(Received for publication, May 13, 1997, and in revised form, June 18, 1997)
From the Centre de Recherche en Rhumatologie et Immunologie, The human phospholipase D1 (hPLD1) has recently
been cloned. Although recent data have implicated PLD in
receptor-stimulated secretion, the regulation of the activity of PLD
enzymes remains to be clarified. Purified hPLD1 is activated by several
cytosolic cofactors among which are protein kinase C Phospholipase D (PLD)1
plays an important role in signal transduction through the hydrolysis
of phosphatidylcholine to choline and phosphatidic acid (PA). The
latter is a second messenger implicated in the regulation of many
signaling proteins (1). PA can also be dephosphorylated by PA
phosphohydrolases to diacylglycerol, which is an activator of certain
isoforms of protein kinase C (2). PLD activation following interaction
of agonists with G protein-coupled receptors and receptor tyrosine
kinases has been observed in many cells and tissues (1). Although the
precise physiological function of PLD in cells is poorly understood,
the receptor-stimulated PLD activity has been implicated in a broad range of human granulocyte cellular responses, including stimulated superoxide production and secretion (3).
Several studies have demonstrated in cell free-systems, or in
permeabilized cells, the GTP An ARF-regulated PLD enzyme (hPLD1) has recently been cloned (16). The
gene contains several consensus sequences highly conserved in PLDs from
yeast and plants (16-20). The recombinant enzyme is regulated by ARF1
and Rho proteins (21). Protein kinase C HL-60 cells were purchased from the American Type
Culture Collection (Rockville, MD). Fetal bovine serum was from HyClone (Logan, UT). L-Glutamine, penicillin/streptomycin, and
bicarbonate-free medium RPMI 1640 were from Life Technologies, Inc.
(Burlington, Ontario, Canada). Sephadex G-10 and protein A were
purchased from Pharmacia Biotech (Dorval, Québec, Canada).
Nonidet P-40, sodium orthovanadate (NaVO4), and all other
reagents were from Sigma. Catalase was purchased from Boehringer
Mannheim (Laval, Quebec, Canada). Molecular weight standards were
purchased from Bio-Rad (Mississauga, Ontario, Canada).
V4+-OOH was prepared essentially as described by Bourgoin
and Grinstein (25). The monoclonal anti-phosphotyrosine 4G10 (UB
05-321) antibody was obtained from UBI (Lake Placid, NY). Human PLD1
peptides (1-16, MSLKNEPRVNTSALQK; 144-162, RRQNVREEPREMPS; 967-981,
DDPSEDPIQDPVSDK; and 1027-1040, KEDPIRAEEELKKI) were synthesized by
HUKABEL (Ville St Laurent, Québec, Canada). Horseradish
peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG, and the enhanced
chemiluminescence (ECL) Western blotting system were obtained from
Amersham Corp. (Oakville, Ontario, Canada). Recombinant human PLD1
(rPLD1) and mouse PLD2 (rPLD2) were generous gifts from Drs. M. Frohman and A. Morris.
PLD1 antisera were raised against a
mixture of the PLD1 peptides coupled to keyhole limpet hemocyanin with
glutaraldehyde. Two rabbits were immunized each with 250 µg of the
four peptides.
HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum,
L-glutamine (2 mM), streptomycin (100 units/ml), and penicillin (100 µg/ml), and differentiation to
granulocytes was induced with 1.25% (v/v)
Me2SO4 as described (25). Differentiated cells
were harvested by centrifugation and resuspended in bicarbonate-free,
Hepes-buffered RPMI 1640 to be used for experiments.
HL-60 cell
suspensions (6 × 107 cells/ml) were either stimulated
with 100 µM V4+-OOH or treated with the same
volume of appropriate diluents. After selected times, cell suspensions
were mixed to an equal volume of non-denaturing lysis buffer (2×)
containing 50 mM Tris-HCl buffered to pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM
NaVO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin (final concentrations). The
lysates were incubated on ice for 15 min before centrifugation at
13,000 rpm for 15 min. The supernatant (2 × 107 cell
eq/ml) were incubated with 7 µl of the anti-PLD1 serum 04 and 0.5%
bovine serum albumin, 1.2% Nonidet P-40, 20 µg/ml aprotinin, and 20 µg/ml leupeptin for 3 h at 4 °C on a rotator platform. Where
indicated, the anti-PLD1 serum was neutralized with 10 µg/ml immunizing peptides for 2 h at 4 °C before use. This was
followed by an incubation with 20 µg of protein A-Sepharose beads for
1 h at 4 °C. The beads were washed three times with ice-cold
lysis buffer containing 1% Nonidet P-40 and boiled for 7 min at
100 °C in 2 × Laemmli's sample buffer. Immunoprecipitated
proteins were electrophoresed on 8% SDS-PAGE.
