From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received for publication, October 2, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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It has been suggested that protein-protein
interaction is important for protein kinase C (PKC) Phospholipase D (PLD)1
is a ubiquitous enzyme that hydrolyzes phosphatidylcholine to
phosphatidic acid (PA) and choline (1). PA can be metabolized to
diacylglycerol (DAG) by PA phosphohydrolase. PA and DAG are involved in
receptor-mediated intracellular signal transduction, secretion,
cytoskeletal reorganization, and the respiratory burst (2). To date two
isoforms of mammalian PLD (PLD1 and PLD2) have been cloned. These
isoforms share about 50% amino acid similarity, but exhibit quite
different regulatory properties (2). PLD1 has a low basal activity and
responds to protein kinase C (PKC) and to members of the Rho and Arf
families of small G proteins (3-6), whereas PLD2 exhibits a high basal activity and shows little or no response to PKC, Rho, or Arf in vitro (7-9). The intracellular localization of PLD1 remains
ambiguous. Most reports indicate it is localized in the perinuclear
region, including the Golgi apparatus, and some have reported its
presence in caveolae (10-12).
PKC However, other groups have provided evidence that phosphorylation of
PLD1 is needed for its activation. It was found that PMA-dependent PLD1 activation required ATP in cell-free
systems from neutrophils and HL60 granulocytes (20). However, a
possible role for ATP in PIP2 synthesis was not excluded.
PLD1 was also found to be phosphorylated by PKC In this study, we determined the requirements for PKC Materials--
4 Plasmid Construction--
The rat PLD1 was cloned into
PcDNA3.1(His) vector with the N-terminal Xpress tag. The rat PKC Cell Culture and Transfection--
COS-7 cells were maintained
in DMEM supplemented with 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10% fetal bovine serum in 10% CO2.
Six-well plates were seeded with 2 × 105 cells/well,
and 10-cm dishes were seeded with 8 × 105 cells
24 h before transfection with FuGENE6 according to the manufacturer's instructions.
In Vivo PLD Assay--
After 5 h of transfection, cells in
six-well plates were serum-starved overnight (0.5% fetal bovine serum
in DMEM) in the presence of 1 µCi/ml [3H]myristic acid.
PLD activity was assayed by incubating the cells with 0.3% 1-butanol
for 20 min and measuring the formation of [3H]PtdBut as a
percentage of total labeled lipids as described before (23).
Subcellular Fractionation--
After transfection and starvation
overnight, 10-cm dishes of COS-7 cells were washed once with ice-cold
phosphate-buffered saline and then harvested using lysis buffer (25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and protease
inhibitor mixture). After 10-s sonication for two times, the cell
lysate was first centrifuged at 500 × g for 10 min to
remove unbroken cells. The supernatant was then spun at 120,000 × g for 45 min at 4 °C to separate the cytosolic and crude membrane fractions.
In Vitro PLD Assay--
For in vitro assay, the cells
were either untransfected or separately transfected with PLD1 or PKC Immunoprecipitation and Western Blotting--
COS-7 cells
cultured in 10-cm plates were transfected and starved overnight as
described above. The cells were washed once with ice-cold
phosphate-buffered saline and harvested using immunoprecipitation (IP)
buffer containing 25 mM Hepes pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM KCl, 10 mM NaF, 10 mM
Na4P2O7, 1.2 mM
Na3VO4, 1% Nonidet P-40 and protease
inhibitors mixture. The cell suspensions were sonicated for 10 s
and then spun at 120,000 × g for 45 min to pellet the
detergent-insoluble fraction. The supernatant was then precleared by
mixing it with 1 µg of affinity purified mouse IgG and 20 µl of a
1:1 slurry of protein G beads for 1h at 4 °C. The mixture was then
spun and the supernatant was incubated with 2 µl of anti X-press
antibody and 20 µl of protein G beads overnight. The
immunoprecipitates were washed four times with the immunoprecipitation buffer and then resuspended in SDS sample buffer. The samples were
analyzed by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). The blots were then blocked with
1% bovine serum albumin and incubated with primary antibody and then
with horseradish peroxidase-conjugated secondary antibody. The bands
were detected using ECL.
