Phospholipase D

Phospholipase D catalyses the hydrolysis of phosphatidylcholine to generate phosphatidate. The regulation of PLD activity is complex involving a number of small GTP binding proteins, but in particular Arf and Rho, phosphatidylinositol 4,5-bisphosphate and protein kinase C. The cDNA for PLD1 has recently been cloned and shows homology to the yeast and plant genes but only within four domains. Domains I and IV each contain a putative catalytic triad. PLD activity has been detected in plasma membranes, Golgi membranes and in nuclear membranes; it is unclear if different isoenzymes are responsible for this variation, or if the PLDs are differently regulated. The product of PLD activity, PA, appears to be a messenger molecule regulating the actin cytoskeleton and maybe playing a role in the control of membrane traffic and secretion.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms.

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4, 5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Calpha as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4,5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Cα as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

Specific inhibition of rat brain phospholipase D by lysophospholipids

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorly understood. This is primarily due to a lack of known specific inhibitors of PLD. We herein report that the activity of partially purified rat brain PLD is inhibited by certain lysophospholipids, such as lysophosphatidylinositol, lysophosphatidylglycerol, and lysophosphatidylserine in a highly specific manner. Inhibition of PLD by lysophospholipids was dose-dependent: the concentration of lysophosphatidylinositol required for half-maximal inhibition was about 3 micrometer. An analysis of the enzyme-kinetics suggested that lysophospholipids act as non-competitive inhibitors of PLD activity. As expected, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibition of PLD by lysophospholipids, however, was not affected by the presence or absence of Arf or by an increase in PIP(2) concentration. A protein-binding assay suggested that lysophospholipids bind directly to PLD. These results indicate that the observed inhibition of PLD by lysophospholipids is due to their direct interaction rather than to an interaction between lysophospholipids and either Arf or PIP(2). The present study suggests that certain lysophospholipids are specific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by lysophospholipids, presumably liberated by phospholipase A(2) activation, in mammalian cells.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.     

Signalling molecules and the regulation of intracellular transport

A variety of signalling molecules has been implicated over the past 8 years in the regulation of intracellular transport pathways. Those molecules include heterotrimeric GTP binding proteins, members of the protein kinase C family, and members of the Rho subfamily of small GTPases. Until recently, no common theme among the three classes of regulators was apparent. The finding that all three can influence the activity of phospholipase D (PLD), and the fact that members of the Arf subfamily of GTPases (with established roles in intracellular transport) are potent activators of PLD suggests the hypothesis that PLD is a focal point for integration of cellular responses to hormone signalling and for membrane homeostasis. Work during the past 2 years is beginning to uncover some transport pathways where PLD involvement is inferred. It is proposed that, if signalling is required to monitor and adjust transport rates to and from the various membrane organelles, the most economical way to achieve this would be to regulate recycling and allow the concentration of cargo receptors to determine forward transport. BioEssays 20 :495–504, 1998. © 1998 John Wiley & Sons, Inc.     

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.     

A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.     

Substrate specificities and activities of AZAP family Arf GAPs in vivo

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401-413, 2004), are characterized by the presence of at least one NH(2)-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599-610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH(2)-terminal sterile-alpha motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH(2)-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.     

A role for rho-kinase in rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor.

Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway.     

Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.     

The yeast Arf GTPase-activating protein Age1 is regulated by phospholipase D for post-Golgi vesicular transport

Vesicular transport shuttles cargo among intracellular compartments. Several stages of vesicular transport are mediated by the small GTPase Arf, which is controlled in a cycle of GTP binding and hydrolysis by Arf guanine-nucleotide exchange factors and Arf GTPase-activating proteins (ArfGAPs), respectively. In budding yeast the Age2 + Gcs1 ArfGAP pair facilitates post-Golgi transport. We have found the AGE1 gene, encoding another ArfGAP, can in high gene-copy number alleviate the temperature sensitivity of cells carrying mutations affecting the Age2 + Gcs1 ArfGAP pair. Moreover, increased AGE1 gene dosage compensates for the complete absence of the otherwise essential Age2 + Gcs1 ArfGAP pair. Increased dosage of SFH2, encoding a phosphatidylinositol transfer protein, also allows cell growth in the absence of the Age2 + Gcs1 pair, but good growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by lipid composition and can provide ArfGAP function for post-Golgi transport.     

