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(Hypertension. 1998;32:668-675.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Role of Calcium-Sensitive Tyrosine Kinase Pyk2/CAKß/RAFTK in Angiotensin II–Induced Ras/ERK Signaling

Satoshi Murasawa; Yasukiyo Mori; Yoshihisa Nozawa; Hiroya Masaki; Katsuya Maruyama; Yoshiaki Tsutsumi; Yasutaka Moriguchi; Yasunobu Shibasaki; Yoko Tanaka; Toshiji Iwasaka; Mitsuo Inada; ; Hiroaki Matsubara

From the Department of Medicine II, Endocrine Hypertension, Metabolism, and Renal Division, Kansai Medical University, Osaka (S.M., Y. Mori, H. Masaki, K.M., Y. Tsutsumi, Y. Moriguchi, Y.S., Y. Tanaka, T.I., M.I., H. Matsubara); and the Pharmacological Laboratory, Taiho Pharmaceutical Co, Ltd, Kawanai, Tokushima (Y.N.), Japan.

Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Endocrine Hypertension, Metabolism, and Renal Division, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8507, Japan. E-mail matsubah{at}takii.kmu.ac.jp

	Abstract

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Abstract—In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increase in extracellular signal–regulated kinase (ERK) activity in a pertussis toxin–insensitive manner. This ERK activation was abolished by the G_q-associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca²⁺ chelation by BAPTA-AM or TMB-8 abolished Ang II–induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca²⁺ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang II stimulation. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the binding of GTP to p21^Ras, which was nearly abolished by genistein and calmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II–induced ERK activation. It was also found for the first time that cardiac fibroblasts abundantly expressed Ca²⁺-sensitive tyrosine kinase Pyk2/CAKß/RAFTK and that Ang II markedly induced its activation in a Ca²⁺/calmodulin-sensitive manner. Overexpression of the dominant negative mutant of Pyk2 significantly attenuated Ang II– or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, respectively) and ERK tyrosine phosphorylation levels, as well as an increase in the binding of GTP to p21^Ras. These findings demonstrate that in cardiac fibroblasts, Ang II–induced Ras/ERK activation is dominantly regulated by G_q-coupled Ca²⁺/calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II–induced Ras/ERK pathway.

Key Words: angiotensin II • receptors, angiotensin • tyrosine kinase, calcium-sensitive • Pyk2

	Introduction

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Neonatal rat cardiac fibroblasts have abundant high-affinity angiotensin II (Ang II) receptors, which are classified pharmacologically as belonging to the Ang II type 1 receptor (AT₁-R) subtype.^{1 2} AT₁-R stimulation was found to stimulate DNA synthesis and cell proliferation¹ and also increase the synthesis of extracellular matrix proteins,³ suggesting that cardiac fibroblasts contribute to remodeling of the cardiac interstitium in a variety of physiological and pathological conditions. Ang II, acting via AT₁-R, initiates early biochemical events, including rapid production of diacylglycerol and inositol 1,4,5-triphosphate by phospholipase C (PLC)–mediated hydrolysis of inositol phospholipids and activation of protein kinase C (PKC).^{4 5 6} Ang II also induces a rapid increase in expression of the growth-associated nuclear proto-oncogenes similar to cellular events by peptide growth factors and stimulates tyrosine phosphorylation of multiple substrates, including p44 and p42 mitogen-activated protein/extracellular signal–regulated kinases (ERKs).^{7 8 9 10 11 12}

Although it was reported that PKC played a dominant role in the Ang II–induced activation of ERK in vascular smooth muscle cells (VSMCs)¹³ or cardiac myocytes,⁹ other studies indicated that calcium signaling rather than PKC plays a critical role for the ERK activation in these cells.^{10 11 12} Booz et al,⁶ using cardiac fibroblasts, reported that Ang II activates ERK activity by both PKC-independent and -dependent pathways, with increases in intracellular Ca²⁺ playing an important role in the PKC-independent pathway. Thus, because the signal transduction mechanism leading to ERK activation after Ang II stimulation has not been clearly defined, we attempted to examine the roles of various signaling molecules activated by Ang II through AT₁-R using cardiac fibroblasts expressing abundant amounts of AT₁-R. In this study, we propose a novel signaling pathway in cardiac fibroblasts by which AT₁-R signals to p21^Ras and subsequently to ERK mainly through the G_q-coupled Ca²⁺/calmodulin system, and we propose that Pyk2/CAKß/RAFTK activated downstream of Ca²⁺-sensitive tyrosine kinase plays an important role in the efficient activation of the AT₁-R/Ras/ERK signaling pathway.

	Methods

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Materials
GF109203X, U73122, U73343, BAPTA-AM, genistein, ST638, and W7 were purchased from Calbiochem. TMB-8, nifedipine, and calmidazolium chloride were also purchased from Calbiochem. Antibodies were purchased from the following vendors: Upstate Biotechnology (4G10-HRP), Novagen (anti–T7-tag monoclonal antibody), Transduction Laboratories (Pyk2), and New England Biolabs (phospho-specific ERK). The cDNA encoding dominant negative mutant of Pyk2 lacking its kinase domain was from Dr J. Schlessinger (New York Medical Center), T7-epitope tagged ERK1 was from Dr S. Ohno (Yokohama City University), and the dominant negative mutant of Ras (RasN17) was from Dr T. Kurosaki (Kansai Medical University). All cDNAs were subcloned into pRK5 or pRS eukaryotic expression vectors for stable transfection.