Regulatory, Catalytic, and C1-C3 Domains and a C-terminal Deletion
Mutant of PKC
In vitro PLD1 assay results (Fig. 1E) were
consistent with the in vivo PLD results, i.e.
none of the PKC Definition of the C-terminal Residues of PKC Mutation of Phe663 Causes PKC Time Course of PLD1 Activity and Thr Phosphorylation after PMA
Stimulation--
The above results indicated that binding was required
for PKC The PKC Inhibitor Staurosporine and a Kinase-deficient PKC Binding between PKC A major binding site for PKC Because PLD is membrane-associated, it is likely that the interaction
between PKC Previous reports showed that, with deletion of up to 11 aa in the C
terminus, PKC The issue of whether or not phosphorylation is needed for PLD1
activation remains controversial. Several studies have shown that ATP
is not needed for PKC Our results showed that PLD1 becomes Thr-phosphorylated during
PMA treatment of cells expressing wild type PKC To further explore the relationship between PLD1 activation and
phosphorylation, a PKC kinase inhibitor staurosporine and a
kinase-deficient PKC In summary, our results indicate that both the regulatory and catalytic
domains of PKC to activate
phospholipase D1 (PLD1). To determine the one or more sites on PKC
that are involved in binding to PLD1, fragments containing the
regulatory domain, catalytic domain, and C1-C3 domain of PKC
were
constructed and shown to be functional, but they all failed to bind and
activate PLD1 in vivo and in vitro. A
C-terminal 23-amino acid (aa) deletion mutant of PKC
was also found
to be inactive. To define the binding/activation site(s) in the C
terminus of PKC
, 1- to 11-aa deletion mutants were made in this
terminus. Deletion of up to 9 aa did not alter the ability of PKC
to
bind and activate PLDl, whereas a 10-aa deletion was inactive. The
residue at position 10 was Phe663. Mutations of this
residue (F663D and F663A) caused loss of binding, activation, and
phosphorylation of PLD1, indicating that Phe663 is
essential for these activities. Time course experiments showed that the
activation of PLD1 by PMA was much faster than its phosphorylation, and
its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1
phosphorylation in response to 4
-phorbol 12-myristate 13-acetate PMA
and blocked the later decrease in PLD activity. The same results were
found with the D481E mutant of PKC
, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKC
alone can bind to or activate PLD1 and that a residue
in the C terminus of PKC
(Phe663) is required for these
effects. The initial activation of PLD1 by PMA is highly correlated
with the binding of PKC
. Although PKC
can phosphorylate PLD1,
this is a relatively slow process and is associated with inactivation
of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
belongs to the group of conventional PKC isoforms that are
regulated by Ca2+, DAG, and phosphatidylserine. It has a
regulatory domain, which includes the pseudosubstrate, the C1
(phorbol, DAG binding) and C2 (calcium, phosphatidylserine binding)
domains, a catalytic domain, which includes the C3 (ATP binding)
domain, and C4 kinase domain. PKC
is mainly located in the cytosol
and can translocate to membrane fraction upon stimulation by phorbol
esters or certain agonists (13). PKC
is considered to play a major
role in PLD1 activation (2). However, the one or more mechanisms
involved in PLD1 activation are still not clear. Some studies have
shown that PKC
activates PLD1 through a phosphorylation-independent mechanism, because PKC
could activate this PLD isoform in
vitro without ATP (4, 12, 14) confirming earlier work (15, 16).
Furthermore, treatment of phosphorylated PLD1 with Ser/Thr phosphatase
did not affect its activity (17). Instead of phosphorylation, protein-protein interaction has been considered the main mechanism for
PKC
to activate PLD1. Association between PLD1 and PKC
has been
observed in COS-7 cells (18) and in Rat1 fibroblasts (19). Utilizing
proteolytic cleavage of PKC
to separate the regulatory and catalytic
domains, it was suggested that the regulatory domain of PKC
was more
important for PLD1 activation in vitro (16).
at multiple sites
during activation, and the phosphorylation occurred in
caveolin-enriched microdomains within the plasma membrane (21, 22).
