The Role of Phospholipase D in Signaling Cascades1

Phospholipases hydrolyze phospholipids, which are the backbones of biological membranes. The activities of these enzymes not only have a profound impact on the structure and stability of cellular membranes but also play a pivotal role in regulating many critical cellular functions. The activation of phospholipases is involved in many cell-signaling cascades. These enzymes often execute their regulatory functions through the generation of second messengers that transduce biotic and abiotic cues into physiological responses. Sites of cleavage of phospholipids by PLD, PLC, and PLA2, and the products of PLD, PA, and head group (H). Significant progress was made recently toward understanding the structure, regulation, and function of PLD. Plant PLD is an “old” enzyme receiving renewed attention, mainly because of its potential role in transmembrane signaling. It was discovered in plants about one-half century ago, and some distinct and perplexing properties of its activity were soon noted (for review, see Heller, 1978; Wang, 1997). This conventional PLD is widespread in plant tissues and has been purified from several species; however, its regulation and physiological function remained an enigma. The molecular cloning of the first eukaryotic PLD from plants helped to propel the investigation to the molecular realm (Wang et al., 1994; Hammond et al., 1995; Rose et al., 1995). The identification, cloning, and expression of novel types of plant PLDs established that they are a family of heterogeneous enzymes that differ in catalytic and regulatory properties (Pappan et al., 1997a,1997b, 1998; Qin et al., 1997). In addition, the regulated activation of PLD was recently documented in several plant systems, including wounding, hormone action, and plant-pathogen interactions (Ryu and Wang, 1996; Fan et al., 1997; Lee et al., 1997; Ritchie and Gilroy, 1998). The genetic manipulation of PLD in the cell was achieved in plants, mammals, and yeast, and this has provided new insights into the involvement of PLD in cellular functions (Rose et al., 1995; Colley et al., 1997; Fan et al., 1997). With these developments, the role of PLD in signaling cascades has become a topic that attracts increasing attention in various systems (for review, see Wang, 1997; Chapman, 1998; Munnik et al., 1998). Eukaryotic intracellular PLD, which was first cloned from castor bean (Wang et al., 1994), is a highly conserved gene family. The conservation of several regions of the PLD amino acid sequences led to the identification and cloning of PLDs from yeast and animals (Hammond et al., 1995; Rose et al., 1995). All cloned PLDs contain two HxKxxxxD motifs, which are separated by approximately 320 amino acids in plant PLDs. The conserved His, Lys, and Asp residues form a catalytic triad responsible for catalysis. The HxKxxxxD motif was also observed in two phospholipid-synthesizing enzymes, bacterial PS synthase and cardiolipin synthase, in endonucleases, and in other proteins of unknown function in viruses and bacteria. The characteristics of the HxKxxxxD motif are used to define the PLD superfamily (Sung et al., 1997). Plant PLD is encoded by a multiple heterologous gene family. Four PLD cDNAs, designated PLDα, PLDβ, PLDγ, and PLDγ2, were isolated from Arabidopsis (Qin et al., 1997). The Arabidopsis genome project also yielded two PLD genes, for which cDNAs are not yet isolated. Multiple PLDs were cloned in rice and cabbage (Morioka et al., 1997;Pannenberg et al., 1998). Database searches in October 1998 found 15 complete PLD cDNA/gene sequences isolated from eight plant species (castor bean, Arabidopsis, cowpea, cabbage, tobacco, Pimpinella brachycarpa, rice, and maize). Alignments of these PLD sequences revealed several distinct clusters. Cluster I included Arabidopsis PLDα and all of the cDNA cloned to date from other plant species whose sequence identity was 75% to 90%. Therefore, PLDs of cluster I are grouped as PLDα, and this classification takes into account sequence similarity, catalytic properties (described in a later section), and gene structure. Cluster II consists of Arabidopsis PLDβ and the two PLD isologs on chromosomes II and IV (tentatively named PLDβ2 and PLDβ3), which share