From the Department of Pharmacological Sciences and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-8651
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ABSTRACT |
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The mammalian phosphatidylcholine-specific
phospholipase D (PLD) enzymes PLD1 and PLD2 have been proposed to play
roles in signal transduction and membrane vesicular trafficking in
distinct subcellular compartments. PLD1 is activated in a synergistic
manner in vitro by protein kinase C- 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 animal PLD cDNA
sequence (human PLD1) and subsequent studies revealed that an
evolutionarily related PLD superfamily was widespread and encoded
several regions of conserved sequence (1, 3-7). Two distinct mammalian
PLD genes approximately 50% identical have been isolated from humans,
rats, and mice (3, 4, 8-12).
PLD proteins examined thus far from nonmammalian species exhibit
constitutive activity when assayed in vitro, and their
regulation in vivo is effected through mechanisms such as
phosphorylation and translocation (13, 14). Mammalian PLD2 is similarly
constitutively active in vitro (4, 9, 11, 15) and is
regulated in vivo through unknown mechanisms (15, 16). In
contrast, PLD1 exhibits a low basal activity when expressed in tissue
culture cell lines or as a recombinant, purified protein in
vitro and is directly stimulated by 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
(conserved regions II, III, and IV; Ref. 7). 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 (7). 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 NH2-terminal
region of PLD2 and unexpectedly generated a strongly ARF-responsive isoform, which we characterize and discuss in the context of PLD2 regulation in vivo.
General Reagents--
All phospholipids were purchased from
Avanti Polar Lipids. Phosphatidylinositol 4,5-bisphosphate 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 and chimeric PLD1/PLD2 cDNAs 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. For two constructs, a Ras membrane localization sequence was appended to the 3'-end of the open reading frame using a linker encoding the sequence PGCMSCKCVLS.
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.). For in vivo assays, the cells
were washed into serum and phosphoric acid-free Dulbecco's modified
Eagle's medium after transfection and labeled with 5 µCi of
[32P]phosphoric acid (Pi) per dish for
18 h (19).
PLD Assays--
Recombinant mammalian ARF, RhoA, Rac-1, and
PKC- The Amino Terminus of PLD2 Is Required for Its High in Vitro Basal
Activity--
Conservation of protein sequence between yeast PLD and
mammalian PLD1 begins at amino acid 325 of PLD1 (amino acid 308 of PLD2). We previously found that the nonconserved region (denoted the
amino terminus) of PLD1 mediates responsiveness to PKC but is not
required for PLD1's intrinsic enzymatic activity or for small
GTP-binding protein stimulation of PLD1 (Fig.
1).2 We also observed that
deletion of either the amino terminus or the unique "loop" sequence
from PLD1 increased its basal activity, suggesting that these regions
in combination were responsible for at least part of the difference in
the PLD1 and PLD2 states of activation in
vitro.2 These findings prompted us to investigate the
function of the PLD2 NH2 terminus, since PLD2 exhibits high
basal activity and is not PKC-responsive.
The amino termini are well conserved between the different mammalian
PLD2 proteins but exhibit little similarity to the PLD1 amino termini,
particularly over the first 80 amino acids. Weak homology is observed
from amino acid 83 to 196, including a phox domain (Fig. 1,
Ref. 2) that has been proposed to mediate a wide variety of
protein-protein interactions (23). Many membrane-associated proteins
require free amino termini to successfully interact with membrane
surfaces, particularly if they encode prenylation sequences. PLD2 does
not encode such sequences, and it was known from earlier studies that a
free amino terminus is not required, since the protein appears to
behave normally when fused to an NH2-terminal HA-epitope
peptide (4, 7, 15).
To assess the potential role of the PLD2 amino terminus, a 308-amino
acid NH2-terminal deletion analogous to the 325-amino acid
NH2-terminal deletion previously described for PLD1 was
constructed, transfected into COS-7 cells, and assayed for basal and
stimulated PLD activities as described under "Experimental
Procedures" (Fig. 2). As shown in Fig.
2B, the PLD activities in lysates from COS-7 cells
transfected by a control plasmid (Gbx-2, a homeobox protein) and
stimulated by a variety of effectors, or transfected with PLD1 but not
stimulated by exogenous effectors, are relatively low (see also Fig.
