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INTRODUCTION |
Stimulation of the renin-angiotensin system has been shown
previously to contribute significantly to cardiovascular pathology such
as arterial hypertension, left ventricular hypertrophy, and restenosis.
Angiotensin II (AngII)1
exerts its pathological effects by promoting hypertrophic and/or hyperplastic growth responses in vascular smooth muscle cells (VSMC)
(1), cardiomyocytes (2), and cardiac fibroblasts (3). Recently, the MAP
kinase family of serine-threonine kinases has become a focus of
interest in cardiovascular research. In many cell systems MAP kinases
are known to regulate hypertrophic and/or hyperplastic growth by
various stimuli. In VSMC, AngII has been shown to stimulate ERK1/2 (4),
JNK (5), and p38 (6, 7). A positive contribution of ERK1/2 to
AngII-induced protein and DNA synthesis in VSMC was first demonstrated
by Servant et al. (8) and Wilkie et al. (9) and
has been subsequently confirmed by Marrero et al. (10).
Ushio-Fukai et al. (7) recently defined p38 as a critical
component in AngII-mediated protein and DNA synthesis in VSMC.
Furthermore, they showed that inhibition of both ERK1/2 and p38 had an
additive effect on AngII-induced growth in VSMC (7). The above
mentioned studies used PD98059 and SB203580 as specific inhibitors of
ERK1/2 and p38, respectively. However, compounds that specifically
inhibit JNK activation by AngII have not been available up to now.
Therefore, no data are available considering the contribution of JNK to
AngII-mediated growth responses.
The signal transduction events that lead to stimulation of ERK1/2 by
AngII have been elucidated in recent years (11) and have been shown to
include stimulation of the small G-protein Ras (12, 13). The small
G-proteins Rac and Cdc42 are known to be important upstream mediators
of JNK activation since constitutively active mutants of these small
GTPases enhance JNK activity, whereas dominant-negative mutants block
activation of JNK (14, 15). A putative downstream component of Rac and
Cdc42 in the signaling pathway leading to JNK activation is the
serine-threonine kinase p21-activated kinase (PAK) (16), which becomes
activated upon binding to GTP-bound Rac or Cdc42. We have previously
shown that PAK is stimulated by AngII in VSMC in a tyrosine kinase- and
PKC-dependent manner (5). Furthermore, our data implicated
PAK as an upstream mediator of JNK in VSMC (5).
PAK consists of an NH2-terminal regulatory domain,
comprising the p21-binding domain (PBD), an adjacent auto-inhibitory
domain, 4 putative SH3 domain binding PXXP motifs, and
a COOH-terminal catalytic domain (17, 18). Rho family GTPases Cdc42 and
Rac have been demonstrated to bind to PAK solely in their active forms, i.e. the GTP-bound state, via interaction with the PBD (16). PAK has been shown to be activated by tyrosine kinase receptors (19,
20), cytokines (21), and G-protein-coupled receptors (5, 22). In
tyrosine kinase receptor signaling the adaptor molecule Nck has been
identified as a critical component mediating translocation of PAK to
the plasma membrane (19, 23). Nck consists of 3 SH3 and 1 SH2 domains
(24). PAK binds to the second SH3 domain of Nck (19, 20). Nck is known
to bind stimulated tyrosine kinase receptors such as PDGF receptor
(25), EGF receptor (26), and Eph receptor (23, 27, 28) by its SH2
domain. Recently, signal transduction pathways leading to AngII-induced activation of ERK1/2 in VSMC have been shown to include transactivation of the EGF receptor (29, 30). By analogy to Grb2 and AngII-induced ERK1/2 activation, we surmised that the adaptor molecule Nck might play
a role in AngII-mediated JNK activation by mediating translocation of
PAK to the plasma membrane.
In the present study we present evidence that AngII stimulates Rac1 in
VSMC. Furthermore, we demonstrate that AngII promotes translocation of
Nck and PAK to the plasma membrane in VSMC and that AngII-stimulated
PAK activity is greatly enhanced in membrane fractions compared with
the cytosol fraction. Overexpression of dominant-negative Nck mutants,
in CHO cells stably expressing the AT1 receptor (CHO-AT1 cells), led to
inhibition of PAK and JNK activation by AngII and significantly
inhibited AngII-induced DNA synthesis. In addition, overexpression of a
PAK mutant (PAK-A13) that cannot bind Nck could not be stimulated by
AngII in CHO-AT1 cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Antibodies were purchased from the following
vendors: Upstate Biotechnology Inc. (rabbit polyclonal antibodies: Nck
(06-288), monoclonal anti-Myc tag (clone 9E10, 05-419); Transduction
Laboratories (monoclonal Nck (N15920), Rac1 (R56220), Cdc42 (C70820); Santa Cruz Biotechnology (rabbit polyclonal PAK1 (sc-881), EGF receptor
(sc-03), PDGF receptor-
(sc-338), PDGF receptor-
(sc-432), p38
(C-20) (sc-535-G), JNK-1 (C-17) (sc-474-G), monoclonal antibody PY-99
(sc-7020); New England Biolabs (monoclonal phospho-p44/42 MAP kinase
(Thr-202/Tyr-204) E10). Cell signaling technology (polyclonal phospho-c-Jun (Ser-63) II. c-Jun-(79), and ATF-2-(1-96)
substrate was purchased from Santa Cruz Biotechnology, Inc. Genistein,
PP1, phorbol 12,13-dibutyrate, and GF109203X (GFx) were purchased from Biomol Research Laboratories and AG 1296 and AG 1478 from Calbiochem. MBP was purchased from Sigma. Plasmids containing wild type human PAK1
(pCMV6M-PAK1) and kinase-dead PAK-K299R (pCMV6M-PAK1-K299R) were kindly
provided by Gary Bokoch (20). The PAK-A13 mutant (pCMV6M-PAK-A13),
which lacks Nck binding, was kindly provided by Jonathan Chernoff (31).
