From the Department of Pharmacological Sciences and the Institute for Cell and Developmental Biology, SUNY at Stony Brook, Stony Brook, New York 11794-8651
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ABSTRACT |
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Activation of phosphatidylcholine-specific
phospholipase D (PLD) has been proposed to play roles in numerous
cellular pathways including signal transduction and membrane vesicular
trafficking. We previously reported the cloning of two mammalian genes,
PLD1 and PLD2, that encode PLD activities. We additionally reported that PLD1 is activated in a synergistic manner by protein kinase c- During the past 4 years, phosphatidylcholine-specific
phospholipase D (PLD)1
cDNAs have been cloned from a wide variety of species ranging from
bacteria to humans (reviewed in Refs. 1 and 2). Isolation of the first
PLD cDNA sequence from animals (human PLD1) revealed that an
evolutionarily related PLD family was widespread and encoded several
regions of conserved sequence (3). Use of these conserved regions for
motif searches of sequence data bases then revealed the existence of
additional related genes of known and unknown function from bacteria,
viruses, and mammals (4-8). A representative of this PLD superfamily
(human PLD1) and the regions conserved in it are shown in Fig. 1. Two
separate mammalian PLD genes approximately 50% identical have been
reported (PLD1 and PLD2; Ref. 4), but only a single gene appears to
exist in worms, flies, and yeast (as suggested by searches of
GenBankTM). Sequence comparisons suggest that the mammalian
genes arose through duplication of an ancestral gene after divergence
occurred between the lower eukaryotes and animals (4, 9, 10).
PLD proteins from most species exhibit constitutive activity when
assayed in vitro, and their regulation in vivo is
effected through mechanisms such as phosphorylation and translocation
(11, 12). Mammalian PLD2 is similarly constitutively active in
vitro (4, 13). How endogenous PLD2 is regulated in vivo
is not fully understood (4, 14), although it is clear that the activity of both transfected PLD1 and PLD2 increase after agonist stimulation (15). In contrast, however, to PLD2 and PLDs from nonmammalian species,
it has been reported that PLD1 exhibits a low basal activity when
expressed in tissue culture cell lines or as a recombinant, purified
protein in vitro and that it is stimulated in
vitro in the presence of recombinant, purified protein kinase
C- In our initial studies on this topic, we undertook site-directed
mutagenesis of regions held in common among PLDs from different species
(CRII, CRIII, and CRIV; Ref. 8). We found that these regions were
critical for catalysis in vitro and for PLD function in vivo and developed a hypothetical model for the catalytic
cycle involving a covalent phosphatidyl-enzyme intermediate (8). However, although many of the mutants displayed diminished or no
enzymatic activity, none of them exhibited selective responsiveness to
ARF, Rho, or PKC- In this report, we targeted for analysis the regions of PLD1 that are
the least conserved relative to mammalian PLD2 or PLD genes from other
species. We define several regions in terms of their potential
regulatory roles and suggest a model for PLD1 activation.
General Reagents--
All phospholipids were purchased from
Avanti Polar Lipids. PIP2 was isolated as described (3).
L- Site-directed and Deletion Mutagenesis--
Site-directed
mutagenesis of expression plasmids was carried out using the
Quik-Change kit (Stratagene). Plasmids were sequenced to confirm the
intended mutation and the integrity of the surrounding sequences for at
least 100 base pairs using Sequenase (U. S. Biochemical Corp.).
Deletion mutants were constructed using convenient restriction sites or
polymerase chain reaction-based strategies and were sequenced at all
junctions to ensure that the reading frame was maintained. Insertional
mutants were constructed by linearizing pCGN-PLD1 at unique sites in
the open reading frame and religating in the presence of a linker that
inserted 3 or 4 amino acids while maintaining the open reading frame.
Cell Culture--
COS-7 cells were maintained in Dulbecco's
modified Eagle's medium with 10% fetal calf serum. For transfections,
the cells were grown in 35-mm dishes and then switched into Opti-MEM I
(Life Technologies, Inc.).
Western blot analysis was performed as described previously (4, 8, 16).
In brief, 5 µl of cell lysate was added to 5 µl of 2× sample
loading dye containing 8 M urea but not boiled (if boiled
in standard dye, PLD1 becomes largely retained in the stacking gel;
Ref. 16). After separation by SDS-polyacrylamide gel electrophoresis
and transfer to nylon membrane, detection was preformed according to
the supplier's recommendations using 12CA5 (Boehringer-Mannheim), a
monoclonal antibody that detects the Flu (HA) tag epitope appended by
the pCGN expression plasmid. 12CA5 binding was visualized using a
rabbit anti-mouse secondary and a chemiluminescence kit according to
the supplier's recommendations (Amersham Pharmacia Biotech).
