Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786
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ABSTRACT |
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Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 µM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel Gi-dependent event in the action of LPA.
protein kinase C; G protein-coupled receptor; signal transduction; protein phosphorylation
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INTRODUCTION |
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LYSOPHOSPHATIDIC ACID (LPA), a major bioactive lipid of
serum, elicits a broad spectrum of biological responses, including platelet activation, smooth muscle contraction, changes in neuronal cell shape, and induction of cell proliferation and differentiation (14). LPA binds to a seven-transmembrane domain receptor(s) and
activates several heterotrimeric G proteins, which are responsible for
transducing LPA signals into multiple biological responses (5, 15). LPA
stimulates Ras activation, leading to stimulation of Raf, MEK, and the
ERKs via a pertussis toxin (PTx)-sensitive pathway that involves the
subunits of Gi (2, 3, 11, 12, 26), whereas the
subunit of this trimeric G protein mediates
inhibition of adenylate cyclase activity (5, 15). LPA induces
PTx-insensitive stress fiber formation, assembly of focal adhesions,
and tyrosine phosphorylation of focal adhesion proteins (20) via
activation of G
13 (6, 16). LPA
also stimulates phospholipase C (PLC)-mediated polyphosphoinositide breakdown that leads to generation of inositol 1,4,5-trisphosphate and
diacylglycerol, the second messengers responsible for
Ca2+ mobilization from
intracellular stores and activation of protein kinase C (PKC),
respectively. These PLC-dependent responses are thought to be mediated
by PTx-insensitive G proteins of the
Gq family (14, 15). It is also
recognized that some of the downstream responses induced by LPA,
including the activation of transcription factors NF-
B (21) and
serum response factor (1) are elicited by interaction of complementary
pathways activated by Gq,
Gi and G12.
The PKC family consists of multiple related isoforms, i.e.,
conventional PKCs (,
1,
2, and
), novel PKCs
(
,
,
, and
) and atypical PKCs (
and
), all
of which possess a highly conserved catalytic domain (17). Protein
kinase D (PKD) (25), also named PKCµ (9), is a serine-threonine
protein kinase with distinct structural, enzymological, and regulatory
properties. In particular, PKD can be rapidly activated in intact cells
through a phosphorylation-dependent mechanism (29). Treatment of intact cells with biologically active phorbol esters (29), bryostatin (13), or
neuropeptide agonists, including bombesin, endothelin, and vasopressin
(30), induces PKD activation that persists during cell disruption and
immunoprecipitation. Several lines of evidence, including the use of
selective PKC inhibitors and cotransfection of PKD with constitutively
active mutants of PKC
and
, indicate that PKD is activated by
phosphorylation in living cells through a PKC-dependent signal
transduction pathway (13, 29, 30). More recently,
Ser744 and
Ser748 have been identified as
critical phosphorylation sites in the activation loop of the kinase
catalytic domain of PKD (8). These findings reveal an unsuspected
connection between PKCs and PKD and imply that PKD can function
downstream of PKCs in signal transduction.
In the present study, we examined the effect of the multifunctional agonist LPA on the regulation of PKD activity in intact Swiss 3T3 and Rat-1 cells, which have been used extensively as model systems to elucidate the biological effects of this bioactive lipid. We report for the first time that LPA induces a rapid PKC-dependent PKD activation in these cells. Surprisingly, treatment of the cells with PTx markedly and selectively attenuated PKD activation in response to LPA. Our results identify a novel PTx-sensitive event in the action of LPA and provide the first example of a Gi-dependent pathway leading to PKD activation in any cell type.
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MATERIALS AND METHODS |
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Cell culture. Stock cultures of Swiss 3T3 cells and Rat-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO2 and 90% air at 37°C. For experimental purposes, cells were plated in 100-mm dishes at 5 × 105 cells/dish in DMEM containing 10% FBS and used after 6-8 days when the cells were confluent and quiescent.
Immunoprecipitation. Quiescent cultures of cells, treated as described in the individual experiments, were washed and lysed in 50 mM Tris · HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer A). Cell lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C. PKD was immunoprecipitated at 4°C for 2-4 h with the PA-1 antipeptide antiserum (1:100), as previously described (29). The immune complexes were recovered using protein A coupled to agarose.
