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Molecular structure of phospholipase D and regulatory mechanisms of
its activity in plant and animal cells
Kolesnikov, Y. S.; Nokhrina, K. P.; Kretynin, S. V.; Volotovski, I. D.; Martinec,
J.; Romanov, G. A.; Kravets, V. S.

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https://doi.org/10.1134/S0006297912010014

Biochemistry (Moscow), 77, 1, pp. 1-14, 2012-01-27

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REVIEW
Molecular Structure of Phospholipase D and Regulatory Mechanisms of Its
Activity in Plant and Animal Cells{running title} PLD STRUCTURE AND ACTIVITY IN PLANT AND
ANIMAL CELLS
Y. S. Kolesnikov1, K. P. Nokhrina2, S. V. Kretynin1, I. D. Volotovski3, J.
Martinec4, G. A. Romanov5, and V. S. Kravets1*
KOLESNIKOV et al.
1

Institute of Bioorganic Chemistry and Petrochemistry, National Academyof Sciences of Ukraine, ul. Murmanskaya 1, 02094 Kiev, Ukraine; fax: +38(044) 573-2552; E-mail: kravets@bpci.kiev.ua
2
Plant Biotechnology Institute, National Research Council of Canada, 110
Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada; E-mail:
Kateryna.Nokhrina@nrc-cnrc.gc.ca
3Institute of Biophysics and Cell Engineering, National Academy of
Sciences of Belarus, ul. Akademicheskaya 27, 220072 Minsk, Belarus; Email: volot@biobel.bas-net.by
4Institute of Experimental Botany, Academy of Sciences of the Czech
Republic, Rozvojova 263, 16502 Prague 6, Czech Republic; fax: +420 (225)106-456, E-mail: martinec@ueb.cas.cz
5Institute of Plant Physiology, Russian Academy of Sciences, ul.
Botanicheskaya 35, 127276 Moscow, Russia; E-mail: gar@ippras.ru
Received February 1, 2011
Revision received March 23, 2011
Abbreviations: Gα, α-subunit of heterotrimeric G-protein; PKC, protein
kinase C; PLD, phospholipase D; PtdIns(4,5)P2, phosphatidylinositol-4,5bisphosphate.
* To whom correspondence should be addressed.
Abstract⎯Phospholipase D (PLD) catalyzes hydrolysis of phospholipids
with production of phosphatidic acids, which often act as secondary
messengers on transmission of intracellular signals. This review summarizes
data of various leading laboratories on specific features of organization andregulation of PLD activity in plant and animal cells. The main structural
domains of PLD (C2, PX, PH), the active site, and other functionally
important parts of the enzyme are considered. Regulatory mechanisms of
PLD activity are characterized in detail. Studies associated with molecular
design, analysis, and synthesis of new nontoxic substances capable ofinhibiting different PLD isoenzymes in vivo are shown to be promising forbiotechnology and medicine.
DOI: 10.1134/S000629791201001?

Key words: phospholipase D, domains, calcium, lipids, G-proteins, protein
kinases, protein–protein interactions
Phospholipase D (EC 3.1.4.4) (PLD) is an enzyme widely distributed
in plants [1, 2] and animals [3, 4]; it catalyzes hydrolysis of thephosphodiester bond in structural phospholipids (phosphatidylcholine,
phosphatidyl ethanolamine, etc.) with production of phosphatidic acid andlow molecular weight water-soluble compounds of choline or ethanolamine
type [5]. Phosphatidic acid is considered to be not only a necessary
structural element of membranes but also an important secondary messengerof hormone and stress signaling cascades [6, 7] and as a regulator ofmembrane transport [8]. Phosphatidic acid can also be generated bydiacylglycerol kinases [9].
PLD was found and cloned in plants, and the model plant Arabidopsis
thaliana has been shown to have 12 PLD genes [10, 11]. PLD is very
sensitive to external and internal factors, and its multiformity is favorable
for fine coordination of enzyme activity and features of cell metabolism.
PLD is actively studied on different organizational levels in both plants andanimals.
This review analyzes the data on PLD of plants and animals and also
gives the authors’ concept about the involvement of PLD in transmission of
signals of such important phytohormones as cytokinins. In particular, known
regulatory mechanisms of PLD activity are analyzed, and comparative
characteristics of these enzymes in different cells are presented. A wide
spectrum of factors known to play key roles in PLD regulation isconsidered, the organization of the enzyme is analyzed, its role in theregulation of cell metabolism is determined, and lines for studies on lipid
signaling systems are described.
Studies in biochemistry and molecular genetics are favorable fordetermination of the role of PLD in plant and animal cells [12]. There aredata on the involvement of PLD in malignancies [13], angiogenesis [14],leukocyte adhesion, and chemotaxis [15]; the functions of the enzyme areconsidered in platelets [16], nerve cells [17], myocardium [18], lungs [19],in transfer of intracellular membranes [20, 21], and signal transmission in
animals [6, 22-25] and plants [2, 11]. Moreover, the therapeutic potential of
PLD inhibitors has been considered [4]. However, the published data on theregulatory mechanisms of PLD activity in plants and animals and on the
involvement of PLD in signal transmission have not been analyzed yet. The
purpose of this review is to fill this gap and to inform readers about
researches in this field.
PLD of animal cells is encoded by two genes and exists in two forms:
PLD1 and PLD2 [26, 27]. PLD1 (120 kDa) is mainly present on
endomembranes of the cell and is transported under the influence of
extracellular stimuli. PLD2 (106 kDa) is located on the plasma membrane,
has low in vivo activity, and is moderately stimulated by known activatorsof PLD1. Plants are found to have many genes of various PLD isoenzymes
that encode high molecular weight multidomain proteins with molecularweights of 90-125 kDa. In Arabidopsis plants there are 12 genes of PLD:
PLDα(3), β(2), γ(3), δ, ε, and ζ(2) [2]; in rice there are 17 genes of PLD:
PLDα(8), β(2), δ(3), κ, ζ(2), and ϕ [28]. In Arabidopsis PLDα3 [29], PLDδ[11], PLDζ1 [2], and PLDε [30] are located mainly on the plasma
membrane, PLDζ2 is connected with the tonoplast membrane [31], PLDα1shuttles between the cytosol and membranes [2], whereas PLDγ1 is locatedon intracellular membranes [2]. The location of PLDβ is not strictly
determined [2]. PLDα1 is the most active and PLζ1 and PLDε are the least
active isoenzymes in plants. In the absence of activators and calcium in the
incubation medium all PLD isoenzymes of plant cells are inactive in vitro.