Mutation of three of the sites resulted in a partial reduction in the
ability of PMA to activate the enzyme in vivo (21).
activation of
PLD1. Different fragments of PKC
, including the regulatory and
catalytic domains and one that encompassed the C1-C3 domains, were
constructed to see which domain or domains were required for binding
and activation of PLD1. In addition, several C-terminal deletion
mutants of PKC
were constructed to define the role of the C
terminus. These studies indicted that both the regulatory and catalytic
domains are required and identified Phe663 as a crucial
residue in the binding and activation of PLD1. Studies of the binding
of PKC
with PLD1 were performed and indicated a high correlation
between binding and activation of the phospholipase. The role of
phosphorylation was explored by determining the time courses of PLD1
activation and phosphorylation and the effects of a PKC inhibitor and a
PKC
mutant deficient in kinase activity. The results indicated that
phosphorylation was not required for activation of PLD1 but was
associated with inactivation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phorbol 12-myristate 13-acetate
(PMA), staurosporine, bovine serum albumin, Nonidet P-40 (Nonidet
P-40), phosphatidylinositol 4,5-bisphosphate (PIP2),
and horseradish peroxidase-conjugated secondary antibody were from
Sigma. Dipalmitoylphosphatidylcholine (PC), phosphatidylethanolamine,
and phosphatidylbutanol (PtdBut) standard were from Avanti Polar Lipids
Corp. [methyl-3H]PC and
[3H]myristic acid were from PerkinElmer Life Sciences.
Protein G-agarose beads, Dulbecco's modified Eagle's medium
(DMEM), penicillin, streptomycin, fetal bovine serum, Tris-glycine
SDS-polyacrylamide gels, PcDNA3.1(+), PcDNA3.1(HisA,B,C)
vectors, and anti-Xpress monoclonal antibody were from Invitrogen. The
transfection reagent FuGENE6 and the protease inhibitor mixture
were from Roche Molecular Biochemicals. COS-7 cells were from American
Type Culture Collection. Anti-PKC
monoclonal antibody was from BD
Transduction Laboratories. Anti-PKC
(C-terminal) monoclonal
antibody was from Upstate Biotechnology. Anti-phosphothreonine
polyclonal antibody was from Zymed Laboratories Inc.
Plasmid and PCR-product purification kits were from Qiagen. QuikChange
mutagenesis kit and Pfu Turbo DNA polymerase were purchased from Stratagene. Anti-rabbit IgG, horseradish peroxidase, ECL reagent,
and film were from Amersham Biosciences. All restriction enzymes and T4
DNA ligase were from New England BioLabs. The rat PKC
in PTB vector
and the regulatory domain (1-311) in PCH3 vector were kindly provided
by Dr. Susan Jaken (Eli Lilly).
and its regulatory domain were subcloned at the EcoRI site
into PcDNA3.1(+) vector. Catalytic domain (312-672), C1-C3
(1-468),
23 (1-649),
11 (1-661),
10 (1-662),
9
(1-663),
8 (1-664),
7 (1-665),
6 (1-666),
5 (1-667), and
1 (1-671) were generated by PCR with primers containing the 5'
and 3' EcoRI sites. D481E, F663D, and F663A mutants were
generated using the QuikChange site-directed mutagenesis kit from
Stratagene. All constructs were sequenced to verify the coding regions
and were well expressed in COS-7 cells.
or its mutants. The control supernatant or that containing
overexpressed PKC
or its mutants was used as the PKC fraction, and
the crude membranes containing PLD1 were resuspended in lysis buffer
and used as the PLD1 fraction. The PLD1 activity was measured by the
formation of [3H]PtdBut in vitro as described
previously (14). Briefly, phospholipid vesicles generated from
phosphatidylethanolamine/PIP2/PC (16:1.4:1, v/v) containing
[palmitoyl-3H]PC (0.5 µCi/reaction) were used with
1-butanol (0.6%) as substrate. The reaction mixtures were incubated at
37 °C for 30 min and stopped with chloroform/methanol/HCl
(50:98:2, v/v). The lipids were extracted from the organic phase and
resolved by thin-layer chromatography. Bands co-migrating with a PtdBut
standard were quantitated by liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fail to Stimulate PLD1--
To see if some PKC
domains might be enough to activate PLD1, the regulatory domain (RD,
1-311, 37 kDa) catalytic domain (CATA, 312-672, 43 kDa), C1-C3
domain (1-468, 55 kDa), and a C-terminal 23-aa deletion mutant (
23,
1-649, 76 kDa) were constructed, and their expression, binding, and
activation of PLD1 are shown in Fig. 1.
Fig. 1A shows that none of the PKC
domains
activated the endogenous PLD activity of COS-7 cells in the absence or
presence of PMA. Co-expression of the regulatory and catalytic domains also did not restore the stimulation of PLD activity (data not shown).
Similar results were obtained with overexpressed PLD1 in COS-7 cells
(data not shown). Western blotting (Fig. 1B) shows that all
the domains were well expressed and of the appropriate molecular mass.
