Localization and possible functions of phospholipase D isozymes

Biochimica et Biophysica Acta 1439 (1999) 245^263 www.elsevier.com/locate/bba

Review

Localization and possible functions of phospholipase D isozymes Mordechai Liscovitch *, Malgorzata Czarny, Giusy Fiucci, Yaakov Lavie, Xiaoqing Tang Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel Received 1 February 1999; accepted 5 April 1999

Abstract The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase D; Phosphatidic acid; Subcellular localization; Phosphatidylinositol 4,5-bisphosphate; Membrane microdomain

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246

2.

Identi¢cation and cloning of eukaryotic phospholipase D genes . . . . . . . . . . . . . . . . . . . .

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3.

Regulation of phospholipase D by small GTPases of the ARF and RhoA families . . . . . .

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4.

Membrane-bound and soluble forms of phospholipase D . . . . . . . . . . . . . . . . . . . . . . . . .

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5.

Localization of phospholipase D in speci¢c subcellular 5.1. Plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Exocytic and endocytic vesicles . . . . . . . . . . . . . . 5.5. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ARF, ADP-ribosylation factor; BFA, brefeldin A; CaLB, Ca2‡ ^lipid binding; DAG, diacylglycerol; ER, endoplasmic reticulum; GDI, GDP dissociation inhibitor; GFP, green £uorescent protein; G proteins, guanine nucleotide binding proteins; GTPQS, 5P-O-(3-thio)triphosphate; MDR, multidrug resistance; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP2 , phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; PS, phosphatidylserine ; TGN, trans-Golgi network * Corresponding author. Fax: +972-8-934-4116; E-mail: [email protected] ; URL: http://www.weizmann.ac.il/Vlhliscov/ 1388-1981 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 0 9 8 - 0

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Phosphatidylinositol 4,5-bisphosphate as a cofactor for PLD and a putative membrane targeting signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7.

Localization of phospholipase D in detergent-insoluble membrane microdomains and caveolae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.

Future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Lipid-derived molecules have been implicated in the regulation of cell proliferation, cell di¡erentiation, speci¢c cell functions, and cell senescence and death. Bioactive lipids are known to serve both as extracellular messengers that act on cell-surface receptors and as intracellular messengers that mediate receptor-triggered events [1^4]. Messenger molecules are derived from lipid constituents of biological membranes by a number of signal-activated enzymes, including phospholipases, lipid kinases and acylases. These enzymes, which are normally inactive, are characteristically activated in response to receptor occupancy (or other forms of cell stimulation), resulting in the conversion of a structural membrane lipid constituent into a biologically active messenger molecule. Phospholipase D (PLD) is a signal-activated enzyme that hydrolyses phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), to form phosphatidic acid (PA) and the free polar head group of the phospholipid substrate [5]. Most PLDs catalyze a unique transphosphatidylation reaction, utilizing primary short-chain alcohols as phosphatidyl group acceptors, generating a phosphatidic acid alkyl ester (phosphatidyl-alcohol; Fig. 1). A critical advance in understanding the physiological roles of PLD involved the demonstration that cell stimulation results in a rapid and dramatic activation of PLD (the early studies are reviewed in [6,7]). The nature of the activating signals is diverse. Stimuli that activate PLD include hormones, growth factors, neurotransmitters, cytokines, extracellular matrix constituents, antigens and certain physical stimuli. PLD activation is mediated by several distinct, agonist- and isozyme-speci¢c

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Fig. 1. Phospholipase D-catalyzed reactions. Phospholipase D hydrolyzes the distal phosphodiester bond in phospholipids. A phosphatidyl-enzyme intermediate is normally subject to nucleophilic attack by water. However, primary short chain alcohols (e.g., methanol, ethanol, 1-propanol, n-butanol) can substitute for water in a competing transphosphatidylation reaction. The product of PLD-catalyzed transphosphatidylation is the corresponding phosphatidic acid alkyl ester, or phosphatidyl-alcohol. Unlike PA, which can also be produced by diacylglycerol kinase and by acylation of glycerol-3-phosphate, a phosphatidylalcohol is uniquely formed by PLD. Relative to PA, which can be further metabolized to DAG and lyso-PA, a phosphatidyl-alcohol is metabolically stable and tends to accumulate in cells upon PLD activation. Being an `abnormal' phospholipid, changes in cellular phosphatidyl-alcohol level are readily detectable. Because of these properties phosphatidyl-alcohols are very useful markers of PLD activation in vitro and in vivo. Primary short-chain alcohols attenuate PLD-catalyzed formation of PA by trapping the phosphatidyl moiety in a biologically inactive phosphatidyl-alcohol. `Alcohol trap' experiments have been utilized to establish the role of PLD in various physiological responses. It should be noted that although transphosphatidylation is catalyzed only by PLDs, not all PLDs can catalyze this reaction. Among the non-transphosphatidylating PLDs are certain bacterial PLDs [154], a yeast Ca2‡ -dependent PLD [33,34] and a mammalian mitochondrial PLD [118].

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mechanisms, that involve guanine nucleotide-binding (G) proteins and protein kinases. Under physiological conditions the activation of PLD results in formation of PA. There is increasing evidence that PA acts as an intracellular messenger [8,9]. Collectively, existing data indicate that the activation of PLD is an important regulatory event in eukaryotic cells. Various aspects of PLD research were dealt with in a number of recent reviews [10^13] and are further discussed elsewhere in this issue. This review will focus on the localization, in di¡erent cellular compartments, of PLD activities and isozymes in the context of their proposed functions. 2. Identi¢cation and cloning of eukaryotic phospholipase D genes While signi¢cant progress was made in characterizing PLD activity in vitro and the signal-dependent activation of PLD in vivo, many attempts to purify the enzyme(s) from mammalian sources have foundered, mostly due to progressive loss of enzymatic activity (e.g., [14^16]; V. Chalifa-Caspi, unpublished results). Attempts to purify PC-speci¢c PLD from plant tissues were more successful. Abousalham et al. puri¢ed a V90 kDa PLD from cabbage leaves and determined the sequence of an N-terminal 29amino-acid peptide [17]. Independently, a very similar V90 kDa PLD was puri¢ed from castor bean endosperm that had a nearly identical N-terminal peptide sequence [18]. Based on the peptide sequence, Wang et al. isolated from an endosperm cDNA library a clone encoding the castor bean PLD, the sequence of which included the N-terminal aminoacid sequence of the puri¢ed PLD [19]. When expressed in Escherichia coli the cloned protein exhibited the characteristic hydrolytic and transphosphatidylation activities of PLD. Two additional, highly homologous plant PLDs have been cloned from rice (Oryza sativa) and maize (Zea mays) [20]. The sequencing of the ¢rst eukaryotic PC-speci¢c PLD by Xuemin Wang and his colleagues was a major breakthrough because it opened the way for the discovery of related sequences in the protein and nucleotide sequence databases, and enabled cloning strategies based on the existence of highly conserved

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sequence domains or motifs. Indeed, the cloning of plant PLD quickly led to the identi¢cation of the ¢rst yeast PLD gene SPO14/PLD1 [21^23], and the cloning of the ¢rst cDNA encoding human PC-PLD [24]. To date, eukaryotic PLD genes that have been molecularly cloned include: three plant genes, namely PLDK, PLDL and PLDQ [25,26]; two mammalian PLD genes termed PLD1 [24,27] and PLD2 [28,29]; and a yeast PLD gene ^ SPO14/PLD1 [21^ 23]. In addition, protein sequence databases contain the complete coding sequence of a putative PLD from Caenorhabditis elegans. These PLD genes all belong to an extended gene superfamily that also includes bacterial PLDs, non-PLD phosphatidyltransferases/phospholipid synthases, and certain viral proteins and their mammalian homologs [30^32]. Eukaryotic PC-PLD genes that have been sequenced to date share a relatively conserved core domain £anked with much less conserved N- and C-terminal regions (Fig. 2). Additional forms of PLD may exist in eukaryotes, that do not belong to the PLD/phosphatidyltransferase gene family [33,34]. 3. Regulation of phospholipase D by small GTPases of the ARF and RhoA families The regulation of PLD isoforms by small GTPases that belong to the Ras superfamily, as well as by protein kinase C, was covered in several recent reviews (e.g., [35]) and is dealt with in great detail elsewhere in this issue. Reconstitution experiments, in which membrane fractions or cytosol-depleted cell `ghosts' were combined with cytosol or fractions thereof, have provided the ¢rst clear evidence that cytosol contains one or more factors that can restore stimulation of PLD activity by guanosine 5P-O-(3thio)triphosphate (GTPQS; a non-hydrolyzable analog of GTP) [36^38]. The ¢rst such factor to be puri¢ed was identi¢ed as the small G protein ADP-ribosylation factor (ARF) [39,40]. ARF acts to recruit cytosolic coat proteins to newly forming vesicles in the Golgi [41,42] and is likely be involved in additional membrane tra¤c phenomena [43^45]. Both PLD1 gene products, i.e., the splice variants PLD1a and PLD1b, are regulated by ARF [46,47]. These isoforms probably represent the ARF-depend-

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Fig. 2. Domain structure of eukaryotic PC-speci¢c PLDs. Alignment of four PLD sequences from representative species of vertebrates (Homo sapiens; PLD1a), invertebrates (Ceanorhabditis elegans; putative PLD), fungi (Saccahromyces cerevisiae ; SPO14/PLD1) and plants (Ricinus communis; PLD-K), reveal six domains which are conserved among all eukaryotic PLDs (red boxes). Additional domains are conserved in the yeast, human and worm sequences but are absent from plant PLD-K (blue boxes). Other domains are common to the human and worm PLDs only (green boxes). Plant PLD-K thus seems to represent a distinct, possibly more primitive form of PLD with a minimal set of structural elements required for PLD activity [31]. There is signi¢cant internal homology between a region that corresponds to domains 1^2 and a region that corresponds to domains 4^5 [32], suggesting a gene duplication and fusion event. Domains 2 and 5 contain a short sequence motif (HXKxxxxD, where x represents a hydrophobic amino acid) termed the phosphatidyltransferase (PT) or the HKD motif (azure boxes). The highly conserved histidine, lysine, aspartate, glycine and serine residues in the HKD/PT motif play an important role in catalysis [155,156]. Plant PLDs have an N-terminal Ca2‡ -lipid binding (CaLB) domain (also known as a C2 domain; yellow box). CaLB/C2 domains are found in a wide range of signaling proteins [157]) and are believed to be involved in binding acidic phospholipids in a Ca2‡ -dependent manner [158] and to mediate recruitment of CaLB/C2-containing proteins to speci¢c subcellular compartments [159]. Four black bars drawn above the yeast PLD1/SPO14 gene represent the previously designated conserved regions I through IV [30].

