From the Department of Molecular Physiology and Biophysics and the Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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G13 belongs to the
G12 family of heterotrimeric G proteins, whose effectors
are poorly defined. The present study was designed to test if
phospholipase D (PLD) is regulated by G13 and if Rho-type small GTPases are involved. Expression of the constitutively active Q226L mutant of the -subunit of G13 in COS-7 cells
stimulated the activity of a rat brain phospholipase D isozyme (rPLD1)
co-expressed in the cells. Wild type G
13 was ineffective
unless the cells were incubated with
AlF4
. rPLD1 was
previously shown to be activated by constitutively active V14RhoA in
COS-7 cells (Park, S. K., Provost, J. J., Bae, C. D., Ho, W. T., and Exton, J. H. (1997) J. Biol.
Chem. 272, 29263-29272). When the endogenous Rho proteins of the
cells were inactivated by treatment with C3 exoenzyme from
Clostridium botulinum, the ability of
G
13Q226L to activate rPLD1 was greatly attenuated. Co-transfection of dominant negative N19RhoA and N17Rac-1, but not
N17Cdc42Hs or N17Ras, also inhibited the activation. Expression of
constitutively active G
q in COS-7 cells also activated
rPLD1, but constitutively active forms of G
i2 and
G
s were without effect. These findings support an
effector role for PLD in G13 signaling and demonstrate a
requirement for Rho GTPases in this response.
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INTRODUCTION |
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Phosphatidylcholine (PC)1 specific phospholipase D (PLD) hydrolyzes its substrate into phosphatidic acid (PA) and choline in response to growth factors and G protein-coupled receptor agonists (1). PA may act as a lipid messenger in the cell, inducing cytoskeletal rearrangements or growth-regulatory responses, and is implicated in the regulation of NADPH-oxidase and intracellular membrane trafficking and fusion events (1, 2). PA can also be converted to the protein kinase C (PKC) activator diacylglycerol by phosphatidic acid phosphohydrolase or to the G protein-coupled receptor agonist lysophosphatidic acid by a PA-specific phospholipase A2 (1).
Analysis of PLD activity from various tissues and subcellular fractions supports the existence of biochemically distinct isozymes differing in subcellular localization and responses to Ca2+, phosphatidylinositol 4,5-bisphosphate (PIP2), oleate, and detergents in vitro (1, 3). Studies of signal transduction pathways in intact cells support the involvement of PKC and Rho GTPases in the regulation of PLD (1, 2). In vitro experiments confirm a direct regulatory role for PKC, Rho-family, and ARF GTPases in PLD activation (1, 2). PIP2 also activates PLD in vitro and may be required for PLD regulation in vivo (1).
Mammalian PLD isoforms have been cloned including hPLD1a, hPLD1b, hPLD2, and rPLD2 (4-7). hPLD1a, a 1072-amino acid protein, is specific for PC and is regulated by PKC, ARF, RhoA, and PIP2 (4, 5). hPLD1b encodes a 1034-amino acid splice variant with properties similar to hPLD1a (5). hPLD2 is also PC-specific, has high basal activity both in vitro and in vivo, and is activated by PIP2, but not PKC, ARF, or RhoA (6).
rPLD1, cloned in our laboratory from a rat brain library using a
fragment of hPLD1, is 91% identical to hPLD1b in amino acid sequence
and is expressed in a wide variety of tissues (8). It is stimulated
when co-expressed with constitutively active V14RhoA in COS-7 cells (8)
and also by treatment of the cells with a PKC-activating phorbol ester
(8) or lysophosphatidic acid.2 Like hPLD1, rPLD1 is
activated directly by RhoA, Arf, and PKC in
vitro.2
The Rho-related G proteins Rho, Rac, and Cdc42 are members of the Ras superfamily of low molecular weight GTPases. They regulate multiple downstream effects, including the contractility and organization of the actin cytoskeleton (9-12). Rho is required for cytoskeletal, transcriptional, and PLD responses to some G protein-coupled receptor agonists (2, 12-14), although the pathways involved and the precise nature of the Rho-requirement remain to be defined.
G13, a member of the G12 family of
heterotrimeric G proteins, was first identified by the cDNA cloning
of its -subunit (15, 16). G13 is expressed in most cell
lines and tissues and is especially abundant in human platelets (17).
G13 is coupled to platelet thrombin and thromboxane
A2 receptors (18, 19), suggesting a possible role in
platelet activation. Immediate downstream effectors have not been
identified for the G12 family, although downstream effects
resulting from overexpression of constitutively active mutant forms of
G
12 and G
13 have been described,
including neoplastic transformation of cultured fibroblasts (20, 21), increased amiloride-sensitive sodium-proton exchange (22-24),
induction of immediate early gene expression (13, 25), actin stress fiber formation and focal adhesion assembly (26), and activation of the
c-Jun N-terminal kinase (JNK) cascade (27-29). A number of these
effects can be blocked by dominant negative mutant forms of Rho-related
G proteins, indicating a requirement at some level for these low
molecular weight GTPases (13, 16, 23, 26-30).
