The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains

Four types of phospholipase D (PLD), PLD alpha, beta, gamma, and delta, have been characterized in Arabidopsis, and they display different requirements for Ca(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca(2+)-dependent phospholipid-binding C2 domain and require Ca(2+) for activity. This study documents a new type of PLD, PLD zeta 1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLD zeta 1 has been identified and used to express catalytically active PLD in Escherichia coli. PLD zeta 1 does not require Ca(2+) or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLD zeta 1 requires PIP(2) for activity, but unlike the PIP(2)-requiring PLD beta or gamma, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into alpha, beta, delta, gamma, and zeta based on their gene architectures, sequence similarities, domain structures, and biochemical properties.     

Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.     

Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential

The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2.     

Cloning,overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.     

磷脂酶D的细胞信号转导作用

磷脂酶D(PLD)是一类重要的跨膜信号转导酶类.分别由一个基因家族的不同成员编码.植物PLD的总体域结构相似,只是不同类型之间在某些单元上有重要差异.它们各具独特的生物化学特性.不同的PLD在不同的胁迫类型启动的特定的细胞过程中执行独特的细胞信号转导功能.PLD与其它磷脂酶及Ca2 信使之间有交互作用,形成复杂的信号转导网络.这一网络在不同植物种类、器官、组织和细胞类型中表现出特异性.文章最后讨论了PLD研究中有待揭示的问题并展望了今后的发展方向.     

Modulation of phospholipase D activity in vitro

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP₂). In a sophisticated interplay PIP₂ seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca²⁺ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed.     

Extending the C2 domain family: C2s in PKCs delta, epsilon, eta, theta, phospholipases, GAPs, and perforin.

Various membrane lipid metabolites, generated by phospholipases C and D (PLCs, PLDs), are known to regulate the activities of protein kinases C (PKCs) and GTP-ase activating proteins (GAPs) in a range of cellular processes. Conventional Ca(2+)-dependent PKCs (alpha, beta I, beta II, and gamma), PLCs and various GAPs are all known to contain copies of a phospholipid-binding domain, termed C2 or CalB. Here we recognize that C2 domains are also present in "new" Ca(2+)-independent PKCs (delta, epsilon, eta, and theta), other kinases, a eukaryotic PLD, the breakpoint cluster region (BCR) gene product, and two further GAPS. Twenty-two previously unrecognized C2 domain sequences are presented, which include a single copy in the mammalian poreforming proteins, perforin.     

Evidence for and characterization of Ca2+ binding to the catalytic region of Arabidopsis thaliana phospholipase Dbeta

Most types of plant phospholipase D (PLD) require Ca(2+) for activity, but how Ca(2+) affects PLD activity is not well understood. We reported previously that Ca(2+) binds to the regulatory C2 domain that occurs in the N terminus of the Ca(2+)-requiring PLDs. Using Arabidopsis thaliana PLDbeta and C2-deleted PLDbeta (PLDbetacat), we now show that Ca(2+) also interacts with the catalytic regions of PLD. PLDbetacat exhibited Ca(2+)-dependent activity, was much less active, and required a higher level of Ca(2+) than the full-length PLDbeta. Ca(2+) binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca(2+) binding data yielded an estimate of 3.6 high affinity (K(d) = 29 mum) binding sites on PLDbeta. The Ca(2+)-PLDbetacat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca(2+) binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca(2+)- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca(2+) regulates PLD activity.     

The transphosphatidylation potential of a membrane-bound phospholipase D from poppy seedlings

Plant phospholipases D (PLDs) occur in a large variety of isoenzymes, which differ in Ca(2+) ion requirement, phosphatidylinositol-4,5-bisphosphate (PIP(2)) activation and substrate selectivity. In the present study a membrane-bound PLD has been identified in the microsomal fractions of poppy seedlings (Papaver somniferum). The maximum PLD activity is found after 2 days of germination in endosperms and after 3 days in developing seedlings. In contrast to the four poppy PLD isoenzymes described hitherto, the membrane-bound form is active at lower Ca(2+) ion concentrations (in the micromolar instead of millimolar range) and needs PIP(2) for hydrolytic activity. Remarkable differences are also observed in head group exchange reactions. The reaction rates of the transphosphatidylation of phosphatidylcholine by various acceptor alcohols follow the sequence glycerol>serine>myo-inositol>ethanolamine, whereas ethanolamine is preferred by most other PLDs. Despite the biocatalytic differences, the membrane-bound PLD interacts with polyclonal antibodies raised against α-type PLD, which reveals some structural similarities between these two enzymes.     