SPECIFIC FEATURES OF PRIMARY STRUCTURE OF PLD
Active site of PLD. PLDs from animal and plant cells have a modular
structure where sites responsible for catalysis are surrounded by regulatory
sequences (figure). The active site of PLD consists of four conservative
amino acid sequences (І-ІV) among which ІІ and ІV are especiallyconservative in various organisms. Every motif of II and IV contains thesequence, HxKxxxxDxxxxxxGSxN (abbreviated HKD, where x are
nonconservative amino acids), and due to this sequence PLD can catalyzehydrolysis of phospholipids [32]. Residues of histidine (H), lysine (K), andaspartic acid (D) are directly involved in hydrolysis of phosphodiester bondsof phospholipids. The histidine residue in this case acts as a nucleophile that
attacks the phosphorus atom of the substrate [32-34]. The catalytic site of
PLD is formed when the HKD sequences are in the correct proximity. The
PLD-catalyzed hydrolysis of the phosphodiester bond is supposed to occur
in two stages by the so-called ping-pong mechanism: successive attack of
the substrate by imidazole residues of histidines, each of which is acomponent of the catalytic sequences of HKD. As a result, the phosphatidic
acid residue of the cleaved phospholipid covalently binds transiently with
the imidazole group of histidine of PLD producing a short-lived
intermediate that can be easily cleaved on attack by a water molecule [35,36].
{Figure} Features of organization of phospholipase D of animal and plant
cells. DRY, binding site of α-subunit of the heterotrimeric G-protein; Gβγ,
β- and γ-subunits of the heterotrimeric G-protein; І and ІІІ, conservative
domains; PtdIns(5)P, phosphatidylinositol-5-phosphate; PtdIns(4,5)P2,
phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P3,
phosphatidylinositol-3,4,5-trisphosphate
Key:1) Plants
2) PLD
3) P
4) actin5) Animals
6) acylation
7) PKCδ PKCα8) tubulin9) casein kinase10) cyclin-dependent kinase (PLD2)11) ribosomal S6 kinase 212) interaction with choline,13) translocation to caveola
14) active site sequence15) binding site of oleic acid16) binding domain of lipids and calcium ions17) conservative domain18) binding site of polyphosphoinositides19) plextrin-homologous domain20) binding site of Gα21) phox-homologous domain22) phosphorylation site23) negative regulation and caspase cut site

Lipid-binding PLD sequences. Lipids can modify PLD in plant andanimal cells. In particular, some phospholipids usually act not as substrates
but as cofactors of the enzyme. The PLD activity depends on binding of thecofactor phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). In animals
this is explained by the presence within the active site limits (between
motifs ІІІ and ІV) of a conservative sequence that includes residues of basic
and aromatic amino acids [37, 38]. This sequence ensures high levels of
PLD1 and PLD2 activities and also promotes the translocation of PLD1 tomembranes in response to extracellular signals [39, 40].
The amino acid sequence of PLD enriched by basic and hydrophobic
residues is involved in binding polyphosphoinositides [37]. This structure
and its inverted repeat (RxxxxKxRR and RKxRxxxxR) in PLDβ of
Arabidopsis and tomatoes surround the C-terminal sequence HKD [2, 41].
PLDβ1 contains all four residues of basic amino acids (mainly Arg) binding
polyphosphoinositides, whereas one of them in PLDγ and PLDζ is replaced
by a nonpolar or acidic residue. This structure in PLDδ has two conservative
residues of basic amino acids, whereas in PLDα and PLDε there is only one
such residue and the other residues are replaced by nonpolar or acidic ones
[2, 11]. In addition to inverted repeats that bind polyphosphoinositides(called PBR2), PLDβ also contains another site binding these lipids (PBR1)
that is located near the N-terminal sequence of HKD [2]. PBR1 is believed
to be an independent binding site of PtdIns(4,5)P2 and of some otherphosphoinositides; it consists of highly conservative amino acid residues
and contains many side chains of lysine, arginine, and histidine residues.
The specific binding of PtdIns(4,5)P2 with them plays an extremely
important role in the regulation of plant PLDβ by this phospholipid because
it promotes conformational rearrangements strengthening the enzyme
association with the substrate [2, 41, 42]. PLDα and PLDδ contain aminoacid substitutions in the abovementioned binding site of PtdIns(4,5)P2 [2].
The phosphate group in the 4′-OH position of the inositol ring of
PtdIns(4,5)P2 plays a key role in formation of ionic bonds with lysine
residues K437 and K440 of the PLDβ molecule [2].
Domain C2 interacting with lipid membranes depending on the Ca2+
concentration is another type of phosphoinositide-binding sequences ofplant PLDs. This domain is not strictly specific to PtdIns(4,5)P2. At low
level of Ca2+ negatively charged PtdInsP and PtdIns(4,5)P2 molecules attract
positively charged amino acid residues R and K in the C2 domain and thus
favor the binding with substrates of PLDα of Arabidopsis and catalysis by
PLDβ and PLDγ [2, 43]. Binding sites of PtdIns(4,5)P2 in domain C2 arealso found in PLDα of cabbage [44], poppy [45], and tomatoes [46]. The C2
domain in PLDα of tomatoes binds with high affinity phosphatidic acid but

