From the Department of Medicine, David Geffen School
of Medicine and Molecular Biology Institute, University of
California, Los Angeles, California 90095 and the ¶ Division of
Biology, California Institute of Technology,
Pasadena, California 91125
Received for publication, October 31, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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To examine the contribution of different
G-protein pathways to lysophosphatidic acid (LPA)-induced protein
kinase D (PKD) activation, we tested the effect of LPA on PKD activity
in murine embryonic cell lines deficient in G Protein kinase C (PKC),1 a major target for the tumor
promoting phorbol esters, has been
implicated in the signal transduction pathways regulating a wide range
of biological responses, including changes in cell morphology,
differentiation, and proliferation (1, 2). Molecular cloning has
demonstrated the presence of multiple PKC isoforms (2-5),
i.e. conventional PKCs ( PKD/PKCµ is a serine/threonine protein kinase (6, 7) with distinct
structural, enzymological, and regulatory properties (8). In
particular, PKD is rapidly activated in intact cells through a
mechanism that involves phosphorylation (8). Specifically, exposure of
intact cells to phorbol esters, cell-permeant diacylglycerols, or
bryostatin induces rapid PKD phosphorylation and activation, which is maintained during cell lysis and immunoprecipitation (8-13).
Several lines of evidence, including the use of PKC-specific inhibitors
and co-transfection of PKD with constitutively active PKC mutants
indicate that PKD is activated through a novel
PKC-dependent signal transduction pathway in
vivo (9-11). The residues Ser-744 and Ser-748 in the activation
loop of PKD have been identified as critical phosphorylation sites in
PKD activation (14, 15). Taken together, these results suggest an
important connection between PKCs and PKD and indicate that PKD can
function downstream of PKC in a novel signal transduction pathway.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G
proteins) are composed of Many GPCRs also interact with other heterotrimeric G proteins including
members of the G12 family, which mediate activation of the
low molecular weight G proteins of the Rho subfamily (21-26) via
guanine nucleotide exchange factors that directly link the G Lysophosphatidic acid (LPA), a major bioactive lipid of serum (40),
elicits a plethora of biological responses by activation of its
specific GPCRs (41-43). Thus far, three genes
(lpA1, lpA2, and
lpA3) encoding LPA receptors,
LPA1/EDG-2, LPA2/EDG-4, and
LPA3/EDG-7, have been identified in mammals (43-45). All
LPA receptors are coupled to Gi and Gq and, at
least LPA1 and LPA2 are also coupled to
G12 (42, 45, 46). LPA induces Ras activation leading to
stimulation of Raf, MEK, and the ERKs via a pertussis toxin
(PTx)-sensitive pathway that involves the LPA has also been shown to induce a rapid PKC-dependent PKD
activation in intact Swiss 3T3, Rat-1, and IEC-6 cells (56, 57).
Interestingly, treatment of these cells with PTx markedly attenuated
PKD activation in response to LPA. These results identified the
involvement of an additional Gi-dependent
pathway leading to PKD activation and indicated that the endogenous
Gq pathway is not sufficient to promote PKD activation in
response to LPA. Because our previous studies demonstrated that PKD can
be activated through Gq- and
G12-dependent pathways (see above) and because LPA receptors are also coupled to Gq and G12,
we hypothesized that LPA induces PKD through multiple G protein signal
transduction pathways. Here, we tested this hypothesis using mouse
embryonic cell lines deficient in G Cell Culture--
Mouse embryonic fibroblasts were generated
from genetically engineered mice (58, 59) that contained gene knockout
for rhodopsin kinase, G Cell Transfection and cDNA Constructs Used in
Transfections--
To transfect the cells, RK cells and
G Immunoprecipitation--
Cultures of RK, G Kinase Assay of PKD--
PKD autophosphorylation 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) 100 µM [
Exogenous substrate phosphorylation by immunoprecipitated PKD was
carried out by mixing 20 µl of the washed immunocomplexes with 20 µl of a phosphorylation mixture containing 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK), a peptide based on phosphorylation site two of
glycogen synthase. After 10 min of incubation at 30 °C, the reaction
was stopped by adding 100 µl of 75 mM
H3PO4, and 75 µl of the supernatant was
spotted on P-81 phosphocellulose paper. Free [ Western Blot Analysis for pS744/748 and ERK-2/ERK-1
Activation--
Samples of cell lysates were directly solubilized by
boiling in 2× SDS-PAGE sample buffer. Following SDS-PAGE on 8% gels, proteins were transferred to Immobilon-P membranes (Millipore) and
blocked by 3-6 h incubation with 5% nonfat milk in phosphate-buffered saline, pH 7.2. Membranes were then incubated 3 h with the
respective antibodies. PKD phosphorylation was determined by incubating
the membrane with an antibody that specifically recognizes the
phosphorylated state of serine 744 and serine 748 of PKD at a dilution
of 1:1000 in phosphate-buffered saline, 0.1% Tween 20 containing 5%
bovine serum albumin.
Activation of ERK-2 and ERK-1 occurs through phosphorylation of
specific threonine and tyrosine residues, resulting in slower migrating
forms in SDS-PAGE gels. These activated forms were monitored by using a
specific antiphospho-ERK-1/ERK-2 monoclonal antibody that recognizes
the phosphorylated state of Thr-202 and Tyr-204 of ERL-1/2 at a
dilution of 1:1000 in phosphate-buffered saline, 0.1% Tween 20 containing 5% nonfat dried milk. The same membranes were stripped and
probed in a similar fashion with goat anti-ERK-2 polyclonal antibody.
Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000,
Amersham Biosciences) was then applied for 1 h at room temperature after washing 3 times with phosphate-buffered saline containing 0.05% Tween. Immunoreactive bands were detected by enhanced
chemiluminescence Western blotting ECLTM reagents.
Materials--
[ LPA Induces PKD Activation in RK Cells through a
PKC-dependent Pathway--
To examine the contribution of
different G protein pathways to LPA-induced PKD activation, we decided
to test the effect of this agonist on PKD activity isolated from murine
embryonic cell lines lacking RK, G
The results presented in Fig.
