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Biochem. Soc. Symp. 74, 247–257
(Printed in Great Britain)
© 2007 The Biochemical Society
Regulation of phospholipase D
activity, membrane targeting
and intracellular trafficking by
phosphoinositides
Andrew J. Morris1
Departments of Medicine and Biochemistry, The Gill Heart Institute, University
of Kentucky, 900 South Limestone Street, 326 Charles T. Wethington Building,
Lexington, Kentucky 40536-0200, U.S.A.
Abstract
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.
1
email [email protected]
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A.J. Morris
Introduction
PC (phosphatdidylcholine)-specific PLD (phospholipase D) is a glycerophospholipid phosphodiesterase that catalyses hydrolysis of its substrate to
generate PA (phosphatidic acid) and choline. Although first identified in plants,
PLD activities are now known to be present in essentially every biological
system examined [1]. While PLD probably plays a ‘housekeeping’ role in phospholipid synthesis, turnover and membrane remodelling, the sustained interest in these enzymes in eukaryotes stems from their roles in cell signalling and
cell regulation. PLD activity is tightly controlled. Mammalian PLD activity is
rapidly increased in many cell types in response to activation of cell surface
receptors for a wide range of hormones, neurotransmitters and growth factors.
Direct regulation of PLD by protein kinases, small GTPases and other interacting proteins couples cell surface receptors to activation of intracellular PLD
activity. PLD-generated PA is an intracellular signalling lipid that regulates the
actin cytoskeleton and controls pathways that regulate cell growth and survival.
Perhaps more significantly, a growing body of experimental evidence, which
is rapidly being augmented by mechanistic insights, implicates PLD as a vital
regulator of both constitutive and agonist-promoted intracellular vesicle trafficking [1,2]. Several recent reviews discuss the structure, regulation and functions of PLD. This short article focuses on the role of phosphoinositides as
regulators of yeast and mammalian PLD activity and function and, in particular,
on mechnaisms responsible for targetting these extrinsic membrane proteins to
different intracellular membrane compartments.
Yeast and mammalian PLDs are activated by PtdIns(4,5)P2
In the early 1990s, several groups observed that PLD activity in broken
cell assay systems was stimulated by hydrolysis-resistant GTP analogues [3–6].
Surprisingly, this effect did not appear to directly involve the α-subunits of heterotrimeric G-proteins, but reconstitution studies identified small GTPases of
the ARF (ADP-ribosylation factor) and Rho families as GTP-dependent activators of PLD. The molecular characterization of eukaryotic PLDs identified
PLD1 as the target of these small GTPases while the other widely expressed
mammalian PLD, PLD2, was refractory to stimulation by these activators [7–9].
The prototypic eukaryotic PLD, the product of the budding yeast Spo14 gene
is also insensitive to activation by these GTPases [10,11]. The development of
in vitro assay systems for measurement of PLD using completely artificial substrate preparations was a key step in the ‘reconstitution’ experiments that identified these PLD regulators. Brown, Sternweis and colleagues [5] observed that
mammalian PLD activity was low against sonicated dispersions of mixtures of
the PC substrate and PE (phosphatidylethanolamine) but that activity was significantly increased when PtdIns(4,5)P2 was included in these substrate preparations [5]. This PLD activity (later ascribed to PLD1) was concurrently activated
by ARF. Although PtdIns(4,5)P2 can promote ARF activation by stimulating
guanine nucleotide exchange, the effect of PtdIns(4,5)P2 appeared to be exerted
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directly on PLD activity because stimulation of PLD1 activity by Rho family
GTPases and certain conventional protein kinase C isoforms was also shown
to require PtdIns(4,5)P2 [12,13]. Subsequent work showed that the activity of
PLD2 and of Spo14p, which are both unresponsive to these protein activators,
was similarly dependent on PtdIns(4,5)P2 [9,10]. Stimulation of catalysis by
PtdIns(4,5)P2 is therefore a defining shared functional property of these yeast
and mammalian PLD enzymes.
How do phosphoinositides activate PLD?
Only phosphoinositides with a vicinal 4,5-phosphate pair activate PLD1,
PLD2 and Spo14p. PtdIns(3,4,5)P3 was found to be approximately equipotent
with PtdIns(4,5)P2 but approx. 50% as efficacious. Other phosphoinositides
were ineffective as were a range of anionic lipids including PA. Water soluble
inositol phosphates and glycerophosphoinositol phosphates neither activate
PLD nor inhibit the activation of PLD by PtdIns(4,5)P2 [5,8–10,14,15]. PLD2
expressed in insect cells using a baculovirus vector and purified from the cytosolic fraction of these cells binds weakly to large unilamellar vesicles of pure PC.