Kinase assay of PKD.
The kinase activity of PKD was determined in an in vitro kinase assay
by mixing 20 µl of PKD immunocomplexes with 10 µl of a
phosphorylation mixture containing (final concentration) 10 µM
[-32P]ATP (specific
activity 400-600 cpm/pmol), 30 mM Tris · HCl, pH
7.4, 10 mM MgCl2, and 1 mM
dithiothreitol. After 10 min of incubation at 30°C, the reaction
was stopped by washing with 200 µl of kinase buffer and then adding
an equal volume of 2× SDS-PAGE sample buffer (200 mM
Tris · HCl, pH 6.8, 2 mM EDTA, 0.1 M
Na3VO4, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol), followed by
SDS-PAGE analysis (27, 29). The gels were dried, and the 110-kDa
radioactive band corresponding to autophosphorylated PKD was visualized
by autoradiography. Autoradiographs were scanned in a ScanJet 6100C/T (Hewlett Packard), and the labeled band was quantified using the National Institutes of Health image software program.
Phospholipase B treatment of LPA and FBS.
LPA and lysophosphatidates bound to albumin in serum are inactivated by
treatment with phospholipase B (PLB) (10). Here, 100 µl of a 500-µM
LPA stock solution were dissolved in PBS-0.01% bovine serum albumin
(wt/vol) or 100 µl of FBS were incubated with or without 100 IU of
PLB for 1 h at 37°C. The PLB-treated LPA or FBS were used
immediately, as indicated in Fig. 1. In
control experiments the activity of PLB was destroyed by heating to
75°C for 1 h before incubation with LPA or FBS.
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Measurement of intracellular calcium concentration.
Intracellular calcium concentration
([Ca2+]i)
was measured with the fluorescent indicator fura 2. Confluent and
quiescent cultures of Swiss 3T3 cells, grown on 9 × 22 mm
coverslips, were washed twice with DMEM and then incubated for 10 min
in DMEM containing 1 µM fura 2-AM at 37°C. The cultures were then
washed twice with Hanks' buffered salt solution, pH 7.2, supplemented
with NaHCO3 (35 mM),
CaCl2 (1.3 mM),
MgCl2 (0.5 mM),
MgSO4 (0.4 mM), and 0.1% bovine
serum albumin (calcium buffer). The coverslips were then incubated for
a further 15 min in calcium buffer and then transferred to a quartz
cuvette containing 2 ml of the same buffer, and fluorescence was
monitored using a Hitachi F-2000 fluorospectrophotometer with dual
excitation wavelengths of 340 nm (1) and 380 nm (
2) and an
emission wavelength of 510 nm while the cells were continually stirred
at 37°C.
[Ca2+]i
was determined using the equation
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Phospho p42mapk (ERK-2) and p44mapk (ERK-1) immunoblot. Quiescent cultures of Swiss 3T3 cells grown on 33-mm dishes were washed twice with DMEM and incubated with or without PTx (30 ng/ml) for 3 h in DMEM at 37°C. LPA (5 µM) was then added to the cultures and incubated at 37°C for a further 5 min. The cells were lysed in 2× SDS-PAGE sample buffer, and the lysates were analyzed by SDS-PAGE. After SDS-PAGE, the proteins were transferred to immobilon membranes as per the manufacturers instructions. Membranes were blocked with 5% nonfat milk and incubated overnight with monoclonal anti-phospho ERK-1 and ERK-2 antibody (0.2 µg/ml, E10, Bio-Labs). Immunoreactive p42mapk (ERK-2) and p44mapk (ERK-1) bands were detected by enhanced chemiluminescence Western blotting ECL reagents (Amersham).
Materials.
[-32P]ATP (370 MBq/ml) was from Amersham International. GF-I (also known as GF-109203X
or bisindolylmaleimide I), Gö-7874, Ro-31-8220, U-73122,
U-73343, and PTx were from Calbiochem. LPA, phorbol 12,13-dibutyrate
(PDB), wortmannin, rapamycin, PD-098059, genistein, and PLB were
from Sigma. The monoclonal anti-phospho-ERK-1 and
ERK-2 antibody E10 was purchased from Bio-Labs. Protein
A-agarose was from Boehringer Mannheim. PA-1 antiserum was raised
against the synthetic peptide EEREMKALSERVSIL that
corresponds to the carboxy terminal region of the predicted amino acid
sequence of PKD, as previously described (27, 29). Other items were
from standard suppliers or as indicated in the text.