Some additional immunoreactive proteins were present in all samples,
corresponding to endogenous PKC
and an unknown protein of 46 kDa.
The catalytic domain was also well expressed (Fig. 1B). To
demonstrate that the domains were functionally intact, PMA-induced
membrane translocation of the wild type enzyme, regulatory domain, and
C1-C3 fragment was tested. Fig. 1C shows that all these
proteins were translocated. As expected, the catalytic domain did not
(data not shown). To test the functional intactness of the catalytic
domain, the ability of this fragment and wild type PKC
to increase
the Thr phosphorylation of proteins in COS-7 cell lysates was examined.
Fig. 1D illustrates that both PKC
and its catalytic
domain induced marked phosphorylation of several proteins.
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Fig. 1.
The effects of PKC
domains on binding and activation of PLD1 in COS-7 cells.
A, COS-7 cells were transfected with vector, PKC
wild
type (WT), RD, CATA, C1-C3, and
23, respectively, and
in vivo PLD assay was carried out. B, cells were
lysed using lysis buffer containing antiproteases EDTA and EGTA to
limit protein degradation. The cell lysates were analyzed by SDS-PAGE
on 4-20% gels and Western blotted using anti-PKC
N-terminal
antibody for WT, RD, C1-C3, and
23 and anti-PKC
C-terminal
antibody for CATA, respectively. C, COS-7 cells expressing
WT, RD, C1-C3, and
23 PKC
were fractionated into membranes and
cytosol, respectively, as described under "Experimental
Procedures." Each fraction was analyzed by SDS-PAGE and Western
blotted using anti-PKC
antibody to test the translocation.
C, cytosol; M, membranes. D, COS-7
cells expressing WT, CATA,
9,
10, F663D, and F663A PKC
were
stimulated with PMA and lysed for Western blot analysis using
anti-phospho-Thr antibody. Vector-transfected cells stimulated with PMA
were used as control. E, COS-7 cells, respectively,
expressing PLD1 and WT, RD, CATA, C1-C3, or
23 PKC
were
fractionated into membrane and cytosolic fractions for in
vitro PLD assay. Membranes from PLD1-transfected cells and cytosol
from nontransfected cells were used as control. F and
G, PLD1 was co-expressed in COS-7 cells with WT or RD, CATA, C1-C3, and
23, respectively. Cell
lysates were immunoprecipitated using anti-Xpress antibody. Western
blotting was carried out using both anti-Xpress and anti-PKC
N-terminal antibodies for WT, RD, C1-C3, and
23 (F), or
both anti-Xpress and anti-PKC
C-terminal antibodies for WT and CATA
(G), respectively. Data are representative of at least three
separate experiments.
domains activated PLD1. Fig. 1 (F and
G) shows the results of binding tests between PLD1 and
PKC
or its domains. Before PMA stimulation, there was slight binding
between PLD1 and PKC
, which was greatly increased by PMA. However,
none of the domains was able to bind to PLD1 in the presence or absence
of PMA. As shown in Fig. 1B, all the domains were well
expressed, as was PLD1 (not shown).
Required for
Activation and Binding of PLD1--
Fig. 1 demonstrated that a
C-terminal 23-aa deletion of PKC
resulted in a loss of its ability
to bind and activate PLD1. To further define the residue(s) involved,
C-terminal deletion mutants of 1, 5, 6, 7, 8, 9, 10, and 11 aa were
made and shown to be well expressed in COS-7 cells (data not shown).
Fig. 2A shows the effects of
the different PKC mutants on endogenous PLD activity in
vivo. It is evident that, with deletion of 9 C-terminal residues
(
9), PKC
still retained its ability to activate PLD. However, a
10-aa deletion (
10) caused PKC
to lose this. Similar results were
found with overexpressed PLD1 in COS-7 cells (data not shown). To
confirm these results, the
9 and
10 mutants were examined in
an in vitro PLD1 assay. The results were consistent with the
in vivo data in that
9 mutant retained the ability to activate PLD1, whereas
10 mutant lost it (Fig. 2B).