PLD Assays--
The PLD cDNAs were transiently overexpressed
in COS-7 cells as described previously using the mammalian expression
vector, pCGN (3, 4). The transfection efficiency using LipofectAMINE (Life Technologies, Inc.) was observed to be approximately 5-20%.
Mammalian PLD activity was measured in vitro using the
headgroup-labeled phosphatidylcholine release assay as described
previously (4, 16, 19-21). In brief, after allowing 24 h for
expression of the recombinant plasmids, the transfected cells were
harvested by scraping in 60 µl of phosphate-buffered saline
containing protease inhibitors and then sonicated. 10 µl of lysate
was added to assay buffer, sonicated lipids containing
[3H]phosphatidylcholine, water, and in some cases, PLD
effectors. Recombinant ARF, RhoA, Rac-1, and PKC-
Mammalian PLD activity was measured in vivo using the
transphosphatidylation assay exactly as described previously (4, 8, 28,
21). In brief, COS-7 cells were transfected with PLD1 plasmids, labeled
with [3H]oleate for 24 h, washed, and cultured with
0.3% butanol with or without 100 nM PMA for 30 min. After
removal of the medium, cold methanol was added to stop the reaction,
and the lipids were extracted, dried, resuspended, and separated on a
TLC plate. The phosphatidylbutanol lipids were visualized using iodine
staining, scraped into a vial, and quantitated using a scintillation counter.
PLD1 subcellular localization using Flu epitope or GFP-tagged PLD1 was
visualized as described previously (4). In brief, COS-7 cells were
fixed 24 h after transfection and analyzed using immunohistochemistry to detect the expressed proteins. The commercial monoclonal antibody, 12CA5 (Boehringer-Mannheim), was used to detect
the Flu (HA) tag according to the supplier's recommendations. GFP-PLD1
was visualized directly.
PKC- Rationale for Deletion Analysis--
PLD1 contains several regions
of sequence not found in mammalian PLD2 or PLDs from other species, and
we and others have hypothesized that these regions confer the
unusual regulatory properties of PLD1 (1, 2, 17).
The most unique region is the "loop" sequence in the center of the
protein (amino acids 505-620 in human PLD1a, Fig.
1). Mammalian PLD2 lacks these 116 amino
acids; yeast PLD (Spo14p) lacks an additional 30 amino acids, and the
loop region is similarly absent from all other known PLD proteins
except for one from Caenorhabditis elegans, in which the
loop peptide is increased in size to 300 amino acids (the regulatory
properties of this C. elegans PLD have not been reported;
GenBankTM accession number U55854). Part of the PLD1 loop
region undergoes alternative splicing, removing 33 amino acids to
generate the PLD1b isoform (9, 16). This regulated splicing is
conserved in mouse, rat, and human and confers no obvious changes in
regulation to human PLD1 (16), although it has been suggested that
there are subtle changes in Rho responsiveness for rat PLD1 (17). The
loop region is the least well conserved region when mouse, rat, and
human PLD1 sequences are compared.
The amino termini are well conserved between different mammalian PLD1
proteins but exhibit little similarity to the amino termini of
mammalian PLD2 or PLDs from nonmammalian species. This is particularly
true for the first 100 amino acids. Weak homology is observed from
amino acids 100-330 including a PX domain (2) and a weakly significant
PH domain (22). PX domains have been proposed to mediate a wide variety
of protein-protein interactions (23), and PH domains frequently mediate
inositol lipid and phosphate binding (PIP2 is a requisite
co-factor for PLD1 and PLD2). Many membrane-associated proteins require
free amino termini to successfully interact with membrane surfaces,
particularly if they encode prenylation sequences. PLD1 does not encode
such sequences, and it was known from earlier studies that a free amino
terminus was not required because the protein appears to behave
normally when fused to an amino-terminal Flu epitope peptide (3,
4).
In contrast to the amino terminus, the final 41 amino acids at the
carboxyl terminus are relatively well conserved between PLD1, PLD2, and
some of the PLDs from nonmammalian species (2). To assess the potential
role of PLD1-specific sequences in PLD1 activation by effectors, a
series of deletion mutants were constructed, transfected into COS-7
cells, and assayed for basal and stimulated PLD activities.
The Amino Terminus Is Required for PKC-
Strikingly, however,
Unexpectedly,
These results also suggested that the PH-like region encoded by amino
acids 220-329 is unlikely to underlie the absolute requirement for
PIP2 exhibited by PLD1 (3), because the truncated protein retains wild-type catalytic activity despite the elimination of most of
this sequence. To test this issue, wild-type PLD1 and
The simplest explanation for selective loss of PKC-
In the second set of experiments, we examined the physical interaction
between PKC- The PLD1-specific Loop Region Does Not Mediate Effector Activation
but Does Contain a Negative Regulatory Element--
Two deletion
constructs were generated in the loop region (Fig.