Plasmids containing wild type Nck (pEBB-Nck) and mutant Nck
pEBB-Nck-W38,143,229K with inactivating mutations of all three Nck-SH3
domains (denoted Nck-KSH3all in this paper) and pEBB-Nck-R308K, with an
inactivating mutation in the Nck-SH2 domain (designated Nck-K308 in
this paper), were kindly provided by Bruce Mayer and have been
described previously (32).
Cell Culture and Transfection--
VSMC were isolated from 200- to 250-g male Wistar-Kyoto rats and maintained in Dulbecco's modified
Eagle's medium supplemented with 10% bovine calf serum, as described
previously (33). Passages 8-15 VSMC at 80% confluence were
growth-arrested by incubation in 0.4% calf serum for 24 h prior
to use. Chinese hamster ovary (CHO) cells stably transfected with
angiotensin II type 1 receptor were kindly obtained from Dr. Kenneth
Baker and maintained in F-12 media supplemented with 20 mmol/liter
HEPES, 0.2 mg/ml G418, and 10% fetal calf serum. Transient
transfection of CHO-AT1 cells using PAK1 and Nck plasmids was performed
using LipofectAMINE (Life Technologies, Inc.). Expression of
transfected proteins was checked in each experiment by Western blotting
with anti-Myc-tag antibody 9E10 for detection of PAK1 or monoclonal Nck
for detection of Nck.
Immunoprecipitation, in Vitro Binding Studies, and Western Blot
Analysis--
Growth-arrested VSMC or CHO-AT1 cells were either left
untreated or stimulated by 100 nM to 1 µM
AngII for the indicated times. Cells were lysed with lysis buffer
containing 20 mM HEPES, pH 7.5, 150 mM NaCl,
1% Triton X-100, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The indicated antibodies
or GST fusion proteins were added to equal amounts of protein per
sample and incubated for 12 h at 4 °C. Antibody complexes were
collected by addition of protein A-agarose for 3 h. GST
fusion proteins were collected by addition of glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) for 3 h. Precipitates were washed 5 times in cell lysis buffer, resuspended in SDS sample buffer, and
boiled for 10 min. After centrifugation for 10 min at 10,000 × g, the supernatants were size-fractionated by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with the indicated
antibodies. Secondary antibodies were coupled to horseradish
peroxidase, and Western blot detection was done by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech). Equal loading of
the immunoprecipitated protein of interest was ascertained in every
experiment by Western blotting.
Preparation of Cytosolic and Membrane Fractions--
VSMC were
incubated in hypotonic buffer containing 10 mM HEPES, pH
7.4, 100 mM NaCl, 10% glycerol, and 1 mM EDTA
for 1 h. Cells were scraped off the dishes, and lysates were
precleared by centrifugation at 10,000 × g at 4 °C
for 10 min. Cell lysates were then homogenized with a Teflon Wheaton
Homogenizer by 30 Dounces and centrifuged in an ultracentrifuge at
100,000 × g for 30 min. Supernatants were denoted the
cytosolic fraction. Pelleted proteins were solubilized by addition of
lysis buffer containing 2% n-octyl
-D-glucoside and centrifuged a second time in an
ultracentrifuge at 100,000 × g for 30 min. The
supernatant of the second ultracentrifugation was denoted the membrane
fraction. For determination of PAK activity from membrane and cytosolic
fractions, hypotonic buffer of cytosolic fraction was adjusted in order
to obtain the same buffer conditions as was used for the membrane fraction.
PAK Immunocomplex MBP in-Gel Kinase Assay--
Growth-arrested
VSMC were stimulated, and cells were lysed with lysis buffer containing
10 mmol/liter HEPES, pH 7.4, 0.1% Triton X-100, 5 mmol/liter EGTA, 5 mmol/liter EDTA, 50 mmol/liter NaCl, 50 mmol/liter NaF, 50 mmol/liter
sodium pyrophosphate, 1 mmol/liter sodium orthovanadate, 10 mg/ml
leupeptin, and 1 mmol/liter phenylmethylsulfonyl fluoride. Lysates were
precleared by centrifugation, and protein concentration was measured by
DC protein assay (Bio-Rad). PAK antibody was added to equal amounts of
protein per sample and incubated for 12 h at 4 °C. Antibody
complexes were collected by addition of protein A-agarose for 3 h.
Precipitates were washed 5 times in cell lysis buffer, resuspended in
SDS sample buffer, and boiled for 10 min. After centrifugation for 10 min at 10,000 × g the supernatants were
size-fractionated by SDS-PAGE, and PAK activity was assayed by
32P incorporation into MBP using an in-gel kinase
assay as previously described (4).
PAK Immunocomplex in Vitro Kinase Assay--
CHO-AT1 cells were
co-transfected by Myc-tagged wild type PAK1 (pCMV6M-PAK1) or PAK-A13
(pCMV6M-PAK-A13) and the indicated pEBB-Nck plasmids for 24 h.
Cells were then growth-arrested for 24 h prior to stimulation by 1 µM AngII for 30 min. Cells were lysed with lysis buffer
(see above). Myc tag antibody 9E10 (Upstate Biotechnology Inc.) was
added to equal amounts of protein per sample and incubated for 4 h
at 4 °C. Antibody complexes were collected by addition of protein
A-agarose for 2 h. Precipitates were washed 3 times in cell lysis
buffer and 2 times in kinase reaction buffer. Subsequently, samples
were incubated in 30 µl of kinase reaction buffer containing 50 mM HEPES, pH 7.4, 10 mM MgCl2, 2 mM MnCl2, 50 µM ATP, and 5 µCi
of [
-32P]ATP for 30 min at 30 °C in the presence of
1 µg of MBP as substrate. The in vitro kinase reaction was
stopped by addition of SDS sample buffer and heating of samples to
95 °C for 10 min. After centrifugation for 10 min at 10,000 × g, the supernatants were size-fractionated by SDS-PAGE, and
MBP phosphorylation was assessed by autoradiography.