For immunoprecipitation under reducing conditions, the cell suspensions
(6 × 107 cells/ml) were mixed to an equal volume of
boiling denaturing buffer A (2×) containing 125 mM
Tris-HCl buffered to pH 6.8, 150 mM NaCl, 6% SDS, 5 mM NaVO4, 1 µM pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 4% Samples were
electrophoresed on 8% SDS-PAGE, proteins were transferred on Immobilon
PVDF membrane (Millipore Corp.), and WB was performed as described
(24). Membranes were incubated with anti-PLD1 serum 04 (1:2000) and
exposed to peroxidase-conjugated anti-rabbit IgG (1:20,000) for 1 h at 37 °C in blocking solution or using the 4G10
anti-phosphotyrosine (Tyr(P)) antibody (1:4000) as described previously
(23). The membranes were covered with ECL detection reagents.
Autoradiographs were obtained by exposing Kodak X-Omat film to
membranes.
We generated hPLD1 antipeptide antisera to examine the presence of
hPLD1 in human HL-60 cells. To minimize proteolysis, the cell lysates
were prepared by direct transfer of cell suspensions into boiling
sample buffer. The samples were electrophoresed, transferred to PVDF
membranes, and probed with the anti-PLD1 serum 03 and 04. rPLD1 (Fig.
1, A and B,
lane 1) and rPLD2 (Fig. 1, A and B,
lane 2) were added to the membrane samples before
electrophoresis and immunoblotting with the anti-PLD1 sera. As shown in
Fig. 1 the antibodies revealed a strong band at 120 kDa corresponding to rPLD1. rPLD2 was not recognized by the two anti-PLD1 sera. The
presence of two immunoreactive bands in HL-60 granulocytes at 115 kDa
and 120 kDa were consistently observed (lane 5). The immunoreactivity of the 120- and 115-kDa protein suggested the presence
of two closely related PLD1 enzymes in HL-60 cells. The specificity of
the two anti-PLD1 sera was demonstrated by the absence of the 120- and
115-kDa bands in immunoblots carried out with antigen-preneutralized
antibodies (data not shown). The two immunoreactive bands at 120 and
115 kDa were detected in the membrane (lane 3) and to a
lesser extent in the cytosolic (lane 4) compartments. Searches for hPLD1 isoforms revealed the presence of several closely related human PLD homologues (18, 19). DNA sequence analysis identified
a second PLD1-related protein lacking 38 amino acids in the middle of
the PLD1 sequence (amino acids 584-622). The 120- and 115-kDa PLD
variants were named PLD1a and PLD1b, respectively (21). PLD1a and PLD1b
are generated by alternative splicing of the PLD1 mRNA, and both
forms are similarly sensitive to activation by small GTPases and
protein kinase C
The inhibition of receptor-stimulated PLD activity by selective
inhibitors of tyrosine kinases is well documented (23). Increased PLD
activity was also detected in the anti-Tyr(P) immunoprecipitates of
neutrophil lysates previously primed with granulocyte-macrophage colony-stimulating factor and stimulated with fMLP
(formylmethionylleucylphenylalanine) (29). Furthermore, granulocyte PLD
activity was increased by a treatment with tyrosine phosphatase
inhibitors such as
V4+-OOH.2 The
molecular mechanisms that regulate these processes are, however, poorly understood as it remained unclear whether PLD itself, or one or
more of its cofactors, were subject to regulation by tyrosine phosphorylation. To assess the role of tyrosine phosphorylation in the
activation of PLD, we immunoprecipitated PLD using the anti-PLD1 serum
under nondenaturing and under denaturing conditions. HL-60 granulocytes
were incubated with V4+-OOH (100 µM) or an
equal volume of the diluent for the indicated times. The samples were
prepared by direct transfer of the cell suspensions to an equal volume
of ice-cold 2 × nondenaturing lysis buffer. The lysates were
incubated with the PLD1 antibodies (preneutralized or not with the
peptide antigens), and the immune complexes were precipitated by the
addition of protein A-Sepharose beads. The amounts of precipitated PLD1
and the presence of tyrosine-phosphorylated proteins in the
precipitates were monitored, following transfer to PVDF membranes, by
successive immunoblotting with anti-PLD1 and anti-Tyr(P) antibodies,
respectively. Although PLD1 could be immunoprecipitated by the
anti-PLD1 serum after the second immunization (Fig.