Cell Culture
Cardiac fibroblasts were prepared from ventricles of 1-day-old Wistar rats and grown as previously described; subcultured fibroblasts from passages 4 through 10, used in this experiment, were >99% fibroblasts.^{14 15} Subconfluent cells were serum-starved for 24 hours and used for the experiments.

ERK Activity
ERK activity was determined as previously reported.^{16 17} Briefly, the cells lysed in the lysis buffer were centrifuged after brief sonication, and the supernatant was assayed with an ERK assay kit (Amersham) that measured the incorporation of [-³³P]ATP into synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific ERK substrate. For immunoblot of protein resolved by 9% SDS-PAGE, we used phospho-specific ERK antibody (New England Biolabs Inc) that detects p42^ERK and p44^ERK only when catalytically activated by phosphorylation at Tyr-204.^{16 17}

Analysis of GTP-Bound Ras
Cells were prelabeled with 0.1 mCi/mL carrier-free ³²P-orthophosphate for 18 hours in phosphate-free DMEM. The reaction was terminated by aspirating the media, and cells were lysed in buffer containing 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton-X100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, 1 µg/mL antipain, 0.2% (wt/vol) aprotinin, 1 µg/mL chymostatin, and 1 µg/mL PMSF. The supernatant was immunoprecipitated with anti–Ha-Ras-agarose conjugate (Santa Cruz Biotechnology Inc) for 90 minutes at 4°C. Ras-associated guanine nucleotides were eluted in 2 mmol/L EDTA, pH 8, 2 mmol/L DTT, 0.2% SDS, 0.5 mmol/L GTP, and 0.5 mmol/L GDP for 20 minutes at 65°C. Eluted GTP and GDP were separated on polyethyleneimine-cellulose plates by thin-layer chromatography using 1.2 mol/L ammonium formate and 0.8 mol/L HCL. Labeled nucleotides were quantified by densitometry.

Calcium Analysis
Cells were washed twice and loaded with 3 µmol/L fura 2-AM in PBS containing 20 mmol/L HEPES, pH 7.2, 5.6 mmol/L glucose, 0.025% BSA, and 1 mmol/L CaCl₂. After 45 minutes of incubation at 37°C, cells were washed and diluted to 10⁶ cells/mL with the same buffer. Ca²⁺ levels were measured by exciting the fura 2 at 340 nm and 380 nm and rationing the fluorescence intensities detected at 510 nm. From this ratio, the level of Ca²⁺ was estimated using K_d that is derived from calibration curves. [Ca²⁺]_i was calibrated and computed as described.¹⁸

Transfection of DNA
DNAs (5 to 10 µg) was transfected with Lipofectamine Plus reagent according to the manufacturer's instructions (Gibco BRL) as previously reported¹⁹ ; stably transfected cells were selected with geneticin.

Transfected Epitope-Tagged ERK
Cells were cotransfected with tagged p42^ERK cDNA expression plasmid together with the RasN17 with Lipofectamine. After 48 hours of incubation, cells were disrupted with brief sonication. The supernatant was incubated with anti–T7-tag antibody for 4 hours at 4°C, precipitated using protein A/G agarose, and resuspended in 40 µL kinase buffer containing 18 mmol/L HEPES, pH 7.5, 10 mmol/L Mg(OAc)₂, 50 µmol/L ATP, 2 µCi [-³²P]ATP (Amersham), and 20 µg myelin basic protein for 20 minutes at 30°C. After incubation, the reaction was terminated by adding Laemmli sample buffer; the supernatant was boiled for 5 minutes and subjected to SDS-PAGE. The gel was washed with 7% acetic acid for 30 minutes and with 3% glycerol for 30 minutes, dried, and exposed.

Statistical Analysis
Results are expressed as mean±SE. ANOVA and Fisher's protected least significant difference test were used for multigroup comparisons, with a value of P<0.05 considered significant.