Binding is increased appreciably when acidic lipids are incorporated into these
vesicles, with an additional specific increase in binding affinity observed when
PtdIns(4,5)P2 is included in the vesicles [16]. A similar PtdIns(4,5)P2-dependent
increase in binding affinity was exhibited by an enzymatically active mutant of
PLD1 lacking the N-terminus and inter catalytic domain ‘loop’ region and, more
recently, using another deletion mutant of PLD1 [17,18]. The specific increase
in binding affinity indicates that these PLD enzymes can bind to PtdIns(4,5)P2
in the bilayer surface of these liposomes. Like other phospholipases, PLDs are
interfacial enzymes [18]. High levels of activity require that the enzyme associates with a substrate-containing interface where it can function in a processive
mode of catalysis performing multiple rounds of substrate hydrolysis before it
dissociates. PtdIns(4,5)P2 could therefore activate PLD by simply anchoring the
enzyme to the lipid surface and promoting this processive catalysis in a similar
manner to that described for phosphoinositide-specific PLCδ which associates
with substrate-containing lipid surfaces through its N-terminal PH (pleckstrin
homology) domain [19]. As noted above, PLD1 and PLD2 exhibit significant
binding to liposomes containing acidic phospholipids. To test the hypothesis
that PtdIns(4,5)P2 activates PLD2 solely by anchoring the enzyme to the liposome surface we compared PLD2 activity and binding to liposomes of PC, PE
and PS (phosphatidylserine, molar ratio 1:1:1) containing either no additional
lipid, 5% PtdIns(4,5)P2 or 10% PA. PLD binding to a fixed concentration of
these liposomes (100 µM total lipid) was assessed by measuring PLD activity
remaining in the supernatant after the liposomes had been sedimented by centrifugation. While negligible binding of PLD2 was detected to pure PC liposomes, approx. 45% of the input enzyme bound to the PC/PS/PE liposomes
and this value was increased to approx. 90% when either 5% PtdIns(4,5)P2 or
10% PA were included. In parallel experiments we prepared liposomes containing radiolabelled PC substrate and measured PLD2 activity against this
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Figure 1 Activation of PLD2 by PtdIns(4,5)P2 in vitro is not mediated
by recruitment of the enzyme to substrate-containing lipid surfaces.
PLD2 was expressed in insect cells and purified and sucrose-loaded liposomes
of the indicated compositions were prepared as described previously [16].
PLD2 binding was measured by monitoring PLD2 activity remaining in the
supernatant after sedimentation of the liposomes by ultracentrifugation. PLD
activity was measured against liposomes of identical compositions containing
[3H]PC (PLD activity was not determined against pure PC vesicles in this assay).
The data shown are means ± S.E.M. (n = 3).
substrate by monitoring release of water-soluble choline. PLD2 activity against
substrate in PC/PS/PE liposomes was very low, even though a significant fraction of the enzyme was bound to the liposomes. More dramatically, although
comparable levels of PLD2 were bound to vesicles containing PA as were bound
to vesicles containing PtdIns(4,5)P2, activity against the PA-containing vesicles
was low and comparable to that observed against substrate in the PC/PS/PE
liposomes (Figure 1). These data strongly suggest that the increase in PLD activity
observed using substrate preparations containing PtdIns(4,5)P2 is not simply a
result of increased PLD binding to these vesicles. These observations support
the idea that activation of PLD activity by PtdIns(4,5)P2 involves an as yet undefined allosteric mechanism.
What is the structural basis for phosphoinositide
activation of PLD?
The liposome binding studies described above indicate that PLD1 and
PLD2 bind to PtdIns(4,5)P2 in a bilayer interface. Surface plasmon resonance
studies using supported lipid monolayers also revealed a specific association
between PLD1 and phosphoinositides including PtdIns(4,5)P2 [20]. PLD2 can
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be activated by photoreactive benzophenone derivatives of PtdIns(4,5)P2, and
a radioactive form of one of these derivatives was used to label the protein
in a manner that could be protected by an excess of PtdIns(4,5)P2 but not by
other phosphoinositides or other acidic lipids that do not activate the enzyme
[16]. Taken together, these results strongly suggested that a binding interaction
between the PLD enzymes and PtdIns(4,5)P2 is important for stimulation of
catalysis by this lipid.