5A). The first ( A Mini-PLD1 Protein Lacking the Amino Terminus and Loop Region Is
Active and Responsive to Rho and ARF Effectors--
The amino terminus
and loop deletion findings were extended by constructing a PLD1
cDNA lacking both regions (Fig. 5A). The resulting
cDNA ( The Carboxyl Terminus of PLD1 Is Critical for PLD1
Function--
Unlike the amino terminus, which is quite variable, the
carboxyl-terminal 40 amino acids are well conserved in PLD proteins ranging from C. elegans to mammals. A construct that
truncated the carboxyl-terminal 80 amino acids (
In contrast to the carboxyl terminus, the amino terminus can be
truncated ( Partially purified mammalian PLD activities began to be examined
several years ago and were found to exhibit low basal activities and
the capacity for synergistic activation by PKC- In a previous report, we showed that the PLD catalytic site is composed
of two (CRII and CRIV) and possibly three (CRIII) noncontiguous blocks
of sequence that presumably are folded into apposition in active
protein (8). In this report, we define two regions, the amino terminus
and the loop region, that contribute to the low basal activity of
purified PLD1. The amino terminus is also required for PKC- These findings extend our working model for PLD1 activation in
vitro, which no doubt will continue to undergo modification until
structural data become available. We propose that the amino-terminal region blocks catalytic activity and that for wild-type protein, ARF,
Rho, and PKC- Nonetheless, because basal activity increases only modestly when the
amino terminus is removed, there are clearly other mechanisms that act
to silence PLD1 activity which ARF and Rho also modulate. One such
component appears to involve the loop region, which confers a
significant inhibitory effect on PLD1 and appears to limit the degree
of activation by all three classes of activators. Because the loop
region is relatively poorly conserved even between different mammalian
PLD1 proteins, the precise sequence in it may be unimportant, so long
as it acts as a "spacer" to misalign CRII and CRIV until effector
interaction occurs. Studies in progress on a large set of randomly
mutagenized PLD1 alleles have failed to identify specific sequences in
the loop region important for PLD1
regulation,2 supporting this
proposal. Deletion of the loop region has the strongest effect on some
of the Rho family members, consistent with the modest alteration in Rho
responsiveness reported for rat PLD1a and PLD1b (17), which differ in
the inclusion or exclusion of the carboxyl-terminal 33 amino acids
of the loop region (16).
Finally, because PKC- Our findings also raise the issue of what role the PLD1 PX region
plays, because its function does not appear to involve interaction with
PKC- Although the amino-terminally truncated PLD1 appears useful on the
surface as a tool for dissecting PLD1 regulation and function in
vivo, its high basal activity in vivo and other altered
regulatory properties suggest that interpretation of its effects might
be complicated. It is probable that more appropriate selectively responsive PLDs will be generated through the use of mutagenic scanning
or other approaches.
(PKC-
), ADP-ribosylation factor 1 (ARF1), and Rho family members. We
describe here molecular analysis of PLD1 using a combination of domain
deletion and mutagenesis. We show that the amino-terminal 325 amino
acids are required for PKC-
activation of PLD1 but not for
activation by ARF1 and RhoA. This region does not contain the sole
PKC-
interaction site and additionally functions to inhibit basal
PLD activity in vivo. Second, a region of sequence unique
to PLD1 (as compared with other PLDs) known as the "loop" region
had been proposed to serve as an effector regulatory region but is
shown here only to mediate inhibition of PLD1. Finally, we show that
modification of the amino terminus, but not of the carboxyl terminus,
is compatible with PLD enzymatic function and propose a simple model
for PLD activation.
INTRODUCTION
Top
Abstract
Introduction
References
(PKC-
) or ARF or Rho small GTP-binding protein family members
(3, 16). These findings also demonstrated that the effectors interact directly with PLD1, because no other proteins were present in the
assays. Each class of effectors can act alone to stimulate PLD1, and in
combination they elicit a synergistic activation (16, 17). However,
because PLD1 does not contain motifs frequently found in other kinds of
signaling enzymes, such as C2, SH2, and SH3 domains, Rho binding
sequences, or membrane localization signals, it was not readily
apparent how the small GTP-binding protein and PKC-
interactions
would be mediated. Moreover, PKC-
stimulation of PLD1 is conducted
through a novel mechanism, because the stimulation is ATP-independent
and appears to be mediated by the regulatory domain of PKC-
instead
of its catalytic one (16, 18).
