From the Department of Pharmacology,
§ Department of Cell Biology and Physiology, the
¶ Center for Biological Imaging of the University of Pittsburgh,
Pittsburgh, Pennsylvania 15261 and from the
Department of
Pharmacological Sciences and the Institute for Cell and Developmental
Biology, State University of New York, Stony Brook, New York 11794
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ABSTRACT |
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The primary known function of phospholipase D
(PLD) is to generate phosphatidic acid (PA) via the hydrolysis of
phosphatidylcholine. However, the functional role of PA is not well
understood. We report here evidence that links the activation of PLD by
insulin and the subsequent generation of PA to the activation of the
Raf-1-mitogen-activated protein kinase (MAPK) cascade. Brefeldin A
(BFA), an inhibitor of the activation of ADP-ribosylation factor
proteins, inhibited insulin-dependent production of PA and
MAPK phosphorylation. The addition of PA reversed the inhibition of
MAPK activation by BFA. Overexpression of a catalytically inactive
variant of PLD2, but not PLD1, blocked insulin-dependent
activation of PLD and phosphorylation of MAPK. Real time imaging
analysis showed that insulin induced Raf-1 translocation to cell
membranes by a process that was inhibited by BFA. PA addition reversed
the effects of BFA on Raf-1 translocation. However, PA did not activate
Raf-1 in vitro or in vivo, suggesting that the
primary function of PA is to enhance the recruitment of Raf-1 to the
plasma membrane where other factors may activate it. Finally, we found
that the recruitment of Raf-1 to the plasma membrane was transient, but
Raf-1 remained bound to endocytic vesicles.
Growth factor-mediated activation of
PLD1 has been well documented
and occurs in response to a broad class of mitogens, including insulin,
platelet-derived growth factor, epidermal growth factor, vasopressin,
and phorbol esters (1-4). Activation of PLD occurs through interaction
with the small G-proteins of the ADP-ribosylation factor (ARF) (5, 6)
and Rac/Rho families (7) as well as with protein kinase C (PKC) (8, 9).
The relative contribution of these factors to the activation of PLD is
highly dependent on the cell type and signaling model examined. For
example, stimulation of Rat-1 fibroblasts overexpressing the
human insulin receptor (HIRcB cells) with insulin activates PLD
exclusively through the ARF pathway (10), whereas the activation of PLD
by insulin in adipocytes appears to be primarily Rho-mediated
(11). Activation of PLD has been implicated in a wide variety of
intracellular and extracellular processes, including actin
polymerization, coatomer assembly, vesicle transport, neutrophil
activation, and platelet aggregation (12-16).
Activated PLD catalyzes the hydrolysis of phosphatidylcholine to
generate PA. However, the downstream consequences of PA generation are
not well understood. Although it is clear that the principal effects of
PA in some systems may be mediated by its conversion to diacylglycerol
(DAG) or lysophosphatidic acid (LPA), PA may also be a potent second
messenger. Several laboratories have identified putative targets for PA
in growth factor signal transduction, including a protein tyrosine
phosphatase (17), phospholipase C- Recently, Ghosh et al. (20) reported that PA interacts
directly with the serine-threonine kinase Raf-1, an important component of the MAPK signaling cascade. Here we report that the stimulation of
the MAPK pathway by insulin is dependent on PLD activation, and this
effect is mediated through the induction of Raf-1 translocation to the
plasma membrane by PA. Furthermore, overexpression of a catalytically
inactive variant of PLD2, but not PLD1, blocks insulin-induced phosphorylation of MAPK. We also show that PA is required for complete
activation of Raf-1 in response to insulin. However, PA alone cannot
activate Raf kinase in vivo, does not have any effect on Raf
kinase activity in vitro, and has no effects on the MAPK
cascade. We also show that PA induces Raf-1 translocation to the plasma
membrane and that the generation of PA is essential for the induction
of Raf-1 translocation by insulin. PA also induced Raf-1 translocation
to the plasma membrane in Ha-Ras(Q61L)-transformed Rat-1 fibroblasts,
suggesting that PA may act concurrently with activated Ras in
stimulating Raf-1 translocation. Raf-1 was found associated with
intracellular vesicles containing the insulin receptor and clathrin
after stimulation with insulin. Furthermore, Raf-1 association to
endocytic vesicles was dependent on the generation of PA, suggesting a
model in which Raf-1 migrates along with endocytic vesicles during
receptor-mediated endocytosis via its interaction with PA.
Cell Culture--
Rat-1 fibroblasts overexpressing the human
insulin receptors (HIRcB cells) were grown in Dulbecco's modified
Eagle's medium/Ham's F-12 (1:1) supplemented with 10% fetal bovine
serum and 100 nM methotrexate. Cells were grown to 80%
confluency and serum-starved for at least 18 h prior to
stimulation. Cells were treated with 200 nM insulin and/or
200 µM PA for 10 min. When indicated, cells were
pretreated with brefeldin A (BFA) for 10 min prior to stimulation with
insulin or PA. Ha-Ras(Q61L)-transformed Rat-1 fibroblasts were treated
as described above.
Phospholipase D Activity--
HIRcB cells were labeled with
[3H]palmitate (5 µCi/ml) overnight and treated with
insulin as described above. The reaction was stopped after 1, 2, 5, 10, 15, and 20 min of insulin stimulation by the addition of cold PBS.
Cells were then scraped in cold PBS and pelleted by centrifugation.
Lipids were then extracted with chloroform/methanol (1:1). The lipid
phase was collected and developed by thin layer chromatography (TLC) on
silica gel 60 plates using ethyl acetate/trimethylpentane/acetic acid
(9:5:2) as the solvent. The position of PA was measured by
autoradiography and by the position of lipid standards (Avanti Polar
Lipids). Lipids were then scraped from the TLC plates and counted via
liquid scintillation. In other assays, cells were stimulated with
insulin in the presence of 0.3% butanol for 20 min to determine the
total activity of PLD by the standard transphosphatidylation assay
described by Shome et al. (10). Levels of PA or
phosphatidylbutanol were normalized to total fatty acid label
incorporated into lipid.