Adding higher concentrations of PKC
and the
9 mutant increased
the PLD activity whereas higher concentrations of the
10 mutant did
not, further supporting the conclusion that the
10 mutant is
inactive on PLD1 (data not shown). Fig. 2C shows the binding
between PLD1 and PKC
or its C-terminal 9- and 10-aa deletion
mutants. The
9 mutant showed binding with PLD1 in the presence of
PMA stimulation that was equivalent to that of the intact enzyme,
whereas the
10 mutant showed negligible binding. Importantly, both
the
9 and
10 mutants still retained kinase activity as shown by
the phosphorylation of proteins in the cell lysates (Fig.
1D).
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Fig. 2.
The effects of PKC
C-terminal truncation mutants on binding and activation of PLD1
in COS-7 cells. A, COS-7 cells were transfected with
vector, PKC
(WT), or
1, 5, 6, 7, 8, 9, 10, or 11 C-terminal aa mutants, respectively, for in vivo PLD assay.
B, membranes from PLD1-transfected cells and cytosol from
cells transfected with WT,
9, or
10 PKC
were obtained for
in vitro assay. Cytosol from nontransfected cells was used
as control. C, PLD1 was co-expressed in COS-7 cells with
PKC
or its
9 and
10 mutants, respectively. Cell lysates were
immunoprecipitated using anti-Xpress antibody and Western blotted using
both anti-Xpress and anti-PKC
antibodies. Data are representative of
at least three separate experiments.
to Lose Its Ability to
Activate and Bind PLD1--
The above results indicate that
Phe663, which is 10 aa from the C terminus, is very
important for PKC
to activate PLD1. To prove this, two single-amino
acid mutants (F663D and F663A) were made to test their effects on PLD1.
Both mutants were expressed very well in COS-7 cells and translocated
from the cytosol to membrane fraction after PMA stimulation (data not
shown). In vivo PLD assays showed that both mutants could
not activate endogenous PLD activity in COS-7 cells in the presence or
absence of PMA (Fig. 3A).
Similar results were obtained when overexpressed PLD1 in
vivo (data not shown). In vitro PLD assay results also
showed that both mutants lost the ability to activate PLD1 (Fig.
3B). Similar results were obtained with higher
concentrations of the PKC
mutants were employed (data not shown).
Fig. 3C shows the binding between PLD1 and the two mutants.
The results show that both mutants bound negligibly to PLD1 even in the
presence of PMA stimulation. The phosphorylation of PLD1 by PKC
was
also studied using antibodies to phospho-Ser, phospho-Thr, and
phospho-Tyr, and the results showed that only Thr residues were
detectably phosphorylated after PMA stimulation (data not shown). Fig.
3D shows the results of PLD1 phosphorylation by PKC
and
its mutants. The data show that PKC
greatly increased PLD1
phosphorylation upon PMA stimulation. The
9 mutant could still
phosphorylate PLD1 upon PMA stimulation, whereas the
10 mutant
completely lost this function. The two Phe663 mutants also
showed greatly decreased ability to phosphorylate PLD1. Expression of
higher levels of the PKC
mutants still showed greatly impaired PLD1
phosphorylation (data not shown). As expected from the findings with
the deletion mutants, both the F663D and F663A mutants retained general
phosphorylating ability (Fig. 1D). These results indicate
that PLD1 is phosphorylated by PKC
upon PMA stimulation and indicate
that binding is required for this phosphorylation.
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Fig. 3.
Effects of F663D and F663A mutants on binding
and activation of PLD1 in COS-7 cells. A, COS-7 cells
were transfected with vector, PKC (WT), F663D, and F663A mutants,
respectively, for in vivo PLD assay. B, membranes
from PLD1-transfected cells and cytosol from cells transfected with
PKC
, F663D, or F663A were obtained for in vitro PLD
assay. Membranes from PLD1-transfected cells and cytosol from
nontransfected cells were used as control. C, PLD1 was
co-expressed in COS-7 cells with F663D or F663A. Cell lysates were
immunoprecipitated using anti-Xpress antibody and Western blotted using
both anti-Xpress and anti-PKC
antibodies. D, PLD1 was
co-expressed in COS-7 cells with WT,
9,
10, F663D, and F663A
PKC
, respectively. Cell lysates were immunoprecipitated using
anti-Xpress antibody and Western blotted using anti-Phospho-Thr
antibody (PThrPLD). Data are representative of at least
three separate experiments.
to activate PLD1, because all the deletions and mutations
that caused PKC
to lose its ability to bind to PLD1 also caused a loss of activation. However, because phosphorylation of PLD1 by PKC
must involve some association between the two enzymes, it was ambiguous
whether or not PLD1 phosphorylation was required for its activation. To
further study the role of phosphorylation in PLD1 activation, the time
courses of PLD1 activation and phosphorylation upon PMA stimulation
were studied. This required a modification of the usual protocol,
i.e. COS-7 cells were first treated with PMA for 1, 5, 15, 30, and 60 min, then 1-butanol was added, and the cells were incubated
for another 2 min. The results are shown in Fig.