ent PLD activity measured in membranes and permeabilized cells. Evidence that ARF mediates receptor activation of PLD in situ was provided by employing brefeldin A (BFA), a fungal metabolite that is known to block ARF activation by inhibiting guanine nucleotide exchange (reviewed in [42]). BFA was found to inhibit PLD activation in vitro by exogenously added ARF, indicating that, like other ARF-dependent functions, the activation of PLD is BFA-sensitive [48]. More importantly, BFA was shown to inhibit (albeit partially) m3-muscarinic receptor stimulation of PLD activity in intact human embryonic kidney (HEK) cells [49] and in 1321N1 human astrocytoma cells [50]. We have obtained similar results in GnRHstimulated KT3-1 mouse gonadotrophs, in which BFA inhibited PLD activation at low micromolar concentrations (T. Fuchs, Z. Naor, M. Liscovitch, unpublished results). These data indicate that ARF mediates, at least in part, receptor-induced activation of PLD and are consistent with a model in which activation of PLD is preceded by activation and

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translocation of ARF to cellular membranes. The mechanism(s) of receptor-mediated activation of ARF is unknown, but is likely to involve stimulation of guanine nucleotide (GTP for GDP) exchange onto ARF. ARF guanine nucleotide exchange factors have been isolated recently [51], that may be involved in receptor activation of PLD. Finding how cell surface receptors that activate PLD are coupled to an ARF guanine nucleotide exchange protein is a major challenge for future studies. Recent work has indicated that both receptor tyrosine kinases and heptahelical G-protein-coupled receptors might associate with ARF directly (see Section 5.1). Another small GTPase which has been strongly implicated in PLD activation is RhoA, which, like ARF, appears to regulate PLD1. Initial evidence that a Rho family member is required for PLD activation came from studies employing Rho GDP dissociation inhibitor (GDI), a protein that binds speci¢cally to Rho family small G proteins and thus inhibits Rho-dependent functions. Rho GDI caused a nearly complete inhibition of PLD activation by

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GTPQS in human neutrophil membranes [52]. Similar inhibitory e¡ects of Rho GDI were seen with membranes prepared from rat liver [53], HL-60 cells [54] but cf. [55]), MDCK cell nuclei [56] and human neutrophils [57]. The activation of PLD could be restored in Rho GDI-treated membranes by RhoA, Rac1 and Cdc42 in that order of e¤cacy [53,54,58]. RhoA stimulates PLD also in untreated HL-60 membranes [54,59] and a partially puri¢ed PLD activity extracted from porcine and rat brain membranes [60,61]. The activation by RhoA depends on its isoprenylation [56,61] although isoprenylation seems to be required for guanine nucleotide exchange rather than for PLD activation per se, because recombinant non-prenylated RhoA is fully e¡ective if pre-loaded with GTPQS [57]. Kwak et al. have established that complete depletion of RhoA abrogates the ability of cytosol to restore PLD activation by GTPQS [57]. Hence, RhoA appears to be an essential cytosolic factor required for membrane PLD activation in vitro. It is noteworthy that RhoA was ine¡ective in stimulating an HL-60 cytosolic PLD activity [54]. RhoA is covalently modi¢ed and functionally inhibited by a bacterial ADP-ribosyltransferase, Clostridium botulinum C3 exoenzyme, and a bacterial glucosyltransferase, Clostridium di¤cile toxin B (see [62] for review). In the early studies implicating RhoA in PLD activation, the ADP-ribosylation of RhoA by C3 toxin had no e¡ect on its ability to activate PLD in vitro [52,53]. However, more recent studies with rat brain membranes [61] and MDCK cell nuclear membranes [56] showed that C3 exoenzyme did block PLD activation by RhoA. C3 exoenzyme and toxin B were utilized to examine the role of RhoA in receptor-mediated activation of PLD [63,64]. Schmidt et al. have demonstrated that C. di¤cile toxin B inhibits muscarinic receptor-mediated activation of PLD. Other receptor-mediated responses were left untouched. The speci¢city of the e¡ect was further evinced by the fact that phorbol esterinduced activation was una¡ected, and that disruption of the actin cytoskeleton, by agents that do not act on Rho, had no e¡ect on PLD activation [63]. Hence, the inhibition by toxin B was due neither to inhibition of PLD itself nor was it a secondary consequence of cytoskeletal reorganization. Malcolm et al. have shown that C3 exoenzyme (scrape-loaded into Rat-1 ¢broblasts) attenuated the activation of

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PLD in response to lyso-PA, endothelin-1 and phorbol esters [64]. Again, disruption of the actin cytoskeleton by another agent, cytochalasin D, had only a minimal e¡ect on PLD activation, and activation of phospholipase C was not a¡ected by the C3 toxin. Collectively these data implicate RhoA in signaling from cell surface receptors to PLD. Interestingly, ARF synergizes with RhoA (as well as other factors that were shown to activate PLD in vitro, including PKC-K, and two cytosolic proteins of 50 kDa and 36 kDa) [54,60,61,65,66]. These interactions appear to be physiologically signi¢cant. The extensive characterization of PLD activation by muscarinic stimulation of HEK cells transfected with m3mAChR [49,63,67] clearly demonstrates that both ARF and RhoA are involved in receptor-PLD coupling. Such synergism suggests that ARF and RhoA interact with the same PLD molecule, most likely via di¡erent domains. Characterization of the recently cloned PLD1 gene products indicates that they represent the ARF- and RhoA-sensitive PLD forms. Structural analysis of PLD1 indicates that its N-terminal domain (ca. 300 amino acids) is required for PKC activation [68], whereas RhoA interacts with a 363-amino-acid C-terminal domain [69]. Despite the great strides made in understanding the regulation of PLD, the precise cellular localization and function of the cloned PLD isoforms are still poorly de¢ned. However, the availability of sequence information, recombinant DNA material and speci¢c antibodies is beginning to shed some light on these issues. 4. Membrane-bound and soluble forms of phospholipase D The ¢rst mammalian PLD to be characterized was discovered in brain membrane fractions by Kanfer and colleagues [70]. This enzyme is often referred to as the `oleate-activated' PLD because it is activated by sodium oleate and other unsaturated fatty acids. The oleate-activated PLD behaves as an integral membrane protein as it cannot be extracted from membranes by high salt concentrations [71]. It exhibits a clear preference for PC as substrate [14,72,73], especially when acting upon endogenous phospholipid substrates [74]. The oleate-activated PLD was found in brain microsomes and synaptic

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Table 1 Localization of phospholipase D activities or isozymes in speci¢c subcellular compartments and organelles Cell compartment or organelle

PLD activity or isozyme

Reference(s)

Plasma membrane

Oleate-activated PLD GTPQS-stimulated PLD ARF/RhoA-stimulated PLD PLD2 ARF-dependent PLD PLD1a and PLD1b ARF-stimulated PLD PMA-stimulated PLD (in vivo) ARF/RhoA-stimulated PLD Oleate-activated PLD Ca2‡ -dependent PE-PLD PIP2 -stimulated PC-PLD ARF/RhoA-stimulated PLD PI/PE-preferring PLD ARF-dependent PLD

[75^77] [88] [91,92] [28] [113] [106] [48] [90] [56,79,92,101] [78,79] [118,119] [149] [121] [83^85] [54,86]

Secretory granules Golgi apparatus Endoplasmic reticulum Nucleus Mitochondria DIGs/Caveolae Cytoskeleton Cytosol

membranes [75] and in fractions enriched in rat brain plasma membranes [76]. Its presence in rat liver plasma membranes was also reported [77]. An oleate-activated PLD was found also in rat brain neuronal nuclei [78], rat liver nuclei and rat ascites AH7974 hepatoma cells nuclei [79]. The oleate-activated PLD has been successfully puri¢ed from porcine lung [80], and recently its activity in cardiac muscle was shown to decrease upon induction of diabetes [81]. Nevertheless, the sequence of the oleate-activated PLD, its regulation, and its function remain unknown. Direct evidence for the presence of two forms of PLD in membranes was obtained by heparin a¤nity chromatography of a Triton X-100 extract prepared from rat brain membranes, which resolved two peaks of PLD activity: an oleate-activated form and an ARF-activated form [82]. The latter has characteristics of a peripheral membrane protein because it may be solubilized from membrane, at least in part, at high ionic strength [15,39]. In addition to the membrane-bound PLD forms, a soluble, probably cytosolic PLD was discovered in extracts prepared from various cells [54,83^85]. Some of the reported cytosolic PLD activities di¡er from membrane-bound PLDs in their preference for PI [83,85] or PE [84] as substrate over PC. However, these apparent di¡erences may re£ect di¡erent substrate presentation modalities in the various assays. The soluble PLD activities also exhibited a requirement for, or stimulation by Ca2‡ ions [54,83,85]. The

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cytosolic PLD is likely to be related to the peripheral membrane PLD because both forms are activated by ARF [39,54,86] and therefore are likely to be products of the PLD1 gene. The primary sequence of both PLDs cloned so far reveals no obvious transmembrane domain(s). Yet, PLDs must interact with a phospholipid substrate in cell membranes and thus must either be constitutively associated with, or transiently recruited to the membrane through a membrane-interaction module, the identity of which is still not known. Expression of recombinant rat PLD1a and PLD1b in Schizosaccharomyces pombe revealed that both isoforms are predominantly membrane associated, although signi¢cant activity (10^15%) is found in cytosol [47]. Qualitatively similar results were obtained for human PLD1a expressed in Sf9 cells [24] and rat PLD1b expressed in COS-7 cells [27]. Likewise, expression of recombinant rat PLD2 in S. pombe revealed that the enzyme was relatively enriched in the membrane fraction compared to cytosol, but the latter fraction exhibited signi¢cant levels of activity [29]. Although the above data concern studies in which the isoforms were overexpressed, they are consistent with the biochemical data (Table 1). Together, these results suggest that PLD1 and PLD2 might be `ambiquitous' enzymes [87], namely, enzymes that can be either cytosolic or membrane-associated or both, depending on the physiological state of the cell. In summary, these data imply that multiple PLD