Assessment of MAPK Phosphorylation--
HIRcB cells were treated
as described above or as described in the figure legends. Cells were
washed with cold PBS, scraped into microcentrifuge tubes in Buffer A
(10 mM Hepes, pH 7.4, 2 mM EDTA, 1 mM Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride), and centrifuged. Cells were resuspended
in a 0.5-ml detergent lysis buffer (50 mM Hepes, pH 7.4, 0.1 M NaCl, 1.5% sodium cholate, 1 mM EDTA, 1 mM EGTA, 5 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 1 mg/ml soybean trypsin inhibitor)
and lysed for 30 min at 4 °C. Cell lysates were then boiled in SDS
sample buffer, and equal amounts of protein were resolved by SDS-PAGE
followed by transfer to a nitrocellulose membrane. The nitrocellulose
membrane was then probed with a phospho-specific anti-MAPK antibody
(New England Biolabs). Immunocomplexes were detected by enhanced chemiluminescence.
Phospholipase D Mutants--
Wild-type and catalytically
inactive variants of PLD1 and PLD2 (K898R-PLD1 and K758R-PLD2) were
made as described previously (21) and fused to Aequorea
victoria green fluorescent protein by subcloning into
pEFGP-C1 (CLONTECH). The enzymatic activity of the
GFP-wild-type enzyme chimeras expressed in COS-7 cells was determined
in vitro to confirm that the GFP tag did not generate an
inactive phenotype.2 The
structure of the chimeras was verified directly by sequencing. Cells
were transfected using LipofectAMINE transfection reagent. Transfection
efficiency was assessed by conventional epifluorescence microscopy
prior to experimentation using filters appropriate for the detection of GFP.
Construction of Raf-GFP--
A pUC13 expression vector encoding
the human Raf-1 cDNA was a gift from Dr. Said Sebti. The human
Raf-1 cDNA was amplified by polymerase chain reaction and subcloned
into pEGFP-N1 (CLONTECH) using the EcoRI
and BamHI restriction sites, successfully fusing GFP to the
C terminus of Raf-1. Escherichia coli (DH5- Raf Kinase Assay--
HIRcB cells were transfected with
Raf-1-GFP and treated as described above. After stimulation, the cells
were washed with ice-cold PBS, scraped in 0.5 ml of Buffer A, and
centrifuged. Cell pellets were then resuspended in 0.5 ml of detergent
lysis buffer and lysed for 30 min on ice. 3 µl of monoclonal anti-GFP antibody (CLONTECH) were conjugated to 30 µl of
agarose-anti-mouse IgG (Sigma) for 1 h at room temperature in
detergent lysis buffer and washed twice. Cells lysates were incubated
with agarose-conjugated antibody for 3 h at 4 °C with
end-over-end rotation. Immunocomplexes were pelleted (10 min at
10,000 × g) and washed 3 times with detergent lysis
buffer and 2 times with kinase buffer (50 mM Tris, pH 7.3, 150 mM NaCl, 12 mM MnCl2, 1 mM dithiothreitol, 0.2% Tween 20). The activity of Raf-1
kinase present in Raf-GFP immunoprecipitates was determined using a
standard procedure (22-24). To test whether PA activated Raf-GFP
in vitro, 200 µM PA was included in the
reaction buffer where indicated. Following the kinase assay,
immunocomplexes were boiled in SDS sample buffer and resolved on a 10%
acrylamide gel followed by transfer to a nitrocellulose membrane.
Membranes were probed with an anti-Raf-1 antibody (Transduction
Laboratories) and resolved via an appropriate horseradish
peroxidase-conjugated secondary antibody and ECL. Kinase activity was
normalized to the relative amounts of Raf-GFP in the immunoprecipitate
as determined by densitometry.
Subcellular Fractionation--
HIRcB cells were transfected with
the plasmid encoding Raf-GFP using LipofectAMINE (Life Technologies,
Inc.). Cells were stimulated as described above and were scraped in 0.5 ml of Buffer A, pelleted by centrifugation, and resuspended in Buffer B
(Buffer A + 50 mM NaF, 1 µg/ml aprotinin, and 10 µg/ml
leupeptin). Cells were lysed by sonication with 3 series of 5 × 5-s bursts using a microtip sonicator. The nuclei and cell debris were
subsequently pelleted (3,000 RPM, 10 min, 4 °C). The remaining
supernatant fraction was spun at 100,000 × g for 80 min at 4 °C to separate membrane and cytosolic fractions. Membrane
pellets were solubilized in Buffer B containing 1% Triton X-100 and
incubated for 30 min at 4 °C. Supernatant and pellet fractions were
boiled in Laemmli sample buffer, and equal amounts of protein were run
on a 10% acrylamide gel followed by transfer to nitrocellulose.
Membranes were probed with an anti-Raf-1 antibody (Transduction
Laboratories). Immunocomplexes were then detected chemiluminescence.
Densitometry was performed as described above.
Fluorescence Microscopy--
HIRcB cells or Rat-1 fibroblasts
transformed with Ha-Ras(Q61L) (25) were plated on
poly-L-lysine-coated glass coverslips and transfected with
the plasmid encoding Raf-GFP using Superfect Transfection Reagent
(Qiagen) as per the manufacturer's instructions. Cells were stimulated
as described above. Live cells were imaged at 37 °C using a
Molecular Dynamics 2001 laser scanning confocal microscope equipped
with a 60× oil immersion objective and a microperfusion incubator
(Medical Systems Corp.) attached to the microscope stage. Samples were
excited at a wavelength of 488 nm, and emitted light was filtered
through a 530DF30 double bandpass filter prior to detection with a
photomultiplier tube. Quantitation of fluorescence intensity was
performed with Molecular Dynamics ImageSpace software.
Internalization Assay--
HIRcB cells were stimulated as
described above and treated as described in Shome et al.