(8).
EXPERIMENTAL PROCEDURES
-Dipalmitoyl phosphatidylcholine [choline-methyl-3H]
([3H]phosphatidylcholine) was obtained from American
Radiolabeled Chemicals. All other reagents were obtained from
previously noted standard sources and were of analytical grade unless
otherwise specified (3).
were purified as
described previously (16). The small GTP-binding proteins were used at 1 µM after being EDTA-stripped and preloaded with GTP
S
as described previously (16). PKC-
was activated in the assay tube
by the addition of phorbol ester to a final concentration of 100 nM. ATP was not present in the assay tubes to which PKC was
added. The components were mixed on ice, and the reactions were
initiated by transfer to a 37 °C water bath and then stopped after
30 min by addition of bovine serum albumin and trichloroacetic acid. The samples were centrifuged to precipitate unreacted lipid, and the
supernatants containing the released [3H]choline were
quantitated in a scintillation counter.
-PLD1 Co-immunoprecipitation--
Wild-type or truncated
PLD1 was transiently overexpressed in COS-7 cells in 35-mm dishes as
described above. After 24 h of culture in medium containing serum,
the cells were washed in Opti-MEM I lacking serum and cultured in fresh
Opti-MEM I for an additional 20 min. The medium was then replaced by
Opti-MEM I containing PMA at 100 nM. After an additional 5 min of culture, the cells were washed using ice-cold phosphate-buffered
saline, harvested from the dishes by scraping using ice-cold lysis
buffer (20 mM Tris-HCl, pH 7.5, mM
MgCl2, 150 mM NaCl, 0.1 mM
dithiothreitol, 10 µg/ml leupeptin, and 0.5 mM
phenylmethylsulfonyl fluoride), and sonicated until lysis occurred. All
subsequent steps were carried out at 4 °C unless noted otherwise.
The resulting lysate was precleared using a 3,000 × g
spin for 5 min. The supernatant was then centrifuged at 30,000 × g for 30 min to pellet the membranes. Pilot experiments had
shown that virtually all of the PLD1 segregates to this pellet under
these conditions. The membrane pellet was then resuspended in lysis
buffer containing 2% cholate and immunoprecipitated through incubation
with 10 µl of 12CA5 coupled to protein A-Sepharose on a rotator
(12CA5 is a monoclonal antibody that recognizes the Flu epitope tag
fused to the amino terminus of the PLD1 constructs as expressed in
pCGN). The beads were spun for 5 s at 16,000 × g
and washed three times with 1 ml of 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 150 mM NaCl, 0.1%
Triton X-100. The beads were resuspended in 30 µl of wash buffer
following which an equal volume of 2× SDS sample buffer containing 8 M urea was added (16). The samples were incubated at room
temperature (but not boiled) before protein electrophoresis (16).
Western blot analyses were performed as described previously, using
12CA5 or a rabbit PLD1-specific antiserum (16) to detect PLD1 (4) and
an anti-PKC-
monoclonal antibody (Santa Cruz) to detect PKC-
.
RESULTS
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Fig. 1.
Conserved and unique features for human
PLD1. The PLD1 amino acid sequence encodes regions of sequence
that either are unique to PLD1 (loop region) or are
conserved with mammalian PLD2 and some or all PLDs from nonmammalian
species (other boxed regions). Possible functions that have
been proposed or demonstrated for these regions are listed underneath
each box. See "Results" for details. CT, carboxyl
terminus; LOOP, loop region.
Activation of
PLD1--
As shown in the top line of Fig.
2A (and see figure legend),
PLD activity in COS-7 cell lysates overexpressing wild-type PLD1 increases dramatically (30-60-fold) in the presence of small
GTP-binding proteins or PKC-
. However, three proximal deletion
mutant proteins (
1-100, 1-157, and 1-228) were found to be
inactive (Fig. 2A) and to differing degrees, unstable (Fig.
2B), suggesting that critical regions had been deleted or
that protein misfolding occurred as a result of the missing sequences.
However,
1-325 was both stable and active, demonstrating that the
minimal sequence required for catalysis does not include the
amino-terminal third of the protein. The atypical stability of this
truncated protein may result from the fact that the site surrounding
amino acid 325 appears to constitute a protein domain boundary, as
suggested by the fact that amino acid similarity between yeast and
mammalian PLD dramatically increases carboxyl-terminal to this
point (1, 3).
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Fig. 2.
An amino-terminal PLD1 deletion mutant
exhibits selective loss of responsiveness to
PKC- . A, structure and
in vitro activity of PLD mutants in lysates after expression
in COS-7 cells (not shown at the left is an amino-terminal
20-amino acid Flu epitope tag appended by the pCGN expression plasmid).