1A show that LPA induced a
marked increase in PKD autophosphorylation activity in a
concentration-dependent fashion in RK cells. Half-maximal
and maximal stimulation (6-fold increase) were achieved at 0.07 and 3 µM, respectively. As illustrated in the inset
to Fig. 1, similar results were obtained when PKD activity in
immunocomplexes was determined by phosphorylation of syntide-2 (61,
62), a synthetic peptide previously demonstrated to be an excellent
substrate for PKD (6).
We previously proposed that phosphorylation of Ser-744 and Ser-748
within the PKD activation loop plays a critical role in PKD activation
(14, 15, 20). Recently, a novel antibody recognizing
predominantly the phosphorylated state of Ser-744 (pS744/pS748) in PKD
was used in our laboratory to monitor activation loop phosphorylation
in response to phorbol ester and bombesin (15). To examine whether LPA
induces activation loop phosphorylation in the RK cells, lysates from
these cells treated with increasing concentrations of LPA were analyzed
by SDS-PAGE, followed by Western blotting with the pS744/748 antibody.
As shown in Fig. 1A, LPA induced PKD Ser-744 phosphorylation
in a concentration-dependent fashion in RK cells with
maximal stimulation achieved at 3 µM.
To determine whether LPA induces PKD activation and activation loop
phosphorylation through a PKC-mediated pathway in RK cells, we used
inhibitors that discriminate between PKCs and PKD. RK cells were
treated for 1 h with the inhibitors of phorbol ester-sensitive isoforms of PKC, GF I (also known as GF 109203X or bisindolylmaleimide I) or Ro 31-8220 (64, 65), prior to stimulation with 3 µM LPA. As shown in Fig. 1B (left panels), exposure
to either GF I or Ro 31-8220 prevented PKD activation (top)
and Ser-744 phosphorylation (middle) induced by LPA. In
contrast, the compound GFV, which is structurally related to GF I but
biologically inactive, did not prevent PKD activation and
phosphorylation in response to LPA in RK cells. Similar results were
obtained when PKD activity in immunocomplexes was determined by assays
of syntide-2 phosphorylation (bottom). Previously, we
demonstrated that GF I and Ro 31-8220 do not inhibit PKD activity when
added directly to the in vitro kinase assay at identical
concentrations to those required to block PKD activation by LPA in RK
cells (9, 11). Thus, the results shown in Fig. 1B imply that
GF I and Ro 31-8220 do not inhibit PKD activity directly but interfere
with LPA-induced PKD activation and activation loop phosphorylation in
intact RK cells by blocking PKC.
To determine whether LPA induces PKD activation and phosphorylation
through a PLC-dependent pathway in RK cells, cultures of
these cells were treated with the aminosteroid U73122, an inhibitor of
PLC (66, 67), prior to stimulation with LPA. As shown in Fig.
1B (right, top and middle
panels), 2 µM U73122 markedly reduced PKD activation
and Ser-744 phosphorylation in response to the subsequent addition of
LPA. Similar results were obtained when PKD activity in immunocomplexes
was determined by assays of syntide-2 phosphorylation (Fig.
1B, right bottom panel). The inhibitory effect of
U73122 was selective because treatment with this compound did not
interfere with PKD activation and Ser-744 phosphorylation induced by
PDB (Fig. 1B).
Effect of Treatment with PTx on LPA-induced PKD Activation in RK
Cells--
Previous studies demonstrated that LPA induces PKD
activation through a PTx-sensitive pathway in 3T3 (18) and IEC-6 cells (57) but through a pathway that was only partially attenuated by PTx in
IEC-18 cells (57). Because PTx catalyzes the ADP-ribosylation and
inactivation of members of the G
To assess the contribution of Gi to LPA-induced PKD
activation in RK cells, cultures of these cells were treated with
increasing concentrations of PTx (1-100 ng/ml) for 3 h and then
challenged with 3 µM LPA for 10 min. As illustrated by
Fig. 2A, treatment of RK cells
with PTx at a concentration as high as 100 ng/ml attenuated only
slightly PKD activation and Ser-744 phosphorylation in response to LPA
(the maximal attenuation was only ~25%). Similar attenuation of PKD
activation was obtained when PKD activity in immunocomplexes was
determined by phosphorylation of the exogenous substrate syntide-2 rather than by autophosphorylation (Fig. 2B). These results
suggest that LPA induces PKD activation predominantly through
Gi-independent pathways in RK cells, presumably involving
G LPA-induced PKD Activation Requires Functional
Gq--
Most cell types express multiple members of the
Gq and G12 families with overlapping functions,
thus rendering it difficult to analyze the contribution of these G
proteins to heptahelical receptor signaling. To circumvent this problem
and in view of the results presented above, we next examined whether
LPA induces PKD activation in murine embryonic fibroblasts generated
from double knockout mice for G
In agreement with the results shown in Figs. 1 and 2, treatment of RK
cells with either LPA or PDB induced a marked increase in PKD catalytic
activity measured either by autophosphorylation or syntide-2
phosphorylation assays (Fig. 3B). In striking contrast, addition of LPA to cultures of G
To further confirm the requirement of functional Gq in
LPA-induced PKD activation, we examined whether transfection of
Gq/11 KO cells with wild type G LPA-induced Maximal PKD Activation Requires Functional
G
The results presented in Fig. 5 were
designed to examine in more detail the stimulation of PKD activity in
response to LPA in G
We verified that LPA induces PKD activation in these cells through a
PKC- and PLC-dependent pathway. As shown in Fig.
5B, left, exposure to either GF I or Ro 31-8220 prevented PKD activation and Ser-744 phosphorylation induced by LPA
whereas the biologically inactive analog GFV did not prevent PKD
activation in response to LPA. Similarly, treatment with the PLC
inhibitor U73122 (66, 67) prevented PKD activation induced by LPA but
not by PDB, as shown by assays of autophosphorylation, Western blot
analysis, or syntide-2 phosphorylation (Fig. 5B,
right).
LPA-induced PKD Activation and Phosphorylation Requires Functional
Gi in G
As shown in Fig. 6A, treatment
with PTx dramatically inhibited both PKD activation and Ser-744
phosphorylation elicited by LPA in a
concentration-dependent fashion. Indeed, treatment with 30-100 ng/ml PTx completely abolished PKD activation and Ser-744 phosphorylation in response to LPA. In contrast, treatment with 50 ng/ml PTx did not interfere with PKD activation and Ser-744 phosphorylation induced by PDB in these cells. As illustrated in Fig.