2D, where it is shown that anti-PLD antisera can detect the
overexpressed but not the endogenous proteins). In contrast, lysates
from PLD1-transfected cells exhibit a dramatic increase in activity
when stimulated by mammalian ARF1, Rho family members, or PKC-
Unexpectedly, deletion of the amino terminus of PLD2 (PLD2
We next examined PLD2
Finally, we examined PLD2 ARF1 Acts Directly on PLD2 PLD1 and PLD2
We previously reported that PLD1 is activated by mammalian ARF1 (mARF1)
more effectively than by yeast ARF2 (yARF2) (Fig. 3A, Ref. 20). We examined this
response for PLD2 Activation of PLD1 and PLD2
The first set of mutant ARF proteins examined (set
a in Fig. 4A) consisted of a series of swaps at
species-specific amino acids in region I. None of the mutant proteins
examined were altered for their relative capacity to stimulate PLD1 or
PLD2 Chimeric Analysis of PLD1 and PLD2--
In an analogous manner,
chimeric PLD1/PLD2 proteins were generated and assayed in an attempt to
locate regions conferring isoform-specific activation properties for
PLD1 and PLD2 (Fig. 5). Chimeras A and C
consisted of substitutions of amino-terminal portions of PLD2 for the
equivalent regions in PLD1. Although NH2-terminally
truncated PLD1
PLD2 is characterized by high basal activity in vitro, and
this constitutive activity is largely lost when the first 308 amino acids are truncated. A chimeric protein in which the first 234 amino
acids of PLD2 are substituted by the corresponding region of PLD1
(chimera B) behaves relatively similarly to wild-type PLD2, whereas
substitution of the first 308 amino acids (chimera D) results in a
protein that behaves relatively similarly to PLD2
Finally, the loop region unique to PLD1 was inserted in PLD2 (chimera
G). Perhaps surprisingly, the resulting protein is catalytically active
and was relatively unperturbed by the insertion except for a moderate
general decrease in activity. It was previously reported that the loop
region empirically acts as a negative regulatory element for PLD1,
since its deletion results in increased basal and stimulated
activities.2 The moderate general decrease observed after
insertion of the loop sequence into PLD2 supports the hypothesis that
the relatively lower levels of activity manifested by PLD1 ensue in
part from a negative influence of inserting a peptide sequence into
this site, although the mechanism through which the decrease in
activity is achieved is not known.
The activities observed in vitro were assessed in
vivo using the transphosphatidylation assay (Fig.
6A). No significant
differences were observed for the chimeric proteins between the
in vitro and in vivo basal activities. Similarly,
protein expression was examined using Western analysis (Fig.
6B). All of the chimeric proteins were detected at the
predicted molecular weights, although breakdown products were
additionally observed.
Taken together, analysis of the chimeric PLDs provides some insight
into the role of several regions of the PLD proteins but also reveals
that the many of the functions potentially mediated by these regions
are not readily transferable. For example, the amino terminus of PLD1
is required for response to PKC- A Free Carboxyl Terminus Is Required for PLD2 Activity, but Not
Because the COOH Terminus Undergoes Modification--
As reported
previously, the carboxyl terminus of PLD1 is relatively well conserved
with PLD2 and is required for enzymatic activity. Unlike the amino
terminus, which tolerates peptide tags such as HA, green fluorescent
protein, or His6 quite well (i.e. the resulting
proteins remain enzymatically active), the carboxyl terminus is
refractory. The addition of HA or other peptide tags inactivates
PLD1.3 This suggested that a free-carboxyl terminus is
required for enzymatic activity, as one possibility because it mediates
membrane association, potentially through post-translational
modification of the terminal amino acid, which is a conserved
threonine. The possibility that a free carboxyl terminus was required
to mediate membrane association was tested by fusing a Ras membrane
localization peptide to the carboxyl terminus, which should block the
PLD2 carboxyl terminus but result in the hybrid protein being
transported to the membrane surface regardless. Analysis of the
chimeric proteins revealed that the role of the conserved carboxyl
terminus is unlikely to involve membrane localization because fusion of
the Ras membrane localization tag to the carboxyl terminus inactivated
PLD2 (Fig. 7). The possibility that the
terminal amino acid (threonine) becomes modified was examined by
mutating it to alanine, which is not capable of being modified in the
same manner as threonine. Examination of this mutant PLD2 revealed that
modification of the carboxyl terminus appears unlikely to be critically
important, since PLD2-T933A exhibited essentially wild-type PLD2
activity (Fig. 7).
