Regulation of constitutive protein transit by phospholipase D in HT29-cl19A cells.

Phospholipase D (PLD) plays a central role in the control of vesicle budding and protein transit. We previously showed that in resting epithelial HT29-cl19A cells, PLD is implicated in the control of constitutive protein transit, from the trans-Golgi network to the plasma membrane, and that phorbol ester stimulation of protein transit is correlated with PLD activation (Auger, R., Robin, P., Camier, B., Vial, G., Rossignol, B., Tenu, J.-P., and Raymond, M.-N. (1999) J. Biol. Chem. 274, 28652-28659). In this paper we demonstrate that: 1) PLD is not implicated in the earliest phases of protein transit; 2) PLD controls apical but not basolateral protein transit; 3) HT29-cl19A cells express PLD1b and PLD2a mRNAs and proteins; 4) the expression of a catalytically inactive mutant of PLD2 (mPLD2-K758R) significantly inhibited apical constitutive protein transit whereas expression of a catalytically inactive mutant of PLD1 (hPLD1b-K898R) prevented increases in the rate of apical transit as triggered by phorbol esters; 5) PLD2 appears to be located in a perinuclear region containing the Golgi whereas PLD1, which is scattered in the cytoplasm in resting cells, is translocated to the plasma membrane after phorbol ester stimulation. Taken together, these data lead to the conclusion that in HT29-cl19A cells, both PLDs regulate protein transit between the trans-Golgi network and the apical plasma membrane, but that they do so at different steps in the pathway.     

Transmodulation between phospholipase D and c-Src enhances cell proliferation

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.     

Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.     

Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells

Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl(2))-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl(2) stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE(2)) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl(2)-induced COX-2 expression and PGE(2) formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl(2)-induced COX-2 expression and PGE(2) production. PLD1 enhanced COX-2 expression by CoCl(2) via reactive oxygen species (ROS), p38 MAPK kinase, PKC-delta, and PKA, but not ERK, whereas PLD2 enhanced CoCl(2)-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-delta, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl(2)-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl(2)-induced COX-2 expression through differential signaling pathways in astroglioma cells.     

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis

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

Phospholipase D2: a pivotal player modulating RBL-2H3 mast cell structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells.     

Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells

Phospholipase D (PLD) activity is elevated in response to mitogenic and oncogenic signals. PLD also cooperates with overexpressed tyrosine kinases to transform rat fibroblasts. 3Y1 rat fibroblasts overexpressing the tyrosine kinase c-Src undergo apoptosis in response to serum withdrawal. We report here that elevated expression of either PLD1 or PLD2 in these cells prevents apoptosis induced by serum withdrawal. 3Y1 cells transformed by the activated tyrosine kinase v-Src have elevated PLD activity and are resistant to apoptosis induced by serum withdrawal. However, if PLD activity is blocked, the v-Src-transformed cells underwent apoptosis. MDA-MB-231 cells are a human breast cancer cell line with substantially elevated levels of PLD activity. Inhibiting PLD activity in these cells similarly rendered them sensitive to the apoptotic insult of serum withdrawal. These data indicate that elevated PLD activity generates a survival signal(s) allowing cells to overcome default apoptosis programs.     

Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.     

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.     

Cell invasion of highly metastatic MTLn3 cancer cells is dependent on phospholipase D2 (PLD2) and Janus kinase 3 (JAK3)

MTLn3 cells are highly invasive breast adenoacarcinoma cells. The relative level of the epidermal-growth-factor-stimulated invasion of this cell line is greater than two other breast cancer cell lines (MDA-MB-231 and MCF-7) and one non-small cell lung cancer cell line (H1299). We have determined that the mechanism of cancer cell invasion involves the presence of an enzymatically active phospholipase D (PLD), with the PLD2 isoform being more relevant than PLD1. PLD2 silencing abrogated invasion, whereas ectopic expression of PLD2 augmented cell invasion in all four cell lines, with an efficacy (MTLn3 ± MDA-MB-231 > H1299 ± MCF-7) that correlated well with their abilities to invade Matrigel in vitro. We also report that PLD2 is under the control of Janus kinase 3 (JAK3), with the kinase phosphorylating PLD2 at the Y415 residue, thus enabling its activation. Y415 is located downstream of a PH domain and upstream of the catalytic HKD-1 domain of PLD2. JAK3 knockdown abrogated lipase activity and epidermal-growth-factor-stimulated cell invasion directly. For the purposes of activating PLD2 for cell invasion, JAK3 operates via an alternative pathway that is independent of STAT, at least in MTLn3 cells. We also consistently found that JAK3 and PLD2 pathways are utilized at the maximum efficiency (phosphorylation and activity) in highly invasive MTLn3 cells versus a relatively low utilization in the less invasive MCF-7 cell line. In summary, a high level of cell invasiveness of cancer cells can be explained for the first time by combined high JAK3/PLD2 phosphorylation and activity involving PLD2's Y415 residue, which might constitute a novel target to inhibit cancer cell invasion.     

Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner

Phospholipase D2 (PLD2), one of the two mammalian members of the PLD family, has been implicated in cell proliferation, transformation, tumor progression and survival. However, as precise mechanistic details are still unknown, we investigated here if the PLD2 isoform would signal through the PI3K/AKT pathway. Transient expression of PLD2 in COS7 cells with either the WT or with a Y179F mutant, resulted in an increased basal phosphorylation of AKT in residues T308 and S473, in a PI3K-dependent manner. Transfection of PLD2-Y179F (but not the wild type) caused an increased (>2-fold) DNA synthesis even in the absence of extracellular stimuli. Other signaling mechanisms downstream such PLD/PI3K dependence (that might lead to DNA synthesis regulation) were further studied. PLD2-Y179F caused an increase in phosphorylation of p42/p44 ERK and in the expression of G0/G1 phase transition markers (p21 CIP, PCNA), and these effects, too, were dependent on PI3K. Interestingly, Akt, once activated induced the phosphorylation of PLD2 on residue T175, an effect that was inhibited by LY296004. Lastly, if PLD2-Y179F is further mutated in residue K758 (PLD2 Y179F-K758R), which renders inactive a catalytic site, DNA synthesis is then abrogated, indicating that the activity of the enzyme (i.e. synthesis of PA) is necessary for the observed effects. In conclusion, the unavailability of residue Y179 on PLD2 to become phosphorylated leads to an augmentation of DNA synthesis concomitantly with MEK and AKT phosphorylation, in a process that is dependent on PI3K and independent of any extracellular stimuli. This might be critical for the maintenance of the PLD2-regulated proliferative status.     

Phorbol myristate acetate-induced Egr-1 expression is suppressed by phospholipase D isozymes in human glioma cells

Early growth response-1 (Egr-1) is involved in the regulation of cell growth. Here, we found that overexpression of phospholipase D (PLD) isozymes decreased tumor promoter phorbol myristate acetate (PMA)-induced Egr-1 expression and transactivation in glioma cells. Suppression of PMA-induced Egr-1 was dependent on the expression level of PLD isozymes. Overexpression of catalytically inactive PLD, treatment with PA, and prevention of PA dephosphorylation by 1-propranolol significantly suppressed PMA-induced Egr-1 expression. PLD-induced suppression of Egr-1 was reversed by inhibition of phosphatidylinositol 3-kinase (PI3K). Taken together, these results suggest that elevated expression and activity of PLD attenuate PMA-induced Egr-1 expression via PI3K pathway.     

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.     

Role for phospholipase D in receptor-mediated endocytosis

In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C alpha and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.     

Elevated phospholipase D activity induces apoptosis in normal rat fibroblasts

Elevated expression of phospholipase D (PLD) in rat fibroblasts overexpressing a tyrosine kinase leads to cell transformation. However, it has been difficult to get elevated expression of PLD in normal rat fibroblasts. Using transient transfection and an inducible expression system, we were able to get elevated expression of PLD1 and PLD2 in 3Y1 rat fibroblasts. Elevated expression of either PLD1 or PLD2 in 3Y1 cells led to apoptosis in the absence of serum. Elevated PLD expression resulted in reduced cell viability and the cleavage of the caspase 3 substrates poly-ADP-ribose polymerase (PARP) and protein kinase C delta. Elevated PLD expression also stimulated cytochrome c release, indicating that the mitochondrial apoptosis pathway was activated. Thus, while elevated PLD expression can transform cells with elevated tyrosine kinase expression, elevated expression of PLD activity in normal cells renders cells sensitive to apoptotic insult.     