JNK Activity Assay--
JNK1 was immunoprecipitated from control
and AngII-stimulated CHO-AT1 cell lysate 48 h after transfection
of the indicated plasmids. Agarose beads were collected by
centrifugation and washed three times with lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol,
and 1% Triton X-100) and two times with kinase reaction buffer. Beads
were then incubated for 30 min at 30 °C in 30 µl of kinase
reaction buffer containing 20 mM HEPES, 10 mM
MgCl2, 50 µM ATP in the presence of 2 µg
c-Jun-(1-79) as substrate. The reaction was terminated by the addition
of SDS sample buffer; proteins were separated by SDS-PAGE, and JNK
activity was assessed by Western blotting with phospho-c-Jun-specific
antibodies (Cell Signaling Technology).
GST-PAK-PBD Binding Assay--
PAK-PBD binding assay was
performed essentially as described previously (34). In brief, the
p21-binding domain of human PAK1, comprising amino acids 68-166, was
subcloned into the bacterial expression vector pGEX-2TK (Amersham
Pharmacia Biotech, Inc.) and was expressed in Escherichia
coli as GST-PAK-PBD fusion protein according to the
manufacturer's protocol. 15 µl of a 50% slurry of GST-PAK-PBD
glutathione-Sepharose 4B was added to cell lysates of VSMC constantly
rotating at 4 °C for 60 min. Bound proteins were collected by
centrifugation, and pellets were washed three times in cell lysis
buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 25 mM NaF, 10% glycerol, 0.25% sodium deoxycholate, 10 mM MgCl2, 1 mM EDTA, 1% Triton
X-100) and finally suspended in SDS sample buffer. Proteins were size-
fractionated by SDS-PAGE and binding of Rho family GTPases was
determined by Western blotting with Rac1 and Cdc42 antibodies.
Generation of Nck-SH Domain GST Fusion Proteins--
GST fusion
proteins containing Nck-SH2 and the three Nck-SH3 domains were
generated by subcloning of the corresponding polymerase chain reaction
fragments from a template Nck cDNA (32) into pGEX-2TK (Amersham
Pharmacia Biotech). Nck-SH2 encoded amino acids 275-377; Nck-SH3-1
encoded amino acids 2-68; Nck-SH3-2 encoded amino acids 98-168, and
Nck-SH3-3 encoded amino acids 190-256. Nck-SH fragments subcloned
into pGEX-2TK were sequenced to exclude mutations due to polymerase
chain reaction amplification. GST fusion proteins were isolated using
glutathione-Sepharose 4B according to the manufacturer's instructions
(Amersham Pharmacia Biotech). Fusion proteins were checked by Coomassie
Blue staining and yielded single bands of expected size for all
GST-Nck-SH domains.
[3H]Thymidine Incorporation into DNA--
The
effect of AngII on [3H]thymidine incorporation into cell
DNA was assessed as described previously (35). In brief, CHO-AT1 cells
were grown to confluence in 24-well plates and growth-arrested in
serum-free medium for 24 h prior to stimulation with 1 µM AngII for 24 h. 20 h after addition of AngII
3 µCi/ml [3H]thymidine was added. 4 h later the
experiment was stopped by aspirating the medium and subjecting the
cultures to sequential washes with Dulbecco's phosphate-buffered
saline containing 1 mM CaCl2, 1 mM
MgCl2, 10% trichloroacetic acid (w/v), and ethanol/ether (2:1, v/v). Acid-insoluble [3H]thymidine was extracted
with 0.5 M NaOH (250 µl per well), and 100 µl of this
solution was mixed with 5 ml of scintillator liquid (Ultimagold,
Packard Insturment Co.) and quantified using a liquid scintillation
counter (LS 3801, Beckman Instruments). 50 µl of the residual
solution was used for determination of protein using the Bio-Rad
protein assay according to the method of Bradford (36). Three
independent experiments were performed where triplicate values for each
condition were obtained. Data were calculated as cpm/µg protein.
Densitometry and Statistics--
For quantification of Western
blots or 32P incorporation into MBP, films were scanned and
analyzed by densitometry using arbitrary units. Activation is presented
as the fold increase over the respective control (mean ± S.E.).
Statistical analysis was performed by Student's t test
(unpaired and two-tailed) using StatView 5.0 software. A p
value of <0.05 was considered significant.
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RESULTS |
AngII Stimulates Rac1 in VSMC--
To determine whether AngII
stimulated Cdc42 or Rac in VSMC, we used the GST-PAK-PBD binding assay
recently developed by Bagrodia et al. (37). Cdc42 and Rac1
were expressed in VSMC as determined by Western blotting (data not
shown). Addition of GTP
S to total cell lysates of VSMC increased
binding of endogenous Rac1 and Cdc42 to GST-PAK-PBD, demonstrating
functional integrity of the fusion protein used (data not shown). AngII
stimulated binding of Rac1 to GST-PAK-PBD in a
time-dependent manner, peaking at 1 min (3.1 ± 0.13-fold increase, n = 3) and showing sustained activation up to 10 min (Fig. 1A,
lower panel). The dependence of Rac1 binding to GST-PAK-PBD on
AngII concentration was determined at 1 min. Maximal stimulation was
observed at a concentration of 1 µM AngII (2.6 ± 0.62-fold increase, n = 3) (Fig. 1B, lower panel). However, AngII-induced stimulation of Cdc42 was not
observed (data not shown).

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Fig. 1.