not phosphatidylcholine, but association with phosphoinositides is markedly
weakened on increase in the level of their phosphorylation [46].
Domain PX. PLD of animals and PLDζ of plants contain the Nterminal domain PX (named after the protein p47phox that is a subunit of
NADPH oxidase of phagocytes), which includes a conservative sequenceenriched in proline residues [2, 28]. The PX domain of PLD1 selectively
binds PtdIns(5)P, thus promoting the translocation of the enzyme tomembranes of endocytic vesicles [39], and also interacts with PtdIns(3)Pand PtdIns(3,4)P2 on the membranes [47]. This domain contains twobinding sites of membrane phospholipids: one of them is highly specific to
PtdIns(3,4,5)P3, PtdIns(3)P, PtdIns(5)P, and other phosphoinositides,
whereas the other also binds phosphatidic acid, phosphatidylserine, andother negatively charged lipids. The concurrent binding of phospholipids
with the abovementioned sites synergistically increases the affinity of
domain PX for the membranes [48, 49]. The PX domain of PLD1 plays animportant role in regulation of the enzyme translocation into endocytosis
sites on the membranes under the influence of extracellular signals [40, 49].
In PLD1 and PLD2 this domain can stimulate the GTPase activity of theprotein dynamin [50] and ensure the binding with PLD2 and activation of
PLD2 of phospholipase Cγ1 [51], tyrosine kinase Syk [52], and protein
kinase Cζ [53].
Domain PH. PLD1 and PLD2 of animals [38] and also PLDζ ofplants [2, 28] include the domain PH (plextrin-homologous domain)
responsible for specificity of localization of these enzymes in cells. The PH
domain is located near the C-terminus of the PX domain. Deletions or point
mutations in the domain of PLD1 and PLD2 do not influence the activity invitro [47, 54], but inactivate the enzymes in vivo due to disturbance of theirlocalization in cells [38, 40]. The PH domain of PLD2 binds PtdIns(4,5)P2
with moderate affinity but with high selectivity [38]. Deletion of thisdomain in PLD1 lowers the binding of this phosphoinositide [47], but did
not influence the in vitro activation of PLD1 and PLD2 byphosphoinositides [37, 38]. This suggests the presence in the PLD structureof other sites of association with PtdIns(4,5)P2. The PH domain of PLD1
and PLD2 contains conservative cysteine residues (Cys240 and Cys241)
that can be palmitylated and thus ensure the binding with the membranes
independently of phosphoinositides [55-57], which stabilizes the interactionof the enzyme with the lipid bilayer [56]. Palmitylation promotes thetranslocation of PLD1 into endocytosis sites on the membranes under the
influence of an extracellular stimulus [40] and the translocation of PLD2from early endosomes onto the plasma membrane [38].
Loop-shaped sequence. The primary structure of PLD1 of animalcells is uniquely characterized by the presence of a loop-shaped domain
consisting of 116 amino acid residues (a.a.). Insertion of this sequence into
PLD2 does not change the activity of this isoenzyme. The loop-shaped
domain of PLD1 contains a site of negative regulation, which possibly is
responsible for the low basal in vitro activity of PLD1 as compared to thatof PLD2 [54]. This conservative sequence belonging to PLD1 also contains
sites of cleavage by caspases. The in vitro action of caspases on thissequence weakens the sensitivity of PLD1 to protein kinase C but increases
its sensitivity to small GTPases [58].
Sites of Ca2+ binding. Based on specific features of the primarystructure of the N-terminal domains binding lipids, PLD are subdivided onto

C2-PLD (PLDα, PLDβ, PLDγ, PLDδ, and PLDε of plants) and PX/PH-PLD(PLDζ of plants and PLD1/2 of animals) (figure). The presence of Nterminal conservative domain C2 consisting of 130 a.a. and binding Ca2+
and phospholipids is a unique feature of the majority of plant PLDs [2]. The
binding of Ca2+ with the domain C2 provides for the PLD translocation to
substrate on the membrane and induces changes in the enzyme
conformation [46]. A direct binding of Ca2+ in three-looped structures of theC2 domain [11] has been shown for PLDα and PLDβ of Arabidopsis [2].
Calcium forms a coordination bond with four or five acidic amino acidresidues in the calcium-binding loops (CBL) of the PLD domain C2. The
main role in coordination of Ca2+ in the C2 domain of PLDβ, γ, and δ isplayed by two aspartic acid residues in the Ca2+-binding loop 3 (CBL3), theonly Asn residue in the Ca2+-binding loop 2 (CBL2), and Asp in the Ca2+binding loop 1 (CBL1) [2, 43]. In PLDα two of these amino acids are
replaced in CBL1 and CBL2 by basic or neutral residues [11, 43], whereas
the C2 domain of PLDε does not have any amino acid binding with calcium[30]. In PLDα of other plants coordination bonds with Ca2+ are created withf asparagine and aspartic acid [59], glutamine and glutamic acid [41], andalso their combination [42]. The above-described amino acid substitutions
are responsible for the low affinity of PLDα and PLDε for Ca2+, andtherefore these isoenzymes hydrolyze phospholipids only in the presence ofhigh concentrations of Ca2+ [2] or at low pH values increasing the affinity
for calcium. The Ca2+ binding has been also observed in the active site of
PLDα [60] and PLDβ, where it is stimulated by phosphatidylserine [61].
Other functional regions of PLD. PLDs of animal cells also containother functional regions on the N- and C-termini. In particular, the Nterminal amino acid sequence of human PLD1 (residues 1-49 and 216-318
including the PH domain) plays a key role in the stimulation of this enzyme
by protein kinase Cα [62]. Moreover, the sequence consisting of 155 Nterminal amino acids of human PLD1, especially Ser2, is involved in theinteraction with polymerized F-actin [63]. The actin-binding domain ofplant PLD located between catalytic subdomains in the limits of theconservative domain III contains asparagines and threonine residues that are
directly involved in the actin binding [64]. The C-terminus of PLD canregulate the enzyme localization in the cells, contribute to its interaction
with small GTPases of the RhoA family, and also play an extremely
important role in stabilizing the active conformation of the catalytic site of
PLD [65, 66].
In plants an amino acid sequence DRY located on the N-terminal endof the HKD C-subdomain is responsible for the binding of PLDα1 with theheterotrimeric G-protein subunit Gα. Except for PLDζ and PLDγ2, thissequence is also present in other PLD isoenzymes of Arabidopsis [67].
Potential phosphorylation sites of serine, threonine, and tyrosine residues
are found in the primary structure of PLDγ of Brassica oleracea and also inhomologs of poppy PLDα [45, 68].