Increased cell growth due to a new lipase-GEF (Phospholipase D2) fastly acting on Ras

We report the novel finding that Phospholipase D2 (PLD2), through its PX and PH domains, binds specifically to Ras and catalyzes the GDP/GTP exchange (i.e., is a GEF), with potency comparable to Ras-GRF-1, a known Ras-GEF. Cells overexpressing PLD2-GEF inactive mutants (F129Y and R172C/L173A) fail to stimulate cell proliferation compared to the wild type-expressing cells. The GEF effect on Ras follows a faster kinetics than other GTPase substrates (such as Rac2 or Rac1) and is a better substrate, too. The GEF action is due to PLD2 (protein) itself, independent of the lipase product PA. PA can still have a fine-tuning regulatory effect on Ras-GTP depending upon its cellular concentration. Rapidly growing human breast cancer cells MDA-MB 231 (but not the slow growing MCF7 counterpart) have high levels of endogenous PLD2-GEF which correlates with high Ras activation. The PLD2-“GEF” activity is even higher than the classical “lipase” activity and is abrogated with GEF single point mutants, particularly F129Y, and concomitantly with a slow rate of cell growth. This can be crucial to cancer biology in that not only Ras mutations explain abnormal growth, but the existence of a new GEF for Ras: a GEF molecule that happens to be a phospholipase.     

The exquisite regulation of PLD2 by a wealth of interacting proteins: S6K, Grb2, Sos, WASp and Rac2 (and a surprise discovery: PLD2 is a GEF)

Phospholipase D (PLD) catalyzes the conversion of the membrane phospholipid phosphatidylcholine to choline and phosphatidic acid (PA). PLD's mission in the cell is two-fold: phospholipid turnover with maintenance of the structural integrity of cellular/intracellular membranes and cell signaling through PA and its metabolites. Precisely, through its product of the reaction, PA, PLD has been implicated in a variety of physiological cellular functions, such as intracellular protein trafficking, cytoskeletal dynamics, chemotaxis of leukocytes and cell proliferation. The catalytic (HKD) and regulatory (PH and PX) domains were studied in detail in the PLD1 isoform, but PLD2 was traditionally studied in lesser detail and much less was known about its regulation. Our laboratory has been focusing on the study of PLD2 regulation in mammalian cells. Over the past few years, we have reported, in regards to the catalytic action of PLD, that PA is a chemoattractant agent that binds to and signals inside the cell through the ribosomal S6 kinases (S6K). Regarding the regulatory domains of PLD2, we have reported the discovery of the PLD2 interaction with Grb2 via Y169 in the PX domain, and further association to Sos, which results in an increase of de novo DNA synthesis and an interaction (also with Grb2) via the adjacent residue Y179, leading to the regulation of cell ruffling, chemotaxis and phagocytosis of leukocytes. We also present the complex regulation by tyrosine phosphorylation by epidermal growth factor receptor (EGF-R), Janus Kinase 3 (JAK3) and Src and the role of phosphatases. Recently, there is evidence supporting a new level of regulation of PLD2 at the PH domain, by the discovery of CRIB domains and a Rac2-PLD2 interaction that leads to a dual (positive and negative) effect on its enzymatic activity. Lastly, we review the surprising finding of PLD2 acting as a GEF. A phospholipase such as PLD that exists already in the cell membrane that acts directly on Rac allows a quick response of the cell without intermediary signaling molecules. This provides only the latest level of PLD2 regulation in a field that promises newer and exciting advances in the next few years.     

Transformation of cells overexpressing a tyrosine kinase by phospholipase D1 and D2.

Phospholipase D (PLD) activity is elevated in response to most mitogenic signals. Two mammalian PLD genes (PLD1 and PLD2) have been cloned and their gene products have been characterized. PLD1 is a downstream target of the Ras/RalA GTPase cascade implicated in mitogenic and oncogenic signaling. Consistent with a role in mitogenic signaling, elevated expression of PLD1 transforms cells overexpressing the epidermal growth factor (EGF) receptor (EGFR). However, PLD2 colocalizes with the EGFR in caveolin-enriched light membrane microdomains. We therefore investigated whether PLD2 could also contribute to the transformation of cells overexpressing a tyrosine kinase. We report here that elevated expression of PLD2 transforms rat fibroblasts overexpressing either the EGFR or c-Src. Since overexpression of a tyrosine kinase is a common genetic alteration in several human cancers, these data suggest that elevation of either PLD1 or PLD2 may contribute to the progression to a malignant phenotype in cells with elevated tyrosine kinase activity.