(26). Briefly, stimulated cells were washed with PBS and Dulbecco's
modified Eagle's medium, 0.1% bovine serum albumin (pH 4.0) at
4 °C and incubated with 1 mg/ml trypsin in PBS for 30 min at 4 °C
to cleave the Immunoisolation of Vesicles--
Cells were treated as described
above with BFA, insulin, and/or PA. After stimulation, cells were
placed on ice and washed with cold PBS. Cells were scraped into Buffer
A and pelleted. Cells obtained from two 100-mm dishes were combined and
resuspended in 1 ml of Buffer C (Buffer A + 5% sucrose, 1 µg/ml
aprotinin, and 10 µg/ml leupeptin) and homogenized with 4 passes
through a ball homogenizer as described by Martin (28). Unbroken cells and nuclei were pelleted by centrifugation at 3000 rpm for 10 min
(4 °C). Equal amounts of protein were layered on a 35% sucrose solution (in Buffer A) and centrifuged for 90 min at 150,000 × g (4 °C) in an SW55 TI swing bucket rotor. Following
centrifugation, the vesicle fraction was collected at the interface of
the two layers. Antibody CT-1 was conjugated to agarose-anti-mouse IgG as described above and incubated with the vesicle fractions in order to
isolate internalized vesicles containing the insulin receptor.
Following immunoprecipitation for 3 h at 4 °C,
immunoprecipitates were washed 3× in cold Buffer C and 2× in cold
PBS. The proteins of interest (Clathrin, Raf-1, and the insulin
receptor) were resolved via SDS-PAGE and detected with specific antibodies.
Cell Preparation for Immunoelectron Microscopy--
Cells were
grown to confluence in 100-mm dishes, treated as described above with
insulin and PA, and immediately fixed for 1 h in 2%
paraformaldehyde, 0.01% glutaraldehyde in PBS. The cells were scraped
from the Petri dish, spun, and resuspended in 2% gelatin (300 bloom),
fixed for 10 min in the same fix as above, and cryoprotected overnight
in 2.3 M sucrose in 0.1 M PBS. Subsequently, the cell pellet was diced into 1 mm3, mounted on cutting
stubs, shock-frozen, and stored in liquid nitrogen. Thin sections
(70-100 nm) were cut using a Reichert ultracut S Ultramicrotome with a
FC4S cryo-attachment and lifted in a small drop of sucrose and mounted
on Formvar-coated carbon grids. The sections were washed three times in
PBS containing 0.5% bovine serum albumin and 0.15% glycine, pH 7.4 (Buffer D), followed by a 30-min incubation with purified goat IgG (50 mg/ml) at 25 °C and three additional washes with buffer. All the
preceding steps are designed to ensure minimal nonspecific reaction to
the antibodies used. Sections were then incubated for 60 min in the first primary antibody, a murine IgG1 directed against
Raf-1 (Transduction Laboratories) followed by three washes and a 60-min
incubation with 5 nm of gold-labeled protein G (0.1-2 µg/ml). The
sections were then washed six times (5 min/wash), and the second
primary antibody, a rabbit polyclonal IgG directed against the
The Generation of PA by PLD Is Required for Receptor-induced MAPK
Phosphorylation--
In order to investigate the functional role of
PLD activation in insulin signaling, we used a Rat-1 fibroblast cell
line that overexpresses the human insulin receptor (HIRcB cells).
Insulin-induced activation of PLD in these cells appears to be
exclusively dependent on ARF activation (10). We have shown that BFA,
an inhibitor of ARF activation (29-31), blocks completely
insulin-dependent PLD activation in these cells (10).
Fig. 1A shows the effects of
insulin on the generation of PA by HIRcB cells. Also shown in the
figure is the blockade of the generation of PA by treatment with BFA.
To assess the physiological relevance of insulin-induced PA production,
we examined its effect on the MAPK signaling pathway. Activation of
this cascade is characterized by a series of phosphorylation events,
one of which is the phosphorylation of MAPK. We therefore used an
antibody specific for phosphorylated MAPK as a marker for the
activation of this signaling cascade (Fig. 1B). As shown,
insulin induced the phosphorylation of MAPK. The effect of insulin was
inhibited by pretreatment with BFA at doses consistent with the
inhibition of PLD activity (Fig. 1A (10)). Addition of PA
had no effects on the phosphorylation of MAPK. However, the addition of
PA reversed the inhibitory effects of BFA on insulin-induced MAPK
phosphorylation.
The role of PLD in the insulin-induced phosphorylation of MAPK was
further examined using cells that had been transiently transfected with
catalytically inactive mutants of PLD1 and PLD2. These variants contain
a conservative Lys to Arg mutation in the active site, effectively
disrupting the ability of PLD to hydrolyze phosphatidylcholine. These
mutants have been shown to be devoid of activity in vivo and
in vitro using PC as a substrate and to localize similarly
to the wild-type proteins (21). In these assays, GFP-PLD1 and GFP-PLD2
constructs were used to transfect HIRcB cells. These chimeric proteins
were used instead of the original proteins because the expression of
the GFP constructs facilitated significantly the analysis of the
transfection efficiency. Thus, we verified the expression of these
constructs by conventional epifluorescence analysis prior to
experimentation. A transfection efficiency of approximately 60% was
obtained for each of the constructs (data not shown). As shown in Fig.
2A, overexpression of
GFP-tagged constructs of the catalytically inactive K758R-PLD2 mutant
in HIRcB blocked insulin-induced PLD activity, demonstrating the ability of this mutant to act as a dominant negative. Furthermore, expression of the K758R-PLD2 also blocked insulin-induced MAPK activation (Fig. 2B). In contrast, overexpression of the
catalytically inactive K898R-PLD1 mutant failed to inhibit
insulin-dependent PLD activity and insulin-induced MAPK
phosphorylation in HIRcB cells (Fig. 2, A and B).
This demonstrates that, in HIRcB cells, insulin signals preferentially
through PLD2 and that a functional PLD2 is required for insulin-induced
activation of MAPK. Taken altogether, these data lead us to conclude
that the generation of PA by PLD2 is essential, but not sufficient, to
mediate the effects of insulin on the MAPK signaling cascade.