REGULATORY MECHANISMS OF PLD ACTIVITY
Activity of PLD is controlled by hormones, neurotransmitters, andother physiologically active molecules influencing the enzyme throughspecific regulatory mechanisms. And PLD is often contributes to signal

transmission through production of phosphatidic acid as a secondary
messenger. In particular, PLD is involved in transmission of signals ofclassic plant hormones such as abscisic acid [11] and cytokinin [69].
Regulation of PLD activity by calcium ions. Calcium plays animportant role as a modifier of PLD activity in plant cells, in particularunder the influence of cytokinin [70] and also as a cofactor of PLD
isoenzymes containing the C2 domain [2, 43, 61]. Calcium ions activate
PLDα and PLDβ through binding with the C2 domain. This leads toconformational changes in the enzymes that result in strengthening thesubstrate binding and weakening the association with PtdIns(4,5)P2 [2].
Calcium also binds with the active site of PLDβ, which increases its affinity
for the activator (PtdIns(4,5)P2) as discriminated for the affinity for thesubstrate (phosphatidylcholine) [61]. In turn, the binding of PtdIns(4,5)P2
with the active site of the enzyme increases its affinity for the substrate [2].
In plants there are PLDs with activities depending on millimolar andmicromolar concentration of Ca2+ in vitro.
Phospholipases D of plant cells are active in vitro in the presence of2+
Ca concentrations of 10-100 mM and hydrolyze phospholipids
independently of the presence in the substrate mixture of lipid cofactors(PtdInsP or PtdIns(4,5)P2). In particular, Ca2+ (1-100 mM) in vitro activates
PLDα isolated from cabbage [60], strawberry [42], Vigna sinensis [71],sunflower [72], and poppy [45]. The maximal activity of PLDα1 in
Arabidopsis is observed at the Ca2+ concentration of 25 mM. On using purephosphatidylcholine as a substrate, the activity of this enzyme is stimulated
at Ca2+ concentrations of 20-100 mM [2]. At the Ca2+ concentration of
50 mM, the other PLD isoenzyme, PLDε, is also active [30]. Such highconcentrations of Ca2+ activate those plant PLD isoenzymes that have theC2 domain with low affinity for Ca2+ [2].
Plant PLDs are also stimulated my micromolar concentrations of Ca2+[5]. In the case of PLDβ, this is due to a high affinity of the C2 domain for
Ca2+ [2]. Activities of PLDβ and PLDγ of Arabidopsis are maximal at
50 µM Ca2+. PLDγ is less stimulated by Ca2+ than PLDβ and PLDα.
Increase in Ca2+ concentration (>50 µM) is associated with a decrease in theactivities of PLDγ and PLDβ [2, 43]. Activities of the PLDγ subtypes
PLDγ1 and PLDγ2 depend on the Ca2+ concentration similarly [73].
Moreover, PLDα of castor bean in acidic medium (pH 4.5-5.0) is also
stimulated in vitro at low Ca2+ concentration (50 µM), but on increase in the
pH value the active concentration of Ca2+ increases to millimolar values [2].
The activity of isoenzyme PLDδ of Arabidopsis is observed in vitro at both
micro- and millimolar Ca2+ concentrations. The activity of PLDδ in thepresence of an activator (oleic acid) is maximal at 100 µM Ca2+ [11]. PLDεis also active in vitro at micromolar Ca2+ concentrations, but only in thepresence of oleic acid [30].
Plant cells also contain calcium-independent PLDs: PLDζ1 ofArabidopsis [2] and PLD of mustard seeds [74]. Moreover, Ca2+ does notmarkedly change the in vitro activity of purified PLD1 from animal cells[26].
Modification of PLD activity by lipids. PLDs are regulated byvarious lipids, and among them polyphosphoinositides are considered to be
especially important modifiers of cytoskeleton dynamics, membranetransport, and enzyme activity [75]. PLDs sensitive to these phospholipids
are involved in transmission of intracellular signals of hormones, inparticular of cytokinins in plants [76-78]. In animal cells the catalytic
activities of PLD1 and PLD2 are sensitive to PtdIns(4,5)P2, which acts astheir cofactor [26]. The binding of PtdIns(4,5)P2 with PLD1 and especiallywith PLD2 is important for the stimulation of these enzymes [37, 55, 79].
PLD2 is allosterically activated by phosphatidylinositol-4,5-bisphosphate invitro [37]. Optimal concentrations of this lipid provide for a positive effectof activators (proteins Rho, ARF1, and PKCα) on PLD1 in vitro promoting
the PLD binding with a lipid bilayer [80]. The enzyme of PtdIns(4,5)P2
biosynthesis, phosphatidylinositol-4-phosphate-5-kinase Іα, is favorable for
an increase in PLD activity in Cos7 cells. The PLD2 activity increases underconditions of a simultaneous expression of the genes encoding PLD and theabovementioned phosphoinositide kinase [79]. As discriminated from otherphosphoinositides, PtdIns(3,4,5)P3 binds with residue Arg179 in the PX
domain of PLD1 [49], stimulates the enzyme activity [49, 81], and also
promotes the translocation of PKCζ into the cell nucleus [82], which resultsin activation of the nuclear PLD.
In plants polyphosphoinositides are important cofactors of PLDβ,
PLDγ, and PLDζ, and in their absence these isoenzymes are inactive in
vitro. PLDβ of Arabidopsis displays the most pronounced specificity. This
isoenzyme is stimulated in vitro over a wide range of PtdIns(4,5)P2
concentrations [11]. PLDβ is less sensitive in vitro to otherphosphatidylinositol phosphates [2]. PtdIns(4)P and PtdIns(4,5)P2 areadditive in increasing the activities of PLDβ and PLDγ [2, 43]. As
discriminated from PLDγ2, PLDγ1 is the most sensitive to PtdIns(4,5)P2[73]. PLDα does not depend on polyphosphoinositides in the presence of
optimal Ca2+concentrations but is stimulated by these lipids in vitro in acidic
medium in the presence of micromolar Ca2+ concentrations [2, 11]. The
activity of PLDδ of Arabidopsis depends on PtdIns(4,5)P2 because this lipid
increases the affinity of the enzyme active site for the substrate [11, 83]. The
PLDζ depends only on PtdIns(4,5)P2 [2], whereas PtdIns(3,4)P2 is a
cofactor of PLDε at micromolar concentrations of Ca2+ [30]. Thus, PtdInsP2
acts as a cofactor of PLDβ, PLDγ, PLDε, and PLDζ of Arabidopsis. Notethat PLD activity independent of PtdInsP2 has been detected in mustard
seeds [74].
Other lipids also are negative modifiers of PLD. In particular, C2-, C6-,
C8- and double-chained ceramides inhibit the activity of PLD by affecting
its interactions with protein activators [84], acting on the catalytic site of
PLD or on its binding with PtdInsP2 [85, 86]. Lysophospholipids also
specifically inhibit PLD in vitro independently of the small GTPase Arf andPtdInsP2. They directly bind with PLD and noncompetitively inhibit the
enzyme in animals [87] and plants [88].
Unsaturated fatty acids, products of lipid cleavage, are believed to be
activators of animal PLD2 in vitro. This enzyme can be stimulatedsynergistically by PtdIns(4,5)P2 [89, 90]. PLDδ and PLDε of Arabidopsis
under in vitro conditions depend on oleic acid [11, 30], but this may be
associated with modification of properties of the lipid bilayer with whichthese isoenzymes are associated [83]. The mechanism of the inhibitory
effect in vitro of N-acetylethanolamines (N-laurylethanolamine, Nmyristoylethanolamine) on the activity of PLDα from Arabidopsis, castor
bean, and cabbage [91] is unknown.
Regulation of PLD activity by G-proteins. The cells of animals
contain specific protein factors that are important for activation of PLD.