6B, similar results were obtained when PKD activity in immunocomplexes was determined by assays of syntide-2 phosphorylation. These findings contrast with those shown in Fig. 2 with RK cells (PTx
inhibited LPA-induced PKD activation by only ~25%) and indicate that
Gi plays a critical role in promoting PKD activation in
G
Recently, we demonstrated that in addition to G LPA Induces Activation of Mitogen-activated Protein Kinases
via Gi-dependent but PKC-independent Pathways
in RK, G
In most cell types, LPA stimulates Ras activation leading to
stimulation of Raf, MEK, and the ERKs via a PTx-sensitive
pathway that involves the
Treatment of RK cells with PTx markedly inhibited ERK activation in
response to LPA, whereas exposure to GF I or Ro 31-8220 did not
interfere with ERK activation, indicating that LPA induces ERK through
a PTx-sensitive, PKC-independent in RK cells. Virtually identical
results were obtained with G Concluding Remarks--
LPA promotes a broad range of biological
responses and multiple molecular events in target cells (42).
Consistent with the stimulation of multiple signaling pathways, LPA has
been shown to activate several heterotrimeric G proteins including
Gq, Gi, and G13 in Swiss 3T3 cells
(54). PKC-dependent PKD activation has been identified as
an early event in the action of LPA in intact Swiss 3T3, Rat-1, and
IEC-6 cells (56, 57). Because our previous studies indicated that
G
Based on the results presented in this study, we propose a model that
envisages that LPA induces PKD activation through Gq acting
synergistically with G12 and Gi. Thus,
inhibition of Gi signaling in RK cells by treatment with
PTx only induces a modest attenuation of PKD activation in response to
LPA because the remaining G protein pathways (Gq and
G12) cooperate to mediate a substantial response to LPA.
Similarly, treatment of RK cells with C. difficile toxin B
that inactivates Rho GTPases, the effectors of the G12 pathway, had little effect on PKD activation by LPA because the remaining G protein pathways (in this case Gq and
Gi) are sufficient to mediate PKD activation in response to
LPA. An important result substantiating our hypothesis is that
treatment of RK cells with both C. difficile toxin B
and PTx completely prevented LPA-induced PKD activation, indicating
that endogenous Gq appears necessary but not sufficient to
mediate this response.
The salient features of this model are corroborated by the results
obtained in the cell lines deficient in either G
Recent work from other laboratories has shown that transcriptional
responses induced by LPA also result from synergistic effects between
parallel G protein signaling pathways. For example, the stimulation of
the transcription factor NF-
Here, we propose that PKD activation, which precedes the
transcriptional responses, is also mediated by cooperative effects between events initiated by Gq, Gi, and
G12. In this manner, PKD may constitute an early point of
convergence of Gq, G12, and Gi signaling in LPA-treated cells. The scheme shown in Fig.
9 summarizes the multiple G protein
signal transduction pathways involved in LPA-induced PKD activation
proposed in this study and also illustrates the molecular, cellular,
and pharmacological approaches used in our experiments.
q/11
(G
q/11 KO cells) or G
12/13
(G
12/13 KO cells) and used cells lacking rhodopsin
kinase (RK cells) as a control. In RK and G
12/13 KO
cells, LPA induced PKD activation through a phospholipase C/protein
kinase C pathway in a concentration-dependent
fashion with maximal stimulation (6-fold for RK cells and 4-fold for
G
12/13 KO cells in autophosphorylation activity)
achieved at 3 µM. In contrast, LPA did not induce any significant increase in PKD activity in G
q/11 KO cells.
However, LPA induced a significantly increased PKD activity when
G
q/11 KO cells were transfected with G
q.
LPA-induced PKD activation was modestly attenuated by prior exposure of
RK cells to pertussis toxin (PTx) but abolished by the combination
treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation
in a concentration-dependent fashion in
G
12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an
antibody that detects the phosphorylated state of this residue. Our
results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA
requires additional G-protein pathways to elicit a maximal response
with Gi playing a critical role in G
12/13 KO
cells. We conclude that LPA induces PKD activation through
Gq, Gi, and G12 and propose that
PKD activation is a point of convergence in the action of multiple
G-protein pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
1,
2, and
), novel PKCs (
,
,
, and
), and
atypical PKCs (
and
) all of which possess a highly conserved
catalytic domain.
,
, and
subunits and transduce external signals from heptahelical receptors to intracellular effectors
(16). Mammalian G protein
subunits are classified into four
subfamilies: Gs, Gi, Gq, and
G12. The
subunit of Gq stimulates the
isoforms of phospholipase C (PLC), which catalyze the production of
inositol 1,4,5-trisphosphate that triggers the release of
Ca2+ from internal stores and diacylglycerol that activates
the classical and novel isoforms of PKC (reviewed in Ref. 17). A
variety of neuropeptide agonists that signal through heptahelical
receptors and couple to heterotrimeric G proteins, including bombesin,
bradykinin, endothelin, and vasopressin, induce rapid PKD activation in
normal and neoplastic cells (11, 13, 18, 19). Although each of these
receptors activates Gq and G
q
signaling stimulates PKD activity (20), expression of a
COOH-terminal fragment of G
q that acts in a
dominant-negative fashion, attenuated (but did not eliminate) PKD
activation in response to agonist stimulation of bombesin receptor
(20). These results suggested that G protein-coupled receptors (GPCRs)
stimulate PKD activation not only via G
q but also
through other G protein-mediated signaling pathways.
subunits to Rho (27-29). Rho plays a major role in promoting cytoskeletal responses including formation of actin stress fibers, assembly of focal adhesions, and tyrosine phosphorylation of focal adhesion proteins and has been implicated in gene expression, cell
migration, proliferation, and transformation (30-32). Interestingly, a
number of recent studies have suggested a convergence between Rho- and
PKC-mediated signaling in yeast and mammalian cells (33-38). For
example, Slater et al. (37) have demonstrated that Rho-GTP potently stimulates PKC
activity in vitro using
recombinant proteins and Sagi et al. (38) reported that
G
q and PLC signaling are synergistic with Rho. Recently,
we demonstrated that in addition to G
q, Rho- and
G
13-mediated signaling can promote PKD activation in
intact cells and that endogenous Rho and G
13 contribute
to PKD activation in response to bombesin GPCR stimulation (39). Thus,
our results identified PKD as a novel downstream target in
G
13 and Rho signaling and indicated that GPCR
stimulation promotes PKD activation via both Gq- and
G
13/Rho-dependent pathways.