PA Induces Raf-GFP Translocation, but Does Not Activate Raf Kinase
Activity--
It has been shown that Raf-1 binds PA in
vitro (20). Therefore, one putative site of action for PA in the
MAPK signaling cascade is the activation of Raf-1. To assess the role
of PA on the regulation of Raf-1 activity, we constructed a Raf-GFP
fusion protein and transiently transfected HIRcB cells. In order to
demonstrate that the fusion protein behaved as the native kinase,
enzymatic assays were performed on materials immunoprecipitated with an antibody specific for the GFP epitope. Treatment of HIRcB cells with
insulin induced a 2-fold activation of Raf kinase activity, whereas
treatment with PA did not have statistically significant effects on Raf
kinase activity (Fig. 3A). As
predicted by our hypothesis, the activation of Raf by insulin was
inhibited by BFA. This inhibition was reversed by the addition of PA,
suggesting that the generation of PA by PLD is necessary for Raf-1
activation and that PA alone cannot activate Raf-1. Because Ghosh
et al. (20) showed a direct interaction between Raf-1 and
PA, we tested the ability of PA to activate directly immunoprecipitated
Raf-GFP. As shown, PA was unable to stimulate Raf kinase activity in
immunoprecipitated Raf-GFP. Interestingly, some endogenous Raf-1
co-immunoprecipitated with Raf-GFP, suggesting that Raf-GFP can
oligomerize with endogenous Raf-1 in vivo (data not shown).
This supports the idea that the Raf-GFP construct is physiologically
active.
The effects of anionic phospholipid-protein interactions, such as those
between phosphatidylinositol 4,5-bisphosphate (PIP2) and
proteins containing pleckstrin homology domains, have been shown to
stimulate the recruitment of proteins to cellular membranes with or
without stimulation of catalytic activity. Since activation of Raf-1
in vivo is highly dependent upon the translocation of Raf-1
to the plasma membrane, we examined the relationship between PA-induced
Raf-1 translocation and the activation of Raf-1. In these experiments,
we fractionated cells expressing GFP-tagged Raf-1 by centrifugation and
separated cytosolic (supernatant) and membrane (pellet) fractions. PA
alone was sufficient to induce Raf-GFP translocation from the
supernatant to the pellet fraction (Fig. 3B).
Insulin-induced Raf-GFP translocation was inhibited by BFA in a
dose-dependent manner consistent with the inhibition of
PLD. Addition of PA to cells treated with BFA and insulin restored Raf-GFP translocation into the pellet fraction, efficiently reversing the effects of BFA. PA was also found to be at least as efficient as
insulin in the recruitment of Raf-1 to cell membranes. In contrast, as
shown above, PA failed to stimulate Raf kinase activity. This suggests
the hypothesis that the recruitment of Raf to cell membranes is
essential but not sufficient to completely activate the kinase.
Live Cell Dynamics of Raf-GFP Translocation--
Raf-GFP
translocation in live cells was studied using a Molecular Dynamics
confocal microscope equipped with a microperfusion incubator to
maintain a constant temperature of 37 °C. Confocal sections of the
plasma membrane adjacent to the coverslip in HIRcB cells expressing
Raf-GFP were taken in order to track Raf-GFP translocation to the
plasma membrane. Cells were imaged every 1 min following the addition
of insulin or PA. Representative plasma membrane sections of a cell
stimulated with insulin are shown in Fig.
4A. Total intensity of the
section was then quantitated using Molecular Dynamics ImageSpace
software, and the results were normalized for background intensity.
Insulin induced a transient Raf-1 translocation to the plasma membrane
peaking at approximately 10 min (Fig. 4B).
Pretreatment of HIRcB cells with BFA did not alter endogenous levels of
membrane-bound Raf-GFP but blocked insulin-induced Raf-GFP
translocation (Fig. 4C, black line). However, the
addition of PA restored Raf-1 translocation to cells treated with BFA
and stimulated with insulin. Furthermore, Raf-GFP translocation was dependent on the addition of PA. When PA was included along with insulin, Raf-GFP translocation occurred immediately (Fig.
4C, gray line). PA alone was also found to induce
Raf-1 translocation, and this effect was insensitive to the addition of
BFA (data not shown).
PA Induces Raf-GFP Translocation in Ras-transformed
Cells--
Raf-1 translocation to the plasma membrane has been
previously shown to be stimulated by the activation of the small
G-protein Ras. Ras is constitutively bound to the plasma membrane and
binds Raf-1 upon the exchange of GDP for GTP. In order to examine
whether PA-induced translocation of Raf-1 can occur in the presence of activated Ras, we imaged confocal plasma membrane sections in Rat-1
cells transformed with the GTPase-deficient (constitutively active)
Ha-Ras(Q61L) (25). PA was found to induce rapid and transient Raf-GFP
in these cells (Fig. 5). Levels of
Raf-GFP at the plasma membrane were sustained until approximately 8 min
and decreased to control levels by approximately 12 min after
stimulation with PA. Thus, PA also stimulated the transient recruitment
of Raf-1 to the plasma membrane in Ras-transformed
cells.
Insulin and PA Induce Raf-1 Translocation to Endocytic
Vesicles--
In mid-cell confocal sections of insulin and PA-treated
HIRcB cells, it was noted that Raf-1 accumulated in vesicular
intracellular structures in addition to the plasma membrane.
Interestingly, BFA did not inhibit PA-induced translocation to the
plasma membrane or intracellular structures, whereas the effects of
insulin were sensitive to BFA. PA has been implicated in the generation
of transport vesicles from the endoplasmic reticulum and
Golgi (12, 32), and recent work by Chung et al.
(33) has further suggested that PLD-generated PA promotes formation of
endocytic vesicles and vesicle coat assembly via a positive feedback
mechanism with PIP2 biosynthesis. These findings suggested
to us the hypothesis that the generation of PA is important for
receptor-mediated endocytosis and formation of endocytic vesicles and
that Raf-1 leaves the plasma membrane while still bound to an endocytic
vesicle via its association with PA.