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isozymes that exhibit distinct substrate speci¢cities and a di¡erential dependence on Ca2‡ ions are expressed in mammalian cells. Furthermore, these biochemical studies have indicated the existence of membrane-bound and cytosolic enzymes, suggesting a di¡erent subcellular localization of the di¡erent PLD isoforms. The bimodal distribution of PLD isoforms raise three interrelated questions. First, with what cellular membranes is PLD associated? Second, is the association of PLD with the membrane transient (and therefore signal-regulated), or constitutive? Third, what is the nature of the membrane-interaction module and the membrane targeting signal that together mediate the association of PLD with the membrane? Possible answers to the above questions are discussed below. 5. Localization of phospholipase D in speci¢c subcellular compartments Subcellular fractionation and characterization studies do indeed demonstrate the presence of PLD activities in many cellular membranes, including the nuclear envelope, endoplasmic reticulum (ER), Golgi apparatus, transport/secretory vesicles and plasma membrane (Table 1). 5.1. Plasma membrane Ribbes et al. have measured PLD activity in subcellular fractions from human neutrophils utilizing [3 H]1-alkyl-2-acylglycero-3-phosphocholine as a liposomal substrate, in the presence of cytosol and GTPQS. PLD activity was found predominantly in the plasma membrane [88]. Activation of PLD in [3 H]1-alkyl-2-lysophosphatidylcholine-prelabeled cells by phorbol ester resulted in generation of [3 H]PA and [3 H]phosphatidylethanol, both of which were found exclusively in the plasma membrane fraction [88]. The subcellular distribution of [3 H]phosphatidyl-alcohol was also examined in phorbol estertreated, [3 H]palmitate-prelabeled HeLa cells, where this PLD product was found both in plasma membrane-enriched and intracellular membrane fractions [89]. However, a similar study, carried out in [3 H]acetate-prelabeled baby hamster kidney cells, provided evidence for localization of [3 H]phospha-

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tidyl-alcohol mainly in endoplasmic reticulum membranes [90]. Localization of [3 H]phosphatidyl-alcohol in both plasma membranes and intracellular membranes was observed in subcellular fractions from [3 H]acetate-prelabeled HL-60 cells, stimulated in vitro with ARF-1 and GTPQS [91]. Since relatively long periods of incubation with phorbol ester are required, the possibility cannot be ruled out that [3 H]phosphatidyl-alcohol that is produced in one compartment is then transported to another compartment(s). The identity of the PLD isoform which is responsible for the production of [3 H]phosphatidyl-alcohol in intact cells could not be determined in these studies; however, later studies have indicated that the phorbol ester-responsive PLD is likely to be PLD1 [27,28]. PLD1 gene products are regulated by ARF and RhoA. The distribution of ARF- and RhoA-stimulated PLD activity was examined in rat liver subcellular fractions in vitro [92]. This study demonstrated that both ARF- and RhoA-dependent activities (likely representing the same enzymatic entity, namely PLD1) are highest in the plasma membranes, although signi¢cant activity was also found in nuclei and the Golgi apparatus. The plasma membrane localization of PLD, reported in certain cells, is consistent with the signalactivated nature of the enzyme and suggests that, in some cases, PLD activation could be tightly coupled to cell surface receptors. Whereas PKC is often stimulated upstream to PLD, a PKC-independent mode of activation was extensively documented (see [93] for review). A recent example is provided by lyso-PA, which activates PLD rapidly and transiently (20^60 s) in the absence of any activation of PI-speci¢c phospholipase C, phospholipase A2 , phosphoinositide 3-kinase or the mitogen-activated protein kinase cascade [94]. Direct receptor-PLD coupling mediated by G proteins has recently been suggested or demonstrated for both receptor tyrosine kinases and heptahelical G protein-coupled receptors. Certain members of the latter family of receptors were shown to associate with the PLD activator ARF, thus enabling BFA-sensitive stimulation of PLD [50]. Insulin, acting via the insulin receptor tyrosine kinase, causes activation of ARF, stimulates PLD in an ARF-dependent manner and promotes ARF association with the insulin receptor [95]. Recent evidence indicates that PLD2 is constitutively associated

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with the EGF receptor and that it undergoes tyrosine phosphorylation upon ligand-induced receptor activation [96]. Indeed, recombinant mouse PLD2 exhibits a plasma membrane localization under basal conditions, and is redistributed into submembranous vesicles upon serum stimulation [28]. In all the cases discussed above the implication is that PLD, which is localized in, or is recruited to the plasma membrane, could be activated by cell surface receptors directly and independently of other signaling cascades. The function of this receptor-direct activation of PLD at the plasma membrane is likely to be as a classical e¡ector enzyme, generating PA which acts as a second messenger. Discussion of the possible messenger functions of PA is beyond the scope of this review. Nevertheless it is important to note a novel signaling function of PA in facilitating the recruitment of Raf1 to the plasma membrane [97,98]. Whereas these data support a role for plasma membrane, ARF-dependent PLD in signal transduction, another function of PLD in endocytosis (possibly regulated by ARF-6) cannot be ruled out. 5.2. Nucleus The presence of PLD activity in cell nuclei was demonstrated in several recent studies. In addition to the above mentioned work showing ARF- and RhoA-dependent PLD activities in rat liver nuclei [92], a PLD activity was identi¢ed in MDCK cells nuclei which was stimulated in a synergistic manner by ATP (but not by non-phosphorylating ATP analogs) and by GTPQS [56,99]. A nuclear resident RhoA was implicated in the activation of this PLD, which resulted in elevation of nuclear PA and diacylglycerol (DAG) levels [56]. Signal-dependent activation of nuclear PLD was demonstrated also in thrombin-stimulated IIC9 ¢broblasts, which is likely mediated by thrombin-induced translocation of RhoA to the nucleus [100]. In the IIC9 ¢broblasts, however, the activation of PLD has no apparent role in generating DAG, possibly re£ecting the absence of a nuclear PA phosphohydrolase in these cells [100]. Thus, the ability of nuclear PLD to generate DAG (in conjunction with a nuclear PA phosphohydrolase) may well be cell type-speci¢c (compare [56,100]). The RhoA-dependent nuclear PLD is likely to be one of the mammalian PLD1 gene products,

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which can be stimulated by both RhoA and ARF. Indeed, in addition to the oleate-activated PLD, Banno et al. have identi¢ed in liver and hepatocyte nuclei an ARF-dependent PLD [79]. Interestingly, activity of the ARF-dependent PLD (but not of the oleate-activated enzyme) was increased nearly fourfold during liver regeneration concomitant with an increase in nuclear ARF level [79]. The ARF-dependent PLD was suggested to reside in the nuclear envelope since it was decreased upon treatment with Triton X-100 [79]. Similar results were obtained in nuclei prepared from HL-60 myelomonocytic leukemia cells, although in this case an ARF-insensitive activity remained associated with the nuclear matrix [101]. The nuclear ARF/RhoA-activated PLD may be involved in nuclear envelope signal transduction and/or nuclear vesicle dynamics during mitosis. The former possibility is consistent with the notion of a nuclear signaling apparatus, independent of that which operates in the plasma membrane (reviewed in [102,103]). The suggested role of ARF in nuclear envelope disassembly and reassembly during mitosis [44] raises the possibility that nuclear ARF/RhoAactivated PLD is involved in this process. It is unlikely however, that ARF-activated PLD plays an essential role in nuclear envelope dynamics in all eukaryotic cells (cf. [21]). 5.3. Golgi apparatus Various forms of ARF (ARF-1 through ARF-6) have been implicated in a variety of intracellular transport events from the nucleus to the plasma membrane [43^45]. The role played by ARF-1 in vesicular tra¤c in the Golgi apparatus is very well characterized [41,42]. ARF-1 is believed to associate with Golgi membranes upon activation (exchange of GDP with GTP), inducing the recruitment of coatomer proteins and the budding-o¡ of transport vesicles. The involvement of PLD in this process is suggested by the fact that ARF-dependent PLD activity was reported to be higher in Golgi-enriched membranes, compared to ER-enriched membranes or plasma membranes [48]. Furthermore, this study showed that Golgi membranes from BFA-resistant PtK1 cells exhibit 5- to 10-fold higher basal (i.e., non-ARF-dependent) PLD activity, which could be explained by BFA-resistant retention of active ARF

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in these membranes [48]. Ktistakis et al. provided additional evidence in favor of a role for PLD in ARF-dependent Golgi transport, by showing that formation of COPI-coated vesicles is inhibited by ethanol and that the requirement for ARF in coatomer binding can be bypassed by adding an exogenous, constitutively active bacterial PLD [104]. Primary alcohols inhibited also protein transport from the ER to the Golgi, an e¡ect which was reversed by exogenous addition of PA liposomes [105]. These studies suggested a model in which the accumulation of a PA facilitated the recruitment of coatomer to the site of vesicle budding in the Golgi [104]. Interestingly, in two studies in which the localization of recombinant tagged PLD1 was examined, a localization in the Golgi was not observed. HA-tagged PLD1a, expressed in REF-52 rat embryo ¢broblasts, was localized in the ER and, possibly, in late endosomes [28]. Likewise, recombinant PLD1a and PLD1b, tagged with green £uorescent protein (GFP) and expressed in COS-1 and RBL-2H3 cells, did not colocalize with a Golgi marker (TGN38) but, rather, with secretory granules and lysosomal markers [106]. Immuno£uorescence localization experiments of endogenous PLD1 gene products have not been reported as of yet. It should also be noted that the activation of PLD may not be an essential part of the basic ARF-mediated vesicle budding machinery of the Golgi in eukaryotic cells because in yeast, for instance, such a functionality does not exist. Furthermore, recent work demonstrates that COPI-coated vesicles can be formed in vitro from chemically de¢ned synthetic liposomes using reconstituted puri¢ed coatomer and ARF-1 [107,108]. PLD appears to be involved also in formation of nascent secretory vesicles in the trans-Golgi network (TGN). The evidence includes the fact that either immunopuri¢ed recombinant PLD1 [109] or a puri¢ed bacterial PLD [110] stimulated secretory vesicle formation from the TGN. In addition, inhibition of PLD-catalyzed formation of PA by n-butanol attenuated nascent vesicle release, whereas neither secnor tert-butanol (which do not support transphosphatidylation by PLD) were ine¡ective in this regard [109]. It was further shown that PLD-independent generation of PA, by the combined addition to permeabilized cells of bacterial phosphatidylinositol phospholipase C and diacylglycerol kinase, is su¤-