Values in parentheses on the right beneath the top
line indicate the fold activation of wild-type PLD1 when
stimulated by a series of effectors as compared with basal activity.
The values represent the average of four experiments and were
calculated after subtraction of the background activity observed
subsequent to transfection of a non-PLD cDNA (Gbx2) expressed from
pCGN and stimulated under the same conditions (actual cpm: basal
activity in cell lysates from Gbx2-transfected cells (basal
background), 541 cpm; basal activity in cell lysates from wild-type
PLD1-transfected cells, 871 cpm; ARF1-stimulated activity in
Gbx2-transfected cells (ARF-stimulated background), 937 cpm;
ARF1-stimulated activity in PLD1-transfected cells, 20250 cpm; see also
Figs. 2C and 6A as examples). The background-corrected wild-type PLD1
activities were then normalized to 100% for each category and used as
a base to calculate relative activities of the mutants shown below
(after similar background corrections). Mutant activities shown
represent the average of four to nine separate experiments. The
intra-assay variance was 7%, and the inter-assay in some cases was
higher due to day-to-day differences in assay parameters. B,
Western blot analysis of wild-type and mutant proteins. Lysates were
electrophoresed in SDS-polyacrylamide gel electrophoresis gels, and
recombinant proteins were visualized using a monoclonal antibody to
detect the Flu epitope tag as described previously (8) and under
"Experimental Procedures." Black arrowhead, PLD1 is
detected at a range of sizes from 122 to 130 kDa due to
post-translational modifications. Infrequently, a breakdown product is
observed (white arrowhead). Asterisk, the
monoclonal antibody detects an 80-kDa cytoplasmic nonspecific protein
in COS-7 cell lysates, which coincides with the truncated but correctly
sized protein observed for
1-325. Black dots indicate
the expected bands for wild-type and truncated PLD1 proteins.
C, in vivo analysis of
1-325 PLD1. In
vivo assays were carried out as described previously and under
"Experimental Procedures." The results shown are representative of
four separate experiments conducted. PLD1-K898R is a catalytically
inactive point mutant as described previously (8). In all experiments,
the
1-325 basal activity was higher than the wild-type PLD1 basal
activity. The left panel depicts the absolute levels of PLD
activity observed in separate experimental dishes. In the right
panel, the averaged basal (endogenous) PLD activities (K898R) were
subtracted from the averaged experimental values to show the activity
specifically generated by the overexpressed wild-type and mutant PLD
proteins. The numbers indicate the fold induction in the presence of
PMA. D, PLD activity in vivo was assessed after a
4-h preincubation with PMA. The basal and +PMA results shown were
averaged from three separate experiments. The values were corrected for
the background activities observed in cells transfected with pCGN and
stimulated under the same conditions (i.e. raw values in a
single, representative experiment: basal: pCGN (7781 cpm), wild-type
PLD1 (11383 cpm);
1-325 (15474 cpm). PMA-stimulated: pCGN (39251 cpm), wild-type PLD1 (73851);
1-325 (44799 cpm)).
1-325 was capable of being activated only by
ARF and Rho small GTP-binding protein family members; no response to
PKC-
was observed (the in vitro assay was also conducted on
1-325 generated and purified from baculovirus with similar results; data not shown). The result demonstrates that a sequence uniquely required for activation of PLD1 by PKC-
is present in the
amino terminus of the protein. To extend this observation,
1-325
was expressed in COS-7 cells and assayed using the
transphosphatidylation assay (Fig. 2C). A low endogenous PLD
basal activity and a modest response to PMA was observed in cells
transfected with K898R, a catalytically inactive PLD1 allele (8),
whereas a robust response to PMA was observed for wild-type PLD1
(11-fold above basal, after subtraction of the endogenous component of
the response). However, no PMA-stimulated increase in activity was
observed for PLD1-
1-325, confirming that the amino terminus appears
to be required for PLD1 to respond to PKC-
. Similar results were
observed after down-regulation of PKC-
through a 4-h
pretreatment with PMA (Fig. 2D).
1-325 assayed in vivo exhibited a
significantly and reproducibly increased (2-fold) basal activity in
comparison with wild-type PLD1 (Fig. 2, C and D),
suggesting that a component of the normal physiological regulation of
PLD1 in vivo requires the amino terminus to be intact. The
mechanism underlying the increased basal activity in vivo is
not apparent. Possibilities include increased sensitivity in
vivo to small GTP-binding proteins or changes in subcellular
location to sites more conducive to PLD1 activation.