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RESULTS |
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LPA induces PKD activation in Swiss 3T3 and Rat-1 cells.
Quiescent Swiss 3T3 fibroblasts have proved to be a useful model system
for elucidating signal transduction pathways in the action of multiple
agonists, including LPA (19). To examine whether LPA induces PKD
activation, confluent and quiescent cultures of these cells were
stimulated with 5 µM LPA for 10 min and lysed. The extracts were
immunoprecipitated with the PA-1 antibody raised against a peptide
composed of the 15 carboxy terminal amino acids of PKD. The
immunocomplexes were incubated with
[-32P]ATP and then
analyzed by SDS-PAGE and autoradiography to examine the level of
autophosphorylation. As illustrated in Fig. 1, stimulation of Swiss 3T3
cells with LPA induced a marked PKD activation that was maintained
during cell disruption and immunoprecipitation. In 25 independent
experiments, addition of 5 µM LPA to cultures of Swiss 3T3 cells
induced a 7.6 ± 0.47 (means ± SE)-fold increase in PKD activity.
PKC mediates LPA-stimulated PKD activation.
Next, we determined the role of PKCs in PKD activation induced by LPA.
Quiescent cultures of Swiss 3T3 cells were treated with various
concentrations of GF-I (also known as GF-109203X or bisindolylmaleimide
I), a potent inhibitor of phorbol ester-sensitive isoforms of PKC (24)
but not PKD (29, 30), before PDB stimulation. As shown in Fig.
2, treatment of the cells with GF-I
potently blocked PKD activation induced by subsequent addition of LPA, in a concentration-dependent fashion. In contrast, GF-I added directly
to the in vitro kinase assay, even at the concentrations (0.5-2.5
µM) required to abrogate LPA-mediated PKD activation in intact 3T3
cells, did not inhibit PKD activity.
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LPA induces PKD activation via PLC.
To determine whether LPA induces PKC-dependent PKD activation through a
PLC-dependent pathway, Swiss 3T3 cells were treated with the
aminosteroid U-73122, an inhibitor of PLC, prior to stimulation with
LPA. As shown in Fig.
3A,
U-73122 markedly reduced PKD activation in response to the subsequent
addition of LPA in a concentration-dependent fashion. Maximal
inhibition of LPA-stimulated PKD activation was achieved at 2.5 µM.
The inhibitory effect of U-73122 was selective because this agent, at
similar concentrations, did not interfere with PKD activation induced
by PDB. Furthermore, U-73343, an inactive analog of U-73122, did not
affect LPA stimulation of PKD activation when added at an identical
concentration (Fig. 3B).
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LPA stimulates PKD activation via a PTx-sensitive pathway.
LPA activates several heterotrimeric G proteins including
Gq and
Gi, which are responsible for
transducing LPA signals into multiple biological responses (6, 15).
PLC-mediated polyphosphoinositide breakdown, leading to rapid
Ca2+ mobilization and PKC
activation, is thought to be mediated by PTx-insensitive G proteins of
the Gq family, at least in rodent cell lines (14). We verified that treatment of Swiss 3T3 cells with 30 ng/ml PTx for 3 h, a condition known to promote ADP ribosylation and
inactivation of Gi in these cells
(22), did not interfere with the rapid and transient increase in
[Ca2+]i
induced by LPA (Fig.
4A). In
contrast, a similar treatment with PTx markedly inhibited ERK
activation in response to LPA, as shown by Western blot analysis using
an antibody that recognizes the dually phosphorylated and active
p42mapk (ERK-2) and
p44mapk (ERK-1) (Fig.
4A).
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DISCUSSION |
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LPA promotes a broad range of biological responses and multiple molecular events in target cells (15). Consistent with the stimulation of multiple signaling pathways, LPA has been shown to bind to several heptahelical receptors (5) and activate several heterotrimeric G proteins, including Gq, Gi, and G12 in Swiss 3T3 cells (6) and in Rat-1 cells (14). The results presented here demonstrate that LPA rapidly induces PKD activation in intact Swiss 3T3 and Rat-1 cells and thus identify a novel molecular response in LPA action. Our results also suggest that LPA is a major factor in serum that mediates PKD activation in these cell types.