Prior to the molecular cloning of PLD1 and PLD2, it was
anticipated that there would be at least two different PLD isoforms (reviewed in Ref. 26). There was general agreement that PLD was
activated by agonist stimulation, but several groups had reported that
PLD activities originating from different subcellular compartments exhibited significantly different regulatory characteristics. Most
frequently, such reports described Rho-selectively responsive or
ARF-selectively responsive PLDs. With the cloning of PLD1 and PLD2, it
became clear that PLD1 was unexpectedly capable of responding in
vitro to all Rho and ARF family members examined as well as PKC- Since the original description of PLD2, several hypotheses have been
developed to resolve the issue of in vitro constitutive activity versus presumed regulation in vivo.
These included PLD2-specific inhibitors (15), sequestration of PLD2 by
unstimulated tyrosine kinase receptors (16), and regulation of
phosphatidylinositol 4,5-bisphosphate levels (27). We suggest here that
PLD2 may be regulated by ARF family members in vivo either
through the action of unknown factors that render it strongly
ARF-dependent or by being truncated to an ARF-responsive isoform.
In a prior report, we described the finding that the amino terminus of
PLD1 is required for PLD1 to respond to PKC- The present report demonstrates a number of similarities between PLD1
and PLD2. The amino terminus is not required for the intrinsic activity
of either protein, but it does mediate regulation. In contrast, the
carboxyl terminus is critical for enzymatic activity. Both PLDs can be
made ARF-responsive, although the functional significance of this
stimulation is not clear for either protein. Finally, it was previously
proposed that the low basal activity of PLD1 derives at least in part
from the combined effect of a negative regulation imposed by the amino
terminus and the PLD1-specific loop region. The current work extends
and supports this finding by showing that insertion of the
PLD1-specific loop sequence into PLD2 decreases its basal activity. It
is possible that specific sequences in the PLD1 loop mediate negative
regulation. Alternatively, and more probably, the specific sequence is
not critical, since it is relatively poorly conserved in PLD1 between
different mammalian species. The origin of the PLD1-specific loop is
not clear. PLD1 and PLD2 have a very similar intron-exon organization,
and the PLD1-specific loop is derived from two additional exons present in PLD1 but not in PLD2 (28, 29). Unrelated loop sequences are present
in Drosophila and in Caenorhabditis elegans PLDs, but their roles in regulation have not yet been determined (reviewed in
Ref. 2). Analysis of PLD genes from additional higher eukaryotes will
be required to correlate the presence of loop sequences with the
evolution of lowered basal activity and the capacity for effector stimulation.
PLD1 and PLD2 have been proposed to localize to different subcellular
regions (4). The finding that PLD1 and PLD2 preferentially respond to
different ARFs supports and extends this finding. PLD1 is best
activated by ARF1, consistent with the prior reports that both are
located in perinuclear compartments (4, 25). PLD2 was previously
reported to localize to the plasma membrane in serum-starved cells and
to submembranous vesicles potentially part of an endocytic cycle after
serum stimulation (4). PLD2 is not activated well by ARF6, which has
been reported to localize to the plasma membrane (25), but it is
activated well by ARF5, which may mediate several endocytic steps (30).
Nonetheless, since all of the ARF proteins activate both PLDs, no
specific types of interactions are ruled out by our findings.
The ARF and PLD proteins are likely to interact through the two ARF
switch regions (amino acids 41-55 and 70-80). Such regions mediate
effector actions for all small GTP-binding proteins. The species-specific activation of PLD1 and PLD2 by mARF1 and yARF2 does
not appear to originate from species-specific amino acids in the ARF
switch regions, since, with the exception of a single difference that
does not alter the ARF responsiveness (P76S, Fig. 4A), the
switch regions are conserved. Instead, a site adjacent to the second
switch region (amino acids 82 and 83) appears to be critical and may
cause a subtle conformational change in the switch:effector
orientation. Although ARF5 was the most potent stimulator tested for
PLD2, there may be other ARFs that are better. In addition to the three
ARFs that were not tested, ARF-like proteins exist as well that also
stimulate endogenous PLD (31, 32).
, ADP-ribosylation
factor 1 (ARF1), and Rho family members. In contrast, PLD2 is
constitutively active in vitro. We describe here molecular
analysis of PLD2. We show that the NH2-terminal 308 amino
acids are required for PLD2's characteristic high basal activity.
Unexpectedly, PLD2 lacking this region becomes highly responsive to ARF
proteins and displays a modest preference for activation by ARF5.