1-325 remains
membrane-localized similar to PLD1 as determined by subcellular
fractionation (data not shown), but the specific sites to which it is
targeted may be altered.
1-325 were
assayed in vitro in the presence of ARF and in the presence
or absence of PIP2. In the absence of PIP2,
wild-type PLD1 retained only 7% of the activity that it exhibited in
the presence of PIP2, and
1-325 was completely inactive
(data not shown). These results clearly demonstrate that the activity
manifested by
1-325 is PIP2-dependent and
thus that the PIP2-interacting site lies outside the amino
terminus. As one possibility, a region present between the loop region
and CRIII (Fig. 1) also contains a minimal PH domain basic amino acid motif.
responsiveness
would be that the sole PKC-
interaction site lies in the amino
terminus and that the amino terminus functions as a regulatory domain
for PKC-
. However, two sets of experiments argue against this model.
The first line of argument arises from our attempts to generate PLD1
point mutants that are selectively nonresponsive to PKC-
. To
accomplish this, we targeted highly conserved residues in the PX domain
(Figs. 1 and 3). The PX domain has been
proposed to be a protein-protein interaction motif that can act as a
SH3- or Pak kinase-binding site (23). Three different mutant proteins
(two point mutants and one insertional mutant) were generated and
assayed in vitro (Fig. 3A). The proteins were stably expressed (data not shown), but surprisingly, not only was
PKC-
-responsiveness lost, but all three proteins were largely unresponsive to ARF and Rho. This result suggests that although the
amino terminus is not required for catalysis or for activation by small
GTP-binding proteins, it is nonetheless capable of locking the protein
into an "off" state if nonfunctional. This implies that one step in
the small GTP-binding protein activation of PLD1 is to mediate a
conformational change in the amino terminus that permits catalysis.
Nonetheless, when the PX point mutants were expressed in
vivo, they were active, exhibited a high level of basal activity,
and responded to PKC-
(PMA; Fig. 3B). These results confirm that the role of the PX domain is not to mediate interaction with PKC-
. Moreover, the results suggest that loss of PX domain functionality accounts for the increased basal activity observed for
1-325.
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Fig. 3.
PX domain mutants are largely inactive
in vitro but exhibit a higher basal activity in
vivo and respond to PMA. A, PLD1 residues
matching amino acids conserved in PX subdomains (23) and mutant
activities. The mutations generated were made to the most highly
conserved residues of the PX motif and are listed above the subdomain
boxes. Relative activities were calculated as described in
the legend to Fig. 2. Mutant activities shown represent the average of
three separate experiments. Intra-assay variance was 7%. B,
mutant PLD1 activities in vivo. PLD activity in intact cells
was assayed as described in the legend to Fig. 2. The experiment is
representative of three separate experiments performed in duplicate,
and the values shown were corrected for endogenous PLD activity as
described in the legend to Fig. 2C. Higher basal activity and a
response to PMA was observed for the PX mutants in each experiment.
Numbers above each bar signify the fold increase
of nonstimulated mutant PLD1 activities over the nonstimulated
wild-type PLD1 activity.
and PLD1. It was previously reported that PKC-
co-immunoprecipitates with PLD1 in a PMA-dependent manner
(24). Using a modification of the published protocol (24), we examined
whether
1-325 fails to co-precipitate PKC-
(Fig.
4; a representative experiment is shown).
A modest increase in the co-immunoprecipitation of PKC-
with PLD1 in
a PMA-dependent manner was observed, consistent with the
previously reported finding (24). However, PKC-
also
co-immunoprecipitated very effectively with
1-325, revealing that
at least one PKC-
interaction site lies outside of the amino
terminus. Taken together, the PX mutagenesis and PKC-
-PLD1
co-immunoprecipitation experiments demonstrate that the amino terminus
has a complex function that involves more than simply mediating PKC-
activation of PLD1 and that PKC-
most likely interacts with multiple
sites on PLD1.
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Fig. 4.
PKC-
co-immunoprecipitates with an amino-terminally truncated,
PKC-
-nonresponsive PLD1. Flu
epitope-tagged wild-type and mutant PLD1 proteins overexpressed in
COS-7 cells and stimulated by PMA for 5 min were immunoprecipitated as
described under "Experimental Procedures" using protein A-Sepharose
beads coupled to 12CA5 (an anti-Flu tag monoclonal antibody). Western
blot analysis was performed on parallel blots to visualize PKC-
(left panel) and PLD1 (right panel). In each of
three separate experiments, PKC-
co-immunoprecipitated with
wild-type PLD1 and
1-325 at levels above background (the pCGN
vector background control nonspecifically pulled down a small amount of
PKC-
). A faint band can be observed in the PLD1 Western
blot in the pCGN lane in the position corresponding to
1-325. This
band corresponds to the nonspecific protein identified by the 12CA5
monoclonal antibody in COS-7 cells that is readily seen in whole cell
lysates (Fig. 2B), not contaminating endogenous or
recombinant PLD1. Lane C, pCGN; lane WT,
wild-type PLD1.
