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INTRODUCTION |
Bradykinin (BK)1 is
rapidly generated following inflammation or injury. The released BK is
known to mediate multiple proinflammatory effects including smooth
muscle contraction, pain, vasodilatation, increased vascular
permeability, eicosanoid synthesis, and neuropeptide release. We
recently reported that BK can also activate the transcription factor
NF-
B and stimulate proinflammatory cytokine synthesis in human
fibroblasts and epithelial cells (1, 2). BK-stimulated NF-
B
activation was shown to be mediated through the G protein-coupled B2 BK
receptor and was pertussis toxin sensitive (1). The small GTPase RhoA
was also shown to be both necessary and sufficient for BK-stimulated
NF-
B activation (2).
Phosphatidylinositol 3-kinase (PI 3-kinase) is a ubiquitous lipid
kinase that phosphorylates the 3-position of the inositol ring of
inositol phospholipids to generate such lipid messengers as
phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The exact role and signal molecular
targets of these lipid products has not yet been determined, however, increasing evidence suggests that they may serve as intracellular second messengers (3-5). PI 3-kinase is a heterodimer consisting of a
p85 regulatory subunit with SH2 domains and a p110 catalytic subunit
(5, 6). In the regulation of receptor-mediated intracellular pathways,
including G-protein-coupled receptor, PI 3-kinase has been reported to
be directly activated by 
subunits released from activated G
proteins (7).
We therefore investigated the role of PI 3-kinase in the signaling
events that lead to NF-
B activation in BK-stimulated A549 human
transformed epithelial cells. In this report we show that BK stimulates
PI 3-kinase activity in A549 cells and that inhibition of PI 3-kinase
blocks BK-stimulated NF-
B activation. These results indicate that PI
3-kinase is a novel signal transducer for BK-induced NF-
B activation
in airway epithelial cells.
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EXPERIMENTAL PROCEDURES |
Reagents--
BK was obtained from Peninsula Laboratories
(Belmont, CA). The human lung adenocarcinoma cell line, A549, (distal
respiratory epithelium-like) was obtained from the American Type
Culture Collection (Manassas, VA). A549 cells were maintained in Ham's
F12K medium containing 10% fetal bovine serum at 37 °C in a
humidified 5% CO2 environment. Immediately before
stimulation, A549 cells were changed into serum-free RPMI 1640 (Irvine
Scientific). Oligonucleotides and their complementary strands for
electrophoretic mobility shift assays (EMSA) were from Promega
(Madison, WI) and Santa Cruz Biotechnology. The sequences were a
consensus
B site (underlined),
5'-AGTTGAGGGGACTTTCCCAGGC-3' (NF-
B) (8), and a mutant
B site with the G to C substitution (underlined) in the
B binding
motif, 5'-AGTTGAGGCGACTTTCCCAGGC-3'. [
-32P]ATP (>5000 Ci/mmol) was from Amersham Pharmacia
Biotech. The plasmid pmTNF
(a gift from Vladimir Kravchenko, The
Scripps Research Institute La Jolla, CA) was used for preparation of
recombinant murine TNF
from Escherichia coli. The
specific activity of TNF
purified by ion-exchange chromatography was
7 × 107 units/mg protein. The p85 dominant negative
plasmid, p85
N-C
478-514, was constructed using a previously
published method (9).
EMSA--
Nuclear extracts were prepared from A549 cells plated
at a density of 1 × 106 cells in a 6-well plate using
a modified method of Dignam et al. (10), and EMSA were
performed using 2.5 µg of the nuclear extract as described previously
(2).
PI 3-Kinase Assay--
Aliquots of cell lysates normalized for
protein content were incubated for 3 h with anti-PI 3-kinase
antibodies directed against the 85-kDa regulatory subunit (Upstate
Biotechnology, Lake Placid, NY). The immune complexes were absorbed
onto protein A-Sepharose and washed as described (11). PI 3-kinase
assays were performed directly on beads. Briefly, the reaction was
carried out for 10 min in a buffer containing 40 mM HEPES,
pH 7.2, 6 mM MgCl2, 1 mM EDTA, 10 µg of PI
(Avanti Polar Lipids, Alabaster, AL), 10 µM ATP, and 10 µCi [
-32P]ATP (6,000 Ci/mmol; NEN Life Science
Products). Adenosine (0.2 mM) was added to the reaction
mixture to inhibit residual PI 4-kinase activity. After the incubation,
the reaction was stopped with methanol plus 2.4 N HCl (1:1, v/v), and
lipids were extracted and analyzed by thin-layer chromatography.