AngII stimulates Rac1 in VSMC,
time course, and concentration dependence. A, VSMC
were stimulated by 1 µM AngII for the indicated times,
and binding of activated Rac1 to GST-PAK-PBD (PAK-PBD) was determined
by Western blotting (lower panel). B, VSMC were
stimulated by 0.1-1000 nM AngII for 1 min, and binding of
activated Rac1 to GST-PAK-PBD (PAK-PBD) was determined by
Western blotting (lower panel). Equal loading of proteins
for the precipitation assays was assessed by Rac1-Western blotting of
10 µl total cell lysate (TCL) (upper panels).
Arrows indicate Rac1.
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AngII-induced Activation of Rac1 Is Dependent on Tyrosine Kinases
and PKC--
Previous results obtained in VSMC (5) demonstrated that a
tyrosine kinase other than Src is involved in AngII-mediated activation
of PAK and JNK. To test for involvement of tyrosine kinases in Rac1
activation by AngII, VSMC were pretreated by 100 µM
genistein for 16 h or 10 µM PP1 for 15 min prior to
stimulation by 1 µM AngII, and Rac1 binding to
GST-PAK-PBD was determined. We have shown previously that 10 µM PP1 effectively inhibited Src activation by AngII in
VSMC (33). Genistein inhibited AngII-mediated binding of Rac1 to
GST-PAK-PBD (fold increase of Me2SO = 2.47 ± 0.31 versus genistein = 1.21 ± 0.12, p = 0.02, Fig. 2A,
lower panel), whereas PP1 had no significant effect (fold increase
of Me2SO = 2.47 ± 0.31 versus
PP1 = 2.63 ± 0.37, p = 0.74, Fig. 2B, lower panel), indicating that a tyrosine kinase other than Src is
involved in AngII-induced Rac1 activation.

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Fig. 2.
AngII-induced Rac1 activation is dependent on
tyrosine kinases. VSMC were pretreated by 100 µM
genistein for 16 h (A) or 10 µM PP1 for
15 min (B) and then stimulated by 1 µM AngII
for 1 min. Binding of activated Rac1 to GST-PAK-PBD
(PAK-PBD) was assessed by Western blotting (lower
panels). Equal loading of proteins for the precipitation assays
was assessed by Rac1-Western blotting of 10 µl total cell lysate
(TCL) (upper panels). Arrows indicate
Rac1. C, quantitative densitometry of Rac1 immunoblots was
performed. Values shown are mean ± S.E. of three independent
experiments. *, p < 0.05 versus
Me2SO (DMSO).
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We next examined the effect of PKC inhibition on Rac1 activation by
AngII. Down-regulation of phorbol ester-sensitive PKC isoforms
(PKC-
, -
, -
, -
, -
, -
, and -
) by pretreatment of VSMC with 1 µM phorbol 12,13-dibutyrate for 24 h
inhibited AngII-mediated activation of Rac1 (fold increase of
Me2SO = 2.8 ± 0.63 versus phorbol
12,13-dibutyrate 0.97 ± 0.21, p = 0.05, Fig.
3A, lower panel). However, the
PKC inhibitor GFx, which is thought to inhibit preferentially PKC-
,
-
, and -
(38), did not have a major effect on Rac1 activation by
AngII (fold increase of Me2SO = 2.8 ± 0.63 versus GFx = 2.1 ± 0.25, p = 0.48, Fig. 3B, lower panel).

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Fig. 3.
Rac1 stimulation by AngII is dependent on PKC
activation. VSMC were pretreated by 1 µM phorbol
12,13-dibutyrate for 24 h (A) or 1 µM
GF109203X (GFx) for 10 min (B) and then
stimulated by 1 µM AngII for 1 min. Binding of activated
Rac1 to GST-PAK-PBD (PAK-PBD) was assessed by Western
blotting. Equal loading of proteins for the precipitation assays was
assessed by Rac1-Western blotting of 10 µl of total cell lysate
(TCL) (upper panels). Arrows indicate
Rac1. C, quantitative densitometry of Rac1 immunoblots was
performed. Values shown are mean ± S.E. of three independent
experiments. *, p < 0.05 versus
Me2SO (DMSO). Contr., control.
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Nck-PAK Interaction--
PAK has been shown to bind Nck by
interaction of its first proline-rich region with the second Nck-SH3
domain (19, 20). Whereas Galisteo et al. (19) reported
constitutive Nck-PAK interaction in L6 cells treated by PDGF, Bokoch
et al. (20) observed an increase in the amount of PAK bound
to Nck after stimulation of Swiss 3T3 cells with PDGF. In contrast,
Zhao et al. (39) reported that PAK autophosphorylation
negatively regulated Nck-PAK interaction in COS-7 cells transfected by
recombinant PAK. To determine whether PAK interacted with Nck in VSMC,
we immunoprecipitated Nck from control and AngII-stimulated VSMC and
evaluated binding of PAK by immunoblotting. At base line PAK was bound
to Nck, showing no increase after AngII treatment of VSMC (Fig.
4A). To characterize further
Nck-PAK interaction in VSMC, we performed in vitro binding studies using GST fusion proteins comprising the various Nck-SH domains
and immunoblotted for PAK. As reported previously (19, 20), Nck-PAK
interaction in VSMC was mediated by binding of PAK to Nck-SH3-2 domain
(Fig. 4B). Essentially the same results as shown in Fig. 4
were obtained for CHO-AT1 cells (data not shown).

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Fig. 4.
Nck-PAK interaction. A,
growth-arrested VSMC were treated by 100 nM AngII for the
indicated times. Nck was immunoprecipitated (IP) by
polyclonal Nck antibody, and PAK binding was detected by Western
blotting with polyclonal PAK1 antibody (sc-881). N indicates
immunoprecipitation by rabbit preimmune serum, and TCL
indicates total cell lysate. PAK binds constitutively to Nck in VSMC.
Contr., control. B, total cell lysates of VSMC
were prepared, and in vitro binding assays using GST or the
indicated GST-Nck-SH domain fusion proteins were performed. Bound
proteins were visualized by polyclonal PAK1 antibody. Arrows
to the right indicate PAK in VSMC that migrates as a
doublet. Endogenous PAK binds to the second Nck-SH3 domain.