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cient to stimulate nascent vesicle release from the TGN [111]. Whereas these data support a role for PA in this process, the exact role of PA in the budding of nascent secretory vesicles is a matter for speculation. In analogy to its proposed role in the Golgi stacks, the activation of PLD in the TGN is suggested to result in formation of PA-enriched membrane microdomains that promote recruitment of TGN-speci¢c coat protein(s) such as the AP-1 complex [109]. This, however, is inconsistent with other results showing that recruitment of AP-1 adaptors onto the TGN is not a¡ected by either exogenously added bacterial PLD or by addition of neomycin [112]. 5.4. Exocytic and endocytic vesicles The studies discussed above suggest that, depending on cell type and experimental protocol, PLD activity is localized in plasma membranes, intracellular membranes, or both. One explanation for this apparent multimodal localization could be that PLD activity is recruited onto more than one cellular compartment upon the mobilization and membrane association of known PLD activators such as ARF, RhoA and PKC. The translocation of PLD activity was exempli¢ed in a study in which the neutrophil plasma membrane-enriched fraction was further separated from Golgi/secretory vesicle markers, resulting in complete segregation of ARF-dependent PLD activity from the plasma membrane marker [113]. In resting neutrophils most PLD activity colocalized with a secretory vesicle marker, but, upon stimulation by fMet-Leu-Phe, PLD activity colocalized with the superimposed plasma membrane and secretory vesicle markers, suggesting the mobilization of ARF-dependent PLD to the plasma membrane [113]. Localization of PLD1b to secretory granules, and its translocation to the plasma membrane upon cell stimulation, were observed in RBL-2H3 rat basophilic leukemia cells transfected with GFP-PLD1a and GFP-PLD1b [106]. In resting cells, the GFPtagged enzymes colocalized with markers to lysosomes and secretory granules, but upon stimulation by cross-linking of high-a¤nity IgE receptors, GFPPLD1 was recruited to the plasma membrane [106]. PLD has recently been implicated as a possible e¡ector for ARF-6 in the exocytotic pathway of

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chroma¤n cells [114]. This study shows that chroma¤n cell secretagogues stimulate PLD activity, the phosphatidylethanol product of which is colocalized with a plasma membrane marker. Stimulation resulted also in redistribution of ARF-6 which was translocated from the secretory granule fraction to the plasma membrane [114]. The activation of PLD correlated in time and in its Ca2‡ dependence with the secretory response, and the latter was attenuated by primary (but not secondary) alcohols. In the chroma¤n cells, signi¢cant PLD activity seems to be constitutively associated with plasma membrane [114]. Indeed, immunoblot analysis of chroma¤n cell membrane fractions indicates that PLD1 is predominantly found in plasma membranes whereas it is completely absent from the chroma¤n granules (M. Liscovitch, T. Scha«¡er, unpublished observations). Thus, the localization of PLD1 in secretory granules (as observed in neutrophils and RBL cells) is not a general property of PLD1 in secretory cells. Nevertheless, together, the studies in neutrophils [113], RBL cells [106] and chroma¤n cells [114] clearly suggest that PLD activation could play a role in regulated exocytotic secretion. It is interesting to note that an essential PLD cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2 ; see below) is required for regulated secretion in PC-12 cells (see [115,116] for review). It remains to be seen whether one function of PIP2 might be that of a cofactor in ARF-dependent activation of PLD during the exocytotic vesicle-plasma membrane fusion event, as previously suggested [117]. 5.5. Mitochondria A mammalian mitochondrial PLD-like activity was recently reported, that has several unusual properties [118,119]. Mitochondrial enzyme activity was monitored by measuring PA and headgroup production from endogenous mitochondrial phospholipids. The results suggest that Ca2‡ stimulates the hydrolysis of PE by a PLD-like activity that does not catalyze a transphosphatidylation reaction. The purity of the mitochondrial preparation has not been reported, and the results have not yet been con¢rmed using an exogenous substrate. The possible existence of this putative mitochondrial PLD is of interest, because some of its properties are reminiscent of a recently characterized yeast PLD activity [33,34]. Previous

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work has indicated the signi¢cant presence of an oleate-activated PLD in mammalian mitochondria [72], which more recently was shown to be down-regulated in diabetic cardiac muscle [81]. 5.6. Cytoskeleton One of the proposed functions of PLD in mammalian cells is in mediating agonist-induced reorganization of the cytoskeleton. A RhoA-dependent PLD was implicated in lyso-PA-induced formation of stress ¢bers [94,120]. Very recently, PLD1 was shown to associate with an octylglucoside-insoluble cytoskeletal fraction upon activation by GTPQS in permeabilized U937 cells [121]. This was associated with co-translocation of ARF and RhoA, and the cytoskeletal-associated activity was reduced by the RhoA-selective C. botulinum C3-exotoxin [121]. Also PLD2 might be involved in regulation of the cytoskeleton, as its overexpression results in reorganization of the cortical plasma membrane cytoskeleton [28]. Another example for relocalization of PLD to the cytoskeleton in response to altered physiological state is provided by the yeast SPO14/PLD1 gene product which, during meiosis, undergoes a dramatic translocation ¢rst to the spindle pole bodies and later to the new spore membrane [122]. It is interesting to note that cytoskeletal proteins such as fodrin inhibit PLD activity [123], whereas a lipid regulator of the cytoskeleton, PIP2 , also serves as a PLD cofactor (see below). Together, these data provide tentative yet tantalizing evidence supporting a role for PLD in regulation of the cytoskeleton. As in many of the proposed PLD functions, the target proteins which are presumably regulated by PA have yet to be identi¢ed. 6. Phosphatidylinositol 4,5-bisphosphate as a cofactor for PLD and a putative membrane targeting signal Several lines of evidence suggest that PIP2 is a cofactor required for PLD activation in vitro and in vivo (Fig. 3) [39,55,59,82,117,124,125]. PIP2 is a very potent activator, exhibiting an EC50 6 0.5 mol% regardless of the assay system used [126]. Previously we have shown that neomycin (which binds polyphosphoinositides with high a¤nity) inhibits PLD

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Fig. 3. Role of phosphatidylinositol-4,5-bisphosphate in PLD activation. In vitro experiments indicate that exogenous PIP2 can stimulate PLD activity [117] and that it is required for ARF- and RhoA-mediated PLD activation [39,125]. Neomycin, which is a high-a¤nity ligand of PIP2 , inhibits PLD activity in membranes and in permeabilized cells [127]. ATP potentiates the activation of PLD by GTPQS in permeabilized cells, concomitantly stimulating PIP2 biosynthesis [124]. Inhibition of PIP2 biosynthesis by antibodies to PI 4-kinase or a PI 4-kinase inhibitor adenosine [124] blocks PLD activation. Brain fodrin, a PIP2 -binding protein that decreases cellular PIP2 synthesis has a similar e¡ect [128]. Synaptojanin (a PIP2 5-phosphatase) inhibits PLD activation by promoting PIP2 degradation [160]. C. di¤cile toxin B inhibits Rho family GTPases, reducing PIP2 biosynthesis and level and consequently inhibiting PLD activation [129]. See text for more details. GTPQS, guanosine 5P-3-O(thio)triphosphate ; PA, phosphatidic acid; PI, phosphatidylinositol; PI4K, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol-4-phosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PIP2 , phosphatidylinositol-4,5-bisphosphate; PLD, phospholipase D.

activity in brain membranes, as well as GTPQS-induced PLD activation in permeabilized cells [127]. This e¡ect is likely due to the binding of neomycin to endogenous membrane PIP2 , because the inhibitory action of neomycin is lost upon puri¢cation of the enzyme, but can be restored by including PIP2 in the assay [117]. Similarly, PIP2 was shown to reverse neomycin-induced inhibition of PLD activity in HL60 cell membranes [59]. The activation of PLD by ARF depends on the inclusion of PIP2 in the substrate liposomes [39,82]. In addition, PIP2 is required for the activation by RhoA of rat brain salt-extractable PLD and of PLD in HL-60 membranes [55,125]. Using speci¢c inhibitory antibodies directed against PI 4-kinase, as well as the PI 4-kinase inhibitor adenosine, we have shown that the activation of PLD by GTPQS in permeabilized cells depends upon active synthesis of PIP2 by the phosphoinositide kinases PI 4-kinase and PI 4-phosphate 5-kinase [124]. Similarly, fodrin (non-erythroid spectrin) was found to inhibit PLD activation in permeabilized cells by causing a decrease in cell phosphoinositide levels through inhibition of phosphoinositide biosynthesis

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[128]. Schmidt et al. have demonstrated that C. di¤cile toxin B inhibits m3-receptor-mediated activation of PLD, concomitant with a marked decrease of membrane PIP2 levels. Interestingly, supplementation of exogenous PIP2 restored both basal and GTPQS-induced activation of PLD to normal levels, indicating that PIP2 is necessary for optimal PLD activation [129]. Thus, PIP2 appears to be a required cofactor for PLD activation by G proteins both in vitro and in situ. Three mammalian PLD isoforms, namely PLD1a, PLD1b and PLD2, are activated by PIP2 in vitro [28,29,46,47]. Further, the activity of other eukaryotic PLDs, including the yeast SPO14/ PLD1 gene product (M. Waksman, M. Liscovitch, unpublished observations) and the plant PLD-L and PLD-Q, are stimulated by PIP2 [26,130]. There are several open questions regarding the role played by PIP2 in PLD activation and function. First, does PIP2 activate PLD by direct interaction, or indirectly via modulation of ARF, RhoA or PKC? So far, no obvious PIP2 -binding domain (e.g., a pleckstrin homology domain) was identi¢ed within the PLD sequences, with the exception of plant PLDs that contain a C2/CaLB domain [25]. However, Yokozeki et al. demonstrated that PLD activity can associate with PIP2 -containing liposomes, even in the absence of ARF or RhoA, and that interference with PIP2 ^PLD interaction (using neomycin) releases PLD from the membranes [125]. These data suggest that PLD can interact with PIP2 directly, raising the possibility that PIP2 (possibly in cooperation with PE; cf. [131]) constitute a binding site for PLD on the cytoplasmic face of cellular membranes. This conclusion is supported by studies with plant PLDs, that demonstrate the direct binding of recombinant bacterially expressed PLD-K, -L and -Q with 3 H-labeled PIP2 [26]. PIP2 binding was shown to be speci¢c, concentration-dependent and saturable, and the extent of binding correlated with PIP2 dependent activation. Of note, activation of PLD-K by PIP2 was not inhibited by neomycin nor by high Ca2‡ concentrations, whereas the activation of PLD-L and PLD-Q were sensitive to these conditions. Qin et al. concluded that PIP2 binds to PLD-L and PLD-Q via a polybasic motif near the phosphatidyltransferase motif, while the binding to and activation of PLD-K are mediated by the C2/CaLB domain [26].

