Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck.

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.

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 GTPbound Rac or Cdc42. We have previously shown that PAK is stimulated by AngII in VSMC in a tyrosine kinase-and PKCdependent manner (5). Furthermore, our data implicated PAK as an upstream mediator of JNK in VSMC (5).
PAK consists of an NH 2 -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 AngIIinduced 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.
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 M 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 g/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 ϫ 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 ϫ 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 ϫ 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 ϫ 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 ϫ g the supernatants were size-fractionated by SDS-PAGE, and PAK activity was assayed by 32 P 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 M 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 l of kinase reaction buffer containing 50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 2 mM MnCl 2 , 50 M ATP, and 5 Ci of [␥-32 P]ATP for 30 min at 30°C in the presence of 1 g 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 ϫ 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 MgCl 2, 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 l of kinase reaction buffer containing 20 mM HEPES, 10 mM MgCl 2 , 50 M ATP in the presence of 2 g 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 l 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 MgCl 2 , 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.
[ 3 H]Thymidine Incorporation into DNA-The effect of AngII on [ 3 H]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 M AngII for 24 h. 20 h after addition of AngII 3 Ci/ml [ 3 H]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 CaCl 2 , 1 mM MgCl 2 , 10% trichloroacetic acid (w/v), and ethanol/ether (2:1, v/v). Acid-insoluble [ 3 H]thymidine was extracted with 0.5 M NaOH (250 l per well), and 100 l 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 l 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/g protein.
Densitometry and Statistics-For quantification of Western blots or 32 P 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 Ϯ S.E.). Statistical analysis was performed by Student's t test (unpaired and two-tailed) using StatView 5.0 software. A p value of Ͻ0.05 was considered significant.

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 Ϯ 0.13-fold increase, n ϭ 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 M AngII (2.6 Ϯ 0.62-fold increase, n ϭ 3) (Fig. 1B, lower panel). However, AngII-induced stimulation of Cdc42 was not observed (data not shown). (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 M genistein for 16 h or 10 M PP1 for 15 min prior to stimulation by 1 M AngII, and Rac1 binding to GST-PAK-PBD was determined. We have shown previously that 10 M PP1 effectively inhibited Src activation by AngII in VSMC (33). Genistein inhibited AngII-mediated binding of Rac1 to GST-PAK-PBD (fold increase of Me 2 SO ϭ 2.47 Ϯ 0.31 versus genistein ϭ 1.21 Ϯ 0.12, p ϭ 0.02, Fig. 2A, lower panel), whereas PP1 had no significant effect (fold increase of Me 2 SO ϭ 2.47 Ϯ 0.31 versus PP1 ϭ 2.63 Ϯ 0.37, p ϭ 0.74, Fig. 2B, lower panel), indicating that a tyrosine kinase other than Src is involved in AngII-induced Rac1 activation.
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).
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 ϳ2-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).
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 Ϯ 0.03-fold (n ϭ 3), whereas there was a 3.6 Ϯ 0.3 (n ϭ 3)-fold increase in the membrane fraction (p ϭ 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 tyrosinephosphorylated 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).
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

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. 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 M 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 AngIIinduced PAK activation (Fig. 7).
Overexpression of Dominant-negative Nck Inhibits AngII-induced Activation of PAK and JNK in CHO-AT1 Cells-We have shown previously (5)  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 ϳ150-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).
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).
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 kinasedead 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).

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 AngIIinduced 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 membranetargeted 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 AngIIinduced 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 AngIIstimulated 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 AngIImediated 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 AngIIpromoted 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.