Chloramphenicol Acetyltransferase (CAT) Assay--
The plasmids
p0.2kb[WT]CAT (WT-I
B-CAT) and p0.2kb[MU]CAT (MU-I
B-CAT),
which contain the wild-type and mutant
B enhancers from the I
B
gene, respectively, were obtained from P. Chiao (The University of
Texas M. D. Anderson Cancer Center) (12). The pSVL-CAT plasmid
(Amersham Pharmacia Biotech) was used as a positive control for CAT
expression. The plasmid pCMV
(CLONTECH) was used as a control for monitoring the transfection efficiency by the expression of
-galactosidase. Transient transfection of the A549 cells and measurement of CAT activity were performed exactly as described previously (2).
Isolation of Transfected A549 Cells--
Transfected A549 cells
were specifically isolated using the Capture-Tec pHook-2 system
(Invitrogen) according to the protocol of the manufacturer. Briefly,
4 × 106 A549 cells were transfected with the pHook-2
plasmid that directs the synthesis of a fusion protein containing the
platelet-derived growth factor receptor transmembrane domain fused to a
single-stranded cell surface antibody recognizing the hapten phOx.
Transfected cells were then recovered by incubating the cells in
suspension for 30 min at 37 °C with 2 × 106
magnetic beads coupled to phOx followed by magnetic separation.
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RESULTS |
BK-induced Activation of NF-
B Is Blocked by PI 3-Kinase
Inhibitors--
To assess the role of PI 3-kinase in BK-induced
NF-
B activation, we examined the consequences of pre-incubating
cells with PI 3-kinase inhibitors. Wortmannin and LY294002 have both
been shown to specifically inhibit PI 3-kinase in multiple cell types with distinct and different modes of action (13). Following pretreatment with wortmannin, LY294002, or media control, A549 cells
were stimulated with BK or TNF
, and NF-
B activation was assessed
by EMSA. Whereas the DNA binding activity of NF-
B was potently
induced by BK and TNF
(Fig. 1,
lanes 2-3), BK-induced NF-
B activation was completely
inhibited in A549 cells pretreated with wortmannin (Fig. 1, lane
4) or LY294002 (Fig. 1, lane 6). In contrast, neither
wortmannin nor LY294002 had an effect on TNF
-induced NF-
B
activation (Fig. 1, lanes 5 and 7). These results suggest that PI 3-kinase activity is required for BK- but not TNF
-induced NF-
B activation.

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Fig. 1.
The PI 3-kinase inhibitors wortmannin and
LY294002 abolish BK-induced NF- B
activation. A549 cells were pre-incubated with media alone
(lanes 1-3), 100 nM wortmannin (lanes
4-5), or 50 µM LY294002 (lanes 6-7) for
15 min, then stimulated with 20 nM BK (lanes 2, 4, and 6), or 40 ng/ml TNF (lanes 3, 5, and
7) for 40 min. Nuclear extracts were prepared, and NF- B
activation was measured by EMSA as described under "Experimental
Procedures." The EMSA autoradiograph is shown with the DNA-protein
complex marked with a bracket, and the unbound probe is
indicated by an arrow. These results are representative of
two separate experiments. Med, media; WM,
wortmannin; LY, LY294002.
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We next examined the dose-response and timing of the inhibitory effect
of wortmannin on BK-induced NF-
B activation. As shown in Fig.
2A, wortmannin at doses
greater than or equal to 50 nM significantly inhibited
BK-induced NF-
B activation. To effectively inhibit BK-induced
NF-
B activation, wortmannin needed to be added to the A549 cells at
least 5 min prior to stimulation with BK (Fig. 2B).

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Fig. 2.