Chimeric analysis of PLD1 and PLD2 suggests that the ARF-responsive
region is in the PLD carboxyl terminus. We also inserted into PLD2 a
region of sequence unique to PLD1 known as the "loop" region, which
had been proposed initially to mediate effector stimulation but that subsequently was shown instead to be required in part for the very low
basal activity characteristic of PLD1. The insertion decreased PLD2
activity, consistent with the latter finding. Finally, we show that the
critical role undertaken by the conserved carboxyl terminus is unlikely
to involve promoting PLD association with membrane surfaces.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
(PKC-
) or ARF or Rho small GTP-binding
protein family members (3, 9, 17). Each class of effectors can act
alone to stimulate PLD1 and in combination to elicit a synergistic
activation, suggesting that for each there is a separate site of
interaction on PLD1 (10, 17).
(7). We subsequently undertook molecular analysis
of PLD1 and found that the amino terminus conferred PKC-
responsiveness.2 In addition,
we showed that both the amino terminus and the central loop region
mediated a negative regulatory effect that maintained PLD1's low basal
activity and finally that a conserved carboxyl-terminal region is
critical for PKC-
-mediated catalysis.2
EXPERIMENTAL PROCEDURES
-[choline-methyl-3H]dipalmitoyl
phosphatidylcholine was obtained from American Radiolabeled Chemicals,
and [32P]phosphoric acid was from ICN pharmaceuticals.
All other reagents were obtained from previously noted standard sources
and were of analytical grade unless otherwise specified (3).
were purified and activated using GTP
S or phorbol
12-myristate 13-acetate as described previously (17). Yeast ARF, mutant
mammalian ARFs, and chimeric mammalian/yeast ARFs were generated,
prepared, and activated as described previously (20). Mammalian PLD
activity was measured using the in vitro head group release
assay and the in vivo transphosphatidylation assay as
described previously (4, 17, 21, 22). PLD cDNAs were transiently
overexpressed in COS-7 cells as described previously using the
mammalian expression vector, pCGN (3, 4), or immunoaffinity-purified
from baculovirus-infected Sf9 cells as described previously (4,
15, 17). The transfection efficiency was observed to be approximately
5-20%. Western blots were performed as described previously, using
affinity-purified rabbit anti-PLD peptide antisera to detect PLD1 and
PLD2 (4, 15, 17) or the monoclonal antibody 12CA5 to detect HA-tagged PLD1 and PLD2 (7).
RESULTS
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Fig. 1.
Conserved and unique features for mammalian
PLD1 and PLD2. The PLD amino acid sequences encode regions that
are either unique to PLD1 (loop region) or that
are conserved among mammalian PLD and some or all PLDs from
nonmammalian species (other boxed regions).
Possible functions that have been proposed or demonstrated for these
regions are listed. See "Results" for details. PX,
phox; CR, conserved region; CT,
carboxyl terminus.
.
Lysates from PLD2-transfected cells exhibit a high level of basal
activity, which increases slightly (less than 2-fold) in the presence
of the exogenous effectors, in particular ARF1. A portion of this
increase can be accounted for by stimulation of endogenous PLD1 by the
effectors. The remaining very modest ARF-dependent increase
is variably observed both for PLD2 expressed in COS-7 cell lysates and
for immunopurified native and baculovirus-generated PLD2 (see Table
I and Refs. 4, 12, and 15). The finding that purified PLD2 is largely constitutively active in vitro
has led to the proposal that PLD2 is regulated in vivo
through negative regulatory mechanisms that are released upon agonist
stimulation (4, 15, 16). The modest and variable response of PLD2
in vitro to ARF suggested that ARF stimulation of PLD2
in vivo might play a more significant role in some unknown
context.
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Fig. 2.