2A, lane 2)
immunoprecipitation of PLD1 was more efficient after the fifth
immunization (Fig. 2A, lanes 3-7). As shown in
Fig. 2A, two major bands corresponding to PLD1a and PLD1b
were detected at 120 and 115 kDa, respectively. The two forms of PLD1
were recovered in the supernatant when the immunoprecipitations were
carried out with a normal rabbit serum (Fig. 2A, lane
1) or with the preneutralized PLD1 antibodies (Fig. 2A,
lane 8). The membrane was then stripped and reprobed
with anti-Tyr(P) antibodies (Fig. 2B). A 120-kDa
tyrosine-phosphorylated protein was reproducibly observed in PLD1
immunoprecipitates. Several other tyrosine-phosphorylated peptides
of 160, 140, 135, 90, and 75-80 kDa were also detected in PLD1 immune
complexes. Phosphorylation of the 120-kDa band as well as the presence
of the other phosphorylated proteins in the PLD1 immune complexes was
time-dependent reaching near-maximal levels between 5 and
10 min after the addition of V4+-OOH. PLD1a, PLD1b, and the
associated tyrosine-phosphorylated proteins were not detected in the
immunoprecipitates carried out with a normal rabbit serum (Fig. 2,
A and B, lane 1) or with the preneutralized PLD1 antibodies (Fig. 2, A and B,
lane 8).
To examine whether the 120-kDa tyrosine-phosphorylated protein is PLD1
itself or an associated protein, cell lysis and immunoprecipitation with the anti-PLD1 serum were conducted under reducing conditions as
described under "Experimental Procedures." A representative blot
for PLD1 under these conditions is shown in Fig.
3A. Both PLD1a and PLD1b were
detected in the PLD1 immunoprecipitates. Similar amounts of PLD1a and
PLD1b were immuprecipitated from control (lane 1) and
V4+-OOH treated cells (lane 2). As compared with
unstimulated cells, a major 120-kDa tyrosine-phosphorylated protein was
observed in the immmunoprecitates derived from
V4+-OOH-stimulated cells (Fig. 3B, lane
2) but not from unstimulated granulocytes (Fig. 3B,
lane 1). Neutralization of the anti-PLD1 serum with the
immunizing peptides specifically eliminated PLD1a and PLD1b in the
immunoprecipitates (Fig. 3A, lane 3) as well as
the 120-kDa phosphorylated protein (Fig. 3B, lane
3). Tyrosine phosphorylation of PLD1b was hardly detectable
probably because of the small amounts of PLD1b in the PLD1
immunoprecipitates. These experiments were also performed using the
reverse protocol, immunoprecipitation with anti-Tyr(P) antibodies and
blotting with anti-PLD1 antibodies (Fig.
4). Because of the large amounts of tyrosine-phosphorylated proteins in lysates derived from
V4+-OOH-treated cells and the low abundance of the PLD1
enzymes, we conducted two rounds of immunoprecipitations. A first
immunoprecipitation was carried out with the anti-PLD1 serum under
nonreducing conditions because the efficiency of immunoprecipitation of
PLD1 was higher under these conditions (data not shown). The PLD1
immune complexes were collected and boiled in lysis buffer containing
SDS and
Biochemical studies have detected PLD activities in both the membrane
and the cytosolic fractions of HL-60 cells (11). The differential
regulation of the membrane-associated and of the cytosolic PLD activity
by Rho and ARF proteins suggested the presence of distinct PLD
isoforms. We analyzed the presence and the distribution of PLD1a and
PLD1b in the different cellular compartments of HL-60 granulocytes. In
these experiments the membranes and the cytosols were prepared (24).
PLD1 was immunoprecipitated from the lysates derived from 2 × 107 cell eq with the anti-PLD1 serum, and the amounts of
PLD1a and PLD1b in the immunoprecipitates were determined by blotting
with the anti-PLD1 antibodies. A representative blot from three
different experiments with similar results is shown in Fig.