These factors are small monomeric GTPases of the Rho and ARF familiesof the Ras superfamily. Rho GTPases play an important role in theregulation of various processes in animal cells, including transcription, cell
cycle, secretion, cell transformation, and cytoskeleton rearrangements [92].
Small GTPases of the Rho family are also considered as modifiers of PLD
activity. Some of these proteins — Rac1, Cdc42, and especially RhoA —allosterically activate PLD1 increasing its catalytic activity by lowering the
Michaelis constant [80]. In the presence of nonhydrolyzable analogs of
GTP, small GTPases Rac1, Cdc42, and RhoA activate PLD1 [26, 93, 94]
and PLD2 [95] in vitro. RhoA [96, 97], Rac1 [98], and Cdc42 [99] are
PLD1 stimulators in vivo. The C3-exoenzyme from Clostridium botulinum,
which inhibits the functioning of Rho proteins via their ADP-ribosylation,
decreases the activity of PLD1 [82].
The binding of RhoA with the C-terminus of PLD1 [33, 80, 100]
depends on residues of basic amino acids of the sequence 946-962 of the
enzyme and on activation site І of the RhoA protein. A similar site of Cdc42
also plays an important role in the mechanism of binding and activation of
PLD1 [101]. Other important binding sites of RhoA on the C-terminus ofhuman PLD1 are residues Ile870, Gln975, and Asp999 [102]. The Rho
proteins interact with PLD1 only in an active GTP-bound state [103]. Some
Rho GTPases (RhoA, Rac1, and Cdc42) stimulate PLD1 synergisticallywith ARF1, but not with PKCα [80]. Although RalA GTPases cannot
directly stimulate PLD in vitro and in vivo, they play an important role in
the activation of PLD1 and PLD2 [104, 105] by forming a complex with
Rho and ARF proteins [106, 107]. This confirms the synergic influence of
two monomeric GTPases on PLD activity.
PLD activity also depends on some cytosolic GTPases representing
ADP-ribosylation factors (ARF). The PLD1 activity in vitro increases 1015-fold on incubation with ARF proteins (ARF1, ARF3, ARF4, ARF5, andARF6) [26, 47, 108], whereas the activity of PLD2 increases 1.5-2-fold (butindependently of GTP) [54]. ARF1 and ARF6 activate PLD1 in vivo [109,110], whereas ARF6 activates PLD2 [111]. Comparatively to ARF6, ARF1
preferentially activates PLD1 in vitro [112], whereas ARF6, asdifferentiated from ARF1, markedly stimulates PLD2 in vivo [108]. ARF1
stimulates PLD1 in vitro synergistically with Rho proteins (RhoA, Rac1,
Cdc42) [80, 112] and PKCα [80]. Synergistic effect of ARF1 and Rho isobserved only at optimal concentrations of PtdInsP2 [80].
The translocation of ARF to membranes plays an important role in themechanism of activation of PLD. The translocation of ARF6 stimulates
PLD1 [113] and PLD2 [108] in vivo. The importance of ARF protein
exemplified by ARF1 is proved by its necessity for the activation of PLDs.
ARF1 activates PLD1 in vitro by increasing the catalytic constant withoutchanges in the Km value [80]. The replacement of GDP by GTP in ARF6 isimportant for activation of PLD [114], and this explains the stimulatory
effect of guanine nucleotide exchange factors on the activity of PLD1 invivo [115, 116]. The N-terminus of PLD2 (amino acids 1-308) is essential
for modification of this enzyme activity through ARF. The first 73 Nterminal amino acids of ARF are involved in the activation of PLD1 [117],
whereas the sequence of amino acids 35-94 plays an important role in theinteraction of these proteins [118]. Thus, small GTPases of the Rho and
ARF families are key activators of PLD in animals.