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AngII Induces Translocation of the Nck-PAK Complex to the Membrane
Fraction--
Recruitment of PAK to the plasma membrane has been shown
previously to facilitate its activation (40, 41). Lu et al.
(40) have demonstrated in 293T cells that translocation of PAK can be
mediated by the adaptor molecule Nck. In order to evaluate translocation of Nck and PAK by AngII treatment of VSMC, we prepared cytosolic and particulate cell fractions by differential centrifugation of unstimulated and AngII- or PDGF- stimulated VSMC and performed Western blotting using Nck and PAK antibodies. AngII induced an ~2-fold increase of immunoreactive Nck and PAK in the particulate fraction after 5 min (Fig. 5,
A and B). PDGF treatment of VSMC for 5 min showed
a slightly lower increase of Nck in the particulate fraction compared
with AngII (Fig. 5, A and B); however, PAK
immunoreactivity increased by about 3.5-fold over control (Fig. 5,
A and B).

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Fig. 5.
Translocation of Nck and PAK to the plasma
membrane. A, membrane fractions of VSMC that were
either left untreated (Contr., control), or stimulated by
100 nM AngII for 5 min (AngII), or 50 ng/ml
PDGF-AB for 5 min (PDGF) were prepared as described under
"Experimental Procedures." 20 µg of total protein were
size-fractionated by 7,5% SDS-PAGE, transferred to nitrocellulose
membrane, and blotted by Nck (upper panel) or PAK
(lower panel). B, the relative increase of
immunoreactive Nck (open bars) or PAK (closed
bars) over control in the membrane fraction of AngII- and
PDGF-stimulated VSMC was determined by densitometry. Values shown are
mean ± S.E. of three independent experiments. C, PAK
activity was determined by an immunocomplex MBP in-gel kinase assay
from cytosolic and membrane fractions of VSMC that were either left
untreated (Contr., control) or stimulated by 100 nM AngII for 15 min (AngII). Phosphorylation was
assessed by autoradiography. The result shown is representative of
three independent experiments.
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To evaluate further the relevance of membrane localization for
AngII-induced PAK activation, we determined PAK activity by an
immunocomplex MBP in-gel kinase assay from cytosolic and membrane fractions of VSMC that had been stimulated by 100 nM AngII
for 15 min. In the cytosolic fraction AngII stimulated PAK activity by
1.44 ± 0.03-fold (n = 3), whereas there was a
3.6 ± 0.3 (n = 3)-fold increase in the membrane
fraction (p = 0.02, fold increase cytosolic
versus membrane fraction) (Fig. 5C).
AngII Stimulates Tyrosine Phosphorylation of Nck-associated
Proteins--
Tyrosine kinases are important mediators of
AngII-induced Rac1 (see above), PAK, and JNK activation (5). Therefore,
we were interested to determine whether Nck bound
tyrosine-phosphorylated proteins upon AngII stimulation of VSMC. Nck
immunoprecipitates of AngII-stimulated VSMC showed slightly increased
association of a 100-kDa tyrosine-phosphorylated protein (Fig.
6A, lower arrow). Tyrosine
phosphorylation of a 150-kDa protein was only inconsistently seen (Fig.
6A, upper arrow). To define further the interaction of the
100-kDa protein with Nck, we generated a GST-Nck-SH2 fusion protein.
In vitro binding studies using GST-Nck-SH2 did not detect association of the 100-kDa tyrosine-phosphorylated protein with Nck-SH2
(Fig. 6B) (nor with the various Nck-SH3 domains, data not
shown). Thus binding of the 100-kDa protein to Nck is either indirectly
or is mediated by interaction with an non-SH domain region of Nck.
However, we detected increased association of 25- and 30-kDa
tyrosine-phosphorylated proteins with Nck-SH2 upon AngII stimulation of
VSMC that was not detected by immunoprecipitation (Fig. 6, A
and B).

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Fig. 6.
AngII induces tyrosine phosphorylation of
Nck-associated proteins. A, growth-arrested VSMC were
either left untreated (Contr., control) or stimulated by 100 nM AngII for the indicated times. Nck was
immunoprecipitated (IP) by polyclonal anti-Nck; bound
proteins were size-fractionated by 7.5% SDS-PAGE, transferred to
nitrocellulose membrane, and blotted by anti-phosphotyrosine antibodies
(PY99). N indicates immunoprecipitation by rabbit
preimmune serum. AngII induced tyrosine phosphorylation of a 100-kDa
protein (lower arrow). Increased tyrosine phosphorylation of
an ~150-kDa protein was only inconsistently seen (upper
arrow). Molecular mass markers are indicated to the
right (kDa). B, growth-arrested VSMC
were either left untreated (Contr., control), stimulated by
100 nM AngII for the indicated times, or treated by 50 ng/ml PDGF-AB for 5 min (PDGF). Cell lysates were prepared,
and an in vitro binding assay using GST-Nck-SH2 or GST alone
was performed. Proteins were size-fractionated by SDS-PAGE transferred
to nitrocellulose membrane, and an anti-phosphotyrosine immunoblot was
performed. AngII stimulated binding of a 30-kDa protein to Nck-SH2
(B, middle arrow). A longer exposure of the blot revealed
additional binding of a 25-kDa protein (B, lower arrow).
PDGF-AB treatment stimulated binding of a 180-kDa phosphotyrosine
protein to Nck-SH2 that corresponded to PDGF receptor (B, upper
arrow). Molecular mass markers are indicated to the
right (kDa).
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Binding of Shc (30) and Grb2 (29) to transactivated EGF receptor has
been shown to be a critical step in ERK1/2 activation by AngII.