Structural insights into phospholipase D function

Phospholipase D (PLD) and its metabolic active product phosphatidic acid (PA) engage in a wide range of physiopathologic processes in the cell. PLDs have been considered as a potential and promising drug target. Recently, the crystal structures of PLDs in mammalian and plant have been solved at atomic resolution. These achievements allow us to understand the structural differences among different species of PLDs and the functions of their key domains. In this review, we summarize the sequence and structure of different species of PLD isoforms, and discuss the structural mechanisms for PLD interactions with their binding partners and the functions of each key domain in the regulation of PLDs activation and catalytic reaction.     

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.     

The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis

Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD-PX are vital for this GAP function. Moreover, wild-type PLD-PX, but not mutated PLD-PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity - a novel function in addition to its role as a binding motif for proteins or lipids.     

Phospholipase D as a key modulator of cancer progression

The phospholipase D (PLD) family has a ubiquitous expression in cells. PLD isoforms (PLDs) and their hydrolysate phosphatidic acid (PA) have been demonstrated to engage in multiple stages of cancer progression. Aberrant expression of PLDs, especially PLD1 and PLD2, has been detected in various cancers. Inhibition or elimination of PLDs activity has been shown to reduce tumour growth and metastasis. PLDs and PA also serve as downstream effectors of various cell‐surface receptors, to trigger and regulate propagation of intracellular signals in the process of tumourigenesis and metastasis. Here, we discuss recent advances in understanding the functions of PLDs and PA in discrete stages of cancer progression, including cancer cell growth, invasion and migration, and angiogenesis, with special emphasis on the tumour‐associated signalling pathways mediated by PLDs and PA and the functional importance of PLDs and PA in cancer therapy.     

Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

Plant phospholipase D mining unravels new conserved residues important for catalytic activity

Phospholipase D (PLD) is a key enzyme involved in numerous processes in all living organisms. Hydrolysis of phospholipids by PLD allows the release of phosphatidic acid which is a crucial intermediate of multiple pathways and signaling reactions, including tumorigenesis in mammals and defense responses in plants. One common feature found in the plant alpha isoform (PLDα), in some PLD from microbes and in all PLD from eukaryotes, is a duplicated motif named HKD involved in the catalysis. However, other residues are strictly conserved among these organisms and their role remains obscure. To gain further insights into PLD structure and the role of these conserved residues, we first looked for all the plant PLDα sequences available in public databases. With >200 sequences retrieved, a generic sequence was constructed showing that 138 residues are strictly conserved among plant PLDα, with some of them identical to residues found in mammalian PLDs. Using site-directed mutagenesis of the PLDα from Arabidopsis thaliana, we demonstrated that mutation of some of these residues abolished the PLD activity. Moreover, mutation of the residues around both HKD motifs enabled us to re-define the consensus sequence of these motifs. By sequential deletions of the N-terminal extremity, the minimum length of the domain required for catalytic activity was determined. Overall, this work furthers our understanding of the structure of eukaryotic PLDs and it may lead to the discovery of new regions involved in the catalytic reaction that could be targeted by small molecule modulators of PLDs.     

Regulation of phospholipase D activity, membrane targeting and intracellular trafficking by phosphoinositides

Generation of PA (phosphatidic acid) by PLD (phospholipase D)-catalysed hydrolysis of phosphatidylcholine plays a pivotal role in cellular signalling pathways that regulate organization of the actin cytoskeleton, vesicular transport and exocytosis and stimulation of cell growth and survival. PLD regulation and function are intimately linked with phosphoinositide metabolism. Phosphatidyl 4-phosphate 5-kinase is stimulated by PA in vitro and this enzyme is the downstream effector of a significant subset of PLD signalling pathways. Yeast and mammalian PLDs are potently and specifically activated by the product of this kinase, PtdIns(4,5)P2, through interactions mediated by a polybasic motif within the catalytic core of the enzyme. Integrity of this motif is critical for agonist activation of mammalian PLD and for PLD function in secretion, sporulation and exocytosis in vivo. Although dispensable for catalysis in vitro, these PLD enzymes also contain N-terminal PH (pleckstrin) and PX (phox) homology domains. Binding studies using recombinantly expressed PLD fragments indicate that the PH and PX domains also interact specifically with distinct phosphoinositide ligands. Both the PX and PH domains are important for PLD function by controlling the dynamic association of the enzyme with the plasma membrane and its intracellular trafficking by the endocytic pathway. These results identify two distinct modes of regulation of PLD by phosphoinositides: stimulation of catalysis mediated by the polybasic domain and dynamic regulation of membrane targeting mediated primarily by the PH and PX domains.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp.

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

The differential regulation of phosphatidylinositol 4-phosphate 5-kinases and phospholipase D1 by ADP-ribosylation factors 1 and 6

Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.