5. Although PLD1a and PLD1b could be
detected in the immunoprecipitates of both membrane and cytosolic
compartments (Fig. 5A), the two PLD1 proteins were
predominantly recovered in the membrane fractions. A similar
distribution of PLD1a and PLD1b in the membrane fractions of
V4+-OOH-stimulated and nonstimulated HL-60 cells was
observed, excluding a redistribution of the proteins upon cell
stimulation. The same blot was subsequently probed for the presence of
tyrosine-phosphorylated proteins. As shown in Fig. 5B, no
tyrosine-phosphorylated protein could be detected in the PLD
immunoprecipitates from unstimulated HL-60 cells. In contrast,
stimulation with V4+-OOH resulted in an increase of several
tyrosine-phosphorylated proteins in cytosolic and membrane PLD
immunoprecipitates. In the cytosolic fractions, two bands at 135 and
~85 kDa were detected but we did not observe the presence of a
120-kDa tyrosine-phosphorylated protein. The protein could not be
detected by WB because of the small amount of PLD1 proteins in the
immunoprecipitates obtained from cytosolic protein fractions (Fig.
5A). In the membrane fractions, a major 120-kDa
phosphotyrosine protein was detected and was superimposable with PLD1
(Fig. 5, A and B). Several major unidentified
tyrosine-phosphorylated proteins in the 160-, 85-100-, and 75-80-kDa
regions were also recovered in the PLD1 immunoprecipitates. We also
immunoprecipitated the membrane-bound and cytosolic PLD1 under
denaturing conditions (28). A doublet at 120 kDa and 115 kDa in the
membrane fractions of V4+-OOH-stimulated or unstimulated
HL-60 cells was detected by the anti-PLD1 serum (Fig. 5C).
The cytosolic PLD1 was hardly detectable by WB (data not shown). In
this particular experiment the membrane-bound PLD1b was not clearly
detected in the immune complexes obtained from
V4+-OOH-treated cells but was present in two other similar
experiments (see Fig. 3 and data not shown). These same membranes were
probed for the presence of phosphotyrosine. In the membrane PLD1
immunoprecipitates, a 120-kDa tyrosine-phosphorylated band was detected
in cells stimulated with V4+-OOH. The
tyrosine-phosphorylated band was perfectly superimposable with PLD1a
and showed a similar shape (Fig. 5, C and D). The
data indicate that the membrane-bound PLD1a is tyrosine-phosphorylated. Because of the low amount of cytosolic PLD1 proteins, it was not possible using this protocol to determine whether or not they were
tyrosine-phosphorylated. Although these observations indicate that PLDs
with similar antigenic properties can exist as soluble and
membrane-associated proteins, the presence of additional PLD isoforms
not recognized by our PLD1 antibodies is not excluded.
In summary, the anti-peptide antibodies raised against PLD1 recognize
PLD1 but not PLD2. PLD1a and its spliced form PLD1b are expressed in
HL-60 granulocytes. Both enzymes are associated with the membrane
fractions, and only very small amounts of PLD1 are detected in the
cytosolic fractions. Stimulation of HL-60 cells with
V4+-OOH increased the phosphorylation of PLD1 and its
association with several tyrosine-phosphorylated proteins. This finding
is consistent with the hypothesis that PLD is complexed to several proteins in stimulated cells. PLD1 stimulation requires many cofactors for activation in vitro including the small GTPase RhoA and
ARF in vitro (21). The increased levels of PLD activity in
the membrane compartment is, at least in part, the result of the small
GTPase translocation to membranes upon cell activation with various
agonists (24), but the specific effect of protein tyrosine
phosphorylation on PLD functionality, biochemical properties, and
cofactor requirement is yet to be defined. The availability of
PLD1-selective antibodies should help resolve this issue.
Medicine and
§ Physiology,
, ARF, and RhoA.
In human granulocytes, a strong correlation between tyrosine
phosphorylation of proteins and PLD activity has been established. In
this study, the presence of hPLD1 in HL-60 granulocytes and its
phosphorylation on tyrosine residues have been studied. We generated
antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown
by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs
identified the presence of hPLD1 in HL-60 cells with the bulk of it
being detected in the membranes and only a minor fraction in the
cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a
minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two
forms of hPLD1 were consistently detected by immunoprecipitation under
nondenaturing and denaturing conditions following a WB analysis with
hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate
(V4+-OOH), an inhibitor of tyrosine phosphatases,
hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60
cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1
comigrated with a 120-kDa tyrosine phosphorylated protein by gel
electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes.
hPLD1 and the associated tyrosine-phosphorylated proteins were not
immunoprecipitated by neutralized hPLD1 Abs. Using denaturing
conditions, the PLD immunoprecipitates were sequentially immunoblotted
with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were
detected, and the major PLD1a protein was superimposable with a major
tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a
and PLD1b were recovered, at least in part, in the anti-phosphotyrosine
immunoprecipitates. These results provide evidence that two PLD1 forms
are expressed in human granulocytes. Furthermore, in response to
stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated
and associated with several, presently undefined,
tyrosine-phosphorylated proteins.