An NH2-terminal PLD2 deletion
mutant becomes ARF-responsive. A, structure and
in vitro activity of PLD mutants in lysates after expression
in COS-7 cells (not shown at the left is an
NH2-terminal 20-amino acid HA epitope tag appended by the
pCGN expression plasmid). B, activities exhibited by the
PLDs. The values represent the average of three experiments. The
intraassay variance was 7%, and the interassay variance in some cases
was higher due to small differences in transfection efficiency for
individual plasmids in the separate experiments. The basal values were
relatively variable due to the smaller numbers involved. The ARF and
Rho effectors were preloaded with GTP S, and PKC-
was activated
using phorbol ester as described previously (17). The stimulation
triggered by the addition of GTP
S alone is not shown, but the lack
of response of PLD2
(1-308) to GTP
S-loaded Rho family members
demonstrates that the response to ARF1 is specific and not due to an
unknown GTP
S effect on the assay. C, in vivo
analysis of PLD2
(1-308). In vivo assays were carried out
as described previously and under "Experimental Procedures." The
results shown are representative of three separate experiments
conducted. PLD2-K758R is a catalytically inactive point mutant as
described previously (7). CTL, control (pCGN). D,
Western analysis of wild-type and mutant proteins. Lysates were
electrophoresed in SDS-polyacrylamide gel electrophoresis gels, and
recombinant proteins were visualized as described under "Experimental
Procedures" using a monoclonal antibody to detect the HA epitope tag
as described previously (7) or an affinity-purified polyclonal rabbit
antisera directed against PLD1-specific or PLD2-specific peptides (17,
18). Predicted sizes were as follows: PLD1, 120-124 kDa; PLD2, 106 kDa; PLD1
(1-325), 84 kDa; PLD2
(1-308), 66 kDa. Breakdown or
truncated products are observed for PLD2. The monoclonal antibody
nonspecifically detects unknown 50- and 80-kDa cytoplasmic proteins in
COS-7 cell lysates. The latter coincides with the truncated but
correctly sized protein observed for PLD1
(1-325). The goat
anti-rabbit secondary antiserum nonspecifically detects an unknown
45-kDa protein. The anti-PLD peptide antisera, which were generated
using peptides chosen from the amino termini and the middle of PLD1 and
PLD2, detect the amino termini much more effectively than the sites in
the middle of the proteins. Thus, the NH2-terminally
truncated PLDs are recognized relatively poorly by the anti-peptide
antisera as compared with detection by the anti-HA epitope tag
monoclonal antibody. SK, pBluescript.
Stimulation of purified recombinant full-length and
NH2-terminally truncated PLD2 by ARF1
(1-308) were expressed and purified from
baculovirus-infected Sf9 cells as previously described (4, 15).
Hydrolysis of 3H-labeled phosphatidylcholine was assayed
in vitro in the presence of the PLD proteins alone or in
combination with activators or controls as depicted above. The cpm
shown above represent an averaging of four experiments and were
adjusted by subtracting the counts observed in the assay blank (~1030
cpm). Each experiment was performed in duplicate, and the average
duplicate variability was 2%.
(1-308))
eliminates 85% of the high basal activity in COS-7 cell lysates and
renders the resulting truncated protein almost as ARF1-responsive as
PLD1 (Fig. 2B). No response to PKC-
was detected, consistent with the prior observation that the PKC-
-responsive region in PLD1 is in its NH2 terminus. Less anticipated was
the observation that no response to Rho family members was observed (Fig. 2B), although the site of interaction of PLD1 with Rho
family members is in the carboxyl terminus
(7),3 and this region is well
conserved between PLD1 and PLD2. Nonetheless, since it is
extraordinarily unlikely that PLD2 would encode a cryptic capacity for
response to ARF in vitro unless it was employed in some
context in vivo, the finding suggests three potential hypotheses. First, ARF might stimulate PLD2
(1-308) indirectly by
acting on some other factor present in the COS-7 cell lysates. Second,
ARF might derepress full-length PLD2 inhibited via some unknown
mechanism in vivo. Third, PLD2 might undergo truncation in vivo in some setting, generating an ARF-responsive isoform.
(1-308) activity in vivo (Fig.
2C). PLD2
(1-308) exhibited moderate activity in
vivo in unstimulated cells, although less so than wild-type PLD2
(see also Fig. 6A). Nonetheless, the relatively robust
activity observed suggests that the truncated protein is subcellularly
localized to sites containing activated ARF (in contrast to PLD1) or
that negative regulatory regions active only in vivo are
present in the NH2 terminus. Since PLD1 and PLD2
subcellularly localize to different regions (4) and since PLD1 encodes
an in vivo specific negative regulatory region in its amino
terminus,2 both hypotheses are tenable.
(1-308) protein expression (Fig.