approximately 75% amino acid sequence identity. Cluster III has two members, Arabidopsis PLDγ1 and PLDγ2, which share more than 85% sequence identity. The overall sequence identity shows that PLDβ and PLDγ are more similar to each other than either is to PLDα. Multiple PLD genes also occur in other systems. In mammalian cells, two distinct PLDs were cloned, PLD1 and PLD2. PLD1 has two alternative splicing variants, PLD1a and PLD1b (Hammond et al., 1995; Colley et al., 1997). Two PLDs were reported in yeast, but only the sequence of PLD1 was identified (Rose et al., 1995; Waksman et al., 1997). Domain structures of PLDα, PLDβ, and PLDγ in Arabidopsis. XX in the PLDα C2 marks the loss of two acidic residues potentially involved in Ca2+ binding; XX in the PPI-binding motifs marks the loss of the number of basic residues potentially required for PPI binding. Molecular analyses have documented not only the occurrence of multiple PLDs but also the structural variations that may underlie distinct biochemical properties. PLD activities from plants can be divided into three groups based on their differing requirements for Ca2+ in vitro. The first group is the conventional plant PLD that displays a striking Ca2+ requirement; it is most active at millimolar concentrations of Ca2+, with the optimal concentration ranging from 20 to 100 mm (Heller, 1978). PLDα expressed from the castor bean PLDα cDNA exhibits the characteristic activity of conventional PLD purified from plants (Dyer et al., 1994; Wang et al., 1994; Pappan et al., 1998). Antisense suppression of PLDα in Arabidopsis led to the loss of this conventional PLD activity (Pappan et al., 1997a), so the PLDα gene product must have been responsible for it. Additionally, three isoforms and two cDNAs of the conventional PLD were also identified in some plant species (Dyer et al., 1994; Young et al., 1996; Pannenberg et al., 1998). The second group of PLDs includes those that are the most active at micromolar levels of Ca2+. The presence of such PLD activity was documented in transgenic Arabidopsis, in which the expression of PLDα was suppressed by an antisense gene (Pappan et al., 1997a). The cloning and analysis of PLDβ from Arabidopsis provided unequivocal, molecular evidence for the new type of PLD (Pappan et al., 1997b). The PLDγ that was cloned later also exhibited a Ca2+ dependence similar to that of PLDβ (Qin et al., 1997). These PLDs are PPI dependent and are stimulated by PIP2 and to a lesser extent by PIP, but not by other acidic phospholipids such as PI, PS, PG, and PA. Although the above PLDs require Ca2+ for activity, a third type that is independent of cations was reported inCatharanthus roseus suspension cells (Wissing et al., 1996). Another unique property of this PLD is its lack of transphosphatidylation activity: Two membrane-associated and two soluble variants of this activity have been noted. However, to our knowledge, it has not been purified to homogeneity, and no PLD cloned thus far exhibits such activity. This third type of PLD also differs in substrate specificity and preferences. Conventional PLD uses more than one phospholipid as a substrate. In general, PC, PE, and PG are good substrates, whereas PI, PS, cardiolipin, and plasmalogens are much less efficiently used, if at all (Heller, 1978; Dyer et al., 1994; Abousalham et al., 1997). PLDα, PLDβ, and PLDγ all use PC, PE, and PG as substrates, but the reaction conditions required for PLDβ and PLDγ are strikingly different from those for PLDα (Pappan et al., 1998). PLDβ and PLDγ, but not PLDα, can use PS and NAPE as substrates. Although PLDβ and PLDγ hydrolyze the same substrates, PLDγ prefers ethanolamine-containing PE and NAPE to other lipids, but PLDβ does not. The Ca2+-independent PLD from C. roseus exhibits a unique substrate specificity (Wissing et al., 1996). It is PI specific, which is in contrast to cloned PLDα, PLDβ, and PLDγ, which do not hydrolyze PI. These varied substrate specificities and preferences suggest that the activation of different PLDs may result in selective hydrolysis of membrane phospholipids. Their distinct structural and biochemical properties suggest that PLD isoenzymes are subject to unique controls and activation mechanisms. The different Ca2+ requirements