Both α- and β/γ-subunits of heterotrimeric G-proteins (Gq, Gi, G12)
contribute to the PLD activation by various hormones in animal cells,
although this process occurs in vivo with direct involvement of proteins
Rho, protein kinase C [119, 120], ARF6, or tyrosine kinase Src [121, 122].
The direct influence of heterotrimeric G-proteins results in inactivation of
PLD. Combination of Gβ1γ1 subunits suppresses PLD1 and PLD2, whereas
Gβ1γ1 and Gβ2γ2 inhibit PLD1 stimulated in vitro and in vivo. These
protein complexes interact with the N-terminal region of PLD within limitsof the PX and PH domains (amino acid residues 3-311) [123]. In plant cells
the binding of PLDα with the G-protein α-subunit (Gα) in the GDP-form
suppresses PLD activity in vitro [67, 124]. Addition of GTP into the system
restores the PLD activity. And the interaction of PLDα1 with Gα stimulates
the GTPase activity of Gα that leads to disintegration of the PLDα–Gα
complex [67]. Therefore, the inactive heterotrimeric G-protein is thought tosuppress the membrane-associated PLDα, whereas stimulation of the Gprotein by an extracellular signal promotes PLDα activation. Henceheterotrimeric G-proteins are thought to be important regulators of PLD inplant and animal cells.
Phosphorylation as a mechanism of PLD regulation.
Phosphorylation/dephosphorylation of proteins at specific amino acidresidues plays an important role in mechanisms of signal transmission,
regulation of enzyme activities, and formation of protein complexes in bothplant and animal cells [125-127]. Mainly Ser, Thr, His, Asp, and Tyr
residues are phosphorylated. The Tyr residue is most frequently studied in
the works in this field [127].
The stimulation of PLD in the cells by phorbol esters (in particular, by
PMA, which is an analog of diacylglycerol activating protein kinase C) andalso its suppression by specific inhibitors confirm the role of PKC as a key
factor of PLD regulation in vivo [128]. PKCα and PKCβ1 stimulate PLD[129, 130], but PKCδ and PKCε can act as both positive and negative
regulators of PLD1 and PLD2 in vivo. PKCδ can displace PLD1 from thecomplex with PKCα [131, 132]. Results of in vitro studies have shown thathuman and rat PLD1 are activated under the influence of PKCα, PKCβ1,
and PKCβ2 independently of their kinase activity [26, 47, 130]. The
activation of PLD through protein–protein interactions can also be realized
by atypical PKCs: PKCι (PLD2) [133] and PKCζ (PLD1, PLD2) [82, 134].
Phorbol esters as PKC activators induce the PLD1 binding with PKCαthat leads to activation of PLD1 [130, 135]. PLD2 can also bind with PKCα[136] and PKCδ [137]. The N- and C-terminal sequences of PLD are
involved in the activation of PLD1 on binding protein kinase Cα [80].
PKCα decreases the Michaelis constant and increases the catalytic constantof the PLD1 reaction. PKCα activates PLD1 in vitro synergistically with
Arf1, as discriminated from Rho. This occurs only at optimal concentrationsof PtdInsP2 [80]. The binding of active PKCα with PLD2 also stimulates
PLD2 in vivo, whereas the phosphorylation of PLD2 in vitro in many Ser
and Thr sites by PKCα inactivates it [136, 138]. However, thephosphorylation of Thr566 under the influence of PKCδ is accompanied by
activation of PLD2 [131, 137]. Phosphorylation by PKCα and PKCβ of
PLD2 at Ser and Thr leads to translocation of PLD2 to membranes and its
activation in vivo [139]. However, PKCα and PKCβ phosphorylate and as aresult inactivate PLD1 in vitro [140]. The phosphorylation of PLD1 at Thr
under the influence of PKCα suppresses PLD1 in vivo [141], and thetranslocation of PLD1 to plasma membranes and its resulting activation are
due to phosphorylation of the enzyme by protein kinase Cα at Ser2, Thr147,
and Ser561 [142].
Activation of growth factor receptors stimulates soluble tyrosine
protein kinases of the Src family. Inhibitors of Src are known to suppress
the PLD2 activation in PC12 cells caused by depolarization [143]. Membersof the Src family p60Src and p56Lyn mediate PLD activation in platelets[144]. As discriminated from PLD1, PLD2 is phosphorylated on Tyr and isactivated by other tyrosine kinases of the Src family – Fyn and Fgr [145].
However, tyrosine kinase v-Src phosphorylates PLD1 and PLD2 on Tyr in
COS-7 cells without influencing their activity [146]. Thyroid gland
carcinoma protein kinase RET/PTC also directly phosphorylates PLD2 on
Tyr without its activation [147].
The PLD2 isoform expressed as a constitutively active enzyme invarious cells [148] is a phosphotyrosine protein in vivo [149] and in vitro[150]. Its phospholipase activity is regulated dually byphosphorylation/dephosphorylation and also can induce cell proliferation by
the abovementioned human PLD isoenzyme with involvement of Tyr179
and Tyr511 [150]. Mutations of this and other tyrosine residues – Tyr296
and Tyr415 – result in a decrease in the PLD2 activity [151], whereas thephosphorylation degree of each catalyzed by different enzymes caninfluence the functioning of PLD2. The phosphorylation of PLD2 at Y296
in the PH domain by the epidermal growth factor receptor provides for the
low activity of the enzyme in vitro only in tumor cells. However,tyrosinases JAK3 and Src phosphorylating PLD2, respectively, at Tyr415
and Tyr511 are responsible for the high activity of this enzyme in vitro inless invasive cells and thus prevent the negative effect of the Tyr296
phosphorylation. The Tyr511 residue is a dual site relative to regulation of
the PLD2 activity. Dephosphorylation at Tyr511 and Tyr296 by tyrosine
phosphatases CD45 and PTP1B stimulates PLD, whereas dephosphorylation
at Tyr415 decreases the activity of this enzyme in vitro [150].
Phosphorylated Tyr179 and Tyr511 in PLD2 promote its binding with theSH2 domain of the adapter protein Grb2, which ensures a high lipase
activity of the enzyme in COS-7 cells [151]. Thus, it is concluded that
protein kinases/phosphatases modify PLD through direct
phosphorylation/dephosphorylation of this enzyme on Tyr residues.
PLD activity sharply increases in tumor cell lines in response to stresscaused by removal of serum. Dephosphorylation of Tyr179 and Tyr511 in
PLD2 can act as a signal directed to decrease the activity of PLD and also of
the accompanying proliferative potential capable of preventing cellmalignization [151, 152]. The increased expression and activity of PLD
have been found in cells of many human tumors, which suggests afunctional association of the PLD activity with oncological signals andtumorigenesis [153]. Results of these studies have allowed new nontoxic
PLD inhibitors to be created as promising antitumor preparations [153,154].
Ser/Thr-protein kinases also play an important role in the regulation of
PLD in animal cells. The mechanism of activation of PLD by casein kinase
II (PLD2) [155], cyclin-dependent protein kinase 5 (PLD2) [156], ribosomal
S6-kinase 2 (PLD1) [157], and also by AMP-activated protein kinase(PLD1) [158] is largely due to phosphorylation of the specific residues
Ser134 [156], Thr147 (in the PX domain) [157], and Ser505 [158].