Furthermore, Heenemann et al. (42) recently demonstrated in
VSMC AngII-induced binding of Shc to transactivated PDGF receptor. By
analogy, we were interested to examine whether AngII stimulated binding
of Nck to transactivated EGF or PDGF receptor. Western blotting of Nck
immunoprecipitates from unstimulated and AngII-stimulated VSMC with EGF
receptor or PDGF receptor antibodies yielded negative results (data not
shown). Corresponding in vitro binding studies using
GST-Nck-SH2 fusion proteins also showed no binding of EGF receptor or
PDGF receptor to Nck-SH2 upon AngII stimulation (data not shown),
whereas PDGF treatment of VSMC stimulated binding of GST-Nck-SH2 to
PDGF receptor (Fig. 6B). To substantiate further that EGF or
PDGF receptor transactivation plays no role in AngII-induced PAK
stimulation, we pretreated VSMC for 30 min with 250 nM
AG1478 (to inhibit EGF receptor) or 10 µM AG1296 (to
inhibit PDGF receptor) and determined AngII-induced PAK activity by an
immunocomplex MBP in-gel kinase assay. The concentrations used for
AG1478 and AG1296 have been shown previously to inhibit effectively EGF
and PDGF receptor tyrosine kinase activity in VSMC (30). Neither inhibition of EGF or PDGF receptor by the indicated tyrphostins influenced AngII-induced PAK activation (Fig.
7).

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Fig. 7.
Inhibition of EGF and PDGF receptor kinase
does not affect AngII-induced activation of PAK. Growth-arrested
VSMC were pretreated for 30 min by 250 nM AG 1478 to
inhibit EGF receptor kinase or by 10 µM AG 1296 to
inhibit PDGF receptor kinase. Me2SO (DMSO) was
used as a control. AngII-induced PAK activity was determined by an
immunocomplex MBP in-gel kinase assay as described under
"Experimental Procedures." Phosphorylation was assessed by
autoradiography. The result shown is representative of three
independent experiments. Contr., control.
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Overexpression of Dominant-negative Nck Inhibits AngII-induced
Activation of PAK and JNK in CHO-AT1 Cells--
We have shown
previously (5) that overexpression of kinase-dead PAK (PAK-K299R) did
inhibit AngII-mediated stimulation of JNK in CHO cells stably
expressing the angiotensin II type I receptor (CHO-AT1 cells). To
elucidate further the role of Nck in AngII signal transduction, we
expressed dominant-negative Nck mutants, exhibiting inactivating
mutations in either the Nck-SH2 domain (Nck-K308) or all Nck-SH3
domains (Nck-KSH3all). The plasmids containing the various
dominant-negative Nck mutants were kindly provided by Bruce Mayer and
have been extensively characterized by his group (32). CHO-AT1 cells
were co-transfected with Myc-tagged human wild type PAK1 (pCMV-PAK1)
and either control vector (pEBB-
Gal), pEBB-Nck-K308, or
pEBB-Nck-KSH3all. Expression of transfected plasmids was checked by
immunoblotting using Myc tag antibodies (for PAK expression) or
monoclonal Nck antibody (data not shown). 48 h after transfection
cells were stimulated by 1 µM AngII for 30 min, and PAK
was immunoprecipitated by Myc tag antibody (9E10, Upstate Biotechnology
Inc.). Subsequently, an in vitro kinase assay was performed
using MBP as substrate. AngII-stimulated PAK activity in CHO-AT1 cells
could be significantly inhibited by co-expression of Nck-KSH3all (fold
increase of
-galactosidase = 2.47 ± 0.4 versus Nck-KSH3all = 1.01 ± 0.05, p = 0.03, Fig. 8) and by
Nck-K308 (fold increase of
-galactosidase = 2.47 ± 0.4 versus Nck-K308 = 1.04 ± 0.10, p = 0.03, Fig. 8). To determine the effect of
dominant-negative Nck on AngII-mediated JNK activation, we performed a
JNK immunocomplex in vitro kinase assay using c-Jun as
substrate. Activation of JNK was determined by immunoblotting with
phospho-c-Jun-specific antibodies. Parallel to inhibition of PAK
activation, we found inhibition of AngII-induced JNK by co-expression
of Nck-KSH3all (fold increase of
-galactosidase = 2.87 ± 0.49 versus Nck-KSH3all = 1.57 ± 0.24, p = 0.05, Fig. 8) and Nck-K308 (fold increase of
-galactosidase = 2.87 ± 0.49 versus
Nck-K308 = 1.03 ± 0.05, p = 0.06, Fig.
8).

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Fig. 8.
Overexpression of dominant-negative Nck
inhibits AngII-induced stimulation of PAK and JNK.
A, CHO-AT1 cells were transfected by Myc-tagged wild type
PAK1 together with either control vector (pEBB- -galactosidase),
pEBB-Nck-KSH3all, or pEBB-Nck-K308. PAK activity was examined by an
immunocomplex MBP in vitro kinase assay using anti-Myc tag
antibodies as described under "Experimental Procedures."
Phosphorylation of MBP was determined by autoradiography (A,
upper panel). JNK activity was assessed by an in
vitro kinase assay using GST-c-Jun as substrate and performing
Western blotting with phospho-c-Jun (p-c-Jun)-specific
antibodies as described under "Experimental Procedures" (A,
lower panel). B, quantitative densitometry was
performed as described under "Experimental Procedures." Results
shown are mean ± S.E. * p < 0.05 versus -galactosidase ( -Gal).
Contr., control.
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Since Nck-SH3 domains are known to interact with various other
molecules (39) (e.g. dynamin, NIK, SAM68, and WASP) that might indirectly influence AngII-induced PAK activation, we examined the ability of the PAK-A13 mutant (which does not bind Nck) to be
stimulated by AngII. CHO-AT1 cells were transfected with pCMV6M-PAK-wt or pCMV6M-PAK-A13, and AngII-induced PAK activity was determined by PAK
autophosphorylation and by Myc tag immunocomplex in vitro kinase assay. AngII-induced activation of PAK was completely blocked by
expression of the PAK-A13 mutant (Fig.