PLD1, PLD2 and Spo14p have a common multidomain structure. Their
catalytic core contains tandem HKD or PLD catalytic domains flanked by
PLD-specific sequences that are also critical for catalysis. The C-terminus of
the proteins is divergent in primary sequence and does not contain recognizable
homologies to other proteins. However, the N-termini contain both a PX
(phox homology) and PH domains (Figure 2) [1]. Although there is variation
in their ligand specificity and affinity, examples of both PX and PH domains
have been shown to bind to phosphoinositides and inositol phosphates [21].
However, the entire N-terminus of PLD1 and PLD2 is completely dispensable
for catalysis and, in both cases, deletion mutants of the enzymes are activated
by PtdIns(4,5)P2 with a potency and efficacy that is indistinguishable from that
of their wild-type counterparts [22–24]. Although we now know that the PLD
PX and PH domains can bind phosphoinositides and play important roles in
targeting the enzymes to different membrane compartments in the cell, which
is also a critical determinant of PLD activity and function (see below), they are
however not required for stimulation of catalysis by PtdIns(4,5)P2 in these in
vitro assays. A structurally and functionally diverse range of proteins, most notably regulators of cytoskeletal organization, have been found to bind to and be
regulated by PtdIns(4,5)P2 through interactions that are mediated by sequence
motifs enriched in basic and aliphatic amino acids [25]. PLD1, PLD2 and
Spo14p contain a number of candidate sequences that could potentially function
in this manner. Mutational studies in which these sequences were targeted by
mutation to replace conserved basic amino acids with neutral glycine residues
Figure 2 Domain structure of PLD1, PLD2 and Spo14p. The common
domain organization of PLD1, PLD2 and Spo14p is shown highlighting regions
that may play important roles in membrane targeting and interactions with
phosphoinositides.
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identified residues within a polybasic motif embedded in the catalytic domain
of the enzymes that were required for both association of the enzymes with
and activation by PtdIns(4,5)P2. In the case of PLD2, mutation of this motif
reduced binding affinity of the protein for liposomes containing PtdIns(4,5)P2
and resulted in a significant attenuation of the activation of the enzyme by this
lipid. Mutation of the cognate motif in both PLD1 and Spo14p also reduced
stimulation of catalysis by PtdIns(4,5)P2 [16]. Although difficulties in expressing suitable fragments of any of the PLD enzymes have precluded more detailed
studies of the interaction of this region of the proteins with phosphoinositides,
studies using synthetic peptides corresponding to this polybasic motif support
the idea that this region of the proteins has the capacity to bind PtdIns(4,5)P2.
Like PLD2, the polybasic motif peptides bind weakly to PC vesicles, but with
significant affinity to PC vesicles containing 1% PtdIns(4,5)P2. Energetic calculations indicate that both the basic and aromatic residues present in this motif
must contribute significantly to the observed binding affinity. The polybasic
motif peptide can also sequester PtdIns(4,5)P2 when added to phospholipid
monolayers, as measured by monitoring accessibility of the lipid to hydrolysis by PLC (phosphoinositide-specific phospholipase C) [26]. Taken together,
these results indicate that the polybasic motif is required for activation of PLD1,
PLD2 and Spo14p by PtdIns(4,5)P2 and suggest that this motif could contribute significantly to mechanisms targeting the enzyme to PtdIns(4,5)P2, enriched
intracellular membranes.
Is the requirement for PtdIns(4,5)P2 exhibited by
PLD1, PLD2 and Spo14p observed in vitro important
for regulation of PLD in vivo?
As discussed above, PtdIns(4,5)P2 is clearly an important regulator of
PLD1, PLD2 and Spo14p when these enzymes are assayed in vitro using artificial substrate preparations. The availability of mutant alleles of these enzymes
with attenuated responses to PtdIns(4,5)P2 provided valuable tools to examine
the requirement for PtdIns(4,5)P2 in PLD regulation and function in vivo.