2D). Immunoreactive protein is observed near the predicted
size of 66 kDa, in addition to proteolyzed fragments, which are also observed for wild-type PLD2, but not PLD1 or PLD1
(1-325). The proteolyzed fragments observed for PLD2
(1-308) are unlikely to be
catalytically active, since both larger NH2-terminal
deletions or deletion of any sequence from the COOH terminus yields
inactive protein (data not shown). In the full-length PLD2 COS-7 cell
lysate, however, catalytically active NH2-terminally
truncated proteins could be present that could contribute to the
partial ARF responsiveness observed.
(1-308)--
To rule out indirect
effects of ARF1, we expressed PLD2
(1-308) in baculovirus and
immunoaffinity-purified it using an anti-peptide antiserum (4). Both
full-length PLD2 and PLD2
(1-308) prepared in this manner are
largely intact (unproteolyzed) (data not shown and Ref. 4). As shown in
Table I, whereas full-length PLD2 is minimally stimulated by ARF1 (less
than 2-fold), PLD2
(1-308) exhibits a 13-fold increase. The
stimulation is observed at relatively low levels of ARF1 (0.1 µM), is GTP
S-dependent, and is not
elicited by irrelevant carrier protein (bovine serum albumin). Since
both the ARF1 and PLD proteins were recombinantly expressed and were purified, these data demonstrate that ARF interacts directly with PLD2
(1-308).
(1-308) Respond Best to Different ARFs--
PLD1
and PLD2 localize to different subcellular regions (4). At least six
mammalian ARFs exist, and these also localize to different subcellular
regions (reviewed in Refs. 24 and 25). There are three major groups of
the ARFs, typified by ARF1, ARF5, and ARF6. ARF1 is the best
characterized and localizes to the Golgi and ER. ARF5 localizes to late
endosomes and may translocate to the plasma membrane under some
circumstances. ARF6 localizes to the plasma membrane and possibly early
endosomes. Localizations for the other ARFs are not well defined, and
even the information reported for ARF1, ARF5, and ARF6 remains
controversial. The mechanisms of action of the ARFs also differ. ARF1
and ARF5 are brefeldin A-sensitive and are largely cytosolic until
agonist stimulation and subsequent GTP loading occurs. In contrast,
ARF6 is brefeldin-insensitive, and the majority of it is plasma
membrane-bound even in the absence of cellular stimulation.
(1-308) and unexpectedly found that
PLD2
(1-308) is more efficiently stimulated by yARF2 than by mARF1
(Fig. 3A). The preference is not readily attributable to the
deletion of the amino terminus, since PLD1
(1-325) behaves similarly
to wild-type PLD1, i.e. it responds better to mARF1. This
finding prompted the question of the identity of the mammalian
homologue to yARF2. To address this, we examined activation by mARF1,
mARF5, and mARF6 and found that mARF6 was a relatively poor stimulator
of both PLDs but that mARF5 stimulated PLD2
(1-308) more effectively
than mARF1 did (Fig. 3B). This finding was examined in more
precision by establishing a dose-response curve (Fig. 3C).
We confirmed that although mARF1 and mARF5 stimulated both PLDs
effectively, mARF5 consistently activated PLD2
(1-308) at a greater
potency than mARF1 and possibly with greater efficacy as well. In
contrast, mARF5 and mARF1 stimulation of PLD1 were not significantly
different. mARF5 stimulated PLD1
(1-325) slightly more effectively
than did mARF1 in three separate experiments, but the magnitude of the
difference was variable and considerably smaller than was observed for
PLD2
(1-308) (Fig. 3C and data not shown).
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Fig. 3.
PLD2 preferentially responds to yARF2 and
mARF5 instead of mARF1. A, COS-7 cells transfected with PLD
constructs were lysed and assayed for PLD activity in the presence of 1 µM GTP-loaded ARF proteins. The values represent the
average of three experiments. Basal activity (after subtraction of pCGN
background) was defined as 1.0 for each individual PLD. -Fold
stimulation is shown above each bar
relative to the appropriate basal activity. The intraassay variance was
7%, and the interassay variance in some cases was higher due to small
differences in transfection efficiency for individual plasmids in the
separate experiments. The basal values were relatively variable due to
the smaller numbers involved. B, PLD1 and PLD2 (1-308)
stimulation by a panel of GTP-loaded ARFs (1 µM each) was
measured. Values are presented relative to the stimulation observed for
mARF1 after subtraction of background. C, dose-response
curves for GTP-loaded mARF1 and mARF5 stimulation of PLD1,
PLD1
(1-325), and PLD2
(1-308). A representative experiment (of
three) is shown.