Wortmannin inhibits BK-stimulated
NF- B activation in a dose- and
time-dependent manner. A, A549 cells were
pre-incubated with varying concentrations of wortmannin (WM)
as shown for 15 min, then stimulated with BK (20 nM) for 40 min. NF- B activation was determined as described for Fig. 1 with the
DNA-protein complex marked with a bracket and the unbound
probe indicated by an arrow. B, A549 cells were
preincubated with 100 nM wortmannin for the indicated
times, then stimulated with BK (20 nM) for 40 min. NF- B
activation was determined as described for Fig. 1 with the DNA-protein
complex marked with a bracket and the unbound probe
indicated by an arrow. These results are representative of
three separate experiments.
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BK Stimulates a Rapid but Transient Increase in PI 3-Kinase
Activity--
The results presented above demonstrate that inhibition
of PI 3-kinase activity abrogates BK-induced NF-
B activation. We next examined whether BK would induce increased PI 3-kinase activity in
A549 cells. PI 3-kinase activity was measured using an in
vitro kinase assay using phosphatidylinositol as the substrate
(11). A549 cells were lysed at varying times following stimulation with BK, and the cellular extracts were collected for analysis for PI
3-kinase activity. BK stimulated a time-dependent increase in PI 3-kinase activity (Fig. 3). The
BK-induced increase of PI 3-kinase activity was seen within 5 min of
stimulation and peaked at 20 min. The kinetics of BK-induced PI
3-kinase activation preceded that of BK-induced NF-
B activation,
consistent with a role for PI 3-kinase in the activation of
NF-
B.

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Fig. 3.
BK-stimulates PI 3-kinase in a
time-dependent manner. A549 cells were treated with BK
(20 nM) for the indicated times. The whole cell lysates
were subjected to immunoprecipitation with a rabbit polyclonal antibody
against the p85 subunit of PI 3-kinase. PI 3-kinase activity in the
immunoprecipitated fraction was determined using an in vitro
kinase assay as described under "Experimental Procedures." The
products of the kinase assay were separated by thin-layer
chromatography as described under "Experimental Procedures."
Activity of PI 3-kinase is presented as production of
phosphatidylinositol phosphate (PIP), indicated by an
arrow. Experiments were repeated two times with essentially
identical results.
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Although the PI 3-kinase inhibitors did not effect TNF
-induced
NF-
B activation, TNF
, like BK, stimulated increased PI 3-kinase activity (Fig. 4, lane 6).
Wortmannin and LY294002 each abolished the PI 3-kinase activity
stimulated by either BK (Fig. 4, lanes 1 and 4)
or TNF
(Fig. 4, lanes 2 and 5). Thus BK
stimulates PI 3-kinase activity, and this activity is required for
subsequent NF-
B activation.

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Fig. 4.
Wortmannin and LY294002 inhibit BK- and
TNF -stimulated PI 3-kinase activity. A549
cells were preincubated with 100 nM wortmannin
(WM, lanes 1 and 2), media alone
(lanes 3 and 6), or LY294002
(LY, lanes 4 and 5) for 15 min, then
stimulated (Stim.) with 20 nM BK (lanes 1, 3, and 4), or 40 ng/ml TNF (lanes 2, 5, and
6) for 40 min. Inhib., inhibitor. Activity of PI
3-kinase, measured as described for Fig. 3, is presented as production
of phosphatidylinositol phosphate (PIP), indicated by an
arrow.
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BK-induced PI 3-Kinase and NF-
B Activation Involves B2 BK
Receptors Coupled to Pertussis-sensitive Heterotrimeric G
Proteins--
We previously demonstrated that BK-induced NF-
B
activation in WI-38 and A549 cells is mediated through the B2 BK
receptor (1, 2), a member of the seven transmembrane G protein-coupled receptor superfamily (14). We also showed that the B2 BK receptor is
coupled to a Gi protein in WI38 cells, based on the sensitivity of
BK-induced NF-
B activation and interleukin-1
gene expression to
pertussis toxin treatment (1). The identity of the heterotrimeric G
proteins coupling the B2 BK receptor to PI 3-kinase activity and
NF-
B activation in A549 epithelial cells has not been elucidated. We
therefore examined the effect of pertussis toxin and cholera toxin on
BK-induced NF-
B activation and PI 3-kinase. A549 cells were
pre-treated with pertussis and cholera toxins separately, then
stimulated with BK. Pertussis toxin (0.5 µg/ml) markedly reduced
BK-stimulated NF-
B activation (Fig.