PLD2 mutants with non-conservative substitution of lysine residues within the
polybasic motif were expressed in several mammalian cell lines at comparable
levels with the wild-type enzyme. The wild-type and mutant enzymes were
predominantly membrane associated and exhibited indistinguishable subcellular localization being predominantly found at the plasma membrane and associated with cytoplasmic vesicles. Both wild-type and polybasic motif mutant
PLD2 also exhibited partial localization to detergent-resistant membrane domains. Whereas the wild-type enzyme had readily measurable activity against
endogenous substrates that could be further stimulated by phorbol esters, the
polybasic motif mutant PLD2 was inactive [16]. Polybasic domain mutants of
PLD1 also exhibit low activity when expressed in several cell types. However, in
contrast with PLD2, polybasic motif mutants of PLD1 exhibit a different localization pattern to that observed with wild-type PLD1, being predominantly cytosolic and refractory to agonist or phorbol ester promoted recruitment to the
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plasma membrane [27]. Whereas overexpression of wild-type PLD1 enhances
exocytosis in adrenal chromaffin cells, the polybasic domain mutant of PLD1
fails to promote this process, which may be a consequence of this mislocalization or of its reduced activity [28,29]. Polybasic motif mutants of Spo14 appear
to localize normally in vegetative and sporulating yeast, in the latter case, localizing to the prospore membrane. However, polybasic motif spo14 mutants
could not complement the sporulation defect of Spo14 null strains or fulfill the
essential requirement of Spo14 for secretion during ‘sec14 bypass’ [16]. Taken
together, these results identify a critical role for stimulation of catalysis of these
PLD enzymes mediated by the polybasic domain in PLD function in yeast and
mammalian cells.
What is the function of the N-terminal PX and PH domains?
Although not recognized in the initial reports of the enzymes, PLD1, PLD2
and Spo14p contain tandem N-terminal PX and PH domains [1] (Figure 2). The
PX domains of PLD1 and PLD2 are highly homologous to PX domains that
have been shown to bind phosphoinositides and contain cationic residues in the
ligand binding pocket that, in other examples of this motif, form electrostatic
interactions with the phosphate groups of phosphoinositide ligands [21]. Studies
conducted using surface plasmon resonance or a liposome sedimentation
assay indicate that the isolated PLD1 PX domain binds to PtdIns(3,4,5)P3
with high specificity and has a more moderate non-specific affinity for other
acidic lipids [30,31]. In contrast, the PLD1 PX domain was suggested to bind
selectively to PtdIns5P in binding assays using phospholipids immobilized on
nitrocellulose [28]. Similar studies have not yet been conducted using the PLD2
or Spo14 PX domains.
The PH domains of PLD1, PLD2 and Spo14p also exhibit considerable
primary sequence similarity to PH domains from other proteins that
bind phosphoinositides. Deletion of the PLD1 PH domain was reported
to reduce binding of the enzyme to PtdIns(4,5)P2-containing lipid
monolayers [20]. A purified PLD2 PH domain-containing fragment bound to
PtdIns(4,5)P2-containing liposomes with moderate affinity but high selectivity
[26]. Taken together, these results suggest that, although not obligatory for
stimulation of catalysis by phosphoinositides, the PX and PH domains could
play critical roles in targeting these PLD enzymes to phosphoinositide-rich
membrane compartments. Several lines of evidence support this idea. PLD1
mutants, in which conserved lysine or arginine residues within the PX domain
that were postulated to be important for phosphoinositide binding were
substituted with alanine residues, exhibited reduced activity when expressed
in fibroblasts. Deletion of the PLD1 PH domain or mutation of the PLD2 PH
domain to either disrupt integrity of the motif or replace residues postulated
to play critical roles in phosphoinositide binding resulted in enzymes that
were catalytically inactive when expressed in several different cell types
[20,26,28].
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What role do phosphoinositides play in membrane
targeting and intracellular trafficking of PLD?
PLD1 and PLD2 behave as tightly bound extrinsic membrane proteins.
Biochemical fractionation studies indicate that almost all endogenous or overexpressed PLD1 or PLD2 protein and activity is membrane associated in many
cells. Subcellular localization studies conducted using epitope tagged variants
of the proteins, in some cases augmented by analyses using PLD1 or PLD2
selective antibodies, reveal that the proteins exhibit complex and cell-specific
localization patterns. As a broad generalization, PLD1 exhibits variable distribution between the plasma membrane and intracellular membrane compartments
that include the endoplasmic reticulum, Golgi apparatus and cytoplasmic vesicles that have previously been identified as secretory lysosomes or endosomes.