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crodomains during cell stimulation. Recent work indicates that such microdomains do exist in cell membranes; they may be isolated as low density detergent-insoluble particles, and they are enriched in PLD activity. 7. Localization of phospholipase D in detergentinsoluble membrane microdomains and caveolae

Fig. 4. Localization of phospholipase D in Triton-insoluble, low-density, caveolin-rich membrane domains prepared from HaCaT human keratinocytes. Low-density, Triton-insoluble membrane domains were prepared from HaCaT cell lysates by £otation in a discontinuous sucrose density gradient. Fractions were analyzed for protein content, PIP2 -dependent PC-PLD activity (panel A) and caveolin-1 immunoreactivity (panel A, inset). Speci¢c PLD activity is shown in panel B. Fractions 4^6 (Cav) and 9^12 (NCM) were pooled separately and immunoblotted using two di¡erent anti-human PLD1 antibodies (panel B, inset). Reproduced from Czarny et al. [149] with permission.

Taken together, the above data clearly demonstrate the important role played by PIP2 in PLD activation, and raise the possibility that PLD isoforms may either be constitutively associated with or be translocated to PIP2 -enriched membrane mi-

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Biological membranes may contain microdomains that are laterally segregated in the plane of the bilayer, whose unique lipid and protein composition may re£ect speci¢c functions as signaling platforms and vesicle tra¤c terminals at the plasma membrane [132,133]. Membrane microdomains have been variously termed DIGs (detergent-insoluble glycosphingolipid-rich complexes), LDTI (low-density Tritoninsoluble) domains, CHIFF (CHAPS-insoluble £oating fraction) or GEMs (glycosphingolipid-enriched membranes), based on their composition and physicochemical properties. DIGs exhibit a characteristic lipid composition rich in sphingolipids and cholesterol [133,134]. A subset of such domains comprises speci¢c, morphologically and biochemically well-de¢ned cellular structures termed caveolae [135]. Caveolae are non-clathrin-coated plasma membrane invaginations, 50^100 nm in size [136], that have a characteristic striated coat structure decorated with a 21-kDa integral membrane protein called caveolin [137]. The similar lipid composition of DIGs and caveolae facilitates their isolation as low density, Triton-insoluble membrane particles on discontinuous sucrose density gradients [134,138]. DIGs are present in most if not all cell types, whereas caveolae are mostly (though not exclusively) found in epithelial cells, and are generally absent from hematopoietic cells [137]. Caveolae have been implicated in transport processes such as endocytosis and transcytosis (see [139] and citations therein), as well as cholesterol e¥ux [140]. Both caveolae and DIGs are thought to play an important role in cellular signal transduction [141,142]. Pike et al. have reported recently that the low-density Triton X-100-insoluble DIGs/caveolae fraction is enriched in polyphosphoinositides, especially PIP2 [143,144]. Subsequent work con¢rmed this ¢nding [145,146] and showed that PIP2 -enriched domains

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are present also in cells which are devoid of caveolae [147]. In view of the proposed function for PIP2 as a required cofactor for PLD we examined the presence of PLD activity in DIGs/caveolar membranes. We have found that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in the low-density Triton X-100-insoluble DIGs/caveolae fraction that contains the caveolar marker protein caveolin-1 (Fig. 4A,B) [148,149]. Similar to other PLD activities, the PLD activity in this membrane fraction is stimulated by PIP2 and is inhibited by neomycin. Immunoblot analysis indicates that DIGs/caveolae membranes do not contain the PLD1 isoform (Fig. 4B, inset). In contrast, recombinant mouse PLD2 expressed in stably transfected CHO cells is preferentially targeted to these membrane domains. PLD activity is enriched in low-density Triton-insoluble membrane microdomains also in U937 promonocytes, even though these cells do not express caveolin. In these U937 cells, too, PLD1 is largely excluded from low-density Tritoninsoluble membrane domains. Overexpression of caveolin-1 in v-Src-transformed ¢broblasts resulted in elevation of PLD activity in the DIGs/caveolae membranes [149], suggesting that caveolin may modulate PLD activity in caveolae. This is consistent with the fact that a 20-amino-acid peptide that corresponds to the caveolin-1 sca¡olding domain (caveolin-182ÿ101 ) modulated the DIGs/caveolar PLD activity, causing stimulation at concentrations of 1^10 WM and inhibition at concentrations above 10 WM [149]. In accord with these results, we have found that the activity of PLD in DIGs/caveolar membranes was substantially elevated in multidrug-resistant HT-29MDR and MCF-7-MDR human cancer cells (G. Fiucci, M. Czarny, Y. Lavie, M. Liscovitch, manuscript in preparation). MDR cells express very high levels of caveolin-1 [150]. These results suggest that a PLD activity (most likely PLD2) is enriched in lowdensity Triton-insoluble membrane domains and caveolae. The DIGs/caveolar PLD may utilize resident PIP2 as a cofactor. Otherwise, the regulation of the DIGs/caveolar PLD in situ is unknown. Likewise, the function(s) of this enzyme remain to be elucidated. One possibility is that the DIGs/caveolar PLD is involved in signal transduction processes that have been localized to these membrane micro-

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domains [141,142]. Another intriguing possibility is that the caveolar PLD may have an analogous function to that which had been postulated for ARFdependent PLD in the Golgi. 8. Future perspective Currently, research on the localization of PLD isozymes is at a watershed. Following the cloning of distinct isoforms, the question of subcellular localization is beginning to be addressed using immunological and molecular biological tools. These ¢rst studies will certainly be followed by many more. Therefore, we may realistically expect to acquire a coherent picture of PLD localization in di¡erent cell types and under di¡erent physiological conditions in the near future. Immunological and molecular tools will also be invaluable in establishing the functions of di¡erent PLDs in their various locales. Together, advances in these two ¢elds will provide a fuller understanding of the downstream events that occur in consequence of PLD activation, particularly the identity of target proteins that are directly regulated by PA. Recent work strongly suggests that PA might act to regulate the activity of protein kinases (e.g., [151,152]) as well as protein phosphatases [153]. It is therefore reasonable to assume that in due time, colocalization of PLD with upstream regulatory elements and downstream targets, in speci¢c cellular compartments, will be established and linked to relevant site-speci¢c functions. Acknowledgements The work carried out in our laboratory was supported in part by grants from the Israel Science Foundation (Jerusalem), the U.S.^Israel Binational Science Foundation (Jerusalem), the Minerva Foundation (Munich), the Levine Fund for Applied Research, and the Forchheimer Center for Molecular Genetics (Rehovot). M.L. is the incumbent of the Harold L. Korda Professorial Chair in Biology. Y.L. was a recipient of a Fellowship from the Israel Ministry of Absorption. M.C. is a recipient of a FEBS Long-term Fellowship.

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References [1] M. Liscovitch, L.C. Cantley, Lipid second messengers, Cell 77 (1994) 329^334. [2] N. Divecha, R.F. Irvine, Phospholipid signaling, Cell 80 (1995) 269^278. [3] Y. Nishizuka, Protein kinase C and lipid signaling for sustained cellular responses, FASEB J. 9 (1995) 484^496. [4] S. Ghosh, J.C. Strum, R.M. Bell, Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling, FASEB J. 11 (1997) 45^50. [5] M. Heller, Phospholipase D, in: R. Paoletty, D. Kritchevsky (Eds.), Advances in Lipid Research, vol. 16, Academic Press, New York, 1978, pp. 267^326. [6] J.H. Exton, Signaling through phosphatidylcholine breakdown, J. Biol. Chem. 265 (1990) 1^4. [7] M.M. Billah, J.C. Anthes, The regulation and cellular functions of phosphatidylcholine hydrolysis, Biochem. J. 269 (1990) 281^291. [8] D. English, Y. Cui, R.A. Siddiqui, Messenger functions of phosphatidic acid, Chem. Phys. Lipids 80 (1996) 117^132. [9] M.N. Hodgkin, T.R. Pettitt, A. Martin, R.H. Michell, A.J. Pemberton, M.J. Wakelam, Diacylglycerols and phosphatidates: which molecular species are intracellular messengers?, Trends Biochem. Sci. 23 (1998) 200^204. [10] V.E. Natarajan, Phospholipase D and signal transduction in mammalian cells, Chem. Phys. Lipids 80 (1996) 1^142. [11] J.H. Exton, New developments in phospholipase D, J. Biol. Chem. 272 (1997) 15579^15582. [12] A.J. Morris, S.M. Hammond, C. Colley, T.-C. Sung, J.M. Jenco, V.A. Sciorra, S.A. Rudge, M.A. Frohman, Regulation and functions of phospholipases D, Biochem. Soc. Transact. 25 (1997) 1151^1157. [13] J. Gomez-Cambronero, P. Keire, Phospholipase D: A novel major player in signal transduction, Cell. Signal. 10 (1998) 387^397. [14] T. Taki, J.N. Kanfer, Partial puri¢cation and properties of a rat brain phospholipase D, J. Biol. Chem. 254 (1979) 9761^ 9765. [15] H.A. Brown, S. Gutowski, R.A. Kahn, P.C. Sternweis, Partial puri¢cation and characterization of ARF-sensitive phospholipase D from porcine brain, J. Biol. Chem. 270 (1995) 14935^14943. [16] A.M. Vinggaard, H.S. Hansen, Characterization and partial puri¢cation of phospholipase D from human placenta, Biochim. Biophys. Acta 1258 (1995) 169^176. [17] A. Abousalham, M. Riviere, M. Teissere, R. Verger, Improved puri¢cation and biochemical characterization of phospholipase D from cabbage, Biochim. Biophys. Acta 1158 (1993) 1^7. [18] X. Wang, J.H. Dyer, L. Zheng, Puri¢cation and immunological analysis of phospholipase D from castor bean endosperm, Arch. Biochem. Biophys. 306 (1993) 486^494. [19] X. Wang, L. Xu, L. Zheng, Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L, J. Biol. Chem. 269 (1994) 20312^20317.

