA in Bcl-2 induction and show compelling evidence for the implication of phosphatidylinositol 3 kinase and protein kinase C.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

Hydrogen peroxide-induced phospholipase D activation in rat pheochromocytoma PC12 cells: possible involvement of Ca2+-dependent protein tyrosine kinase

The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.     

Leishmania donovani attachment stimulates PKC-mediated oxidative events in bone marrow-derived macrophages

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O2- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O2- and NO production and stimulation of O2 consumption. Optimal NO and O2- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O2- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment Activation of O2- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.