Phospholipid signalling and lipid-derived second messengers in plants

Phospholipid signalling is mediated by phospholipid breakdown products generated by phospholipases. The enzymes from animals and plants generating known or potential lipid-derived second messengers are compared. Plants possess a phospholipase C and a phospholipase A₂ both of which are agonist-activated. These agonists (auxin, elicitors, perhaps others) bind to the external surface of the plasma membrane. The target enzyme for potential plant lipid-derived second messengers is lipid-activated protein kinase but the possibility that other enzymes may be also lipid-modulated should not be precluded.Abbreviations DAG diacylglycerol - CDPK calmodulin-like domain protein kinase - PLA2 phospholipase A₂ - PLC phospholipase C - PLD phospholipase D - PKC protein kinase C - PS phosphatidylserine     

Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O(2)(-) production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O(2)(-) production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.     

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils

Abstract Protein kinase C (PKC) appears to have a central role in the O₂⁻ response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP₂)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling of PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O₂⁻. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.     

A novel phospholipase D2-Grb2-WASp heterotrimer regulates leukocyte phagocytosis in a two-step mechanism

Phagocytosis is a primary innate response of both macrophages and neutrophils involving the formation of filamentous actin (F-actin)-rich protrusions that are extended around opsonized pathogens to form a phagocytic cup, resulting in their subsequent internalization. The molecular mechanism for this is still not completely understood. We now show for the first time that phospholipase D2 (PLD2) binds to growth factor receptor-bound protein 2 (Grb2) and to the Wiskott-Aldrich syndrome protein (WASp) to form a heterotrimer complex, PLD2-Grb2-WASp, and present the mechanism of interaction. Grb2 binds to the Y169/Y179 residues of PLD2 using its only SH2 domain, and it interacts with the poly-proline region of WASp using its two SH3 domains. The PLD2-Grb2-WASp heterotrimer can be visualized in early phagocytic cups of macrophages ingesting opsonized red blood cells, where it associates with polymerized actin. Cup colocalization and phagocytosis are disrupted with mutants that alter binding at either of the two proteins or by silencing Grb2 with RNA interference (RNAi). WASp association to PLD2-K758R, a lipase-inactive mutant, still occurs, albeit at lower levels, indicating that PLD2 plays a second role in phagocytosis, which is the production of phosphatidic acid (PA) and activation of phosphatidylinositol 5-kinase (PI5K) with subsequent synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)). The latter can be blocked with RNAi, which negates phagocytosis. Lastly, a constitutively "open" active form of WASp (WASp-L270P) brings phagocytosis to its maximum level, which can be mimicked with WASp-WT plus PLD2 or plus PA. Since neither a protein-protein disruption nor lack of PLD activity completely negates cup formation or phagocytosis, we posit a two-step mechanism: PLD2 anchors WASp at the phagocytic cup through Grb2 following protein-protein interactions and also activates it, making key lipids available locally. The heterotrimer PLD2-Grb2-WASp then enables actin nucleation at the phagocytic cup and phagocytosis, which are at the center of the innate immune system function.     

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.     

ras-p21 Activates phospholipase D and A2, but not phospholipase C or PKC,in Xenopus laevis Oocytes

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras-p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras-p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA-hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras-p21 induced the late production of lysophosphatidylcholine on a p42^MAPK-dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD- or ras-induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras-p21 is functionally dependent for its mitogenic activity on TPA- and staurosporine-sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras-p21 was independent of PKC, while PLC-induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras-p21 proteins, while no effect on PLC was observed.     

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.     

Regulation of phospholipase D by tyrosine kinases

Activation of phospholipase D (PLD) represents part of an important signalling pathway in mammalian cells, Phospholipase D catalyzed hydrolysis of phospholipids generates phosphatidic acid (PA) which is subsequently metabolized to lyso-PA (LPA) or diacylglycerol (DAG). While DAG is an endogenous activator of protein kinase C (PKC), PA and LPA have been recognized as second messengers as well, Activation of PLD in response to an external stimulus may involve PKC, Ca²⁺, G-proteins and/or tyrosine kinases. In this review, we will address the role of protein tyrosine phosphorylation in growth factor-, agonist- and oxidant-mediated activation of PLD. Furthermore, a possible link between PKC, Ca²⁺, G-proteins and tyrosine kinases is discussed to indicate the complexity involved in the regulation of PLD in mammalian cells.     

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells.

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.     

Cholecystokinin-evoked Ca(2+) waves in isolated mouse pancreatic acinar cells are modulated by activation of cytosolic phospholipase A(2), phospholipase D, and protein kinase C.

We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca(2+) signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8-induced Ca(2+) signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca(2+) waves elicited by stimulation of high-affinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a. CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca(2+) release was the same at both CCK-8 concentrations, suggesting that the secondary Ca(2+) release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca(2+) waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a. CCK-R are linked to phospholipase C (PLC) and phospholipase A(2) (PLA(2)) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA(2) and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca(2+) wave propagation by yet unknown mechanisms.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Role of PLD−PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase‐mediated superoxide (O₂^.?) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII‐induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C‐α (PKC‐α), ‐δ, ‐ε, and ‐ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐ζ, but not PKC‐α, ‐δ, and ‐ε, attenuated ANGII‐induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII‐induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC‐ζ inhibited ANGII‐induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane‐associated NADPH oxidase). Overall, PLD?PKCζ?p47phox signaling axis plays a crucial role in ANGII‐induced increase in NADPH oxidase‐mediated O₂^.? production in the cells.     

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.     

Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.     

Roles of phosphatidylinositol 3-kinase and phospholipase D in temporal activation of superoxide production in FMLP-stimulated human neutrophils

To determine the temporal roles of phosphatidylinositol 3-kinase (PI3-kinase) and phospholipase D (PLD) during human neutrophil activation stimulated by a chemotactic peptide, we examined the kinetics of these enzymes and related them to a neutrophil function (superoxide production). Both wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), potent and specific inhibitors of PI3-kinase, inhibit PI3-kinase activity in human neutrophils and significantly inhibit superoxide production from the early phase. Ethanol has no effect on PI3-kinase and markedly inhibits superoxide production at the late phase. Although these agents are inhibitory to different degrees, when neutrophils are simultaneously treated with ethanol and PI3-kinase inhibitors, superoxide is not produced. These results suggest that PI3-kinase and PLD play a pivotal role in the signal transduction pathway of the chemo-attractant-receptor involved neutrophil activation. These enzymes produce second messengers which are required for subsequent superoxide production in human neutrophils. NADPH oxidase is activated in a PI3-kinase-dependent manner at the early phase, and PLD activity follows it and is related to superoxide production at the late phase in human neutrophils by stimulation with FMLP.     

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.     

Targeting of protein kinase C-epsilon during Fcgamma receptor-dependent phagocytosis requires the epsilonC1B domain and phospholipase C-gamma1

Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilonC1 and epsilonC1B domains, or the epsilonC1B point mutant epsilonC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilonC259G, epsilonC1, and epsilonC1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilonC1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma1, and PI-PLC-gamma2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma2 is higher than that of PI-PLC-gamma1, PI-PLC-gamma1 but not PI-PLC-gamma2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma2-/- mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma1 provides DAG that binds to epsilonC1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.