5A, lane 5),
whereas cholera toxin had no such inhibitory effect in A549 cells (Fig. 5A, lane 2). Neither of the toxins inhibited
TNF
-induced NF-
B activation in the same cells (Fig.
5A, lanes 3 and 6). BK-induced PI
3-kinase activity was also blocked by pertussis toxin (Fig. 5B, lane 5) but not cholera toxin (Fig.
5B, lane 2), whereas neither pertussis nor
cholera toxin inhibited TNF
-induced PI 3-kinase activity (Fig.
5B, lanes 3 and 6). Thus, our results
indicate that BK stimulates both PI 3-kinase and NF-
B activation
through B2 BK receptors that are coupled to pertussis toxin-sensitive heterotrimeric G proteins.

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Fig. 5.
Pertussis toxin inhibits BK-stimulated
NF- B activation and PI 3-kinase activity.
A549 cells were preincubated for 4 h with either pertussis toxin
(PTX, 400 ng/ml; lanes 4-6) or cholera toxins
(CTX, 5 µg/ml; lanes 1-3) before stimulation
with BK (20 nM) or TNF (40 ng/ml) for 1 h.
A, NF- B activation was determined as described for Fig. 1
with the DNA-protein complex marked with a bracket. B,
activity of PI 3-kinase, measured as described for Fig. 3, is presented
as production of phosphatidylinositol phosphate (PIP),
indicated by an arrow. These results are representative of
two separate experiments. Med., media.
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PI 3-Kinase Activity Is Required for BK-induced NF-
B
Activation--
Further demonstration of the necessity for PI 3-kinase
activity in BK-induced NF-
B activation was obtained by
overexpressing a dominant negative PI 3-kinase (p85
N-C
478-514)
in A549 cells. The deletion of codons 478-514 from the regulatory p85
component of PI 3-kinase has been shown to confer PI 3-kinase dominant
negative activity (9). A549 cells were co-transfected with
p85
N-C
478-514 together with an I
B promoter-CAT reporter
construct. Overexpression of the dominant negative PI 3-kinase mutant
protein abolished BK-induced
B-mediated CAT activity but had no
effect on TNF
-mediated CAT activity (Fig.
6, A and B). To
confirm that overexpression of p85
N-C
478-514 mutant protein
inhibited PI 3-kinase activity in A549 cells, transfected cells were
recovered using the plasmid pHook-2, which encodes a single-stranded
cell surface antibody that can be bound to magnetic beads coated with
antigen (phOx). Co-transfection of A549 cells with the
p85
N-C
478-514 plasmid inhibited BK-stimulated PI 3-kinase 83%
compared with A549 cells transfected with pHook-2 plasmid alone (Fig.
6C).

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Fig. 6.
PI 3-kinase is necessary for BK-stimulated
NF- B activation. A, A549 cells
were co-transfected with 2.5 µg of the WT-I B-CAT plasmid
(lanes 1-6), 0.5 µg of pCMV (lanes 1-6),
and 2.0 µg of either p85 N-C 478-514 (dominant negative mutant
of the p85 subunit of PI 3-kinase; lanes 4-6) or empty
vector (lanes 1-3). After a 48-h incubation in normal
culture media, the transfected cells were stimulated with media alone
(lanes 1 and 4), 20 nM BK
(lanes 2 and 5), or 40 ng/ml TNF (lanes
3 and 6) for 1 h and then harvested. CAT activity
was measured in the crude cell lysates using
[14C]chloramphenicol as a substrate, separated by
thin-layer chromatography as described under "Experimental
Procedures." All results were normalized for transfection efficiency
using the expression of -galactosidase. A PhosphorImager screen was
exposed, and the autoradiograph of the separated native and acetylated
[14C]chloramphenicol is shown. B, relative CAT
activity of the samples shown in panel A is expressed as the
percentage of acetylated [14C]chloramphenicol in each
lane. These results are representative of two separate
experiments. C, A549 cells were co-transfected with 1 µg
of the pHook-2 plasmid (lanes 1-3) and 1.5 µg of either
empty vector (lanes 1-2) or p85 N-C 478-514
(lane 3). After a 60-h incubation in normal culture media,
the transfected cells were isolated using the Capture-Tec pHook-2
system, then stimulated (Stim.) with media alone
(Med, lane 1) or 100 nM BK
(lanes 2-3) for 5 min and harvested. Activity of PI
3-kinase, measured as described for Fig. 3, is presented as production
of phosphatidylinositol phosphate (PIP), indicated by an
arrow.