To study the relationship between PA production and endocytosis, we
first examined the effects of BFA on the internalization of the insulin
receptor. The internalization of the insulin receptor was measured
using a limited trypsin proteolysis assay described previously (26).
Pretreatment of cells with BFA blocked insulin-induced internalization
of the insulin (Fig. 6A). This
is consistent with a role of ARF proteins and PA production in the
formation of the endocytic vesicle (33). To confirm that the
internalization of the insulin receptor proceeded via the classic
endocytic pathway, we immunoisolated vesicles using a specific antibody
that recognizes the C terminus of the
Ultrastructural data using dual staining electron microscopy against
the insulin receptor and Raf-1 epitopes in insulin- and PA-treated
HIRcB cells were obtained to confirm the co-localization of Raf-1 and
the insulin receptor in endocytic vesicles. Both insulin (Fig.
7, B and C) and PA
(Fig. 7D) induced co-localization of Raf-1 with the insulin
receptor in endosomes, indicating that Raf-1 migrates along with
endocytic vesicles in response to stimulation with insulin or PA.
Therefore, both the internalization of the insulin receptor and the
recruitment of Raf-1 to the plasma membrane are dependent on the
generation of PA. Furthermore, PA alone can induce the internalization
of insulin receptors and recruitment of Raf-1 to vesicles containing
insulin receptors, suggesting that Raf-1 is bound to the membranes of
endocytic vesicles via its association with PA.
The activation of Raf-1 kinase activity by growth factors requires
its translocation from the cytoplasm to the plasma membrane where it is
activated through a complex mechanism which includes the interaction
with the GTP-bound form of Ras, and possibly phosphorylation by PKC,
and tyrosine kinases (34-36). Whereas the precise nature of the events
occurring at the plasma membrane remains unresolved, it is clear that
the translocation of Raf-1 is crucial. Targeting Raf-1 to the plasma
membrane by attaching a protein prenylation motif to the C terminus of
Raf-1 is sufficient for activation of the kinase (37), whereas trapping
Raf-1 in the cytoplasm with cytosolic Ras prevents activation (22).
However, translocation itself does not bring about the full activation
of Raf-1. Mineo et al. (38) used a mutant Ras protein that
is deficient in binding to wild-type Raf-1, but binds Raf-1(257L), to
show that the interaction between Ras and Raf-1 stimulates Raf-1 kinase
activity 3-fold better than targeting Raf-1 to the membrane alone.
Furthermore, Roy et al. (39) showed that recruitment of
Raf-1 to the plasma membrane by Ras was not sufficient for full
activation of Raf-1 and that a second interaction between the
cysteine-rich domain (CRD) on Raf-1 and the GTP-bound form of Ras was
necessary for full activation. They also showed that deletion of the
CRD from membrane-targeted Raf-1 abrogated Raf-1 kinase activity,
suggesting that plasma membrane localization of Raf-1 by itself is
insufficient for activation of Raf-1 but that a second regulatory event
affecting the CRD must occur for Raf-1 activation. Thus, Raf-1
translocation and Raf-1 kinase activation are closely related but
distinct phenomena.
It has been assumed for some time that the interactions of Raf-1 with
Ras are the primary mechanism driving the recruitment of Raf-1 to the
cell membrane. Recently, other mechanisms that may play an important
role in the recruitment of Raf-1 to the membrane have been
investigated. For instance, Ghosh et al. (20) have explored
the interactions of Raf-1 with phosphatidylserine and PA in
vitro. Phosphatidylserine appears to bind to the cysteine-rich domain (CRD) of Raf-1, which is analogous to the zinc finger on PKC.
Luo et al. (40) replaced the Raf-1 CRD with the analogous zinc finger domain found on PKC and found that DAG activated this chimera independently of Ras activation, demonstrating that interaction of an effector with the CRD is critical in the activation of Raf-1. Other effectors, such as ceramide (41) and Rap1A (42), interact with
Raf-1 at this site and consequently have effects on its activation. The
PA-binding site proposed by Ghosh et al. (20) does not lie in this crucial lipid binding regulatory domain on Raf-1 but on a
second lipid-binding site near the catalytic domain of Raf-1. The
influence of effector binding at this site on Raf-1 kinase activity, if
any, has not been fully characterized at the present time. It has also
been shown that inhibition of PLD-mediated generation of PA with
ethanol inhibited phorbol ester-induced Raf-1 translocation to cell
membranes (20). This suggests that the generation of PA by a
receptor-sensitive PLD may play an important role in the recruitment
and/or activation of Raf-1 kinase.
The data reported here strongly support this view. By taking advantage
of the fact that insulin-dependent PLD activation in HIRcB
cells is mediated by ARF proteins in a BFA-sensitive manner (10), we
have shown that the blockade of PLD-dependent generation of
PA disrupts the activation of Raf-1, the translocation of Raf-1 to
membranes, and the phosphorylation of MAPK. We also demonstrated that
overexpression of a catalytically inactive variant of PLD2 blocks
insulin-induced activation of PLD and MAPK phosphorylation. In
consistency with the hypothesis that the effects of BFA are a
consequence of the blockade of the generation of PA by
receptor-sensitive PLD, we have also shown that all the effects of BFA
can be reversed by the addition of exogenous PA. However, our data show
that PA alone cannot activate MAPK phosphorylation in live cells and
that it cannot activate Raf-1 in vitro or in cultured cells.
Taking all these data together, we conclude that the generation of PA is required but not sufficient for the activation of Raf-1 by insulin.
On the other hand, we show here that PA is sufficient for induction of
Raf-1 translocation and reverses the blockade of insulin-induced Raf-1
translocation by BFA to intracellular vesicles. These facts suggest a
model in which PA directly facilitates Raf-1 translocation but does not
activate the kinase and is insufficient to completely stimulate Raf-1
kinase activity in intact cells. Furthermore, we show that PA induces
Raf-1 translocation in Ha-Ras(Q61L)-transformed cells, suggesting that
PA and Ras may act concurrently and by parallel pathways in stimulating
Raf-1 translocation to the plasma membrane. We therefore conclude that
the main role of PA in the activation of the MAPK cascade is the
induction of Raf-1 translocation to the cell membrane.