S dependence of PLD activation (4-8).
Reconstitution experiments using porated HL-60 cells or partially
purified PLD from HL-60 cells or brain tissues led to the discovery of
several regulatory proteins among which are the ADP-ribosylation factor
(5, 6) and the small GTPase RhoA (7, 8). Rac1 and Cdc42 are also
involved in the activation of PLD in liver and brain (8, 9). While the
synergistic activation by RhoA and ARF suggests convergence of
regulatory mechanisms on one single PLD isoform (8), additional
biochemical studies indicate the presence of distinct RhoA- and
ARF-regulated PLD isoforms in HL-60 cells and liver (10, 11). RhoA- and ARF-responsive PLD activities are observed in the plasma membrane, nuclei, Golgi, and endoplasmic reticulum (10, 12-15).
can also activate hPLD1 in a
kinase-independent mechanism independently of and synergistically with
the ARF and the Rho families of small GTPases (21, 22). In
granulocytes, receptor-stimulated PLD activity is regulated by tyrosine
phosphorylation of proteins (23). Protein kinase activities regulate,
at least in part, the recruitment and the membrane association of ARF
and RhoA, thereby potentiating the response to GTP
S in HL-60 cell
membranes (24). Furthermore, inhibitors of tyrosine phosphatases such as peroxides of vanadate (V4+-OOH), stimulate PLD activity
by G protein-independent (25) and -dependent mechanisms
(26). The present study was undertaken to examine the presence and
distribution as well as the potential tyrosine phosphorylation status
of hPLD1 in HL-60 granulocytes. We provide evidence that
V4+-OOH induces the tyrosine phosphorylation of hPLD1 and
stimulates its association with several tyrosine-phosphorylated
proteins.
Materials
-mercaptoethanol
(final concentrations) and incubated for 7 min at 100 °C. The
lysates were centrifuged at 12,000 rpm for 2 min at room temperature.
The supernatants were then filtered through Sephadex G-10 columns to
remove the denaturing agents (27). 0.1% Nonidet P-40, 20 µg/ml
aprotinin, 20 µg/ml leupeptin, and 5 µl of bovine serum albumin
(0.01% w/v) were added to the eluates which were precleared with
protein A-Sepharose and subsequently used for immunoprecipitation with
the anti-PLD1 serum 03 as described above. Cytosolic and membrane
fractions were prepared (24) and processed for immunoprecipitation of PLD1 as described previously by Grinstein and Furuya (28).
(21). It is likely that the two bands revealed by
the anti-PLD1 sera correspond to PLD1a and PLD1b.
Fig. 1.
Immunoblot of the PLD1 from HL-60 cells.
HL-60 cells (2 × 106 cells), cytosolic
(C), and membrane (M) proteins (100 µg) were electrophoresed on 8% SDS-PAGE, transferred to a membrane, and probed
with the anti-PLD1 serum 03 (A) and 04 (B). Where
indicated, rPLD1 or rPLD2 were added to the samples. The autoradiograms
were obtained after a 30-min exposure with the membrane. The blot
illustrated is representative of three similar experiments.
[View Larger Version of this Image (75K GIF file)]
Fig. 2.
Immunoprecipitation of PLD1 from HL-60
cells. Cell suspensions were stimulated in the absence or the
presence of 100 µM V4+-OOH (lanes
4-8) for the indicated times. Immunoprecipitations were performed
with the normal rabbit serum (NRS, lane 1), the anti-PLD1 serum obtained after two (lane 2) or five
immunizations (lanes 3-7), and with the peptide-neutralized
anti-PLD1 serum (lane 8). The PLD1 immunoprecipitates
(ip) and their supernatants (sup) were
electrophoresed on 8% SDS-PAGE and transferred to a membrane.
A, immunoblotting with the anti-PLD1 serum 04. B,
the membrane was stripped and probed with anti-Tyr(P) antibodies. One
experiment representative of three with similar results is presented.
[View Larger Version of this Image (41K GIF file)]
-mercaptoethanol. These manipulations would be expected to
disrupt most, if not all, protein-protein interactions. After removing
the denaturing agents (27), the tyrosine-phosphorylated proteins were
immunoprecipitated with agarose-conjugated anti-Tyr(P) antibodies, and
the amounts of PLD1 in the immunoprecipitates were assessed by WB. A
representative blot of these experiments is shown in Fig.