Regulation of mTORC1 by the Rab and Arf GTPases

The mammalian target of rapamycin (mTOR) is a key cell growth regulator, which forms two distinct functional complexes (mTORC1 and mTORC2). mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. mTORC1 is regulated by a wide range of extra- and intracellular signals, including growth factors, nutrients, and energy levels. Precise regulation of mTORC1 is important for normal cellular physiology and development, and dysregulation of mTORC1 contributes to hypertrophy and tumorigenesis. In this study, we screened Drosophila small GTPases for their function in TORC1 regulation and found that TORC1 activity is regulated by members of the Rab and Arf family GTPases, which are key regulators of intracellular vesicle trafficking. In mammalian cells, uncontrolled activation of Rab5 and Arf1 strongly inhibit mTORC1 activity. Interestingly, the effect of Rab5 and Arf1 on mTORC1 is specific to amino acid stimulation, whereas glucose-induced mTORC1 activation is not blocked by Rab5 or Arf1. Similarly, active Rab5 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds and activates mTORC1. Our data demonstrate a key role of Rab and Arf family small GTPases and intracellular trafficking in mTORC1 activation, particularly in response to amino acids.     

SCAMP2 interacts with Arf6 and phospholipase D1 and links their function to exocytotic fusion pore formation in PC12 cells

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5'-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.     

Mastoparan selectively activates phospholipase D2 in cell membranes

Both known isoforms of phospholipase (PL) D, PLD1 and PLD2, require phosphatidylinositol 4,5-bisphosphate for activity. However, PLD2 is fully active in the presence of this phospholipid, whereas PLD1 activation is dependent on additional factors such as ADP-ribosylation factor-1 (ARF-1) and protein kinase Calpha. We find that mastoparan, an activator of G(i) and mast cells, stimulates an intrinsic PLD activity, most likely PLD2, in fractions enriched in plasma membranes from rat basophilic leukemia 2H3 mast cells. Overexpression of PLD2, but not of PLD1, results in a large increase in the mastoparan-inducible PLD activity in membrane fractions, particularly those enriched in plasma membranes. As in previous studies, expressed PLD2 is localized primarily in the plasma membrane and PLD1 in granule membranes. Studies with pertussis toxin and other agents indicate that mastoparan stimulates PLD2 independently of G(i), ARF-1, protein kinase C, and calcium. Kinetic studies indicate that mastoparan interacts synergistically with phosphatidylinositol 4,5-bisphosphate and that oleate, itself a weak stimulant of PLD2 at low concentrations, is a competitive inhibitor of mastoparan stimulation of PLD2. Therefore, mastoparan may be useful for investigating the regulation of PLD2, particularly in view of the well studied molecular interactions of mastoparan with certain other strategic signaling proteins.     

High glucose-induced membrane translocation of PKC βI is associated with Arf6 in glomerular mesangial cells

Protein kinase C (PKC)-induced changes in glomerular mesangial cell (MC) phenotypic behavior has been implicated in diabetes. The activity of diacylglycerol-sensitive PKC isoforms in MCs is altered by ambient changes in glucose, but the regulation of PKC activity and subsequent intracellular signaling events are not yet clearly defined. Small GTP-binding proteins of the ADP-ribosylation factor (Arfs) family, may regulate protein kinase membrane recruitment and hence its activity in signaling events of non-polarized cells. Members of the ARF family may coordinate membrane dynamics and other cellular functions through their interaction with PKC. We studied the activation of Arf, PKC betaI and phospholipase D (PLD) in MCs cultured under normal or high glucose conditions. MCs cultured in high glucose medium exhibited predominantly cytosolic localization of PKC betaI, Arf3 and Arf6. However, phorbol ester (PMA) stimulation of cells cultured in high glucose significantly enhanced membrane association of PKC betaI and Arf6, but not Arf3. Using [3H]choline chloride to prelabel MCs and measuring [3H]choline-containing metabolite release as PLD activity, PMA stimulated a significant increase of PLD activity under high glucose condition. Our data suggest that Arf6 plays a specific role in activation of PKC betaI and PLD under high glucose condition, and may be a significant intracellular event in the change of the mesangial cell phenotype associated with diabetic nephropathy.     

Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D

Macroautophagy (in this paper referred to as autophagy) and the ubiquitin-proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane-bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and in inducing phospholipase D (PLD) activity. PIP(2) and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.