497-645)
replaced 150 amino acids unique to PLD1 when compared with yeast PLD
(Spo14p) with the 11 amino acids present in the corresponding region of
Spo14p. This mutant protein was stably expressed (data not shown) but
was inactive under all conditions tested (Fig. 5A). In the
second construct (
505-621), the 116 amino acids unique to PLD1 when
compared with PLD2 were excised.
505-621 exhibited basal activity
that was increased 3-fold both in vitro and in
vivo as compared with wild-type PLD1 (Fig. 5, A and
B).
505-621 was stimulated effectively in vitro by all three classes of activators and consistently
exhibited approximately a 2-fold higher level of activation in
vitro than wild-type PLD1. These findings demonstrate that the
loop region does not act as an effector regulatory region and suggests
that it accounts for part of the low basal activity characteristic of
wild-type PLD1.
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Fig. 5.
Analysis of central and carboxyl-terminal
regions in PLD1. PLD1 cDNAs lacking the loop region as defined
relative to yeast PLD ( 497-645) or PLD2 (
505-621), lacking the
loop region and the amino terminus, or lacking the carboxyl terminus
were constructed, expressed in COS-7 cells, and assayed as described in
the legend to Fig. 2. The values were derived by averaging five
separate experiments. A, in vitro assays.
B, representative in vivo assay (of two performed
in duplicate; the values shown were corrected for endogenous PLD
activity as described in Fig. 2C). n.d., not
determined. Numbers above each bar signify the
fold increase of the mutant PLD1 activities over the wild-type PLD1
activities as described in the legend to Fig. 3B.
1-325,505-621) encodes a 633-amino acid protein that is
only slightly larger than bacterial (Streptomyces) PLD (556 amino acids).
1-325,505-621 was expressed in COS-7 cells and
assayed. The in vitro basal activity was not elevated, and the extent of activation by ARF and Rho and the lack of activation by
PKC-
was comparable with that observed for
1-325 (Fig.
5A). In vivo, again similar to
1-325, the
basal activity was elevated (2.4-fold), and no response was observed to
PMA (Fig. 5B). Because the small GTP-binding protein
activation was decreased relative to
505-621, and high basal
activity was not observed in vitro, it is possible that the
mini-PLD1 protein is not fully functional. However, the fact that it is
activated well by ARF relative to wild-type PLD1 suggests that it could
be employed for structural determination or in settings in which a PLD1
primarily responsive to ARF would be useful for dissecting PLD1
activation or function.
976-1074) was
generated and assayed (Fig. 5A).
976-1074 was stably
expressed (data not shown) but was inactive under all conditions
tested. This result suggested that unlike the amino terminus, an intact
carboxyl terminus is required for PLD function. Although it is
theoretically possible that a construct truncated at a different or
more distal site might be active, other studies involving
transposon-mediated scanning of PLD1 at the extreme carboxyl terminus
or in which Flu epitope or Ras membrane localization tags were fused to
the carboxyl termini of yeast PLD or PLD2 resulted in similar losses of
activity, supporting this conclusion (data not shown). In contrast, an
amino acid insertion into PLD1 just outside of the conserved
carboxyl-terminal region (1028E-DRRV-D1029) did not inactivate the
enzyme (Fig. 5A), although the absolute levels of activity
observed were 2-fold lower. This suggests that the critical sequence
lies within the final 45 amino acids.
1-325) or fused to heterologous peptide tags without loss of activity. In addition to the Flu epitope tag described previously (3, 4, 8, 16) and used in this report, GFP-tagged PLD1 is
also active and exhibits wild-type basal and simulated activities and
subcellular localization (Fig. 6).
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Fig. 6.
A GFP-PLD1 is active, is regulated normally
by ARF1, and is targeted to the same subcellular localization as is
HA-tagged PLD1. A, GFP was fused to the amino terminus
of PLD1 or inactive PLD1 (K898R). The hybrid cDNAs were transfected
into COS-7 cells and assayed in vitro as described above.
B-E, Flu (HA)-tagged PLD1 and GFP-PLD1 proteins expressed
in COS-7 cells were visualized. Although some staining is observed in
regions potentially corresponding to trafficking membrane vesicles, the
majority resides in perinuclear regions as previously reported for
HA-tagged PLD1 overexpressed in serum-starved rat embryo fibroblasts
(4). F, as a control for the pCGN leader sequence, Gbx-2, a
homeodomain-encoding protein, localizes to the nucleus as would be
expected.