subunits of
Gi (47-51) while the
subunit of this trimeric G
protein mediates inhibition of adenylate cyclase activity (42, 46). LPA
stimulates PLC-mediated generation of inositol 1,4,5-trisphosphate and
diacylglycerol, leading to Ca2+ mobilization from
intracellular stores and PKC activation, respectively. These
PLC-dependent responses are mediated by the
subunit of Gq and/or the
subunits of the PTx-sensitive
Gi (52). LPA also promotes PTx-insensitive stress fiber
formation, assembly of focal adhesions, and tyrosine phosphorylation of
focal adhesion proteins (53) via activation of G
13 and
Rho (24, 54). Because LPA receptors are expressed in most cell types,
this bioactive lipid is a prototype agonist that promotes the
activation of multiple endogenous G protein pathways that are
responsible for transducing LPA signals into a broad spectrum of
biological responses (43, 45, 55).
q/11
(G
q/11 KO cells) or G
12/13
(G
12/13 KO cells), and used a cell line lacking
rhodopsin kinase (RK cells), as a control. Based on our results, we
conclude that LPA induces PKD activation through the cooperation of
Gq with either Gi or G12 and that
PKD activation is a point of convergence in the action of multiple G
protein pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
q/11, and G
12/13.
Stock cultures of these cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 250 µg/ml G418 in a humidified atmosphere containing 10% CO2
and 90% air at 37 °C. For experimental purposes, cells were plated
in 60- or 100-mm dishes at about 30% confluency in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum and 250 µg/ml G418 and were allowed to grow to near confluency (6-7 days)
and then changed to serum-free Dulbecco's modified Eagle's medium for
8-24 h prior to the experiment.
q/11 KO cells were subcultured to 80-90% confluency
in 10-cm dishes. All transfections and cotransfections were carried out
with equivalent amounts of DNA (20 µg/dish). Transfections were
carried out in Opti-MEM using LipofectAMINE 2000 reagent according to
the protocol from the manufacturer (Invitrogen). Cells were used for
experiments 48-72 h after of transfection. Chimeric fusion proteins
between GFP and PKD (GFP-PKD) have been described previously (69,
70).
q/11,
and G
12/13 cells, treated as described in the individual
experiments, were washed and lysed in lysis buffer (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). 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
antiserum (1:100) or with GFP antibody for GFP-PKD transfected cell
lysates, as previously described (9, 10). The immune complexes were recovered using protein A coupled to agarose.
-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 1 ml 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 (9, 60). The gels
were dried and the 110- or 140-kDa radioactive band corresponding to
autophosphorylated PKD or GFP-PKD was visualized by autoradiography.
Autoradiographs were scanned in a GS-710 Calibrated Imaging
Densitometer (Bio-Rad), and the labeled band was quantified using the
Quantity OneTM software program.
-32P]ATP
was separated from the labeled substrate by washing the P-81 paper four
times for 5 min, in 75 mM H3PO4.
The papers were dried, and the radioactivity incorporated into
syntide-2 was determined by Cerenkov counting.
-32P]ATP (370 MBq/ml),
horseradish peroxidase-conjugated donkey anti-rabbit IgG, and enhanced
chemiluminescence reagents were from Amersham Biosciences. Protein-A
agarose and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride
were from Roche Molecular Biochemicals. LPA, PDB, GF 109203X, GF V,
U-0126, PD 098059, and Clostridium difficile toxin B were
obtained from Sigma. Ro 31-8220, U73122, and PTx were purchased from
Calbiochem (La Jolla, CA). Opti-MEM and LipofectAMINE 2000 reagent were
from Invitrogen. PA-1 antiserum was raised against the synthetic
peptide EEREMKALSERVSIL that corresponds to the carboxyl-terminal
region of the predicted amino acid sequence of PKD, as previously
described (9, 60). Phospho-PKD Ser-744/748 antibody was obtained from
Cell Signaling Technology (Beverly, MA). Anti-phospho-ERK-1/2
monoclonal antibody was obtained from New England Biolabs.
Anti-G
q/11, anti-G
12/13, anti-GFP, and
anti-ERK-2 polyclonal antibodies were from Santa Cruz Biotechnologies (Palo Alto, CA). Other items were from standard suppliers or as specifically indicated.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
q/11, and
G
12/13. Initially, we determined whether LPA induces PKD
activation in RK cells, used as controls. Cultures of these cells were
stimulated with increasing concentrations of LPA (0.01-10
µM) for 10 min and lysed. PKD was immunoprecipitated from
the extracts of these cells and the immune complexes were incubated
with [
-32P]ATP, subjected to SDS-PAGE, and analyzed by
autoradiography to detect the prominent 110-kDa band corresponding to
autophosphorylated PKD.
View larger version (40K):
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Fig. 1.
LPA activates PKD in a
dose-dependent and PLC/PKC-dependent manner in
RK cells. A, LPA activates PKD in a
dose-dependent manner. Confluent and quiescent cultures of
RK cells were incubated with various concentrations of LPA for 10 min
at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated
with PA-1 antiserum. Top panel, in vitro kinase
(IVK), PKD activity was determined by an in vitro
kinase assay as described under "Experimental Procedures." followed
by SDS-PAGE and autoradiography. The autoradiogram shown is
representative of three independent experiments. Middle
panel, pS744, cell lysates from the experiments described
above were analyzed by SDS-PAGE and transferred to Immobilon membranes.
Western blot analysis was carried out using pS744/748 PKD antibody.