(1-308) by ARFs Occurs through
Overlapping but Discrete Domains--
The finding that mARF1 and yARF2
differentially stimulated PLD1 was used previously to define a central
portion of mARF1 as the mARF1-specific region responsible for effective
stimulation of PLD1 (20). We extended this type of analysis to
PLD2
(1-308) using the previously described mARF1/yARF2 chimeras and
some additional ARF mutant proteins altered at amino acids differing in
this region between mARF1 and yARF2 (Fig.
4).
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Fig. 4.
Identification of the regions of mARF1 and
yARF2 that confer species specificity to PLD activation.
A, COS-7 cell lysates overexpressing the PLD proteins listed
were stimulated with 1 µM wild-type or mutant mARF1 or
yARF2. Activities are presented relative to the stimulation observed
for mARF1, after subtraction of background. The data were compiled from
nine experiments. B, minimal regions identified as being
involved in the species-specific stimulation of PLD1 and PLD2.
(1-308), suggesting that the amino terminus was not responsible
for the species-specific activation. The next three groups of mutant
proteins (sets b-d) consisted of swaps between regions I,
II, and III. Set b demonstrated that the species-specific stimulation
was not conferred by region I, confirming the results observed for the region I point mutants. Set c revealed that region III was not required
for the mARF1-specific stimulation of PLD1 but does contribute to the
yARF2-specific stimulation of PLD2
(1-308). Set d confirmed that the
critical region for mARF1-mediated stimulation of PLD1 is region II and
that both regions II and III contribute to the yARF2-specific
stimulation of PLD2
(1-308). Finally, in set e, a series of swaps at
species-specific amino acids in region II was examined for PLD1
stimulation. Only one site, amino acids 82 and 83, was found to be
important for the differential stimulation. The critical regions are
depicted in Fig. 4B.
(1-325) is enzymatically active (although refractory
to PKC stimulation),2 chimeras A and C were essentially
inactive. Chimera F exhibited decreased activity, although the ARF
responsiveness retained was preferential for yARF2, suggesting that the
species-specific interaction site for ARF may lie in the carboxyl
terminus.
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Fig. 5.
Analysis of PLD1/PLD2 chimeras. Chimeric
cDNAs were generated as described under "Experimental
Procedures." COS-7 cell lysates overexpressing the hybrid proteins
were assessed for basal and stimulated PLD activities. Values for
wild-type PLD1 and PLD2 were normalized to 100%, and mutant
stimulations were expressed as a percentage of the wild-type response.
The median stimulation of PLD1 by ARF1 was 12-fold. For PLD2, the
median stimulation was 1.1-fold. The average S.D. was 13%. The values
shown were compiled from assays of duplicate samples from 3-9
experiments (depending on the construct).
(1-308). This
finding suggests that the region that mediates the high basal activity
of PLD2 is located between amino acids 235 and 308. The overall
activity of chimera B is nonetheless decreased somewhat except for the
PKC response. This suggests that the NH2-terminal PLD1
fragment may be conferring some negative regulation and PKC responsiveness to the chimera, which is consistent with the role proposed for this region (see the PLD1
(1-325) construct
above).2 However, PKC responsiveness is not observed for
chimera D, which lessens the support for this possibility. Chimera E,
for which the carboxyl terminus of PLD1 was substituted for the
corresponding region in PLD2 (the reciprocal to chimera F) exhibited
only low levels of activity, although its relative stimulation by mARF1 was higher than by yARF2, consistent with the hypothesis that the
species-specific ARF binding site is in the carboxyl terminus.
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Fig. 6.
In vivo and Western analysis of
chimeric PLD proteins. A, in vivo assays
were carried out as described previously and under "Experimental
Procedures." The results shown are representative of three separate
experiments conducted, except for the activity for PLD1, which is
higher than was normally observed (see Fig. 2C).
PLD1 (505-621) lacks the PLD1-specific loop region as described
previously.2 Control was pGbx-2. B, Western
analysis. Lysates were electrophoresed in SDS-polyacrylamide gel
electrophoresis gels, and recombinant proteins were visualized using a
monoclonal antibody to detect the HA epitope tag as described
previously (7) and under "Experimental Procedures." Predicted sizes
were as follows: PLD1, 120-124 kDa; PLD2, 106 kDa; PLD1
(1-325), 84 kDa; PLD2
(1-308), 66 kDa. Breakdown or truncated products are
observed for PLD2 and all chimeras. The monoclonal antibody
nonspecifically detects 50- and 80-kDa cytoplasmic nonspecific proteins
in COS-7 cell lysates. The latter coincides with the truncated but
correctly sized protein observed for PLD1
(1-325).