BBAMCB 55483 16-7-99

[20] J. Ueki, S. Morioka, T. Komari, T. Kumashiro, Puri¢cation and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.), Plant Cell Physiol. 36 (1995) 903^ 914. [21] K. Rose, S.A. Rudge, M.A. Frohman, A.J. Morris, J. Engebrecht, Phospholipase D signaling is essential for meiosis, Proc. Natl. Acad. Sci. USA 92 (1995) 12151^12155. [22] M. Waksman, Y. Eli, M. Liscovitch, J.E. Gerst, Identi¢cation and characterization of a gene encoding phospholipase D activity in yeast, J. Biol. Chem. 271 (1996) 2361^2364. [23] K. Ella, J.W. Dolan, C. Qi, K.E. Meier, Characterization of Saccharomyces cerevisiae de¢cient in expression of phospholipase D, Biochem. J. 314 (1996) 15^19. [24] S.M. Hammond, Y.M. Altshuller, T.-C. Sung, S.A. Rudge, K. Rose, J. Engebrecht, A.J. Morris, M.A. Frohman, Human ADP-ribosylation factor-activated phosphatidylcholinespeci¢c phospholipase D de¢nes a new and highly conserved gene family, J. Biol. Chem. 270 (1995) 29640^29643. [25] X. Wang, Molecular analysis of phospholipase D, Trends Plant Sci. 2 (1997) 261^266. [26] W. Qin, K. Pappan, X. Wang, Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDQ and regulation of plant PLDQ, -L, and -K by polyphosphoinositides, J. Biol. Chem. 272 (1997) 28267^28273. [27] S.K. Park, J.J. Provost, C.D. Bae, W.T. Ho, J.H. Exton, Cloning and characterization of phospholipase D from rat brain, J. Biol. Chem. 272 (1997) 29263^29271. [28] W.C. Colley, T.-C. Sung, R. Roll, J. Jenco, S.M. Hammond, Y. Altshuller, D. Bar-Sagi, A.J. Morris, M.A. Frohman, Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provoke cytoskeletal reorganization, Curr. Biol. 7 (1997) 191^201. [29] T. Kodaki, S. Yamashita, Cloning, expression and characterization of a novel phospholipase D complementary DNA from rat brain, J. Biol. Chem. 272 (1997) 11408^11413. [30] A.J. Morris, J. Engebrecht, M.A. Frohman, Structure and regulation of phospholipase D, Trends Pharmacol. Sci. 17 (1996) 182^185. [31] G. Ribbes, J. Henry, C. Cariven, P. Pontarotti, H. Chap, M. Record, Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D, Biochem. Biophys. Res. Commun. 224 (1996) 206^211. [32] C.P. Ponting, I.D. Kerr, A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases : Identi¢cation of duplicated repeats and potential active site residues, Protein Sci. 5 (1996) 914^ 922. [33] J.A. Mayr, S.D. Kohlwein, F. Paltauf, Identi¢cation of a novel, Ca2‡ -dependent phospholipase D with preference for phosphatidylserine and phosphatidylethanolamine in Saccharomyces cerevisiae, FEBS Lett. 393 (1996) 236^240. [34] M. Waksman, X. Tang, Y. Eli, J.E. Gerst, M. Liscovitch, Identi¢cation of a novel Ca2‡ -dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1, J. Biol. Chem. 272 (1997) 36^39.

Cyaan Magenta Geel

Zwart

M. Liscovitch et al. / Biochimica et Biophysica Acta 1439 (1999) 245^263 [35] J.H. Exton, Phospholipase D, Biochim. Biophys. Acta 1436 (1998) 105^115. [36] J.C. Anthes, P. Wang, M.I. Siegel, R.W. Egan, M.M. Billah, Granulocyte phospholipase D is activated by a guanine nucleotide dependent protein factor, Biochem. Biophys. Res. Commun. 175 (1991) 236^243. [37] S.C. Olson, E.P. Bowman, J.D. Lambeth, Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5P-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol, J. Biol. Chem. 266 (1991) 17236^17242. [38] B. Geny, A. Fensome, S. Cockcroft, Rat brain cytosol contains a factor which reconstitutes guanine-nucleotide-binding-protein-regulated phospholipase D activation in HL-60 cells previously permeabilized with streptolysin O, Eur. J. Biochem. 215 (1993) 389^396. [39] H.A. Brown, S. Gutowski, C.R. Moomaw, C. Slaughter, P.C. Sternweis, ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity, Cell 75 (1993) 1137^1144. [40] S. Cockcroft, G.M.H. Thomas, A. Fensome, B. Geny, E. Cunningham, I. Gout, I. Hiles, N.F. Totty, O. Truong, J.J. Hsuan, Phospholipase D: a downstream e¡ector of ARF in granulocytes, Science 263 (1994) 523^526. [41] R.A. Kahn, J.K. Yucel, V. Malhotra, ARF signaling: A potential role of phospholipase D in membrane tra¤c, Cell 75 (1993) 1045^1048. [42] J. Moss, M. Vaughan, Structure and function of ARF proteins: Activators of cholera toxin and critical components of intracellular vesicular transport processes, J. Biol. Chem. 270 (1995) 12327^12330. [43] M.J. Vidal, P.D. Stahl, The small G proteins Rab4 and ARF are associated with released exosomes during reticulocyte maturation, Eur. J. Cell Biol. 60 (1993) 261^267. [44] A.L. Boman, K.M.C. Sullivan, K.L. Wilson, Regulation of nuclear envelope assembly and disassembly by ARF and other GTP-binding proteins, in: V.W. Hu (Ed.), The Cell Cycle: Regulators, Targets, and Clinical Applications, Plenum, New York, 1994, pp. 197^202. [45] J.M. Lehnard, M.I. Colombo, P.D. Stahl, Heterotrimeric GTP-binding proteins (G proteins) and ADP-ribosylation factor (ARF) regulate priming of endosomal membranes for fusion, Arch. Biochem. Biophys. 312 (1994) 474^479. [46] S.M. Hammond, J.M. Jenco, S. Nakashima, K. Cadwallader, Q. Gu, S. Cook, Y. Nozawa, G.D. Prestwich, M.A. Frohman, A.J. Morris, Characterization of two alternately spliced forms of phospholipase D1. Activation of the puri¢ed enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTPbinding proteins and protein kinase C-alpha, J. Biol. Chem. 272 (1997) 3860^3868. [47] K. Katayama, T. Kodaki, Y. Nagamachi, S. Yamashita, Cloning, di¡erential regulation and tissue distribution of alternatively spliced isoforms of ADP-ribosylation-factor-de-

BBAMCB 55483 16-7-99

[48]

[49]

[50]

[51] [52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

259

pendent phospholipase D from rat liver, Biochem. J. 329 (1998) 647^652. N.T. Ktistakis, H.A. Brown, P.C. Sternweis, M.G. Roth, Phospholipase D is present in Golgi-enriched membranes and its activation by ADP-ribosylation factor is sensitive to brefeldin A, Proc. Natl. Acad. Sci. USA 92 (1995) 4952^4956. U. Rumenapp, M. Geiszt, F. Wahn, M. Schmidt, K.H. Jakobs, Evidence for ADP-ribosylation factor-mediated activation of phospholipase D by m3 muscarinic acetylcholine receptor, Eur. J. Biochem. 234 (1995) 240^244. R. Mitchell, D. McCulloch, E. Lutz, M. Johnson, C. MacKenzie, M. Fennel, G. Fink, W. Zhou, S.C. Sealfon, Rhodopsin-family receptors associate with small G proteins to activate phospholipase D, Nature 392 (1998) 411^414. J. Moss, M. Vaughan, Molecules in the ARF orbit, J. Biol. Chem. 273 (1998) 21431^21434. E.P. Bowman, D.J. Uhlinger, J.D. Lambeth, Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein, J. Biol. Chem. 268 (1993) 21509^21512. K.C. Malcolm, A.H. Ross, R.-G. Qiu, M. Symons, J.H. Exton, Activation of rat liver phospholipase D by the small GTP-binding protein RhoA, J. Biol. Chem. 269 (1994) 25951^25954. A.R. Siddiqui, J.L. Smith, A.H. Ross, R.-G. Qiu, M. Symons, J.H. Exton, Regulation of phospholipase D in HL60 cells. Evidence for a cytosolic phospholipase D, J. Biol. Chem. 270 (1995) 8466^8473. A. Martin, F.D. Brown, M.N. Hodgkin, A.J. Bradwell, S.J. Cook, M. Hart, M.J.O. Wakelam, Activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase in HL60 membranes is mediated by endogenous Arf but not Rho, J. Biol. Chem. 271 (1996) 17397^17403. M.A. Balboa, P.A. Insel, Nuclear phospholipase D in Madin-Darby canine kidney cells. Guanosine 5P-O-(thiotriphosphate)-stimulated activation is mediated by RhoA and is downstream of protein kinase C, J. Biol. Chem. 270 (1995) 29843^29847. J.Y. Kwak, I. Lopez, D.J. Uhlinger, S.H. Ryu, J.D. Lambeth, RhoA and a cytosolic 50-kDa factor reconstitute GTP gamma S-dependent phospholipase D activity in human neutrophil subcellular fractions, J. Biol. Chem. 270 (1995) 27093^27098. K. Ohguchi, Y. Banno, S. Nakashima, Y. Nozawa, Activation of membrane-bound phospholipase D by protein kinase C in HL 60 cells: Synergistic action of a small GTP-binding protein RhoA, Biochem. Biophys. Res. Commun. 211 (1995) 306^311. K. Ohguchi, Y. Banno, S. Nakashima, Y. Nozawa, Regulation of membrane-bound phospholipase D by protein kinase C in HL60 cells. Synergistic action of small GTP-binding protein RhoA, J. Biol. Chem. 271 (1996) 4366^4372. W.D. Singer, H.A. Brown, G.M. Bokoch, P.C. Sternweis, Resolved phospholipase D activity is modulated by cytosolic