4. It is evident that PLD1 activity rose
very rapidly after PMA stimulation, reaching a maximum in about 3-5
min. Thereafter the activity decreased to near basal (0.1% PtdBut) in
30 min. Fig. 4B shows that PKC
rapidly translocated from
the cytosol to membrane fraction within 1 min upon PMA stimulation and
its membrane association increased over 30-60 min. Fig. 4C
shows the time course of PKC
binding with PLD1 after PMA treatment.
The binding was also detectable at 1 min and increased during the 1-h
experiment. Fig. 4D shows that the PLD1 phosphorylation was not evident until 5 min but then continuously increased during the 1-h
incubation. The time course results indicated that the activation of
PLD1 by PMA was much faster than its phosphorylation. This suggested
that PLD1 activation was independent of its phosphorylation and raised
the possibility that phosphorylation actually decreased the activity of
the phospholipase.
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Fig. 4.
Time course of PKC
binding to and activation and phosphorylation of PLD1 upon PMA
stimulation. COS-7 cells were co-transfected with PLD1 and PKC
.
After transfection for 5 h, cells were starved for another 24 h. A, for real-time PLD activity time course upon PMA
stimulation, cells were treated with PMA for 1, 5, 15, and 30 min, then
1-butanol was added, and incubation continued for an additional 2 min.
Cells without PMA treatment were incubated with 1-butanol for 2 min as
0-min control. B, cells were treated with PMA for 1, 5, 15, 30, and 60 min, and then membrane and cytosol fractions were separated
as described under "Experimental Procedures." The samples were then
analyzed using SDS-PAGE and Western blotting using anti-PKC
antibodies. C and D, cells were treated with PMA
for 1, 5, 15, 30, and 60 min and then immunoprecipitated using
anti-Xpress antibody. PLD1 and PKC
binding was detected by Western
blotting using both anti-Xpress and anti-PKC
antibodies
(C). Thr phosphorylation of PLD1 was detected by Western
blotting using anti-Phospho-Thr antibody (PThrPLD)
(D). Data are representative of at least three separate
experiments.
Mutant (D481E) Both Eliminate PLD1 Phosphorylation by PMA While
Blocking the Later Decline in Activity--
To see if phosphorylation
of PLD1 does decrease its activity, two kinds of approaches were used:
a PKC kinase inhibitor staurosporine and a kinase-deficient PKC
mutant (D481E) (24). Fig. 5A
shows that staurosporine strongly inhibited PLD1 phosphorylation
induced by PKC
in the presence of PMA. The D481E mutant also showed
barely detectable phosphorylation of PLD1 upon PMA stimulation (Fig. 5B), consistent with its lack of kinase activity. The time
course experiments (Fig. 5C) showed that the inhibitor
partially blocked the peak activation of PLD1 induced by PMA (0-10
min) and diminished the later decline in activity (10-30 min). Similar
results were seen in cells expressing endogenous PKC (data not shown).
Compared with wild type PKC
, in cells expressing the D481E
mutant, PMA induced a smaller initial activation of PLD and a slower
later decline in PLD activity (Fig. 5C). In an effort to
explain the differences in peak PLD activity, the effects of
staurosporine and the D481E mutation on the binding of PKC
to PLD1
were tested. Staurosporine caused a minimal effect on the binding
between PKC
and PLD1 in the absence of PMA but partially reduced the
association in the presence of the phorbol ester (Fig. 5D).
Binding to PLD1 was also less with the D481E mutant compared with wild
type PKC
(Fig. 5E).
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Fig. 5.