However, it is still unclear whether the activation of PLD is caused byprotein kinase Rho depending on the Rho protein [159] or by
calcium/calmodulin-derived protein kinase II [144] phosphorylation of the
enzyme and whether the PLD2 phosphorylation on the active Ser/Thr
protein kinase AKT (Thr175 in the PX domain) [160] or by protein kinase A
in the case of PLD1 [161] changes the enzyme activity. It is also unclear
whether the activation of PLD [162] is associated with the enzyme
phosphorylation on Ser and Thr by MAP kinases p38, ERK1, and ERK2
[163-165] or is caused only by protein–protein interactions.
Incubation of plasma membrane from cells of the plant B. oleracea
with acidic phosphatase in vitro decreases the activity of PtdInsP2dependent PLD by 80%, whereas inactivation of the phosphatase partiallyrestores the activity of the PLD. Moreover, PLDγ of this plant is bound by
antibodies to phosphoserine/threonine/tyrosine [68]. The presence ofphosphorylation sites in the structure of PLDγ of B. oleracea [68]determines the role of this posttranslational modification in the regulation of
PLD activity in plants.
Influence of cytoskeleton and light on PLD activity. The
cytoskeleton proteins are PLD modifiers. PLD activity also depends on the
degree of actin polymerization. In the presence of active monomeric
GTPases, monomeric G-actin inactivates PLD1 in the membrane fractions
or negatively influences this enzyme resulting by immunoprecipitation [166,167]. However, G-actin stimulates a purified recombinant enzyme PLD1
expressed in cells of insects [63]. A highly purified polymerized F-actin
stimulates PLD1 in vitro but only in the presence of activators – G-proteins
ARF and Rac1 [2, 63]. The translocation of PLD1 to actin filaments iscaused by phosphorylation of the Ser2 residue on the PLD N-terminus byprotein kinase C [63].
The polymerized F-actin stimulates plant PLD activity, whereas
monomeric G-actin suppresses it [2]. In tobacco cells PLDβ1 occurs to be apartner of actin, and the interaction with actin is provided for by Asn323
and Thr382 of the phospholipase [64]. Thus, the interaction of actin and
PLDβ1 creates a system of positive feedback in which F-actin activates
phospholipase D and lipase-produced phosphatidic acid, in turn, activates Factin in places of its interaction with cell membranes [2, 64].
The activity of animal PLD2 is suppressed in vitro under the influenceof β-actin, which prevents the access of PLD2 to the substrate [166]. The
protein α-actinin that links actin filaments suppresses PLD2 in vitro byaffecting its binding with ARF1 [167]. The negative effect of β-actin is
inhibited in the presence of ARF1, whereas the binding of β-actin with
PLD2 is prevented by association of α-actinin with this enzyme [166]. The
phosphorylated form of cofilin (a protein promoting actin polymerizationand depolymerization) is specifically bound and stimulates the basal activityof PLD1 in vitro by promoting its translocation to the plasma membrane[168]. Gelsolin (a protein capping actin) suppresses PLD1 and PLD2 invitro [169], but in the presence of nucleotide trisphosphate it binds with
PLD and activates it [170]. The actin-binding protein fodrin [171] and also
dimer and trimer of spectrin suppress PLD activity and are involved in local
regulation of PLD by actin [171]. However, the effect of two latter proteinscan be associated with disorders in the interaction of PLD1 with PtdInsP2[172].