Bottom panel, scanning densitometry. The results shown are
the values (mean ± S.E. n = 3) of the level of
PKD activation by in vitro kinase obtained from scanning
densitometry expressed as a percentage of the maximum increase in
phosphorylation obtained with 3 µM LPA. Inset,
PKD activity in the immunocomplexes was measured by syntide-2
phosphorylation, as described under "Experimental Procedures." The
results expressed as an increased -fold over control in phosphorylation
represent the mean ± S.E. obtained from three independent
experiments, each performed in duplicate. B, LPA induces PKD
activation through a PLC/PKC-dependent pathway. Confluent
and quiescent RK cells were incubated for 1 h with the PKC
inhibitor GF-109203X (GF1, 3.5 µM) or Ro
31-8220 (Ro, 2.5 µM), or with PLC inhibitor,
U73122. Control cells ( ) received equivalent amounts of solvent or
GFV. The cultures were subsequently stimulated for 10 min with 3 µM LPA or 200 nM PDB at 37 °C. Cells were
lysed with lysis buffer and immunoprecipitated with PA-1 antiserum.
Top panels, in vitro kinase (IVK), PKD
activity was determined by an in vitro kinase assay, as
described under "Experimental Procedures." The autoradiogram shown
is representative of three independent experiments. Middle
panels, pS744, cell lysates from the experiments described as
the above were analyzed by SDS-PAGE and transferred to Immobilon
membranes. Western blot analysis was carried out using pS744/748 PKD
antibody. Bottom panels, PKD activity in the immunocomplexes
was measured by syntide-2 phosphorylation, as described under
"Experimental Procedures." The results expressed as an increased
-fold over control in phosphorylation represent the mean ± S.E.
obtained from three independent experiments, each performed in
duplicate.
i family (68), these
results indicated that Gi contributes to PKD activation to
a different degree in different cell contexts.
q/11 and/or G
12/13.
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Fig. 2.
Effect of PTx on the activation of PKD
induced by LPA in RK cells. A and B, confluent
and quiescent RK cells were incubated with different concentrations of
PTx as indicated for 3 h. Cells were then stimulated with (+) or
without ( ) 3 µM LPA (left panels) or 200 nM PDB (right panels) for 10 min at 37 °C.
Cultures were lysed with lysis buffer and then immunoprecipitated with
PA-1 antiserum. A, top panels, in
vitro kinase (IVK), immunoprecipitates were subjected
to in vitro kinase assay, SDS-PAGE, and autoradiography. The
autoradiograph shown is representative of three independent
experiments. Middle panels, pS744, cell
lysates from the experiments described as above were analyzed by
SDS-PAGE and transferred to Immobilon membranes. Western blot analysis
was carried out using pS744/748 PKD antibody. Bottom panels,
scanning densitometry, the results shown are the values (means ± S.E. n = 3) of the level of PKD activation obtained
from scanning densitometry expressed as a percentage of the maximum
increase in phosphorylation obtained with 3 µM LPA for 10 min at 37 °C.
, values corresponding to PKD activity from cells
incubated with PTx, and then stimulated with LPA;
, values
corresponding to PKD activity from cells incubated with PTx but without
LPA. B, PKD activity in the immunocomplexes was measured by
syntide-2 phosphorylation, as described under "Experimental
Procedures." The results expressed as an increased -fold over control
in phosphorylation represent the mean ± S.E. obtained from three
independent experiments, each performed in duplicate.
q/11 and
G
12/13. As illustrated by the Western blot
analysis of cell lysates shown in Fig.
3A, G
q/11 KO
cells did not express either G
q or G
11
whereas the G
12/13 KO cells did not express either
G
12 or G
13 while, as expected, the RK
cells express the
subunits of these G proteins.
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Fig. 3.
Comparison of three cell lines (RK,
Gq/11 KO, or G12/13 KO) in G
subunit expression and PKD activation induced by LPA or PDB.
A, Western blot analysis (W. Blot) of RK,
Gq/11, and G12/13 knockout cell lysates.
Confluent and quiescent cultures of the three type cells were
solubilized with 2× sample buffer. The lysates were analyzed by
SDS-PAGE and transferred to Immobilon membranes. Western blot analysis
was carried out using the antibody against
q/11,
12, or
13. The positions of
immunoreactive G
subunits at apparent Mr
43,000 are indicated by the arrows to the left.
B, confluent and quiescent cultures of RK cells were
incubated with (+) or without (
) 3 µM LPA or 200 nM PDB for 10 min at 37 °C. Cells were lysed with lysis
buffer and immunoprecipitated with PA-1 antiserum. Upper
panel, IVK, PKD activity was determined by an in
vitro kinase assay as described under "Experimental
Procedures," followed by SDS-PAGE and autoradiography. The
autoradiogram shown is representative of three independent experiments.
Middle panel, scanning densitometry. The results shown are
the values (mean ± S.E. n = 3) of the level of
PKD activation by in vitro kinase obtained from scanning
densitometry expressed as a percentage of the maximum increase in
phosphorylation obtained with 200 nM PDB. Lower
panel, PKD activity in the immunocomplexes was then measured
by syntide-2 phosphorylation, as described under "Experimental
Procedures." The results expressed as an increased -fold over control
in phosphorylation represent the mean ± S.E. obtained from three
independent experiments, each performed in duplicate.
q/11 KO cells did not
induce any significant increase in PKD activity, as measured in
immunocomplexes by either autophosphorylation or syntide-2
phosphorylation assays. PDB, which acts directly on PKC and thus,
bypasses the receptor-G protein interaction, induced robust stimulation
of PKD activity in G
q/11 KO cells, indicating that the
block in LPA action in these cells is upstream of the PKC/PKD cascade.
q restores
the ability of LPA to induce PKD activation. Because PKD is abundantly
expressed in Gq/11 KO cells and RK cells, we used
GFP-tagged PKD (GFP-PKD) for these co-transfection experiments and GFP
antibody to immunoprecipitate the ectopically expressed PKD.
Previously, we demonstrated that the GFP tag does not interfere with
the regulatory properties of PKD (69, 70). Gq/11 KO cells
were co-transfected with GFP-PKD, either alone or together with
pcDNA1-G
q, and the RK cells transfected with GFP-PKD
were used as the control. Three days after transfection, the
transfected cells were stimulated without or with 3 µM
LPA or 200 nM PDB. PKD was immunoprecipitated from the
lysates of transfected cells with GFP antibody, and the immune
complexes were incubated with [
-32P]ATP, subjected to
SDS-PAGE, and analyzed by autoradiography to detect the prominent
140-kDa autophosphorylated GFP-PKD. The results presented in Fig.