The present study examines if PLD is a downstream effector of G13 and if Rho family GTPases are involved in the signal transduction pathway.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs--
A partial G13 cDNA,
reverse transcription-polymerase chain reaction amplified from mouse
brain total RNA, was used to clone a full-length cDNA from a mouse
liver cDNA library (Stratagene) using standard methods. A
G
13Q226L mutant was made by overlap-extension polymerase
chain reaction and confirmed by DNA sequencing. Wild type and Q226L
were subcloned into the HindIII and EcoRI sites of the pcDNA3 expression vector (Invitrogen). rPLD1 in pcDNA3 (8), N19RhoA in pcDNA3, and G
qQ209L in pCMV4 were
described previously (31). G
sQL and G
i2QL
in pcDNA3 were provided by Dr. N. Dhanasekaran (Temple University,
Philadelphia, PA), N17Cdc42 in pcDNA3 was provided by Dr. Shubha
Bagrodia (Cornell University, Ithaca, NY), and N17Rac-1 in pCGT was
provided by Dr. Linda Van Aelst (Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY).
Cell Culture and Transfection-- COS-7 cells (ATCC) were maintained in Dulbecco's modified Eagles's medium (Life Technologies, Inc.) plus 10% fetal bovine serum (Life Technologies, Inc.) under 5% CO2. Six-well plates were seeded with 2 × 105 cells/well and transfected with 2 µg of plasmid DNA and 6 µl of LipofectAMINE (Life Technologies, Inc.) per well according to the manufacturer's instructions. Opti-MEM (Life Technologies, Inc.) was substituted for Dulbecco's modified Eagles's medium during reduced serum incubations.
C3 Scrape Loading-- Cells were washed 24 h post-transfection, twice with phosphate-buffered saline (PBS) and once with scraping buffer (114 mM KCl, 15 mM NaCl, 5.5 mM MgCl2, 10 mM Tris-Cl pH 7.4) and then scraped off the plate in the absence or presence of 5 µg/ml Clostridium botulinum C3 exoenzyme (List Biologicals) as described (14). Cells were replated on polylysine and serum-starved 12 h later.
PLD Assay-- Cells were serum-starved (0.5% fetal bovine serum in Opti-MEM) at 24 h post-transfection for 16 h in the presence of 1 µCi/ml [3H]myristate (NEN Life Science Products), washed with PBS, and incubated in serum-free medium (Opti-MEM) for 50 min. PLD activity was then assayed as described previously (14). Cells were incubated in 0.3% 1-butanol for the indicated times. Cells were then washed with ice-cold PBS and stopped with methanol. Lipids were extracted, and the phosphatidylbutanol product was resolved by thin layer chromatography as described (14). Bands co-migrating with a phosphatidylbutanol standard were quantitated by scintillation counting.
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RESULTS |
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PLD Activation by G13--
To study the effect of
G13 signaling on PLD, we expressed wild type
G
13 and GTPase-deficient, constitutively active
G
13Q226L together with a Rho-responsive PLD isoform,
rPLD1, by liposome-mediated transient transfection of COS-7 cells. High
level expression of the proteins and similar levels of rPLD1 expression
between different co-transfections were confirmed by Western analysis
(not shown). Expression of wild type G
13 had little or
no effect on either endogenous PLD activity or that of rPLD1.
G
13Q226L also had no effect on the activity of the
endogenous PLD, but markedly stimulated rPLD1 activity (Fig.
1A). This activation of rPLD1
by G
13Q226L was consistent with an effector role for PLD
in G13 signaling. However, since G
13
signaling was chronically activated by the prolonged expression of the
Q226L mutant in this experiment, it was not clear how direct or
indirect the activation of rPLD1 might be.
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Requirement for Rho Family GTPases--
To investigate the
potential role of Rho in the regulation of rPLD1 by G13,
we inhibited its function by treating the cells with C3 exoenzyme from
C. botulinum, an ADP-ribosyltransferase that inactivates Rho
(33). Efficient ADP-ribosylation was confirmed by in vitro
assays with C3 (33), which showed that cellular Rho from the C3-scraped
cells was efficiently modified as indicated by a subsequent loss of
ADP-ribosylation in vitro (Fig.
2A). We then looked at the
effect of C3 toxin on the activation of rPLD1 by
G
13Q226L. C3 had little or no effect on the endogenous
PLD activity or that of the expressed rPLD1. It did, however,
significantly diminish the effect of G
13Q226L on rPLD1
activity, consistent with a requirement for Rho in the activation of
rPLD1 by active G
13 (Fig. 2B).