Tubulin monomer inhibits PLD2 of animal cells due to direct andreversible binding with the enzyme [173]. In plants stabilizers anddestabilizers of microtubules stimulate the in vivo accumulation ofphosphatidic acids produced by PLD [174]. Therefore, it is concluded thatcytoskeleton proteins can act as regulators of PLD activity in plant andanimal cells and provide the necessary level and specific location in cells ofphosphatidic acid production.
Plant PLDs are known to be so-called dark-induced proteins (dinproteins). The role of a photoregulatory receptor is played by phytochrome:both white and red lights suppress PLD activity in etiolated plantlets,
whereas far red light restores it (a reversible photo-effect) [175, 176]. This
indicates a possible involvement of the phytochrome receptor in theregulation of PLD activity. Moreover, the photo-dependence of PLD
activity is associated with photosynthesis, because diuron stimulates andglucose suppresses, respectively, the enzyme activity in green and etiolated
oat plantlets.
Involvement of PLD in hormone signaling. PLDs are involved inreception and intracellular transmission of hormonal signals in both animals
and plants. In animals PLDs are activated under the influence of hormones(growth hormone, vasopressin, gonadotropin-releasing hormone, etc.) andof various neurotransmitters (histamine, bradykinin, noradrenaline, etc.)[177]. Interesting data were recently obtained in studies on transmembrane
receptors of animal hormones. Phospholipase D was shown to be a key
enzyme for many of them, being involved in their secretion, endocytosis,
and signaling. Thus, the parathyroid hormone 1 type receptor (PTH1R)
increased PLD activity on interacting with the ligand, PLD1 was involved in
the internalization and intracellular traffic, and PLD2 was involved inreceptor endocytosis [178]. The subclass of formyl peptide receptors(FPRL1) induced by an agonist also can activate PLD, and thephospholipase plays an important role in endocytosis, membrane recycling,
and receptor signaling [179, 180]. PLD2 functioning in plasma membrane
similarly simplifies endocytosis of angiotensin II receptor type 1 [40].
The involvement of PLD in the transmission of abscisic acid signal
has been shown in plants [28, 181, 182]. Moreover, there are data on the
involvement of PLD in the action of gibberellins [28] and in auxin transport[184, 185] (table). PLD also plays a role in the activation by cytokinins of
the primary response genes [77, 186, 187], and a rapid increase in PLD
activity is observed under the influence of cytokinins [69, 76]. The PLDs
involved in transmission of signals of different phytohormones are also
different. The abscisic acid signal is transmitted with involvement ofphospholipase D isoforms α and partially β (table) [28, 181], whereas thecytokinin signal is transmitted not with involvement of the PLD α-isoform,
but more likely with the involvement of PLDγ [76]. Thus, PLD is an
important and specific factor in the signal transmission of various hormonesin both animals and plants.
{Table} Features and role of different PLD isoenzymes in regulation ofplant cell metabolism
Key:1) PLD isoenzyme

2) Note: ABA, abscisic acid; Bs, biotical stress; G, gibberellin; Fz, freezing;Dr, drought; IAA, indolylacetic acid; Oxs, oxidative stress; Os, osmotic
stress; W, wound; Ss, saline stress; P, mineral phosphate; Et, ethylene; N,
mineral nitrogen; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE,
phosphatidylethanolamine.
3) Molecular weight, kDa4) Substrate specificity5) PC5’) PE5”) PG
6) Cofactors
7) calcium8) mM8’) µM
9) PtdInsP210) present10’) absent
11) oleic acid12) Regulation mechanisms13) phosphorylation
14) G-proteins15) actin16) Activity pH-optimum17) Location in chromosomes18) Sensitivity to hormones and stress of mutants by PLD19) increase20) decrease21) knockout/knockdown22) superexpression23) ABA Et Glc Ss Os Dr Fz W G Bs Oxs IAA24) shortage of N25) transport of IAA26) shortage of P

PLD is a widely distributed enzyme involved in the fine regulation of
metabolism of cells with different organization. This enzyme is necessary
for growth and development of animals and plants, especially underconditions of stress and illumination. The primary structure of PLD is
known in detail, and regulatory mechanisms of activities of these enzymes
and their interaction with other signaling systems of the cell are intensively
studied. It has been shown that the activity of known forms of animal PLDs
is modified by serine/threonine and tyrosine protein kinases, GTP-binding
proteins (ARF and Rho), the cytoskeleton, polyphosphoinositides, protein–protein interactions, and hormones. In plants PLDs are directly regulated by
Ca2+, polyphosphoinositides, heterotrimeric G-proteins, cytoskeleton, some
phytohormones, and possibly by protein kinases. However, mechanisms ofin vivo regulation of individual PLD enzymes are studied insufficiently,
especially in plants, despite the established biological role of many
molecular PLD forms (table). In the majority of biological processes PLD
activity is concurrently modified by a number of regulators, and their action
can be additive, synergic, or opposing. New data on the role of PLD in theregulation of proliferation and in carcinogenesis has stimulated intensive
searches for new inhibitors of PLDs as promising antitumor preparations.
To better understand the functional role of PLD isoforms in cells, in vivo
studies are important, and these can be useful when associated withmolecular design, analysis, and synthesis of new nontoxic substances
inhibiting different PLD isoforms in vivo.
This work was supported by the State Foundation for Basic Researchof the Ukraine (projects Nos. F40.4/081 and F41.4/042), National Academyof Sciences of Ukraine (No. 18-10), National Academy of Sciences of
Ukraine and Academy of Sciences of Czech Republic (No. 5/1-2011), theState Foundation for Basic Research of Belarus (B09K-061, B07K-058 and
B05K-108), the Russian Foundation for Basic Research (projects 11-0490491-Ukr. and 11-04-00614), and the Russian Academy of Sciences
Presidium program “Molecular and Cell Biology”.

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