, but appending it to an
amino-terminal truncated PLD2 does not confer PKC-
responsiveness to
the hybrid PLD2. Similarly, appending the amino terminus of PLD2 to
PLD1 does not increase the hybrid PLD1's basal activity and in fact
renders it inactive.
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Fig. 7.
A free carboxyl terminus is critical for PLD2
function. The PLD cDNAs depicted were constructed, expressed
in COS-7 cells, and assayed as described in the legend to Fig. 2.
Activities are presented relative to the stimulation observed for
wild-type PLD2 after subtraction of background. The data were complied
from three experiments with an average S.D. value of 8%.
DISCUSSION
(2). In vivo, however, PLD1 regulation is less well
understood, since phorbol 12-myristate 13-acetate (which activates
PKC-
) stimulates it effectively, but activated forms of ARF and Rho excite only minimal PLD1 activation (reviewed in Ref. 2). The in
vitro constitutive activity reported for PLD2 was unexpected, since this type of PLD had not previously been detected in cellular fractions enriched for PLD activity. It was accordingly proposed that
PLD2 would be regulated differently in vivo. This idea was supported by the observation that there was a variable and very modest
response to ARF by full-length PLD2 in vitro
(~1-1.5-fold; e.g. Fig. 2B).
. As part of the present
work, we engineered the corresponding truncated PLD2 protein and found
that it lost most of its basal activity and became ARF-responsive but
not Rho- or PKC-responsive (Fig. 1). Since the Rho interaction site on
PLD1 involves the carboxyl terminus
(7)4 and the
NH2-terminal truncated form of PLD1 is still
Rho-responsive, it is unlikely that PLD2 is Rho-responsive under any
circumstance. This suggests that in vivo in some settings,
PLD2 may encode the biochemical activity previously described as being
selectively ARF-responsive (reviewed in Ref. 26). The mechanism through which this might occur is not yet clear. Both the
NH2-terminal truncated PLD1 and the
NH2-terminal truncated PLD2 exhibit low basal activities
in vitro but elevated activities in vivo,
suggesting that important (negative) regulatory mechanisms for PLD
exist that have not yet been defined. These could include factors that would inhibit full-length PLD2 and thus permit stimulation by ARF.
Identification of factors interacting with the NH2 termini (e.g. through the phox domain) or reconstitution
of this regulation in vitro using cellular extracts would
provide evidence for this hypothesis. On the other hand, truncated
forms of PLD2 are observed when PLD2 is overexpressed in COS-7 cells.
Although full-length, constitutively active PLD2 can be purified from
endogenous tissues (15), smaller immunoreactive proteins are also
detected (unpublished data) that potentially may be both active and
regulated differentially. Demonstration that PLD2 retains activity
after insertion of the PLD1-specific loop region (chimera G, Fig. 5)
indicates that the amino and carboxyl halves of PLD2 can function
independently and raise the possibility that PLD2 might retain activity
even if it was proteolyzed internally. Recovery and analysis of these potentially smaller PLD2 isoforms may provide evidence for this latter
hypothesis. The result also indicates that there must be significant
intramolecular interaction (affinity) between the amino and carboxyl
halves of PLD. Interruption of these interactions would represent a
novel approach toward generating PLD-specific inhibitors.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. O. Liang and S. Kornfeld for generously providing the wild-type and mutant mammalian and yeast ARFs. We thank Y. Zhang and V. Sciorra for valuable discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from Onyx Pharmaceutical Inc. 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.
2 Sung, T.-C., Zhang, Y., Morris, A. J., and Frohman, M. A. (1999) J. Biol. Chem. 274, in press.
3 T.-C. Sung, Y. M. Altshuller, A. J. Morris, and M. A. Frohman, unpublished results.
4 T.-C. Sung, Y. M. Altshuller, A. J. Morris, and M. A. Frohman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PLD, phospholipase
D;
ARF, ADP-ribosylation factor;
mARF, mammalian ARF;
yARF, yeast ARF;
HA, hemagglutinin;
PKC-, protein kinase C-
;
GTP
S, guanosine
5'-3-O-(thio)triphosphate..
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REFERENCES |
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