4 show that Gq/11 KO cells
cotransfected with wild type G
q and PKD exhibit a marked
increase in LPA-induced PKD activity (Fig. 4A) and Ser-744
phosphorylation (Fig. 4B) compared with Gq/11 KO
cells transfected with GFP-PKD alone. The increased PKD activity and
phosphorylation in Gq/11 KO cells cotransfected with wild
type G
q is comparable with that in the control RK cells transfected with GFP-PKD, which express endogenous G
q.
These results demonstrate that a functional G
q/11
pathway is necessary for mediating LPA-induced PKD activation.
View larger version (39K):
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Fig. 4.
LPA induces PKD activation in
Gq/11 KO cells transfected with
G q. A and
B, Gq/11 KO cells co-transfected with GFP-PKD
and pcDNA1 or pcDNA1 encoding wild type G
q. The
control cells (RK cells) were transfected with GFP-PKD and pcDNA1,
as indicated in this figure. Three days after transfection, the
cultures were left unstimulated (
) or stimulated (+) either with 3 µM LPA or with 200 nM PDB for 10 min and
lysed. A, upper panel, the lysates were
immunoprecipitated with GFP antibody and PKD activity in the
immunocomplexes was determined by an in vitro kinase assay
(IVK) as described under "Experimental Procedures,"
followed by SDS-PAGE and autoradiography. A representative
autoradiogram of three independent experiments is shown. Lower
panel, PKD activity in immunocomplexes was measured by
syntide-2 phosphorylation. The results expressed as an increased -fold
over control in phosphorylation represent the mean ± S.E.
obtained from three independent experiments. B, cell lysates
from the experiments described as above were analyzed by SDS-PAGE and
transferred to Immobilon membranes. Western blot (W. Blot)
analysis was carried out using pS744/748 PKD antibody (upper
panel) or GFP antiserum (lower panel).
12/13--
Recently, we demonstrated that
G
13 contributes to PKD activation through a Rho- and
PKC-dependent signaling pathway and hypothesized that PKD
activation is mediated by both G
q and G
13
in response to acute bombesin receptor stimulation (39). To assess the
role of G
12/13 in PKD activation induced by LPA, we
examined the effect of this agonist on PKD activity in
G
12/13 KO cells. As shown in Fig. 3, addition of 5 µM LPA to cultures of G
12/13 KO cells induced a lower level of PKD activation than that induced by either PDB
in these cells or by LPA in RK cells. These results imply that whereas
G
q/11 are necessary for LPA-induced PKD activation, G
12/13 are required for eliciting maximal LPA-induced
PKD activation.
12/13 KO cells. LPA promoted PKD
activation in G
12/13 KO cells in a
concentration-dependent fashion with half-maximal and maximal stimulation achieved at 0.07 and 3 µM. The
maximal increase in PKD autophosphorylation activity (4-fold) was lower
than that achieved by LPA in RK cells (6-fold). These results confirmed that LPA stimulated PKD activation in G
12/13 KO cells
with reduced effectiveness, even at the concentrations that induced a
maximal response, and implied that the expression of
G
12/13 is necessary for maximal stimulation of PKD in
response to LPA. The results shown in Fig. 5A also
demonstrate that LPA induced PKD Ser-744 phosphorylation and syntide-2
phosphorylation activity (inset) in a
concentration-dependent manner in G
12/13 KO
cells.
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Fig. 5.
LPA activates PKD in a
dose-dependent and PLC/PKC-dependent manner in
G12/13 KO cells. A, LPA activates PKD in a
dose-dependent manner. Confluent and quiescent cultures of
G12/13 knockout cells were incubated with various
concentrations of LPA for 10 min at 37 °C. Cells were lysed with
lysis buffer and immunoprecipitated with PA-1 antiserum. Top
panel, PKD activity was determined by an in vitro
kinase (IVK) assay as described under "Experimental
Procedures," followed by SDS-PAGE and autoradiography. The
autoradiogram shown is representative of three independent experiments.
Middle panel, pS744, cell lysates from the
experiments described as above were analyzed by SDS-PAGE and
transferred to Immobilon membranes. Western blot analysis was carried
out using pS744/748 PKD antibody. Bottom panel, scanning
densitometry. The results shown are the values (mean ± S.E.
n = 3) of the level of PKD activation by in
vitro kinase obtained from scanning densitometry expressed as a
percentage of the maximum increase in phosphorylation obtained with 3 µM LPA. Inset, PKD activity in the
immunocomplexes was measured by syntide-2 phosphorylation, as described
under "Experimental Procedures." The results expressed as an
increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in
duplicate. B, LPA induces PKD activation through a
PLC/PKC-dependent pathway. Confluent and quiescent
G12/13 knockout cells were incubated for 1 h with the
PKC inhibitor GF-109203X (GF1, 3.5 µM) or Ro
31-8220 (Ro, 2.5 µM), or with PLC inhibitor,
U73122. Control cells ( ) received equivalent amounts of solvent or
GFV. The cultures were subsequently stimulated for 10 min with 3 µM LPA or 200 nM PDB at 37 °C. Cells were
lysed with lysis buffer and immunoprecipitated with PA-1 antiserum.
Top panels, IVK, PKD activity was determined by
an in vitro kinase assay, as described under "Experimental
Procedures." The autoradiogram shown is representative of three
independent experiments. Middle panels,
pS744, cell lysates from the experiments described as the
above were analyzed by SDS-PAGE and transferred to Immobilon membranes.
Western blot analysis was carried out using pS744/748 PKD antibody.
Bottom panels, PKD activity in the immunocomplexes was
measured by syntide-2 phosphorylation, as described under
"Experimental Procedures." The results expressed as an increased
-fold over control in phosphorylation represent the mean ± S.E.
obtained from three independent experiments, each performed in
duplicate.
12/13 KO Cells--
We hypothesized
that PKD activation and activation loop phosphorylation are elicited by
LPA and other GPCR agonists through interaction of complementary G
protein pathways, namely, Gq, G
12/13, and
Gi (see Introduction for references). Although the effect of Gi is not prominent in LPA-induced PKD activation in RK
cells, our hypothesis predicts that in G
12/13 KO cells,
members of the Gq family interact with members of the
Gi family in mediating LPA-induced PKD activation. To test
the contribution of Gi to LPA-induced PKD activation in
G
12/13 KO cells, cultures of these cells were treated
with increasing concentrations of PTx (1-100 ng/ml) and PKD activity
in immunocomplexes was measured by either autophosphorylation or
syntide-2 phosphorylation assays.