4A. The presence of PLD1a and PLD1b in the anti-Tyr(P)
immunoprecipitates was evident after stimulation with
V4+-OOH (lane 2) but not in unstimulated cells
(lane 1). Moreover, the PLD1 proteins were not detected in
the immune complexes when the first round of immunoprecipitation was
carried out with preneutralized anti-PLD1 serum (lane 3).
The same membrane was stripped and reprobed with anti-Tyr(P) antibodies
(Fig. 4B). A tyrosine-phosphorylated protein detected at 120 kDa in V4+-OOH-stimulated cells (Fig. 4B,
lane 2) was superimposable with PLD1a (Fig. 4A,
lane 2). In contrast, no tyrosine-phosphorylated proteins
could be detected in unstimulated cells (lane 1) or when immunoprecipitations were performed with neutralized anti-PLD1 serum
(lane 3). Taken together these results demonstrate that PLD1a and possibly PLD1b can be tyrosine-phosphorylated upon
stimulation of intact HL-60 cells with V4+-OOH.
Fig. 3.
Immunoprecipitation of a
tyrosine-phosphorylated PLD1. HL-60 cells were stimulated without
(lane 1) or with (lanes 2 and 3) 100 µM V4+-OOH for 10 min. PLD1 was
immunoprecipitated under denaturing conditions. Where indicated,
immunoprecipitation was performed with the peptide-neutralized
anti-PLD1 serum 03 (lane 3). The immune complexes were
electrophoresed on 8% SDS-PAGE, transferred to a membrane, and blotted
with the anti-PLD1 serum 04 (A) and the anti-Tyr(P)
antibodies (B). One experiment representative of three with
similar results is presented.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
Immunoprecipitation of PLD1 with
agarose-conjugated anti-Tyr(P) antibodies. HL-60 cells were
stimulated without (lane 1) or without (lanes 2 and 3) 100 µM V4+-OOH for 10 min.
PLD1 was immunoprecipitated under nondenaturing conditions as described
in Fig. 2. Where indicated, immunoprecipitation was performed with
neutralized anti-PLD1 serum 03 (lane 3). The PLD1 immune
complexes were subjected to a second round of immunoprecipitation with
the anti-Tyr(P) antibodies using denaturing conditions. The Tyr(P)
immune complexes were electrophoresed on 8% SDS-PAGE and transferred
to a membrane. The samples were analyzed for the presence of PLD1
(A) and of phosphotyrosine (B). One experiment
representative of two with similar results is presented.
[View Larger Version of this Image (32K GIF file)]
Fig. 5.
Subcellular distribution of PLD1 in control
and V4+-OOH-stimulated HL60 cells. Cytosol
(C) and membrane (M) were isolated from cells
stimulated with or without 100 µM V4+-OOH for
10 min. Membrane and cytosolic PLD were immunoprecipitated under
nondenaturing (A, B) or denaturing conditions
(C, D). Immune complexes were electrophoresed on
8% SDS-PAGE, transferred to a membrane, and probed with the anti-PLD1
serum 04 (A, C). The membranes were stripped and
probed with anti-Tyr(P) antibodies (B, D). One
experiment representative of three with similar results is
presented.
[View Larger Version of this Image (42K GIF file)]
*
This work was supported in part by grants and fellowships
from the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Arthritis Society of Canada.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: Centre de
Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Room T1-49, 2705 Boulevard Laurier, Ste-Foy, Québec, G1V 4G2, Canada. Tel.: 418-654-2772; Fax: 418-654-2765; E-mail: Sylvain.Bourgoin{at}crchul.ulaval.ca.
1
The abbreviations used are: PL, phospholipase;
hPL, human phospholipase; rPL, recombinant phospholipase; Abs,
antibodies; ARF, ADP-ribosylation factor; GTPS, guanosine
5
-3-O-(thio)triphosphate; PA, phosphatidic acid; Tyr(P),
phosphotyrosine; V4+-OOH, peroxides of vanadate; WB,
Western blotting; PAGE, polyacrylamide gel electrophoresis; PVDF,
polyvinylidene difluoride.
2
As described previously (25), in two independent
experiments the levels of the phosphatidylethanol in intact HL-60 cells increased from 0.011 ± 0.01% of tritium label in control cells to 0.955 ± 0.11% of tritium label in cells stimulated with 100 µM V4+-OOH for 10 min.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.