Much of the attention on receptor-sensitive PLD has focused on PLD1,
primarily because recombinant PLD1 may be activated by ARF, Rho, and
PKC Our data do not rule out the possibility that PA-stimulated Raf-1
translocation is mediated by metabolites of PA, specifically DAG or
LPA. However, we do not believe this to be the case. DAG-mediated activation of PKC results in potent activation of Raf-1 (48), and thus
a significant conversion of PA to DAG would result in MAPK activation
as well as potent activation of Raf-1. Since treatment of cells with PA
alone failed to activate either Raf-1 or MAPK, we conclude that the
generation of DAG does not play a significant role in the mechanism by
which PA modulates the MAPK cascade. Likewise, a significant
accumulation of LPA would also elicit a strong activation of the MAPK
cascade (49). Therefore, conversion of PA to either LPA or DAG should
have dramatic effects on the Raf-1-MAPK signaling cascade. Since these
effects were not seen after the addition of PA in our experiments, it
is likely that the effects of PA on Raf-1 translocation are due to a
direct interaction between PA and Raf-1 and not a consequence of its
conversion to other lipid second messengers.
Previously, immunocytochemical characterization of Raf-1 translocation
has been limited to a few studies in which Raf-1 was microinjected into
Ras-transformed cells (37, 50, 51). However, little work has been done
to characterize growth factor-induced Raf-1 translocation. Here, we
have studied the dynamics of insulin-induced Raf-1 translocation to the
plasma membrane in live cells. Our data demonstrate that Raf-GFP
undergoes growth factor-induced kinase activation and translocation,
indicating that Raf-GFP is an appropriate model for growth
factor-induced Raf-1 dynamics. In order to assess translocation of
Raf-GFP to the plasma membrane in live cells, we imaged a confocal
section of the plasma membrane adjacent to the cover glass. To select
this section, several images along the z axis of the cell
were collected. Because Raf-1 does not enter the nucleus, the plasma
membrane sections adjacent to the coverslip were identified by choosing
a confocal section below the nucleus. These sections were imaged at
37 °C in order to examine the kinetics of Raf translocation in
response to insulin or PA stimulation.
By using this experimental approach, we show here that the association
of Raf-1 to the plasma membrane is transient. However, Raf-1 does not
simply dissociate from the plasma membrane. Dual staining immunogold
labeling for the insulin receptor and Raf-1 resolved by electron
microscopy shows that Raf-1 co-localized with the insulin receptor in
intracellular vesicular structures in response to stimulation with both
insulin and PA. The structure of these vesicles suggests that they are
endosomes. To confirm that Raf-1 binds endocytic vesicles, cells were
treated with insulin, and vesicles containing the insulin receptor were
isolated using a specific anti-insulin receptor antibody. Both Raf-1
and the heavy chain of clathrin were present in these preparations.
Consistent with our model, the association between Raf-1 and the
isolated vesicles also appeared to be dependent on the presence of PA. This conclusion is based on the observation that BFA blocked the internalization of the insulin receptor and abolished the localization of Raf-1 in the immunoisolated endocytic vesicles. Therefore, we
propose that PA is required for endocytosis of the insulin receptor and
that the association between Raf-1 and endosomes is mediated by a
direct interaction between PA and Raf-1. This is consistent with
current models of vesicle formation mediated by acidic phospholipids
(32, 33, 52-55) which suggest that PA may form an integral part of
newly formed vesicles. These models suggest that PLD-mediated
generation of PA, through participation in a positive feedback loop
concurrently with the generation of PIP2, may sufficiently
perturb membrane structure and facilitate the formation of a vesicle
from a planar membrane. Consequently, the membranes of newly formed
vesicles are enriched with the acidic phospholipids PA and
PIP2. This acidic surface may serve as a binding matrix for
a number of signaling molecules such as Raf-1.
Recent work by Daaka et al. (47) also supports this model.
By using dominant suppressor mutants of Fig. 8 depicts the proposed role of
insulin-induced generation of PA. According to this model, PA has the
following two functions: 1) the recruitment of Raf-1 to the plasma
membrane where it is activated by factors which reside on the plasma
membrane, such as activated Ras and PKC, and 2) facilitation of
endocytic vesicle formation. These two effects, acting in conjunction,
may result in the recruitment of many important components of signal
transduction to the plasma membrane and to the membranes of endocytic
vesicles. Among these components are receptor tyrosine kinases, Raf-1
through its association with PA, and proteins that associate with
PIP2 through pleckstrin homology domains. Our work and that
of Daaka et al. (47) further suggest that the
internalization of these signaling components is necessary for full
activation of the MAPK signaling cascade, probably by bringing Raf-1 in
contact with downstream targets such as MEK. In this paper, we
demonstrate that this phenomenon is mediated by phosphatidic acid.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
(18), and Ras-GAP (19). However,
the physiological relevance of these interactions has not been established.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
) were transformed with the ligated vector and selected using
kanamycin-containing LB agar plates. Plasmid DNA was obtained from
transformed bacteria. The presence of the fusion construct was verified
by digestion with restriction enzymes and direct sequencing.
-subunit of insulin receptors on the cell surface.
Trypsin treatment was stopped with buffer containing 0.4% Triton
X-100, 0.5 mg/ml bacitracin, 25 mg/ml soybean trypsin inhibitor, and
100 µg/ml leupeptin and solubilized for 30 min at 4 °C. Antibody
83.7, a monoclonal antibody raised against the
-subunit of the
insulin receptor (27), was conjugated to agarose-anti-mouse IgG as
described above and incubated with cell lysates and used to
immunoprecipitate internalized (i.e. intact, not exposed to
trypsin hydrolysis) insulin receptors as described by Shome et
al. (26). Immunoprecipitates were extensively washed as described
above, resolved by SDS-PAGE, and immunoblotted with CT-1, a monoclonal
antibody that recognizes the
-subunit of the insulin receptor
(27).