could mean that changes in the levels of cytoplasmic Ca2+ activate PLD isoenzymes differentially. However, the fact that the conventional PLD (PLDα) requires millimolar levels of Ca2+ in vitro casts doubt upon the significance of Ca2+ in controlling its activity in vivo. It is important to note that the optimal Ca2+ concentration was determined by using a single class of lipid substrate often in the presence of organic solvents or detergents such as SDS, which are artificial conditions. A recent study showed that PLDα was active at nearly physiological Ca2+ concentrations when it was assayed at an acidic pH (4.5–5.0) and in the presence of mixed lipid vesicles containing PIP or PIP2 (K. Pappan and X. Wang, unpublished data). This suggests that even though the effect of Ca2+ on PLDα is complex, its activity can be increased by elevating cellular Ca2+ levels. On the other hand, PLDβ and PLDγ were inactive at that pH and were most active at a neutral pH. These distinct pH optima may mean that changes in cellular pH have a different effect on PLD isoforms. At near-physiological concentrations of Ca2+, PLDβ and PLDγ are neutral phospholipases, whereas PLDα is an acidic phospholipase that may be activated by cellular acidification. The presence of a C2 domain in plant PLDs points to a specific mode of activation by Ca2+. C2 domains were identified in a number of signal transduction and membrane trafficking proteins, such as PKC, PLC, and PLA2 (Ponting and Parker, 1996). This domain is important in the Ca2+-regulated translocation of proteins to membranes. Indeed, in the wound activation of PLD in castor bean, the Ca2+-mediated translocation of PLD from the cytosol to the membranes had already been proposed before the presence of a C2 domain on PLDs was recognized (Ryu and Wang, 1996). There is also data suggesting Ca2+-mediated activation of PLD in vivo (Munnik et al., 1998). Another potential regulator of plant PLD is PPI. Not only do PLDβ and PLDγ require PPIs for activity, PLDα activity is also stimulated by PPIs when low levels of Ca2+ are present (Qin et al., 1997). Binding assays have shown that PLDβ, PLDγ, and PLDα are able to bind PIP2. Two PLD regions may be involved in PIP2 binding: one is the near N-terminal C2 domain and the other is the near C-terminal PPI-binding motifs that are missing in PLDα. PPIs are minor lipids and their levels are regulated dynamically. The activation of PLD is likely to be interconnected with the metabolism and signaling of PPIs. Schematic diagram of up- and down-regulation of PLD in plants and animals, showing that PLD signaling can be regulated by modulating PLD activity or by removing PA. The proteinaceous stimulators and inhibitors identified are mainly from animal systems. The activation of PLD in animal systems was first identified just over 10 years ago, and it is now documented in more than 30 cell types stimulated by receptor-directed agonists and by other stimuli such as Ca2+ ionophores and phorbol esters (Cockcroft, 1997; Exton, 1997). Although, historically, the activation of PLD was observed first in plants, studies of PLD activation in plants now lag behind those in animals. It has long been known that wounds and other stresses stimulate a rapid increase in PA and other lipid metabolites. These increases were regarded initially as autolysis resulting from the release of PLD and other lipolytic enzymes during cell damage. Recent studies have shown that wounding a tissue triggers a rapid activation of PLD-mediated phospholipid hydrolysis not only at the wound site but also at undamaged areas (Ryu and Wang, 1996). Stimulation of plant PLD has also been shown in response to treatments with ABA, light, fungal elicitors, and bacterial pathogens (Young et al., 1996; Fan et al., 1997; Chapman, 1998; Munnik et al., 1998; Ritchie and Gilroy, 1998). Direct and derived products of PLD activation. LysoPA and free fatty acid (FA) can be formed from PA by nonspecific acyl hydrolase or by PLA. PA is dephosphorylated to DAG by PA phosphatase. CDP-DAG is the precursor for the synthesis of PS, PI, and PG. XOH, Primary alcohol used for transphosphatidylation; Ptd, phosphatidyl; NAE, N-acylethanolamine. Some of the cellular roles of PA may result from its effect on membrane