DISCUSSION
and small GTP-binding proteins (18, 20, 25, 26). With the cloning and
characterization of two mammalian PLDs as well as PLDs from a variety
of other species, two realizations became apparent. First, the
previously characterized partially purified activity was probably
mediated by the enzyme now known as PLD1. Second, PLD1 is atypical when
compared with nonmammalian PLDs, which exhibit regulatory properties
more similar to PLD2 (i.e. constitutive activity in
vitro).
activation of PLD1 but does not contain (all of) the PKC-
interaction site(s). However, a partially truncated (presumably
nonfunctional) amino terminus also blocks PLD1 activation by ARF and
Rho, demonstrating that the function of this region is more complicated
than as a simple PKC-
regulatory domain. Moreover, PLD1 proteins
mutated in the PX domain are inactive in vitro but exhibit a
high basal activity and are responsive to PKC-
in vivo,
confirming that the role of the PX domain is something other than to
mediate interaction with PKC-
. Potential roles include inter- or
intramolecular interactions or subcellular targeting.
induce conformational changes that remove this
inhibition. When made nonfunctional, as for example when mutations are
made to the conserved PX domain, ARF, Rho, and PKC-
are unable to
overcome the inert amino terminus in vitro and activate PLD1. In vivo, however, these proteins exhibit elevated
basal activity and are responsive to PKC-
, suggesting that PLD1
regulation in vivo is more complicated.
binds amino-terminally deleted PLD1
(
1-325), a PKC-
interaction site must be present in the
carboxyl-terminal two-thirds of the protein. It has previously been
reported that PKC-
activates PLD1 in a PMA-dependent but
ATP-independent manner (16). In addition, it has been shown that this
activation is mediated by the PKC-
regulatory subunit, which can
function to activate PLD1 even in the absence of the PKC-
catalytic
subunit, although at greatly reduced potency
(18).3 Nonetheless, PLD1 does
become phosphorylated by PKC-
(16),3 demonstrating that
there is interaction between PLD1 and the PKC-
catalytic domain,
presumably at a site separate from where the PKC-
regulatory domain
interacts. Finally, as shown in this report, PKC-
interacts with the
carboxyl-terminal two-thirds of PLD1, but this does not lead to
activation. Taken together, we think that the most likely explanation
for these findings is that that there are at least two PKC-
interaction sites on PLD1: an amino-terminal one that interacts with
the PKC-
regulatory domain and a central or carboxyl-terminal one
that interacts with the PKC-
catalytic domain. Because the
three-dimensional PLD1 structure most likely places CRII and CRIV in
opposition, it is not unreasonable to propose that that the
amino-terminal and carboxyl-terminal PKC-
interacting sites may be
occupied simultaneously by a single molecule of PKC-
. It has been
reported that the interaction of PLD1 with PKC-
after PMA
stimulation is a transient one that peaks at 5 min and that lasts less
than 20 min (24), even though PLD1 remains fully activated for more
than 1 h (Fig. 2D and Ref. 4). One possibility arising
from these findings is that interaction of PKC-
with PLD1 may lead
to a conformation change in PLD1 that persists subsequent to the
interaction with PKC-
.
. One possibility is that it may interact with vesicular trafficking machinery because this is a documented role for this motif
in other proteins (reviewed in Ref. 23), and PLD1 has been shown to
affect the rate of budding of vesicles from the trans-Golgi (27) and is
negatively regulated by vesicular trafficking machinery components (28,
29). Consistent with this hypothesis, the amino-terminal region in
yeast PLD is also dispensable for enzymatic activity and instead
regulates changes in subcellular localization from the cytoskeleton to
the spindle apparatus during meiosis (11).
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FOOTNOTES |
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* This work was supported by a Onyx Pharmaceutical Inc. grant and by National Institutes of Health Grants GM54813 (to M. A. F.) and GM50388 (to A. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 516-444-3060;
Fax: 516-444-3218; E-mail: michael{at}pharm.som.sunysb.edu.
The abbreviations used are:
PLD, phospholipase
D; ARF, ADP-ribosylation factor; CR, conserved region; PMA, phorbol
myristate acetate; PIP2, phosphatidylinositol
4,5-bisphosphate; PKC-, protein kinase C-
; PX, phox; HA, hemagglutinin; GTP
S, guanosine 5'-3-O-(thio)triphosphate; GFP, green fluorescent protein.
2 Y. Zhang and M. A. Frohman, unpublished observations.
3 S. M. Hammond and A. J. Morris, submitted for publication.
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REFERENCES |
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