9).

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Fig. 9.
A PAK mutant that lacks Nck binding cannot be
stimulated by AngII. CHO-AT1 cells were transfected by Myc-tagged
wild type PAK1 (PAK-wt) or by Myc-tagged PAK1-A13
(PAK-A13). PAK activity was examined by PAK
autophosphorylation and by an immunocomplex MBP in vitro
kinase assay using anti-Myc tag antibodies as described under
"Experimental Procedures." Phosphorylation of PAK (upper
panel) and MBP (lower panel) was assessed by
autoradiography. The result shown is representative of three
independent experiments.
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Recently, Wen et al. (43) demonstrated decreased ERK1/2
activity in CHO-AT1 cells transfected by kinase-dead PAK1, which had
been stimulated by 12-hydroxyeicosatetraenoic acid. To test for
specificity of JNK inhibition, we determined ERK1/2 activity in
AngII-stimulated CHO-AT1 cells expressing kinase-dead PAK1, Nck-K308,
or Nck-KSH3all by Western blotting using phospho-ERK1/2-specific antibodies. Stimulation of ERK1/2 by AngII and phorbol
12-myristate 13-acetate was neither inhibited by expression of
kinase-dead PAK1 nor by expression of Nck-K308 or Nck-KSH3all (data not
shown). Activation of p38 by AngII could not be detected in CHO-AT1
cells by an immunocomplex in vitro kinase assay using ATF2
as substrate, where activity was assessed by Western blotting with
phospho-ATF2-specific antibodies (data not shown).
Inhibition of JNK by Overexpression of Dominant-negative Nck
Diminishes [3H]Thymidine Incorporation in CHO-AT1
Cells--
The role of JNK in AngII-induced growth responses has not
been defined so far. By overexpression of Nck-K308 and Nck-KSH3all, we
specifically inhibited AngII-stimulated JNK with no effect on ERK1/2.
To define the role of JNK in AngII-induced DNA synthesis, we determined
[3H]thymidine incorporation in AngII-stimulated CHO-AT1
cells that had been co-transfected with wild type PAK1 and the various
dominant-negative Nck plasmids. AngII led to a 2.4 ± 0.4-fold
increase in [3H]thymidine incorporation under control
conditions (transfection of pEBB-
-galactosidase). Overexpression of
Nck-KSH3all and Nck-K308 both inhibited AngII-stimulated DNA synthesis
(fold increase over respective control for Nck-KSH3all = 1.41 ± 0.02, p = 0.02 versus
-galactosidase,
and for Nck-K308 = 1.04 ± 0.08, p = 0.08 versus
-galactosidase, Fig.
10).

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Fig. 10.
Overexpression of
dominant-negative Nck inhibits AngII-induced DNA synthesis in
CHO-AT1 cells. CHO-AT1 cells were co-transfected by wild type PAK1
together with pEBB -galactosidase, pEBB-Nck-KSH3all, or
pEBB-Nck-K308. [3H]Thymidine incorporation of control and
AngII-stimulated CHO-AT1 cells was determined, and the fold increase
over respective control was calculated as described under
"Experimental Procedures." *, p < 0.05 versus -galactosidase
( -Gal).
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DISCUSSION |
In the present study, we report for the first time that AngII
stimulates the GTPase Rac1 in a tyrosine kinase- and
PKC-dependent manner. Furthermore, we demonstrate that
AngII-induced activation of PAK and JNK is mediated by the adaptor
molecule Nck. In addition, we show that specific inhibition of JNK by
overexpression of dominant-negative Nck diminishes AngII-stimulated DNA
synthesis in CHO-AT1 cells.
Small G-proteins of the Ras superfamily are known to be critical
regulators of MAP kinase pathways. Ras has been shown to be an
important regulator of ERK1/2, whereas Rac and Cdc42 are thought to
mainly stimulate JNK and p38. In VSMC, AngII has been shown previously
to regulate ERK1/2 via activation of Ras (12). Recently, Haendeler
et al. (44) also implicated Ras and to a lesser extent Rap1
in AngII-induced ERK1/2 activation. However, small G-proteins of the
Rho family have not been demonstrated so far to regulate AngII-induced
JNK activation in VSMC. In the present report, we demonstrated binding
of (GTP-bound) Rac1 to PAK-PBD in VSMC stimulated by AngII.
Furthermore, our data indicate that a tyrosine kinase other than Src is
involved in PAK and JNK activation by AngII. In addition,
down-regulation of phorbol ester-sensitive PKC isoforms inhibited Rac1
activation by AngII. These results are in agreement with formerly
published data obtained in VSMC (5) considering PAK and JNK activation
by AngII. This indicates that AngII-induced PAK and JNK activation in
VSMC is mediated by Rac1. A critical role for Rac1 in AngII signaling
is further supported by recent findings from Murasawa et al.
(45), who showed in cardiac fibroblasts that overexpression of
dominant-negative Rac1, but not Cdc42, inhibited AngII-mediated
activation of PAK and JNK.