-subunit of the insulin receptor (Santa Cruz Biotechnology), was
applied and followed by a similar washing and labeling strategy as
above, although detection was with a 10-nm protein A-gold conjugate. Finally, sections were washed thoroughly in Buffer D (5 changes) and in
PBS (3 changes), followed by a brief fixation step in 2.5% glutaraldehyde in PBS. Subsequent steps were 3 further washes in PBS, 5 washes in water, counterstaining with uranyl acetate and embedment in
1.25% methylcellulose. Observation was with a Jeol 1210CX electron microscope.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
The effects of BFA on insulin-induced PA
generation and MAPK phosphorylation. A, HIRcB cells
were labeled with [3H]palmitate and stimulated with 200 nM insulin ( ) or pretreated with 50 µg/ml BFA 10 min
prior to stimulation with insulin (
). Insulin was added at 0 min,
and the incubation was stopped at the time points indicated. Lipids
were then extracted, separated by TLC, and counted via liquid
scintillation. The percentage of PA was calculated as the percentage of
total counts of PA compared with the total counts of lipid isolated.
Results shown represent at least three separate experiments.
B, HIRcB cells were pretreated for 10 min with a dose of BFA
as indicated prior to stimulation 200 nM insulin or 200 µM PA for 10 min. Cell lysates were resolved via SDS-PAGE
and Western blot-probed with phospho-specific anti-MAPK
antibodies.
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Fig. 2.
The effect of catalytically inactive PLD
mutants on insulin-dependent PLD activity and MAPK
phosphorylation. HIRcB cells were transfected with plasmids
encoding EGFP (Control) or the catalytically inactive
GFP-PLD mutants (K898R-PLD1; K758R-PLD2). Expression of these
constructs was verified by conventional epifluorescence prior to
experimentation. A, cells were serum-starved and labeled
overnight with [3H]palmitate. PLD activity was then
measured as described under "Experimental Procedures" in cells
stimulated with 200 nM insulin (black bars) or
without any stimulation (white bars). The data are expressed
as the number of counts obtained from the phosphatidylbutanol
(PtdBu) spot-normalized by the total counts of lipid.
Results shown represent at least three separate experiments. Samples
denoted with * were significantly different as determined by analysis
of variance Bonferroni multiple comparisons test (p < 0.05). B, cells treated with and without 200 nM
insulin were lysed and resolved via SDS-PAGE and Western blot.
Nitrocellulose membranes were probed with phospho-specific anti-MAPK
antibodies and resolved as described above.
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Fig. 3.
The role of PLD-generated PA on
insulin-induced Raf-GFP kinase activity and translocation. In
order to assess the role of PLD-generated PA on Raf-1 kinase activity,
HIRcB cells were transiently transfected with a plasmid encoding the
Raf-GFP construct. A, transfected cells were stimulated with
200 nM insulin (Ins), 200 µM PA,
and/or 50 µg/ml BFA as indicated. Kinase activity of
immunoprecipitated Raf-GFP was assessed as described under
"Experimental Procedures." In order to test the ability of PA to
activate directly Raf-1 in vivo, immunoprecipitates
(Ippt) from serum-starved cells were treated with 200 µM PA prior to the kinase assay. Results shown represent
at least 3 separate experiments. Samples denoted with * were
significantly different as determined by analysis of variance
Bonferroni multiple comparisons test (p < 0.01).
B, HIRcB cells transiently transfected with the Raf-GFP
construct were stimulated as shown and fractionated by centrifugation
at 100,000 × g for 80 min as described under
"Experimental Procedures." The membrane pellet and supernatant
fractions were analyzed by SDS-PAGE and immunoblotting with an antibody
raised against Raf-1. The pellet fraction was quantitated by
densitometry, and the optical density of the bands was plotted as
shown.
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Fig. 4.
Live cell dynamics of insulin-induced Raf-GFP
translocation. HIRcB cells transiently transfected with Raf-GFP
were imaged using fluorescent confocal microscopy at 37 °C.
A, a confocal section of the plasma membrane adjacent to the
coverslip was obtained by scanning optical sections along the
z axis of the cell. The plasma membrane adjacent to the
coverslip was identified by choosing a z section below the
nucleus of the cell. This allows quantitation of Raf-GFP translocation
from the cytoplasm (out of focus sections) to the plasma
membrane (confocal section in focus). Cells were imaged at
1-min intervals for 20 min following the addition of 200 nM
insulin. Representative sections are shown. B, fluorescence
intensity of the plasma membrane sections as shown in A was
quantitated using Molecular Dynamics ImageSpace software and plotted
versus time after the addition of insulin. The
arrow indicates point that insulin (200 nM) was
administered. Results were representative of at least 4 separate
experiments. C, HIRcB cells imaged as in A were
pretreated with BFA (50 µg/ml) and stimulated with PA (200 µM) and insulin (200 nM) together (gray
lines) or separately (black lines). Arrows
indicate addition of agonists.
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Fig. 5.
PA induces transient Raf-GFP translocation in
Ha-ras(Q61L)-transformed Rat-1 fibroblasts. In order to test the
ability of PA to induce Raf-1 translocation in the presence of
activated Ras, Ha-Ras(Q61L)-transformed Rat-1 fibroblasts were
transiently transfected with the Raf-GFP construct, and a confocal
section of the plasma membrane was imaged as described in Fig. 3. Cells
were treated with 200 µM PA and imaged at 1-min intervals
for 20 min. Sections shown are representative of three separate
trials.
-subunit of the insulin
receptor (27). As shown, these vesicles also contained clathrin heavy
chains (Fig. 6B). Also shown is the effect of BFA, which
reduced substantially the co-immunoprecipitation of the clathrin heavy
chain with the insulin receptor-containing vesicles (Fig.