Cyaan Magenta Geel

Zwart

260

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

M. Liscovitch et al. / Biochimica et Biophysica Acta 1439 (1999) 245^263 factors other than ARF, J. Biol. Chem. 270 (1995) 14944^ 14950. H. Kuribara, K. Tago, T. Yokozeki, T. Sasaki, Y. Takai, N. Morii, S. Narumiya, T. Katada, Y. Kanaho, Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by Clostridium botulinum C3 exoenzyme, J. Biol. Chem. 270 (1995) 25667^ 25671. L.M. Machesky, A. Hall, Rho: a connection between membrane receptor signalling and the cytoskeleton, Trends Cell Biol. 6 (1996) 304^310. M. Schmidt, U. Rumenapp, C. Bienek, J. Keller, C. von Eichel-Streiber, K.H. Jakobs, Inhibition of receptor signaling to phospholipase D by Clostridium di¤cile toxin B. Role of Rho proteins, J. Biol. Chem. 271 (1996) 2422^2426. K.C. Malcolm, C.M. Elliott, J.H. Exton, Evidence for Rhomediated agonist stimulation of phospholipase D in Rat1 ¢broblasts. E¡ects of Clostridium butulinum C3 exoenzyme, J. Biol. Chem. 271 (1996) 13135^13139. W.D. Singer, H.A. Brown, X. Jiang, P.C. Sternweis, Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity, J. Biol. Chem. 271 (1996) 4504^4510. K. Shimooku, T. Akisue, H. Jinnai, T. Hitomi, C. Ogino, K. Yoshida, S.-I. Nakamura, Y. Nishizuka, Reconstitution of GTP-Q-S-dependent phospholipase D activity with ARF, Rho, and a soluble 36-kDa protein, FEBS Lett. 387 (1996) 141^144. M. Schmidt, S.M. Huwe, B. Fasselt, D. Homann, U. Rumenapp, J. Sandmann, K.H. Jakobs, Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors. Evidence for involvement of tyrosine phosphorylation, Eur. J. Biochem. 225 (1994) 667^675. T.C. Sung, Y. Zhang, A.J. Morris, M.A. Frohman, Structural analysis of human phospholipase D1, J. Biol. Chem. 274 (1999) 3659^3666. M. Yamazaki, Y. Zhang, H. Watanabe, T. Yokozeki, S. Ohno, K. Kaibuchi, H. Shibata, H. Mukai, Y. Ono, M.A. Frohman, Y. Kanaho, Interaction of the small G protein RhoA with the C terminus of human phospholipase D1, J. Biol. Chem. 274 (1999) 6035^6038. J.N. Kanfer, The base exchange enzymes and phospholipase D of mammalian tissue, Can. J. Biochem. 58 (1980) 1370^ 1380. M. Danin, V. Chalifa, H. Mo«hn, U.S. Schmidt, M. Liscovitch, Rat brain membrane-bound phospholipase D, in: J.N. Fain (Ed.), Lipid Metabolism in Signaling Systems, vol. 18, Academic Press, San Diego, 1993, pp. 14^24. V. Panagia, C. Ou, Y. Taira, J. Dai, N.S. Dhalla, Phospholipase D activity in subcellular membranes of rat ventricular myocardium, Biochim. Biophys. Acta 1064 (1991) 242^250. J. Horwitz, L.L. Davis, The substrate speci¢city of brain microsomal phospholipase D, Biochem. J. 295 (1993) 793^ 798. H. Mo«hn, V. Chalifa, M. Liscovitch, Substrate speci¢city of neutral phospholipase D from rat brain studied by selective

BBAMCB 55483 16-7-99

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

labeling of endogenous synaptic membrane phospholipids in vitro, J. Biol. Chem. 267 (1992) 11131^11136. M. Kobayashi, J.N. Kanfer, Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D, J. Neurochem. 48 (1987) 1597^1603. J.N. Kanfer, D. McCartney, Phospholipase D activity of isolated rat brain plasma membranes, FEBS Lett. 337 (1994) 251^254. R.A. Siddiqui, J.H. Exton, Oleate stimulation of diacylglycerol formation from phosphatidylcholine through e¡ects on phospholipase D and phosphatidate phosphohydrolase, Eur. J. Biochem. 210 (1992) 601^607. J.N. Kanfer, D. McCartney, I.N. Singh, L. Freysz, Phospholipase D activity of rat brain neuronal nuclei, J. Neurochem. 67 (1996) 760^766. Y. Banno, K. Tamiya-Koizumi, H. Oshima, A. Morikawa, S. Yoshida, Y. Nozawa, Nuclear ADP-ribosylation factor (ARF)- and oleate-dependent phospholipase D (PLD) in rat liver cells. Increases of ARF-dependent PLD activity in regenerating liver cells, J. Biol. Chem. 272 (1997) 5208^5213. S. Okamura, S. Yamashita, Puri¢cation and characterization of phosphatidylcholine phospholipase D from pig lung, J. Biol. Chem. 269 (1994) 31207^31213. S.A. Williams, P.S. Tappia, C.H. Yu, L. Binaglia, V. Panagia, N.S. Dhalla, Subcellular alterations in cardiac phospholipase D activity in chronic diabetes, Prostaglandins Leukot. Essent. Fatty Acids 57 (1997) 95^99. D. Massenburg, J.-S. Han, M. Liyanage, W.A. Patton, S.G. Rhee, J. Moss, M. Vaughan, Activation of rat brain phospholipase D by ADP-ribosylation factors 1, 5, and 6: Separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes, Proc. Natl. Acad. Sci. USA 91 (1994) 11718^11722. J. Balsinde, E. Diez, B. Fernandez, F. Mollinedo, Biochemical characterization of phospholipase D activity from human neutrophils, Eur. J. Biochem. 186 (1989) 717^724. P. Wang, J.C. Anthes, I.M. Siegel, R.W. Egan, M.M. Billah, Existence of cytosolic phospholipase D. Identi¢cation and comparison with membrane-bound enzyme, J. Biol. Chem. 266 (1991) 14877^14880. C. Huang, R.L. Wykle, L. Daniel, M.C. Cabot, Identi¢cation of phosphatidylcholine-selective and phosphatidylinositol-selective phospholipase D in Madin-Darby canine kidney cells, J. Biol. Chem. 267 (1992) 16859^16865. A.M. Vinggaard, J.J. Provost, J.H. Exton, H.S. Hansen, Arf and RhoA regulate both the cytosolic and the membranebound phospholipase D from human placenta, Cell. Signal. 9 (1997) 189^196. M. Nemat-Gorgani, J.E. Wilson, Ambiquitous behavior ^ a biological phenomenon of general signi¢cance?, Curr. Top. Cell Regul. 16 (1980) 45^54. G. Ribbes, A. Cane, V. Planat, M. Breton, H. Chap, G. Bereziat, M. Record, O. Colard, Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil, J. Cell. Biochem. 62 (1996) 56^68.

Cyaan Magenta Geel

Zwart

M. Liscovitch et al. / Biochimica et Biophysica Acta 1439 (1999) 245^263 [89] Y.S. Edwards, A.W. Murray, Accumulation of phosphatidylalcohol in cultured cells: use of subcellular fractionation to investigate phospholipase D activity during signal transduction, Biochem. J. 308 (1995) 473^480. [90] C. Decker, M.J. Miro Obradors, D.J. Sillence, D. Allan, Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells, Biochem. J. 320 (1996) 885^890. [91] J. Whatmore, C.P. Morgan, E. Cunningham, K.S. Collison, K.R. Willison, S. Cockcroft, ADP-ribosylation factor 1regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells, Biochem. J. 320 (1996) 785^794. [92] J.J. Provost, J. Fudge, S. Israelit, A.R. Siddiqi, J.H. Exton, Tissue-speci¢c distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA- and ADP- ribosylation factor (ARF)-regulated isoenzymes, Biochem. J. 319 (1996) 285^291. [93] M. Liscovitch, V. Chalifa, Signal-activated phospholipase D, in: M. Liscovitch (Ed.), Signal-activated Phospholipases, R.G. Landes, Austin, 1994, pp. 31^63. [94] M.J. Cross, S. Roberts, A.J. Ridley, M.N. Hodgkin, A. Stewart, L. Claesson-Welsh, M.J.O. Wakelam, Stimulation of stress ¢bre formation mediated by activation of phospholipase D, Curr. Biol. 6 (1996) 588^597. [95] K. Shome, C. Vasudevan, G. Romero, ARF proteins mediate insulin-dependent activation of phospholipase D, Curr. Biol. 7 (1997) 387^396. [96] R. Slaaby, T. Jensen, H.S. Hansen, M.A. Frohman, K. Seedorf, PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation, J. Biol. Chem. 273 (1998) 33722^33727. [97] S. Ghosh, R.M. Bell, Regulation of Raf-1 kinase by interaction with the lipid second messenger, phosphatidic acid, Biochem. Soc. Trans. 25 (1997) 561^565. [98] M.A. Rizzo, K. Shome, C. Vasudevan, D.B. Stolz, T.C. Sung, M.A. Frohman, S.C. Watkins, G. Romero, Phospholipase D and its product, phosphatidic acid, mediate agonist- dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway, J. Biol. Chem. 274 (1999) 1131^1139. [99] M.A. Balboa, J. Balsinde, E.A. Dennis, P.A. Insel, A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells, J. Biol. Chem. 270 (1995) 11738^11740. [100] J.J. Baldassare, M.B. Jarpe, L. Alferes, D.M. Raben, Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction, J. Biol. Chem. 272 (1997) 4911^4914. [101] J.M. Clark, M.N. Hodgkin, M.J. Wakelam, HL60 nuclei lacking the nuclear double membrane contain a PLD activity which is insensitive to the ADP-ribosylation factor, Biochem. Soc. Trans. 25 (1997) S590. [102] D.M. Raben, M.B. Jarpe, K.L. Leach, Nuclear lipid metabolism in NEST: Nuclear Envelope Signal Transduction, J. Membr. Biol. 142 (1994) 1^7.

BBAMCB 55483 16-7-99

261

[103] N. Divecha, H. Ban¢c, J.E. Treagus, L. Vann, R.F. Irvine, C. D'Santos, Nuclear diacylglycerol, the cell cycle, the enzymes and a red herring (or how we came to love phosphatidylcholine), Biochem. Soc. Trans. 25 (1997) 571^575. [104] N.T. Ktistakis, H.A. Brown, M.G. Waters, P.C. Sternweis, M.G. Roth, Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles, J. Cell Biol. 134 (1996) 295^306. [105] K. Bi, M.G. Roth, N.T. Ktistakis, Phosphatidic acid formation by phospholipase D is required for transport from the endoplasmic reticulum to the Golgi complex, Curr. Biol. 7 (1997) 301^307. [106] F.D. Brown, N. Thompson, K.M. Saqib, J.M. Clark, D. Powner, N.T. Thompson, R. Solari, M.J. Wakelam, Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation, Curr. Biol. 8 (1998) 835^838. [107] A. Spang, K. Matsuoka, S. Hamamoto, R. Schekman, L. Orci, Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes, Proc. Natl. Acad. Sci. USA 95 (1998) 11199^ 11204. [108] M. Bremser, W. Nickel, M. Schweikert, M. Ravazzola, M. Amherdt, H.C. A, T.H. Sollner, J.E. Rothman, F.T. Wieland, Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors, Cell 96 (1999) 495^ 506. [109] Y.G. Chen, A. Siddhanta, C.D. Austin, S.M. Hammond, T.C. Sung, M.A. Frohman, A.J. Morris, D. Shields, Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network, J. Cell Biol. 138 (1997) 495^ 504. [110] O. Tuscher, C. Lorra, B. Bouma, K.W. Wirtz, W.B. Huttner, Cooperativity of phosphatidylinositol transfer protein and phospholipase D in secretory vesicle formation from the TGN ^ phosphoinositides as a common denominator?, FEBS Lett. 419 (1997) 271^275. [111] A. Siddhanta, D. Shields, Secretory vesicle budding from the trans-Golgi network is mediated by phosphatidic acid levels, J. Biol. Chem. 273 (1998) 17995^17998. [112] M.A. West, N.A. Bright, M.S. Robinson, The role of ADPribosylation factor and phospholipase D in adaptor recruitment, J. Cell Biol. 138 (1997) 1239^1254. [113] C.P. Morgan, H. Sengelov, J. Whatmore, N. Borregaard, S. Cockcroft, ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionylleucyl-phenylalanine stimulation of human neutrophils, Biochem. J. 325 (1997) 581^585. [114] A.S. Caumont, M.C. Galas, N. Vitale, D. Aunis, M.F. Bader, Regulated exocytosis in chroma¤n cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D, J. Biol. Chem. 273 (1998) 1373^1379. [115] T.F.J. Martin, Identi¢cation of proteins required for Ca(2+)-activated secretion, Ann. NY Acad. Sci. 710 (1994) 328^332.