12/13 KO cells. We conclude that in
G
12/13 KO cells, Gi cooperates with
Gq in mediating the PKC/PKD phosphorylation cascade in
response to LPA.
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Fig. 6.
Effect of PTx on the activation of PKD
induced by LPA in G12/13 KO cells. A and
B, confluent and quiescent G12/13 KO cells were
incubated with different concentration of PTx as indicated for 3 h. Cells were then stimulated with (+) or without ( ) 3 µM LPA (left panels) or 200 nM PDB
(right panels) for 10 min at 37 °C. Cultures were lysed
with lysis buffer and then immunoprecipitated with PA-1 antiserum.
A, top panels, in vitro kinase
(IVK), immunoprecipitates were subjected to in
vitro kinase assay, SDS-PAGE, and autoradiography. The
autoradiograph shown is representative of three independent
experiments. Middle panels, cell lysates from the
experiments described above were analyzed by SDS-PAGE and transferred
to Immobilon membranes. Western blot analysis was carried out using
pS744/748 PKD antibody. Bottom panels, scanning
densitometry, the results shown are the values (mean ± S.E.,
n = 3) of the level of PKD activation obtained from
scanning densitometry expressed as a percentage of the maximum increase
in phosphorylation obtained with 3 µM LPA for 10 min at
37 °C.
, values corresponding to PKD activity from cells
incubated with PTx, and then stimulated with LPA;
, values
corresponding to PKD activity from cells incubated with PTx but without
LPA. B, PKD activity in the immunocomplexes was measured by
syntide-2 phosphorylation, as described under "Experimental
Procedures." The results expressed as an increased -fold over control
in phosphorylation represent the mean ± S.E. obtained from three
independent experiments, each performed in duplicate.
q,
G
13-mediated signaling contributes to PKD activation in
bombesin-stimulated cells through endogenous Rho (39). C. difficile toxin monoglucosylates the threonine residue at position
35 in Rac and Cdc-42 and threonine 37 in Rho and thereby inactivates
these small G proteins (71, 72). In view of the results shown in Fig. 6
with G
12/13 KO cells, we hypothesized that inactivation
of Rho GTPases with C. difficile toxin B in RK cells should
enhance the sensitivity of LPA-induced PKD activation to treatment with
PTx. To test this hypothesis, RK cells were treated with PTx either
with or without C. difficile toxin B and then stimulated
with either LPA or PDB, as indicated in Fig.
7. Treatment with C. difficile
toxin B markedly enhanced the ability of PTx to inhibit LPA-induced PKD
activation (Fig. 7A) and Ser-744 phosphorylation (Fig.
7B) in RK cells. These results provide an independent line
of evidence indicating that PKD activation induced by LPA is mediated
by cooperation of multiple G protein pathways.
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Fig. 7.
PKD activation induced by LPA in RK cells is
partially blocked by C. difficile toxin B (toxin
B) and almost abolished by the combination treatments of the
cells with toxin B and Ptx. A and B, confluent
and quiescent RK cells were preincubated with (+) or without ( ) PTx
(50 ng/ml) for 3 h and then treated with different concentrations
of toxin B as indicated for 1.5 h. Cells were then stimulated with
(+) or without (
) 3 µM LPA or 200 nM PDB
for 10 min at 37 °C. Cultures were lysed with lysis buffer and then
immunoprecipitated with PA-1 antiserum. A, upper
panel, in vitro kinase (IVK),
immunoprecipitates were subjected to in vitro kinase assay,
SDS-PAGE, and autoradiography. The autoradiograph shown is
representative of three independent experiments. Lower
panel, PKD activity in the immunocomplexes was measured by
syntide-2 phosphorylation, as described under "Experimental
Procedures." The results expressed as an increased -fold over control
in phosphorylation represent the mean ± S.E. obtained from three
independent experiments, each performed in duplicate. B,
pS744, cell lysates from the experiments described above
were analyzed by SDS-PAGE and transferred to Immobilon membranes.
Western blot analysis was carried out using pS744/748 PKD
antibody.
q/11 KO, and G
12/13 KO
Cells--
To substantiate that the different profiles of PKD
activation in response to LPA in the three cell lines used in our study is specific and reflects the participation of multiple G proteins, we
examined other biological effects induced by LPA in these cells.
subunits of Gi (47-51).
Here, we determined the effect of PTx on LPA-induced ERK activation in
these three types of cells. As shown by Western blot analysis using an
antibody that detects the dually phosphorylated and active ERK-1 and
ERK-2, LPA induced robust activation of the ERKs in RK,
G
12/13 KO, and G
q/11 KO cells (Fig.
8). Treatment with PDB also induced
strong ERK activation in the three cell lines, indicating the existence of a PKC-dependent pathway.
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Fig. 8.
LPA induces activation of ERK-2 and ERK-1 in
RK cells and Gq/11 KO or G12/13 KO cells.
Confluent and quiescent RK cells (A) and Gq/11
KO cells (B) or G12/13 KO cells (C)
were incubated with PTx for 3 h or with the other inhibitors,
GF-109203X (GF1, 3.5 µM), or Ro 31-8220
(Ro, 2.5 µM), or U0126 (U, 2.5 µM), as indicated, for 1 h. Control cells ( )
received an equivalent amount of solvent. Cells were then stimulated
with (+) or without (
) 3 µM LPA or 200 nM
PDB for 10 min at 37 °C. Cultures were lysed with lysis buffer. Cell
lysates from the experiments were analyzed by SDS-PAGE and transferred
to Immobilon membranes. A-C, upper panels,
Western blot analysis with specific antiphospho-ERK-1/2 monoclonal
antibody; lower panels, the Western blot was also probed for
total ERK by ERK-2 polyclonal antibody.
12/13 KO cells and
G
q/11 KO cells. Specifically, LPA promoted ERK
activation in both G
12/13 KO cells and
G
q/11 KO cells through a
Gi-dependent but PKC-independent pathway (Fig.