PLD2 generally exhibits a more pronounced plasma membrane localization,
although distribution of the enzyme to intracellular membrane compartments
is also commonly observed [9,32–36]. The subcellular localization of PLD1 has
been reported to be regulated by both physiological and pharmacological agonists in a variety of cell types. Whereas serum starvation drives localization of
this PLD enzyme to intracellular membrane compartments, agonist activation
of cells results in a dramatic recruitment of the enzyme to the plasma membrane
[28,32]. Clearly the PX, PH and polybasic motifs of these proteins are attractive
candidates to mediate the selective recruitment and sorting of PLD1 and PLD2
to different membrane compartments within the cell. However, analysis of the
membrane targeting of PLD1 and PLD2 is complicated by the finding that the
PH domains of both PLD1 and PLD2 contain conserved cysteine residues that
have been reported to be sites of palmitoylation [33,37]. The binding studies
described above were performed using either a truncation mutant of PLD1 that
lacks the PH domain or recombinantly expressed PLD2 that was purified from
the cytosolic fraction of insect cells and is therefore not palmitoylated. However,
based solely on energy considerations, palmitoylation would be sufficient
to anchor the enzymes to a membrane independently of interactions with
phosphoinositides. Palmitoylation is a reversible post-translational modification that determines the membrane targeting of a growing range of extrinsic
membrane proteins. Although the dynamics of palmitoylation of PLD1 and
PLD2 have not yet been explored, any model for the subcellular localization
and trafficking of these enzymes has to consider the potential implications of
these observations.
With one exception, mutation or deletion of the PLD1 PX domain has been
reported to have a very modest effect on the subcellular localization of PLD1.
Like the wild-type enzyme, PLD1 PX domain mutants were predominantly
cytosolic in resting cells and recruited to the plasma membrane upon agonist
activation. However, mutation of the PLD1 PX domain impaired endocytosis
of the enzyme after agonist-stimulated recruitment to the plasma membrane
[28,30]. Deletion of the entire PH domain from PLD1 increases cytoplasmic
localization of the protein and attenuates both agonist-induced plasma membrane recruitment and subsequent endocytosis. This requirement for the PLD1
PH domain in endocytosis was suggested to be mediated by palmitoylation of
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Figure 3 Intracellular trafficking of PLD1 and PLD2. A model for the
intracellular trafficking of PLD1 and PLD2 is presented. In brief, interactions with
phosphoinositides mediated by the polybasic motif perhaps acting in concert
with the PH and PX domains targets PLD to the plasma membrane. Reversible
palmitoylation of the PH domain traps the enzyme in detergent resistant
membrane domains which are sites of endocytosis. Adapted from [28].
cysteine residues within the domain, resulting in recruitment of the enzyme to
cholesterol- and sphingolipid-enriched lipid rafts which are known to be sites
of endocytosis [28]. Consistent with these observations, mutation of the PLD2
PH domain prevents localization of the protein to detergent-resistant membrane domains. However, in contrast with the observations made with PLD1,
unlike wild-type PLD2 which localizes to the plasma membrane, PLD2 PH
domain mutants localize to a cytoplasmic membrane compartment that partially
corresponds to early endosomes [26]. In light of the weak but selective binding of
the PLD2 PH domain to PtdIns(4,5)P2, these results suggest that the PLD2 PH
domain and polybasic motif may function in tandem to target PLD2 to the
plasma membrane. As suggested for PLD1, other determinants, possibly the PX
domain, may control recruitment of PLD2 to sites of endocytosis. Figure 3
presents a model for how coordinated lipid binding activities of the polybasic,
PH and PX domains in conjunction with regulated palmitoylation of the PH
domain may coordinate the distribution of PLD1 and PLD2 between the plasma
membrane and intracellular membrane compartments [28].
Conclusion
Stimulation of catalysis by PtdIns(4,5)P2 is a hallmark feature of yeast and
mammalian PLDs. This aspect of PLD regulation is critical for PLD function in vivo and interactions with PtdIns(4,5)P2 and other phosphoinositides
clearly also determine PLD activity and function by controlling the subcellular distribution and intracellular trafficking of the enzymes through mechanisms that involve coordinated binding of these lipids to both the polybasic
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motif responsible for stimulation of catalysis and to the N-terminal PH and
PX domains. Although significant progress has been made in identifying sequence determinants required for activation of PLD1, PLD2 and Spo14p by
phosphoinositides, further details of this activation mechanism will require high
resolution structures of these enzymes alone and in complex with their lipid
regulators.
I am grateful to Vicki Sciorra, Simon Rudge, JoAnne Engebrecht, Michael Frohman
and Stuart Mclaughlin for their contributions to some of the work described in this
review. Research in the author’s laboratory is supported by grants from the NIH
(National Institutes of Health).
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