Effects of staurosporine and D481E on PLD1
activation and phosphorylation, and binding to
PKC . A, cells were
co-transfected with PLD1 and PKC
and treated with 20 and 100 nM staurosporine (SP) for 30 min before
incubation for 30 min with 100 nM PMA. Cell lysates were
immunoprecipitated using anti-Xpress antibody and Western blotted using
anti-Phospho-Thr antibody (PThrPLD). Cells with no SP
treatment were used as control. B, PLD1 was co-expressed in
COS-7 cells with WT or D481E PKC
. After 30-min treatment with PMA
(100 nM) cell lysates were immunoprecipitated using
anti-Xpress antibody and Western blotted using anti-Phospho-Thr
antibody. C, cells were co-transfected with PLD1 and WT
PKC
(for control and SP time course) or D481E PKC
(for D481E time
course). Cells were treated with PMA for 1, 5, 15, and 30 min (for SP
time course, 100 nM SP was added 30 min before PMA
stimulation). 1-Butanol was then added, and incubation was continued
for additional 2 min. 1-Butanol was also added to cells without PMA
treatment for 2 min as 0-min control. Control, SP, and D481E time
courses are shown as diamonds, squares, and
triangles, respectively. D, cells were
co-transfected with PLD1 and PKC
and treated with SP (20 and 100 nM) for 30 min before 30-min treatment with PMA (100 nM). Cell lysates were immunoprecipitated using anti-Xpress
antibody and Western blotted using both anti-Xpress and anti-PKC
antibodies. E, cells were co-transfected with PLD1 and D481E
and treated with PMA (100 nM) for 30 min. Cell lysates were
immunoprecipitated with anti-Xpress antibody and Western blotted with
anti-PKC
antibody. Data are representative of at least three
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PLD1 was first observed in COS-7 cells
(17) and later in Swiss 3T3 fibroblasts (18). However, these studies
did not examine the relationship between the binding and the activation
of PLD1. In the present study, we provide much evidence that the
activation of PLD1 by PMA is highly correlated with the binding of
PKC
. This was demonstrated by the effects of deletions and mutations
in PKC
on the two parameters (Figs. 2-4) and was supported by the
observation that the initial time course of activation of PLD1 was
correlated with its association with PKC
and not with its phosphorylation.
on PLD1 is located at the N terminus
(25-27), and it is likely that there is an additional site (26).
However, the binding sites on PKC
for PLD1 are still not defined. In
this study, we initially focused on the binding sites for PLD1 in
PKC
. A previous report using proteolysis and chromatography to
separate the regulatory and catalytic domains of PKC
indicated that
the regulatory domain might be the critical region for PLD1 activation
in vitro (16),2 so
we first studied the binding between PLD1 and this domain and showed no
detectable binding. More PKC
fragments were made, including the
catalytic domain and the C1-C3 fragment, but these fragments also
failed to bind and activate PLD1. The fragments were shown to be
functionally intact by either their membrane translocation in response
to PMA or their retention of kinase activity. Thus these data indicated
that PKC
with both the regulatory and catalytic domains intact was
required for association and activation of PLD1.
and PLD1 occurs at a membrane locus. Thus the C1 and C2
domains of the regulatory domain, which are required for membrane
targeting and translocation of PKC
(29-31) would seem to be
essential for activation of PLD1. By this reasoning, the catalytic
domain, which stays in the cytosol irrespective of PMA stimulation
(data not shown), should be unable to activate PLD1. The observation
that the
23 mutant, which possesses the domains for membrane
targeting, was unable to activate PLD1, indicates that PKC
also
requires one or more residues in the C terminus to bind and activate PLD1.
retained kinase activity (32), so we made more
C-terminal truncation mutants to study their effects on PLD1 activity.
The results showed that the
9 mutant still bound to and activated
PLD1, whereas the
10 mutant did not, and that both mutants retained
kinase activity. In support of the conclusion that the C-terminal 10-aa
position (Phe663) is important for PKC
to activate PLD1,
two mutants of this residue (F663D and F663A) also failed to bind and
activate PLD1. In agreement with a previous study (32), the F663D,
F663A, and
10 mutants retained kinase activity (Fig. 1D).
Thus the inability of these mutants to phosphorylate PLD1 (Fig.
3D) reflects their inability to bind to the phospholipase
(Figs. 2C and 3C).
to activate PLD in vitro (4, 12,
14-16). Also, Ser/Thr phosphatase treatment dephosphorylates PLD1
in vitro but does not inhibit its activity (17). However, there is also a report showing that PLD1 is phosphorylated during activation by PKC
(22). Trypsin treatment of the phosphorylated enzyme immunoprecipitated from cells treated with PMA, followed by
two-dimensional peptide mapping, revealed multiple P-peptides (22).