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DISCUSSION |
BK has recently been shown to stimulate activation of the
transcription factor NF-
B (1), an effect that may be an important contributor to the inflammatory actions of BK (15). The cellular signaling pathways required for BK-induced NF-
B activation, however, are not completely understood. We recently demonstrated that nanomolar concentrations of BK stimulated NF-
B activation in A549 epithelial cells and that the small G protein RhoA was necessary to mediate this
effect (2). The present study provides evidence that the lipid products
of PI 3-kinase are an important part of the signaling pathway leading
to activation of NF-
B by BK. Our results demonstrate, for the first
time, that BK stimulates increased PI 3-kinase activity in A549
epithelial cells and further that PI 3-kinase activity is essential for
BK-induced activation of NF-
B.
Compared with BK-induced NF-
B activation, BK-stimulated PI 3-kinase
activity occurred earlier (5 versus 15 min after adding BK)
and peaked earlier (20 versus 60 min after adding BK). To test the possibility that PI 3-kinase is a component of the
BK-stimulated signaling pathway leading to NF-
B activation, we
assessed the effect of inhibiting PI 3-kinase activity on subsequent
BK-induced NF-
B activation. Wortmannin and LY294002 have been shown
to be specific PI 3-kinase inhibitors. Wortmannin irreversibly
inactivates PI 3-kinase by binding to its p110 catalytic subunit (13);
LY294002 is a competitive inhibitor, binding to the ATP-binding site of the PI 3-kinase (16). Pre-incubation of A549 cells with either wortmannin or LY294002 completely abrogated BK-induced NF-
B
activation. Additional proof that PI 3-kinase was required for
BK-induced NF-
B activation was provided by the ability of a dominant
negative mutant form of the p85 subunit of PI 3-kinase to block
BK-induced NF-
B activation.
The role of PI 3-kinase in NF-
B activation has been recently
addressed in several other reports with variable results. Like BK,
interleukin-1-mediated activation of NF-
B and activator protein-1 (AP-1) was shown to require PI 3-kinase in human hepatoma (HepG2) and
epidermoid carcinoma (KB) cell lines (17). In this system, overexpression of the p110 catalytic subunit of PI 3-kinase was necessary but not sufficient to activate NF-
B, however it was both
necessary and sufficient to activate AP-1. Interestingly, we found that
TNF
-induced NF-
B activation was unaffected by pre-incubation with
PI 3-kinase inhibitors or expression of the dominant negative p85 PI
3-kinase mutant. Wortmannin also failed to inhibit activation of
NF-
B in human T-cell blasts following CD28 receptor ligation,
although it did inhibit AP-1 activation (18).
A number of distinct forms of PI 3-kinase have been described in
mammalian cells. The classic PI 3-kinase is linked to receptors with
intrinsic or associated tyrosine kinase activity and is a heterodimer
consisting of a p110 catalytic subunit and a p85 regulatory subunit
containing one SH3 and two SH2 domains. At least 3 isoforms of the p110
and p85 subunits have been described in mammalian cells. The p110/p85
form of PI 3-kinase 3-phosphorylates PtdIns, PtdIns(4)P, and
PtdIns(4,5)P2 in vitro, although its primary product in vivo appears to be PtdIns(3,4,5)P3 (19). A p110 PI
3-kinase that does not bind to p85 has also been described (5) as well as a p170 form of PI 3-kinase that preferentially 3-phosphorylates PtdIns and to a lesser extent PtdIns(4)P and is relatively insensitive to wortmannin (20). A mammalian counterpart to the yeast Vps34p kinase
that only phosphorylates PtdIns has been found and is associated with a
150-kDa protein but not p85 (19, 21). Our results indicate that the PI
3-kinase involved in BK-induced NF-
B activation is the p110/p85
heterodimer. This conclusion is based on: 1) its sensitivity to low
concentrations of wortmannin, 2) the ability of a dominant negative p85
mutant to inhibit the response, and 3) the capacity of an anti-p85
antibody to immunoprecipitate BK-induced PI 3-kinase.