Targeting of proteins to specific cellular sites is increasingly
recognized as a mechanism to regulate signal transduction pathways. A
critical role for recruitment of PAK to the plasma membrane has been
established by Lu et al. (40, 46) and Bokoch et
al. (41), who demonstrated that targeting PAK directly to the
plasma membrane facilitated its activation. Bokoch et al. (41) reported increased activity of membrane-targeted PAK independently of its ability to interact with Rac or Cdc42 and suggested that binding
to membrane lipids facilitates stimulation of PAK. In contrast, data
from Lu and Mayer (46) showed that activation of membrane-localized PAK
could be inhibited by expression of proteins that inhibit Rho family
GTPases (e.g. dominant-negative Cdc42D57Y), indicating that
translocation of PAK to the plasma membrane serves its interaction with
activated Cdc42 or Rac. The adaptor molecule Nck has been identified as
a putative signaling molecule to mediate translocation of PAK to the
plasma membrane. First, Nck has been implicated previously in tyrosine
kinase receptor signaling leading to activation of PAK (19) and JNK
(23, 28). Upon stimulation of tyrosine kinase receptors, a preformed
Nck-PAK complex translocates to the plasma membrane by binding of
Nck-SH2 domain to phosphotyrosine residues of the activated receptor
(19, 25, 26, 28). Second, Lu et al. (40) demonstrated that recruitment of PAK to the plasma membrane by a myristoylated Nck-SH3-2 domain facilitated activation of PAK. Importantly, overexpression of a
non-myristoylated Nck-SH3-2 domain did not mediate PAK activation, indicating that merely facilitating Nck-PAK interaction is not sufficient for PAK activation (40).
In the present report, we demonstrated in VSMC
AngII-induced translocation of Nck and PAK to the particulate fraction.
Furthermore, compared with the cytosolic fraction, we detected
significantly enhanced PAK activity in the membrane fraction of
AngII-treated VSMC. Thus, our data support a critical role for
recruitment of PAK to the plasma membrane, where stimulation by
activated Rac1 presumably takes place. We hypothesized that in analogy
to ERK1/2 activation by AngII (29, 30, 47), AngII-mediated
transactivation of a tyrosine kinase receptor might promote binding of
Nck, thus facilitating membrane localization of PAK. However, we could
not detect AngII-mediated binding of Nck to either EGF or PDGF
receptor. Furthermore, specific inhibition of EGF and PDGF receptor
kinases by tyrphostins did not affect AngII-mediated PAK activation in VSMC. These results are in agreement with recent data from Eguchi et al. (48) who showed that AngII-induced JNK activation in VSMC was not dependent on transactivation of the EGF receptor. Surprisingly, the tyrosine-phosphorylated 100-kDa protein that we
detected in Nck immunoprecipitates of AngII-stimulated VSMC did not
bind to a GST-Nck-SH2 fusion protein. However, AngII stimulated binding
of 30- and 25-kDa tyrosine-phosphorylated proteins to GST-Nck-SH2 that
were not detected in Nck immunoprecipitates. These data indicate that
the polyclonal Nck-antibody used for immunoprecipitation blocked the
Nck-SH2 domain. To date, the role of Nck-SH2 associated 25- and 30-kDa
phosphotyrosine proteins in AngII-mediated PAK activation remains
unclear. Identification of these molecules and characterization of
their function will be the purpose of future studies.
To define further the role of Nck in AngII signaling, we employed
expression of dominant-negative Nck mutants in CHO-AT1 cells. Our data
indicate that blocking Nck-SH2 domain interacting proteins in CHO-AT1
cells by overexpression of Nck-KSH3all or sequestration of PAK by an
inactivated Nck-SH2 domain (Nck-K308) significantly inhibited
activation of PAK and JNK by AngII. Conversely, a PAK mutant that lacks
Nck binding (PAK-A13) could not be stimulated by AngII. Although we
cannot completely rule out that other proteins interacting with either
Nck-SH3 domains or the PAK amino terminus play a role in AngII-mediated
PAK and JNK activation, these results strongly support a role for Nck
in AngII-mediated activation of PAK and JNK. Furthermore, they are the
first to demonstrate that a G-protein-coupled receptor requires Nck for
PAK activation.
Identification of Nck as an upstream mediator of JNK enabled us to
investigate the role of JNK in AngII-mediated growth responses in more
detail. Several in vivo studies have demonstrated previously the activation of JNK by AngII under conditions that led to media hypertrophy (49, 50) or cardiac hypertrophy (51). However, JNK has also
been implicated in apoptosis (52), and it becomes increasingly clear
that regulation of apoptosis by AngII is critical to hypertrophic
growth in cardiovascular tissues (53). In the present study, we used
CHO-AT1 cells as a model system to investigate the effect of JNK
inhibition on DNA synthesis. Overexpression of dominant-negative Nck
molecules inhibited JNK activation by AngII but did not affect ERK1/2
activation. Inhibition of JNK significantly decreased AngII-induced DNA
synthesis, indicating a growth promoting rather than proapoptotic role
of JNK in AngII signaling, at least in CHO-AT1 cells. In CHO-AT1 cells
and VSMC AngII stimulated ERK1/2 and JNK, whereas it did not activate
p38. Ushio-Fukai et al. (7) recently demonstrated a
stimulatory role for p38 in AngII-induced protein and DNA synthesis in
VSMC. However, in contrast to our results (5) they could not find JNK
activation by AngII in VSMC (7). Viedt et al. (54), on the
other hand, reported AngII-induced stimulation of ERK1/2, JNK, and p38
in VSMC. To date, it remains unclear why these differences exist in the
same cell system. However, since JNK and p38 activate common
transcription factors, such as ATF2 (55), it can be speculated that
they might substitute for each other under different conditions. Since
we could not detect AngII-mediated activation of p38 in CHO-AT1 cells
and VSMC, our results do not exclude a role for p38 in AngII-induced
growth responses in general. The development of specific
pharmacological JNK inhibitors will be of great help to address more
specifically the roles of ERK1/2, JNK, and p38 on AngII-promoted growth
in cardiovascular target tissues such as VSMC.
The present report identifies Rac1 and Nck as upstream
mediators of PAK (and hence JNK) in AngII signaling. These findings, together with previous results (5, 45), suggest the following model for
AngII-mediated JNK activation in VSMC: AngII stimulation of VSMC leads
to rapid activation of Rac1. Translocation of PAK to the "activation
compartment" is mediated by Nck, thus facilitating stimulation of PAK
by activated Rac1. Future studies are now aimed at identifying
Nck-interacting proteins that will hopefully further increase our
understanding of AngII-induced growth processes in cardiovascular tissues.