Some of these overlapped with P-peptides generated from the
phosphorylation of PLD1 by PKC
in vitro. These P-peptides were analyzed by mass spectrometry to reveal phosphorylation of PLD1 at
residues Ser2, Thr147, and Ser561
(22). Mutation of these to Ala resulted in a partial loss of PMA-stimulated PLD activity in vivo. However, many other
P-peptides were not analyzed, raising the question of what
phosphorylation of these other residues would do to PLD1 activity. In
addition, the effects of the mutations on the activation of PLD1 by
PKC
in vitro were not tested.
but not the
10,
F663D, and F663A mutants (Fig. 3D). However, to answer the key question of whether or not this phosphorylation is required for
PLD1 activation, we carried out time course experiments to measure the
real-time PLD1 activity after PMA treatment (Fig. 4A) (32).
Once PtdBut is formed in the PLD assay, it is only slowly degraded, so
that the standard assay, in which 1-butanol is added first and then PMA
is added for different time lengths, only reflects the accumulation of
PtdBut, not the real-time PLD1 activity. The real-time results
indicated that the increase in PLD1 activity was much faster than the
phosphorylation increase (Fig. 4, compare A with
D). After the initial peak of PLD1 activation, there
was a slower decrease in activity, consistent with the frequently observed phenomenon that PtdBut accumulation ceases after several minutes of treatment with PMA or some agonists when PLD activity is
measured using the conventional assay (see Ref. 33, and references therein). Thus, the initial activity increase was not associated with detectable phosphorylation, whereas the subsequent activity decrease was correlated with increased phosphorylation (Fig. 4, A and D). In other words, the results were
consistent with PLD1 phosphorylation having an inhibitory effect on
activity. PLD1 activity quickly reached its peak at 1 min, and membrane
translocation and association of PKC
with PLD1 could be detected at
that time (Fig. 4, A-C). It therefore appears that the
initial association of PKC
with PLD1 is sufficient for PLD1 to reach
its full activation.
mutant (D481E) were used. As expected, staurosporine strongly inhibited PLD1 phosphorylation upon PMA stimulation (Fig. 5A). The D481E mutant also caused
negligible phosphorylation of the enzyme (Fig. 5B)
consistent with its lack of kinase activity. When the effects of these
agents on the PLD1 activity time course in response to PMA were
compared with wild type PKC
alone, both the inhibitor and the D481E
mutation induced a lower peak of PLD1 activity, but then slowed the
subsequent decline in activity (Fig. 5C). The latter results
support the conclusion that phosphorylation of PLD1 inhibits its
activity. However, the effects of both staurosporine and D481E on the
PLD1 peak activity were not consistent with our proposed effect of phosphorylation. To study this, we explored the effects of these agents
on the association between PLD1 and PKC
. Fig. 5D shows that staurosporine partly decreased the association in the presence of
PMA, and Fig. 5E shows that the D481E mutant bound PLD1 to a
less extent than wild type PKC
. These changes in the association with PLD1 could explain the reduced initial activation of PLD. The
reasons are unclear why staurosporine and the D481E mutation partly
reduce the association of PKC
with PLD1 and, hence, decrease the
initial activation of PLD1. This is because the domains in PKC
that
are involved in the binding/activation of PLD1 are unknown. Presumably
the D481E mutation partially disrupts this interaction, as does the
binding of staurosporine to the catalytic (ATP binding) domain.
are required for activation of PLD1 and that there is
a required residue (Phe663) in the C terminus.
Surprisingly, our data indicate that phosphorylation is not required
for the stimulatory action of PKC
on the enzyme in vivo
and suggest that phosphorylation is involved in the down-regulation of
PLD activation that is commonly seen at later times in cells treated
with PMA and agonists.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Judy Nixon for help in the preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.: 615-322-6494;
Fax: 615-322-4381; E-mail: john.exton@vanderbilt.edu.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M210093200
2
In a later study (28), the regulatory domain was
also shown to activate PLD1 in vitro, but it was less potent
and considerably less effective than intact PKC.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PLD, phospholipase
D;
PA, phosphatidic acid;
DAG, diacylglycerol;
PKC, protein kinase C;
PIP2, phosphatidylinositol 4,5-bisphosphate;
PMA, 4-phorbol 12-myristate 13-acetate;
PC, phosphatidylcholine;
DMEM, Dulbecco's modified Eagle's medium;
PtdBut, phosphatidylbutanol;
RD, regulatory domain;
CATA, catalytic domain;
aa, amino acid(s);
WT, wild
type.
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