properties and configuration rather than from its direct effect on proteins. PA is a nonbilayer lipid favoring hexagonal phase formation, particularly in the presence of Ca2+ (Cornell and Arnold, 1996). Thus, a rapid increase in the local concentration of PA may destabilize membranes. The activities of a number of signaling proteins, including G-proteins, PKC, PLC, PLA, PA phosphatase, DAG kinase, and PLDs, are sensitive to changes in membrane conformation (Cornell and Arnold, 1996; Pappan et al., 1998). An increase in PA also increases the net negative charge of membranes, which may alter protein-to-membrane interactions and the flux of ions such as Ca2+. In addition, PA-mediated changes in membrane properties may be produced by altering membrane lipid composition, because PA is a central precursor in glycerolipid biosynthesis (Fig. 4). It has been suggested that PLD plays a role in a broad range of cellular responses, but the requirement of PLD for a particular cellular function was not documented conclusively until recently. The molecular cloning of plant PLD helped to identify the first definitive requirement of PLD in a physiological process. It was noted that the sequence of the yeast sporulation-defective mutant SPO14 contains several regions of sequence similarity to the then newly cloned castor bean PLD, and this gene was later found to encode PLD1 (Rose et al., 1995). Both PLD1 activity and its presence in the nucleus are necessary for signaling the completion of meiosis (Sung et al., 1997). Whether plant PLDs are involved in a similar process is not known. Antisense suppression of plant PLD resulted in a loss of more than 90% of the PLDα in Arabidopsis flowers. But the fertility of PLDα-suppressed plants was not affected, indicating that a high level of PLDα is not essential for reproduction (Fan et al., 1997). Recent studies provide strong evidence of a role for PLDα in ABA action. The expression of PLDα is up-regulated by ABA, as indicated by the increased levels of PLDα promoter activity, mRNA, protein, and membrane-associated activity in response to ABA treatments (Fan et al., 1997; Wang, 1997; Xu et al., 1997). Senescence of the leaves detached from the PLDα-deficient transgenic plants was retarded when they were incubated with ABA (Fan et al., 1997). These data indicate that PLDα is a mediator in ABA actions; the loss of PLDα activity in transgenic plants renders Arabidopsis less sensitive to ABA. A role for PLD/PA in ABA signaling was also indicated in an independent study that used a different system (Ritchie and Gilroy, 1998). ABA increased PLD activity after it was applied to barley aleurone protoplasts. Direct application of PA to aleurone protoplasts suppressed the production of α-amylase and increased the synthesis of an amylase inhibitor in a manner that mimicked the ABA antagonism of GA-induced events in barley aleurone. The fact that an ABA-mediated physiological process is changed by the genetic and pharmacologic alteration of PLD activity suggests that PLD constitutes an early step in mediating ABA action. PLD has also been implicated in the action and production of ethylene. Antisense suppression of PLDα decreases the rate of ethylene-promoted senescence in detached Arabidopsis leaves (Fan et al., 1997). In cultured carrot cells, PLD activation is thought to constitute a signaling step in the perception of an ethylene burst that occurs at the early stage of Glc starvation (Lee et al., 1998). LysoPE is proposed to retard senescence by blocking PLDα activity, which may be involved in promoting the burst of ethylene (Ryu et al., 1997). The involvement of PLD in injury-induced lipid hydrolysis is perhaps the earliest result PLD to a cellular process. PLD can be activated by such as wounding, and et al., and Wang, 1996). wound activation of PLD from its translocation to membranes, which is by an increase in cytoplasmic wounding (Ryu and Wang, 1998). PLD activation is proposed to be an early in the response of the plant to and the PA may as an or as a substrate for the production of other such as fatty and in signaling (Ryu and Wang, 1998). The role of PLD in signaling to plant-pathogen In rice leaves with the bacterial PLDα at the of the membranes that into with during interactions