Treatment of the cells with the PKC inhibitors GF-I, Ro-31-8220, and Gö-7874 before stimulation with LPA strikingly prevented PKD activation. Importantly, these PKC inhibitors did not reduce PKD activity when added directly to the in vitro kinase assays, even at concentrations higher than those used in intact cells to block LPA-induced PKD activation. Furthermore, the PLC inhibitor U-73122 selectively prevented PKD activation by LPA. We conclude that LPA-induced PKD activation is downstream to PLC and PKC in Swiss 3T3 cells.
LPA-induced PLC and PKC activation is thought to be mediated by LPA receptor coupling to PTx-insensitive Gq (14). In line with this hypothesis, inositol phosphate production and Ca2+ mobilization in response to LPA is not prevented by treatment with PTx in rodent cell lines, including Swiss 3T3 cells. In addition, LPA has been shown to stimulate phosphorylation of the Rac exchange factor Tiam-1 via a PTx-insensitive PKC-dependent pathway in these cells (4).
Although LPA induces PLC and PKC activation through Gq and PKD activation in response to LPA is downstream to PKC, it could not be excluded that other signaling inputs also contribute to PKD activation induced by LPA. A surprising feature of our results is that PKD activation in response to LPA is attenuated markedly and selectively by prior treatment of either Swiss 3T3 cells or Rat-1 cells with low concentrations of PTx. These results indicate that the Gq pathway is not sufficient to promote PKD activation in response to LPA in these cells and identify for the first time the involvement of an additional Gi-dependent pathway leading to PKD activation in any cell type.
Interestingly, the concentration of LPA required for stimulation of PKD
activation in Swiss 3T3 cells
(EC50 1 µM) is similar to that
needed for activation of the transcription factors NF-B (21) and
serum response factor (1). Recent evidence indicates that LPA leads to
the activation of these transcription factors through parallel signal
transduction pathways. For example, the stimulation of NF-
B by LPA
is mediated by Gq and
Gi pathways (21), and the
activation of the serum response factor is mediated by cooperative
effects between Gi and
G
13-Rho pathways (1). We
propose that LPA-induced PKD activation, which precedes the activation
of these transcription factors, is also mediated by complementary
pathways initiated by Gi and
Gq.
It is well established that treatment with PTx almost completely blocks
LPA-induced mitogenesis in a variety of cell types. LPA signaling
through PTx-sensitive Gi activates
the Ras-Raf-ERK kinase cascade (2, 3, 11, 12, 26) and phosphoinositide 3-kinase activity in cultured fibroblasts (18). Recently, Takeda et al.
(23) demonstrated that LPA induces phosphoinositide 3-kinase-dependent activation of PKC via Gi. The
results presented here identify PKD activation as a novel PTx-sensitive
early molecular response in the action of LPA, which can be dissociated
from either Ras-Raf-ERK or phosphoinositide 3-kinase signaling
pathways. In addition, our previous results showed that cotransfection
of PKD with a constitutively active form of PKC
does not lead either
to PKD activation (29) or to the formation of stable molecular
complexes between PKC
and PKD (28). The role of PKD in the
PTx-sensitive biological responses induced by LPA warrants further
experimental work.
In conclusion, our results demonstrate that LPA induces PKC-dependent PKD activation in Swiss 3T3 cells. In contrast to the model of PKD regulation by neuropeptide agonists through Gq (30), we propose that LPA stimulates PKD activation through both Gi and Gq in these cells. Our results identify a novel PTx-sensitive molecular response in the action of LPA and demonstrate for the first time the involvement of a Gi-dependent pathway leading to PKD activation in any cell type.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55003-01 to E. Rozengurt. L. Paolucci was supported by a fellowship of the University of Chieti (Italy) and by a short-term fellowship from Boehringer Ingelheim Fonds.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Ave., Warren Hall, Rm. 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1786 (E-mail: erozengu{at}med1.medsch.ucla.edu).
Received 4 June 1999; accepted in final form 23 August 1999.
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