8). These results demonstrate that the lack of expression of these G
proteins does not interfere with other biological effects of LPA
mediated by Gi and provide further support to the
conclusion that LPA utilizes different G protein pathways to activate
parallel phosphorylation cascades leading to PKD and ERK activation in RK cells.
q- and G
13-mediated signaling can
promote PKD activation in intact cells (20, 39), it was surprising that
treatment of these cells with PTx markedly inhibited PKD activation in
response to LPA (56, 57). Because PTx catalyzes the ADP-ribosylation
and inactivation of members of the G
i family (68), these
results identified the involvement of an additional Gi-dependent pathway leading to PKD activation
in response to LPA. However, other results also demonstrated that LPA
induces PKD activation through a pathway that was only partially
attenuated by PTx in IEC-18 cells (57) indicating that Gi
contributes to PKD activation to different degrees in different cell
contexts. In the present study, we tested the hypothesis that LPA
induces PKD activation through multiple G protein signal transduction pathways, including Gq, Gi, and
G13, using mouse embryonic cell lines deficient in
G
q/11 and G
12/13 and using a cell line
deficient in rhodopsin kinase (RK cells), as a control.
q/11 or G
12/13. A crucial role of G
q/11 in
mediating LPA-induced PKD activation is clearly demonstrated by the
fact that LPA-induced PKD activation was completely abrogated in
G
q/11 KO cells and that transfection of
G
q into the Gq/11 KO cells restored
LPA-induced PKD activation to the similar level as the control cells.
In G
12/13 KO cells, LPA induced substantial PKD
activation, a response predicted by the model to be mediated by
cooperation of Gq and Gi. In line with this
interpretation and in sharp contrast to the results obtained with RK
cells, treatment of G
12/13 KO cells with PTx dramatically inhibited PKD activation in response to LPA. These results
are also consistent with the notion that endogenous
G
q/11 is not sufficient to mediate PKD activation in
response to LPA. Thus, our model integrates the PTx-sensitive and
-insensitive effects obtained in G
12/13 KO cells and RK
cells with the striking suppression of LPA-induced PKD activation in
G
q/11 KO cells and leads to the notion that LPA induces
PKD activation through the synergistic interaction of pathways
initiated by Gq, G12, and Gi. A
corollary of these results is that the level of expression of the
different
subunits of heterotrimeric G proteins can be identified
as one of the important molecular elements that determine the influence
of cell context on agonist-induced PKD activation.
B by LPA is mediated by Gq
and Gi pathways (73) and the activation of the serum
response element is mediated by cooperative effects between
Gi and G
13/Rho pathways (74). Furthermore,
recent genetic studies indicate that the Gq-mediated
signaling pathway functionally interacts with the
G12-mediated signaling pathway to promote embryonic
survival (63).
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Fig. 9.
Signal transduction pathways involved in
LPA-induced PKD activation. The scheme proposes a model that
envisages that PKD activation in response to LPA receptor signaling is
mediated through interaction of complementary G protein pathways,
namely, Gq/11, Gi, and G12/13, and
also illustrates the molecular, cellular, and pharmacological
approaches used in this study. As shown in this scheme,
G q/11 plays a crucial role (expressed by thick
arrows) in mediating LPA-induced PKD activation through a PLC- and
PKC-dependent pathway, which is clearly demonstrated by the
fact that LPA-induced PKD activation and phosphorylation were
completely blocked (
) in Gq/11 knockout
(Gq/11KO) cells and that transfection of G
q
into the Gq/11 KO cells restores (
) LPA-induced PKD
activation to the similar level as the control cells. However, LPA
induced maximal PKD activation requires the synergistic interaction of
pathways initiated by Gq, G12, and
Gi. G12/13 mediates PKD activation through Rho
and PKC, and Gi mediates PKD activation through PLC and
PKC. In G12/13 knockout (G12/13 KO) cells, or
in the RK cells treated with C. difficile toxin B (which
inactivates Rho GTPases), or with Gi inhibitor, PTx,
i.e. when either G12/13/Rho pathway alone or
Gi pathway alone is blocked, LPA induces a reduced but
substantial PKD activation. But, treatment of G12/13 KO
cells with PTx, or a combination treatment of RK cells with Ptx and
C. difficile toxin B, which resulted in the block of both
G12/13 and Gi pathways, completely prevented
LPA-induced PKD activation. In addition, this scheme also shows that
LPA-induced signal transduction pathways initiated by Gq,
G12, and Gi leading to PKD activation is PLC
and PKC-dependent, which has been verified by the fact that
in either RK cells or G12/13 KO cells, LPA induced-PKD
activation and phosphorylation could be blocked (
) by U73122 (PLC
inhibitor) or GF1 or Ro 31-8220 (Ro) (selective inhibitors
of PKC). Therefore, PKC/PKD activation is a point of convergence in the
action of multiple G protein pathways. (Also see the text for details
and abbreviations). Ins(1,4,5)P3, inositol
1,4,5-trisphosphate.
![]() |
ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. Melvin I. Simon, Division of Biology, California Institute of Technology, for the generous gift of fibroblast cell lines. We also thank Steven H. Young for assistance in cell transfection and J. Sinnett-Smith, Cliff Hurd, and Osvaldo Rey for helpful discussions and careful reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Health Institute Grants DK 55003, DK56930, DK 17294, and NCI Grant P50 CA 90388-01.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.
§ Supported by National Institutes of Health National Research Service Award F32 CA84658-01A1.
To whom all correspondence should be addressed: Dept. of
Medicine, UCLA School of Medicine, 900 Veterans Ave., Warren Hall Rm.
11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399; E-mail: erozengurt@mednet.ucla.edu.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M211175200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PKC, protein kinase
C;
LPA, lysophosphatidic acid;
G proteins, guanine nucleotide-binding
regulatory proteins;
GPCRs, G protein-coupled receptors;
GFP, green
fluorescent protein;
PDB, phorbol 12,13-dibutyrate;
PKD, protein kinase
D;
RK cells, murine embryonic cell line with rhodopsin kinase gene
knockout;
Gq/11 KO cells, murine embryonic cell line
with G
q/11 gene knockout;
G
12/13 KO
cells, murine embryonic cell line with G
12/13 gene
knockout;
PTx, pertussis toxin;
PLC, phospholipase C;
ERK, extracellular signal-regulated kinase.
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