but not in the interactions (Young et al., 1996). In cells with the fungal a rapid release of was noted which resulted from hydrolysis by PLDγ or PLDβ but not by PLDα, because the two hydrolyze and PLDγ prefers NAPE or PE over other phospholipids (Pappan et al., 1998). potential by which PLD in is the regulation of which is involved in In the activation of PLD is known to an and PA is a of et al., 1997). is a of and proteins. It active when its to the and the translocation of is by Recent studies that is a substrate for the newly identified in animals et al., 1997). Plant and to have the same In addition, and translocation of plant and also occur in cells with fungal et al., 1997). However, PLD and PA play a role in regulating plant activity is study using cells to evidence for the involvement of PLD in the production of and 1997). The occurrence of multiple PLDs with distinct regulatory and catalytic properties in the same suggests that each may have unique functions. Some evidence for distinct functions was from the genetic manipulation of PLD in and yeast systems. The of a PLDα antisense cDNA into Arabidopsis resulted in the loss of more than of PLDα activity, but PLDβ and PLDγ activities in the PLDα-deficient leaves were not (Pappan et al., 1997a). The PLDα antisense leaves a in or ethylene-promoted indicating that the loss of PLDα was not for by PLDβ and PLDγ (Fan et al., 1997). The yeast SPO14 mutant was found to contain PLD activity, designated and thus of the PLD1 function was not for by the gene et al., 1997). of mammalian resulted in whereas an increase in PLD1 expression not alter cell et al., 1997). A the of PLD activation with other lipid and signaling indicate and fatty The of PLD, PLC, and PLA2 several lipid such as PA, and free fatty increases in these lipid have also been found in some plant systems. the of PA was shown to that of and free fatty suggesting a activation of acyl PLC, PA et al., and Wang, 1998; Lee et al., 1997). In addition, PA is a of kinase, which is responsible for the synthesis of PIP2 (Fig. On the other hand, PIP2 is an of plant PLDβ, PLDγ, and some PLDs from animals and yeast (Cockcroft, 1997; Qin et al., 1997). It has been proposed that activation of PLD and in mammalian cells a that to rapid generation of PA and which are involved in In addition, can occur the PLD and the activation of one PLD may stimulate or the function of Recent in the investigation of PLDs in plants, animals, and yeast to an important role for PLD in the of cellular however, many and a understanding of PLD function is yet to be the molecular and cellular by which PLD the cellular functions. An requires identification of the cellular of PLD activation and the that with PLD. if is known about the or proteins, and other of PA, and head groups in plant signaling. The of of the cellular of lipid messengers is the in in The of multiple PLD proteins that the cellular regulation and the of PLDs are The biochemical and genetic data have suggested that the different PLDs may have unique functions. the biochemical properties of each PLD is important to the understanding of its and regulation in the Arabidopsis contains active only three of PLDα, PLDβ, and PLDγ, have been insights into the cellular function of different PLDs can be by the and expression and intracellular and However, such is only for PLDα. Although this is with the role of PLD in signaling it is important to note that PLD can in other cell such as membrane and The early studies of plant PLD functions only with phospholipid during and it was suggested that increases of PLD a phospholipid et al., Fan et al., and This role could be by different PLD proteins, or the same PLD could and signaling on the of the With the of molecular for the various PLDs, PLD and be and be in some of the above In addition, the function of different PLDs can be by and PLD antisense and transgenic of genetic particularly for PLDβ and PLDγ, or may be necessary for an of the role of PLD in cellular With our present of the molecular and of this class of are for in the understanding of the physiological functions of PLDs and the involvement in cascades. I for on and with the and to the whose was not because of PE acid PI phospholipase A phospholipase phospholipase