Cyaan Magenta Geel

Zwart

262

M. Liscovitch et al. / Biochimica et Biophysica Acta 1439 (1999) 245^263

[116] T.F. Martin, K.M. Loyet, V.A. Barry, J.A. Kowalchyk, The role of PtdIns(4,5)P2 in exocytotic membrane fusion, Biochem. Soc. Trans. 25 (1997) 1137^1141. [117] M. Liscovitch, V. Chalifa, P. Pertile, C.-S. Chen, L.C. Cantley, Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D, J. Biol. Chem. 269 (1994) 21403^21406. [118] M. Madesh, K.A. Balasubramanian, Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria, Lipids 32 (1997) 471^479. [119] M. Madesh, S.A. Ibrahim, K.A. Balasubramanian, Phospholipase D activity in the intestinal mitochondria: activation by oxygen free radicals, Free Radic. Biol. Med. 23 (1997) 271^277. [120] K.-S. Ha, E.-J. Yeo, J.H. Exton, Lysophosphatidic acid activation of phosphatidylcholine-hydrolysing phospholipase D and actin polymerization by a pertussis toxin-sensitive mechanism, Biochem. J. 303 (1994) 55^59. [121] S.S. Iyer, D.J. Kusner, Association of phospholipase D activity with the detergent-insoluble cytoskeleton of U937 promonocytic leukocytes, J. Biol. Chem. 274 (1999) 2350^ 2359. [122] S.A. Rudge, A.J. Morris, J. Engebrecht, Relocalization of phospholipase D activity mediates membrane formation during meiosis, J. Cell Biol. 140 (1998) 81^90. [123] S. Lukowski, M.C. Lecomte, J.P. Mira, P. Marin, H. Gautero, F. Marie-Russo, B. Geny, Inhibition of phospholipase D activity by fodrin. An active role for the cytoskeleton, J. Biol. Chem. 271 (1996) 24164^24171. [124] P. Pertile, M. Liscovitch, V. Chalifa, L.C. Cantley, Phosphatidylinositol 4,5-bisphosphate synthesis is required for activation of phospholipase D in U937 cells, J. Biol. Chem. 270 (1995) 5130^5135. [125] T. Yokozeki, H. Kuribara, T. Katada, K. Touhara, Y. Kanaho, Partially puri¢ed RhoA-stimulated phospholipase D activity speci¢cally binds to phosphatidylinositol 4,5-bisphosphate, J. Neurochem. 66 (1996) 1234^1239. [126] V. Chalifa-Caspi, Y. Eli, M. Liscovitch, Kinetic analysis in mixed micelles of partially puri¢ed rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5bisphosphate, Neurochem. Res. 23 (1998) 589^599. [127] M. Liscovitch, V. Chalifa, M. Danin, Y. Eli, Inhibition of neural phospholipase D activity by aminoglycoside antibiotics, Biochem. J. 279 (1991) 319^321. [128] S. Lukowski, J.P. Mira, A. Zachowski, B. Geny, Fodrin inhibits phospholipases A2 , C, and D by decreasing polyphosphoinositide cell content, Biochem. Biophys. Res. Commun. 248 (1998) 278^284. [129] M. Schmidt, U. Rumenapp, C. Nehls, S. Ott, J. Keller, E. Von, C. Streiber, K.H. Jakobs, Restoration of Clostridium di¤cile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate, Eur. J. Biochem. 240 (1996) 707^ 712. [130] K. Pappan, W. Qin, J.H. Dyer, L. Zheng, X. Wang, Molecular cloning and functional analysis of polyphosphoino-

BBAMCB 55483 16-7-99

[131]

[132] [133]

[134]

[135]

[136]

[137] [138]

[139]

[140] [141]

[142]

[143]

[144]

[145]

[146]

sitide-dependent phospholipase D, PLDL, from Arabidopsis, J. Biol. Chem. 272 (1997) 7055^7061. S. Nakamura, Y. Kiyohara, H. Jinnai, T. Hitomi, C. Ogino, K. Yoshida, Y. Nishizuka, Mammalian phospholipase D: phosphatidylethanolamine as an essential component, Proc. Natl. Acad. Sci. USA 93 (1996) 4300^4304. K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569^572. D.A. Brown, E. London, Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes?, Biochem. Biophys. Res. Commun. 240 (1997) 1^7. D. Brown, J.K. Rose, Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface, Cell 68 (1992) 533^544. T. Harder, K. Simons, Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains, Curr. Opin. Cell Biol. 9 (1997) 534^542. N.J. Severs, Caveolae: static inpocketings of the plasma membrane, dynamic vesicles or plain artifact?, J. Cell Sci. 90 (1988) 341^348. R.G. Parton, Caveolae and caveolins, Curr. Opin. Cell Biol. 8 (1996) 542^548. M.P. Lisanti, Z. Tang, P.E. Scherer, M. Sargiacomo, Caveolae puri¢cation and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia, Methods Enzymol. 250 (1995) 655^668. P. Oh, D.P. McIntosh, J.E. Schnitzer, Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven ¢ssion from the plasma membrane of endothelium, J. Cell Biol. 141 (1998) 101^114. C.J. Fielding, P.E. Fielding, Intracellular cholesterol transport, J. Lipid Res. 38 (1997) 1503^1521. M.P. Lisanti, Z. Tang, P.E. Scherer, E. Kubler, A.J. Koleske, M. Sargiacomo, Caveolae, transmembrane signalling and cellular transformation, Mol. Membr. Biol. 12 (1995) 121^124. T. Okamoto, A. Schlegel, P.E. Scherer, M.P. Lisanti, Caveolins, a family of sca¡olding proteins for organizing `preassembled signaling complexes' at the plasma membrane, J. Biol. Chem. 273 (1998) 5419^5422. H.R. Hope, L.J. Pike, Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains, Mol. Biol. Cell 7 (1996) 843^851. L.J. Pike, L. Casey, Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains, J. Biol. Chem. 271 (1996) 26453^ 26456. J. Liu, P. Oh, T. Horner, R.A. Rogers, J.E. Schnitzer, Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains, J. Biol. Chem. 272 (1997) 7211^ 7222. M.G. Waugh, D. Lawson, S.K. Tan, J.J. Hsuan, Phosphatidylinositol 4-phosphate synthesis in immunoisolated cav-

Cyaan Magenta Geel

Zwart

M. Liscovitch et al. / Biochimica et Biophysica Acta 1439 (1999) 245^263

[147]

[148]

[149]

[150]

[151]

[152]

[153]

eolae-like vesicles and low buoyant density non-caveolar membranes, J. Biol. Chem. 273 (1998) 17115^17121. Y. Liu, L. Casey, L.J. Pike, Compartmentalization of phosphatidylinositol 4,5-bisphosphate in low- density membrane domains in the absence of caveolin, Biochem. Biophys. Res. Commun. 245 (1998) 684^690. Y. Lavie, M. Czarny, M. Liscovitch, Localization of phospholipase D in caveolae and its up-regulation in multidrug resistant cancer cells, FASEB J. 11 (1997) A1346. M. Czarny, Y. Lavie, G. Fiucci, M. Liscovitch, Localization of phospholipase D in detergent-insoluble, caveolinrich membrane domains. Modulation by caveolin-1 expression and caveolin-182ÿ101 , J. Biol. Chem. 274 (1999) 2717^ 2724. Y. Lavie, G. Fiucci, M. Liscovitch, Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells, J. Biol. Chem. 273 (1998) 32380^32383. S. Ghosh, J.C. Strum, V.A. Sciorra, L. Daniel, R.M. Bell, Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells, J. Biol. Chem. 271 (1996) 8472^8480. K.A. Waite, R. Wallin, D. Qualliotine-Mann, L.C. McPhail, Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase, J. Biol. Chem. 272 (1997) 15569^15578. Z. Zhao, S.-H. Shen, E.H. Fischer, Stimulation by phospholipids of a protein-tyrosine-phosphatase containing two src homology 2 domains, Proc. Natl. Acad. Sci. USA 90 (1993) 4251^4255.

BBAMCB 55483 16-7-99

263

[154] L.R. Juneja, T. Kazuoka, T. Yamane, S. Shimizu, Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from di¡erent sources, Biochim. Biophys. Acta 960 (1988) 334^341. [155] T.C. Sung, R.L. Roper, Y. Zhang, S.A. Rudge, R. Temel, S.M. Hammond, A.J. Morris, B. Moss, J. Engebrecht, M.A. Frohman, Mutagenesis of phospholipase D de¢nes a superfamily including a trans- Golgi viral protein required for poxvirus pathogenicity, EMBO J. 16 (1997) 4519^ 4530. [156] E.B. Gottlin, A.E. Rudolph, Y. Zhao, H.R. Matthews, J.E. Dixon, Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate, Proc. Natl. Acad. Sci. USA 95 (1998) 9202^9207. [157] C.P. Ponting, P.J. Parker, Extending the C2 domain family: C2s in PKCs N, O, R, a, phospholipases. GAPs and perforin, Protein Sci. 5 (1996) 162^166. [158] J.D. Clark, L.-L. Lin, R.W. Kriz, C.S. Ramesha, L.A. Sultzman, A.Y. Lin, N. Milona, J.L. Knopf, A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2‡ -dependent translocation domain with homology to PKC and GAP, Cell 65 (1991) 1043^1051. [159] D. Mochly-Rosen, Localization of protein kinases by anchoring proteins: A theme in signal transduction, Science 268 (1995) 247^251. [160] J.K. Chung, F. Sekiya, H.S. Kang, C. Lee, J.S. Han, S.R. Kim, Y.S. Bae, A.J. Morris, S.G. Rhee, Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate, J. Biol. Chem. 272 (1997) 15980^15985.

Cyaan Magenta Geel

Zwart