6B). When these vesicles were examined for the presence of
Raf-1 a consistent pattern emerged: very little Raf-1 was associated to
vesicles isolated from untreated cells, whereas a very significant
amount of Raf-1 was found in the vesicles obtained from cells that had
been exposed to insulin (Fig. 6B). In consistency with the
data in Fig. 6, A and B, a short pretreatment
with BFA abolished the co-localization of Raf-1 with clathrin and the
insulin receptor.
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Fig. 6.
A, BFA inhibits the internalization of
the insulin receptor. HIRcB cells treated as described were trypsinized
after stimulation for 30 min at 4 °C in order to cleave the
-subunit insulin (Ins) receptors localized at the cell
surface. Cleaved receptors were separated from intact, internalized
receptors by immunoprecipitation with an antibody raised to the N
terminus of the
-subunit of the insulin receptor. The
immunoprecipitated receptors were detected using a specific antibody
against the
-subunit. Western blots of immunoprecipitates detecting
the
-subunit of the insulin receptor were quantitated via
densitometry and Molecular Dynamics ImageQuant software. The intensity
of the bands is directly proportional to the number of intact,
internalized receptors. Samples denoted with * were significantly
different as determined by analysis of variance Bonferroni multiple
comparisons test (p < 0.01). B, insulin
induces co-localization of Raf-1 and clathrin in vesicles containing
the insulin receptor. Vesicles were isolated from cells following
stimulation as indicated by sucrose gradient purification of cell
lysates. Vesicles containing the insulin receptor were then
immunoisolated with an antibody recognizing the C terminus of the
-subunit of the insulin receptor as described under "Experimental
Procedures." immunoprecipitates were resolved via SDS-PAGE and
Western blot. Nitrocellulose membranes were probed for Raf-1 and
clathrin heavy chain as indicated.
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Fig. 7.
Raf-1 co-localizes with the insulin receptor
in endocytic vesicles. HIRcB cells were left untreated
(A) or stimulated with 200 nM insulin
(B and C) or 200 µM PA
(D). Cells were then fixed and processed for frozen
sectioning prior to labeling for Raf-1 (5 nm gold particles, small
arrow in A) and the -subunit of the insulin receptor (10 nm gold particles, large arrow in A).
Arrows in B-D show vesicles that contain both
insulin receptors and Raf-1, indicating that Raf-1 is internalized with
endocytic vesicles. Bar, 200 nm.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
in vitro (43-45). In vitro studies on
recombinant PLD2, on the other hand, have demonstrated that purified
PLD2 has a high basal activity that is largely insensitive to ARF and Rho (44, 46) and thus was thought to be an unlikely candidate for the
receptor-sensitive PLD activity. However, our findings suggest that in
HIRcB cells PLD2 is the main species involved in
insulin-dependent PLD signaling. This conclusion is based
on the fact that a catalytically inactive variant of PLD2 functions as
a dominant negative and blocks insulin-induced phosphorylation of MAPK,
whereas a catalytically inactive PLD1 does not. Interestingly, this
further suggests that PLD2 is regulated by ARF in vivo,
since the insulin-dependent PLD activity in HIRcB cells
requires ARF activation (10). Recent evidence from the Sung et
al.3 agrees with this
model. Although immunopurified PLD2 was found to be largely
unresponsive to ARF, a preparation of crude membranes containing PLD2
overexpressed in COS-7 cells was activated by ARF preloaded with
GTP
S, suggesting that PLD2 may be regulated by ARF in
vivo. Furthermore, a PLD2 mutant lacking the N-terminal 308 amino
acids displays both reduced in vitro and in vivo
basal activity and is stimulated more than 10-fold by ARF. These
results are consistent with a model for ARF-mediated PLD2 activation in response to insulin activation.
-arrestin or dynamin, they
showed that inhibition of G-protein-coupled receptor endocytosis blocked MAPK phosphorylation. Furthermore, they isolated
Raf-1-containing vesicles that co-isolated with clathrin-coated
vesicles, suggesting that Raf-1 associates with clathrin-coated
vesicles. Our data suggest a very similar model for insulin-mediated
activation of MAPK. We suggest that the activation of the MAPK cascade
by insulin also requires endocytosis of the insulin receptor. Raf-1 is
associated with endocytic vesicles in insulin-treated cells, and PLD
activation appears to be necessary for receptor-mediated endocytosis
and Raf-1 translocation to membranes.
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Fig. 8.
A model for the contribution of PLD and its
product, PA, in mitogenic signaling. Activation of insulin
receptors results in the stimulation of PLD mediated by ARF and in the
activation of Ras via the phosphorylation of insulin receptor
substrate-1 (IRS-1). PLD acts to generate PA which then 1) aids in the
recruitment of Raf-1 to the plasma membrane where it interacts with
GTP-Ras; and 2) facilitates formation of the endocytic vesicle. Raf-1
then remains bound to the endocytic vesicle via its association with
PA. The internalized Raf-1 then propagates the MAPK signaling
cascade.
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ACKNOWLEDGEMENTS |
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We thank Dr. Adrienne D. Cox for the Ha-Ras(Q61L)-transformed cells and Dr. Saïd Sebti for the plasmid encoding c-Raf-1. We also thank Dr. Edwin Levitan for a critical review of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK51183, DK02465, 5-T32-GM08424-04, and GM54813 and American Diabetes Association Grant 96-029.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: Dept. of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-648-9408; Fax: 412-648-1945; E-mail: ggr+{at}pitt.edu.
The abbreviations used are:
ARF, ADP-ribosylation factor; BFA, brefeldin A; CRD, cysteine-rich domain; DAG, diacylglycerol; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein
kinase C; PLD, phospholipase D; Raf-GFP, Raf-green fluorescent protein
fusion protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPS, guanosine
5'-3-O-(thio)triphosphate.
2 T.-C. Sung, Y. Zhang, A. J. Morris, and M. A. Frohman, submitted for publication.
3 T. S. Sung, A. J. Morris, and M. A. Frohman, submitted for publication.
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