BK is an agonist for the B2 BK receptor, and we have previously shown
that B2 BK receptor antagonists completely block BK-induced NF-
B
activation. The B2 BK receptor is a member of the heptahelical superfamily of receptors that are coupled to heterotrimeric G proteins.
To assess the type of heterotrimeric G protein coupling of the B2 BK
receptor to PI 3-kinase and NF-
B, we analyzed the effects of
pertussis and cholera toxins. Pertussis toxin ADP ribosylates G
i and
G
o proteins, whereas cholera toxin ADP ribosylates G
s proteins
(22). BK-stimulated PI 3-kinase activity and NF-
B activation were
both inhibited by pertussis toxin but not by cholera toxin, indicating
that both responses are transduced through the G
i or G
o class of
heterotrimeric G proteins.
Previous studies suggested that G protein-coupled receptors may
activate PI 3-kinase through the p110
, which is independent of p85
independent (5, 23). Other studies, however, provided indirect evidence
that some G protein-coupled receptors activate PI 3-kinase through the
p85/p110 heterodimer (24, 25). By transfecting the A549 cells with a
p85 dominant negative mutant, we showed that the p85/p110 form of PI
3-kinase was activated and required for NF-
B activation following
stimulation with BK, a ligand that acts through a G protein-coupled receptor.
The mechanisms linking PI 3-kinase activation to NF-
B activation are
unknown. D3-phosphorylated phosphatidylinositols are known to play
important roles in cell growth and survival, although the exact role
and immediate downstream molecular targets of PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 are only now emerging (26, 27).
PtdIns(3,4,5)P3 has been shown to activate several of the Ca2+-independent isotypes of protein kinase C (PKC) (28) as
well as the atypical
isotype (PKC
) (29). Interestingly, PKC
has been reported to be important in NF-
B activation (30), an effect that was dependent on p85/p110 PI 3-kinase and protein phosphatase 2A
activity (31). PI 3-kinase was also shown to be required for NF-
B
activation induced by interleukin-4 in transformed human B cells
(32).
Based on our current results and our previous report that RhoA is
required for BK-induced NF-
B activation (2), the relationship between PI 3-kinase activation and activation of the Rho family of
small GTPases appears to be an important issue. Several studies have
demonstrated that PI 3-kinase may be activated downstream of the small
Rho GTPases (33-35). GTP-loaded Rac (but not RhoA) was shown to
directly bind PI 3-kinase (36). Furthermore, inactivation of Rho using
bacterial toxin C3 inhibited lysophosphatidic acid-induced PI 3-kinase
activation in Swiss 3T3 cells (37). Other studies, however, have
suggested that PI 3-kinase may activate the small Rho
GTPases. Expression of a constitutively active PI
3-kinase mutant in Swiss 3T3 cells induced a subset of Rac and
Rho-mediated cellular responses (38). The PI 3-kinase product
PtdIns(3,4,5)P3 has been shown to bind the pleckstrin homology domain
of guanine nucleotide exchange factors, providing a potential mechanism
for PI 3-kinase-mediated regulation of Rho activation (38, 39). Additionally, the p85 regulatory subunit of PI 3-kinase contains a
breakpoint cluster region homology domain (BH) that has been shown to
have GTPase activating protein activity (40).
In summary, we have shown that BK rapidly activates the p85/p110
heterodimeric PI 3-kinase in A549 cells. BK-stimulated PI 3-kinase
activity and NF-
B activation were both inhibited by pertussis toxin
but not cholera toxin, suggesting that the B2 BK receptor mediating
both responses is coupled to the Gi/Go class of G
proteins.
Utilizing both specific inhibitors as well as transient expression of a
dominant negative p85 PI 3-kinase mutant, we further showed that
BK-induced NF-
B activation required PI 3-kinase activity. Although
TNF
also stimulated PI 3-kinase activity, TNF
-stimulated NF-
B
activation was not affected by inhibition of PI 3-kinase activity.
These findings provide evidence that BK-induced NF-
B activation
utilizes a signaling pathway that requires activity of both RhoA and PI
3-kinase and is distinct from the signaling pathway utilized by TNF
.
The relationship between BK-mediated activation of PI 3-kinase and
RhoA, however, remains to be determined.