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rPLD1 Is Activated by Mutationally Active -Subunits of the
G12 and Gq Families--
To determine the
selectivity of G12 family proteins in the activation of
rPLD1, we examined its regulation by other heterotrimeric G protein
-subunits. Accordingly, we expressed mutationally active
-subunits of the G12, Gq, Gi,
and Gs families with and without rPLD1 and then measured
PLD activity (Fig. 4). We found that the activity of rPLD1 was potently stimulated by constitutively active G
13Q226L and G
qQ209L, but not by
G
i2Q205L or G
sQ227L.
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DISCUSSION |
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This report supports the hypothesis that rPLD1, a widely expressed
rat hPLD1b homolog, serves as a downstream effector in G13
signaling. Both mutational activation and fluoride stimulation of
expressed G13 led to potent stimulation of co-expressed
rPLD1 activity (Fig. 1).3 The
acute regulation by fluoride-stimulated G
13 rules out
mechanisms requiring long term activation of the pathway and is
consistent with an effector role for
rPLD1.4
Mutational and fluoride-stimulated activation of G13 had
no effect on endogenous PLD (Fig. 1). This is probably due to
differences between rPLD1 and the endogenous PLD in COS cells. Previous
work in this laboratory has indicated the absence of Rho-reponsive PLD
activity in COS cells (8). This has been observed both in cells
transfected with V14RhoA and also with in vitro assays performed by incubation of COS cell extracts with RhoA and GTPS (8). In
both cases, the Rho response could be reconstituted by expression of
rPLD1 (8).
We found that C3 toxin, which is selective for Rho (33), significantly
blocked rPLD1 activation by G13 (Fig. 2). Dominant negative N19RhoA and N17Rac-1 alone partially but significantly blocked
rPLD1 activation and, in combination, completely blocked the effect
(Fig. 3). These data support a requirement for guanine nucleotide
exchange factor-dependent regulation of Rho family G
proteins such as Rho and Rac in this response (34). Cytochalasin D
treatment of the cells did not block the response (not shown), suggesting that the inhibitory effects of C3 toxin, N19RhoA, and N17Rac
are not simply due to a general disruption of signaling responses as a
consequence of altered cytoskeletal organization or function induced by
these treatments. All together, these data are consistent with the
involvement of Rho family G proteins in the signaling pathway between
G
13 and rPLD1.
We showed that rPLD1 was also activated by mutationally active
Gq, but not by G
i2 or G
s.
This observation is consistent with reports of PLD activation by
Gq-coupled receptors and PKC-activating phorbol esters (1).
rPLD1 is stimulated by the
- and
-isozymes of PKC in
vitro2 and by phorbol ester when expressed in COS-7
cells (8). Our results indicate that rPLD1 is selectively activated by
-subunits of the G12 and Gq families,
although
-subunit mediated regulation of rPLD1 or activation of
other PLD isoforms by heterotrimeric G proteins other than
Gq and G12 family members remain a
possibility.
G13Q226L expression triggers actin stress fiber
formation and focal adhesion assembly (26), JNK activation (27-29),
sodium-proton exchange (23, 24), serum response
element-dependent gene transcription (13), and apoptosis
(30) by pathways also inhibited by dominant negative Rho family G
proteins. For several reasons, rPLD1 activation is likely to occur
either upstream of these effects or by an entirely separate pathway.
First, Rho family G proteins are direct regulators of
rPLD13 (5), while the G
13-mediated effects
listed above are likely to be downstream of a cascade of Rho-initiated
signals. Second, cytochalasin D disrupts cytoskeletal responses, but
did not inhibit rPLD1 activation (not shown). Apoptotic responses and
gene induction typically require hours to develop, while rPLD1 is
activated within minutes by fluoride-stimulated G
13
(Fig. 1). Finally, activation of the JNK cascade by G
13
in COS-7 cells is blocked by N17Ras, but not N19RhoA (27-29), while
rPLD1 activation is blocked by N19RhoA, but not N17Ras (Fig. 3).
Phospholipases are key components of transmembrane signal transduction pathways. PLD-catalyzed production of phosphatidylcholine-derived messengers could mediate critical downstream responses in G13 signaling. Further study is needed to more precisely define the role of PLD in G13-mediated responses. We suggest that G13 may play a role in the regulation of Rho-responsive PLD activity by G protein-coupled receptors.
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FOOTNOTES |
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* This work was supported by Grants DK 47448 and DK 07563 from the National Institutes of Health.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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton{at}mcmail.vanderbilt.edu.
1 The abbreviations used are: PC, phosphatidylcholine; PLD, phospholipase D; PA, phosphatidic acid; PKC, protein kinase C; PIP2, phosphatidylinositol 4,5-bisphosphate; PBS, phosphate-buffered saline; JNK, c-Jun N-terminal kinase.
2 S. K. Park, D. S. Min, and J. H. Exton, unpublished observations.
3
The related G12Q229L was also
found to activate rPLD1 (S. G. Plonk and J. H. Exton,
unpublished observations).
4 Acute activation of rPLD1 was also observed in thromboxane A2-treated COS cells expressing thromboxane A2 receptors (S. G. Plonk and J. H. Exton, unpublished observations).
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REFERENCES |
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