	Results

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AT₁-R–Induced ERK Activation Is Mediated by PLC Activation Through Pertussis Toxin–Insensitive G Protein
Ang II (100 nmol/L) stimulates ERK activity with a maximal increase (about 11-fold) at 5 to 10 minutes followed by a gradual decline (Figure 1A). Ang II–induced ERK activation was increased dose-dependently with a maximal peak at 100 nmol/L (Figure 1B), and this activation was blocked by 10 µmol/L losartan but not by 10 µmol/L PD123319 (data not shown). Subsequent ERK experiments were performed with 100 nmol/L Ang II stimulation for 8 minutes. Treatment with pertussis toxin (PTX) (1 µg/mL) for 24 hours did not affect Ang II–induced ERK activation, whereas it markedly inhibited ERK activation induced by lysophosphatidic acid (LPA), which has been shown to use the G_i-dependent pathway^{20 21} (Figure 1C). These findings were also confirmed by determination of tyrosine phosphorylation level of p42 ERK (Figure 1D). Although a single band was detected in immunoblotting with an anti–phospho-ERK antibody, we found in cardiac fibroblasts used in this study that p42 ERK rather than p44 ERK is dominantly present and tyrosine is phosphorylated by Ang II or EGF, which contrasted with EGF-induced phosphorylation of both p42 and p44 ERK in Cos-7 cells (Figure 1E). A similar pattern of phosphorylation of ERK was also observed when a different commercially available anti–phospho-ERK antibody (Promega) was used for the blot (Figure 1E). Pretreatment with the PLC inhibitor U73122²² nearly abolished Ang II–induced ERK activity dose-dependently (Figure 1F), while a structurally similar derivative of U73122 (U73343), which does not inhibit PLC, failed to exert a similar effect, and ERK activation by 100 nmol/L phorbol 12-myristate 13-acetate (PMA) was not inhibited by U73122 (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 1. Effects of Ang II, PTX, and U73122 on ERK activity in cardiac fibroblasts. A, Fibroblasts were stimulated with Ang II (100 nmol/L) for the indicated periods. B, Fibroblasts were stimulated with Ang II at indicated concentrations for 8 minutes. C and D, Fibroblasts were pretreated with or without 1 µg/mL PTX for 24 hours and stimulated with either Ang II (100 nmol/L) or LPA (1 µmol/L) for 8 minutes. Arrows indicate tyrosine-phosphorylated p42ERK. E, Cardiac fibroblasts or Cos-7 cells were incubated with epidermal growth factor (50 ng/mL) for 5 minutes, and the cell lysate was analyzed with Western blotting using anti-phospho ERK antibodies (lanes 1 through 4 purchased from NEB, lanes 5 through 8 from Promega). Blots shown are representative of 3 separate experiments. F, Fibroblasts were pretreated with indicated concentrations of PLC inhibitor U73122 for 30 minutes and stimulated with Ang II (100 nmol/L) for 8 minutes. Results shown are mean±SE of 3 to 4 separate experiments.

AT₁-R–Mediated ERK Activity Is Not Stimulated by Phorbol Ester–Sensitive PKC But Induced by Ca²⁺/Calmodulin-Dependent Pathway
PLC activation results in generation of inositol triphosphate (IP₃) and diacylglycerol, which induce the release of Ca²⁺ from intracellular stores and PKC activation, respectively.²³ Therefore, we examined the effects of the PKC inhibitors GF109203X and calphostin C on Ang II–induced ERK activation. Pretreatment with GF109203X (1 µmol/L) or calphostin C (50 nmol/L) completely inhibited ERK activation in response to 100 nmol/L PMA with no effects on basal ERK activity, whereas no significant inhibition was observed in Ang II–induced ERK activation (Figure 2A). These drugs also suppressed tyrosine phosphorylation of p42 ERK induced by PMA but not by Ang II (Figure 2B). Depletion of PKC by 24-hour incubation with 1 µmol/L PMA partially inhibited (19±2.7%, P<0.05) Ang II–induced ERK activity (Figures 2A and 2B). These data suggest that PKC-mediated pathways do not play a dominant role in Ang II–induced ERK activation and that GF109203X-, calphostin C–, and phorbol ester–sensitive PKC is not involved in this mechanism.

View larger version (48K): [in this window] [in a new window]   Figure 2. Effects of PKC on Ang II–induced ERK activation. A and B, Fibroblasts were pretreated with or without the PKC inhibitor GF109203X (1 µmol/L) for 30 minutes, calphostin C (100 nmol/L) for 60 minutes, and then stimulated with either Ang II (100 nmol/L) or PMA (100 nmol/L) for 8 minutes. PKC depletion experiments were performed by incubating cells with PMA (1 µmol/L) for 24 hours. Arrows indicate tyrosine-phosphorylated p42ERK. Results are mean±SE of 4 separate experiments. Blots are representative of 3 separate experiments.

Exposure of fibroblasts to Ang II markedly increased intracellular Ca²⁺ levels, and this increase was blocked by pretreatment with the intracellular Ca²⁺ chelator BAPTA-AM (10 µmol/L) (Figure 3A) but not by extracellular Ca²⁺ chelation by EGTA (data not shown). As shown in Figure 3B, Ang II–induced ERK activation was completely inhibited by pretreatment with BAPTA-AM (10 µmol/L) and TMB-8 (100 µmol/L), commonly used as intracellular Ca²⁺ chelators, but not by pretreatment with EGTA or blockade of L-type Ca²⁺ channels with nifedipine. Elevation of cytosolic Ca²⁺ activates a variety of enzymes through interaction with calmodulin.²⁴ To examine whether calmodulin mediates ERK activation in response to Ang II, fibroblasts were preincubated with the calmodulin inhibitors W7 (100 µmol/L) and calmidazolium (10 µmol/L). These drugs completely blocked Ang II–induced ERK activity with no effect on basal ERK activity, and the ERK activation induced by the Ca²⁺ ionophore A23187 was also inhibited by these drugs (Figure 3C). On the other hand, these calmodulin inhibitors had no effect on Ang II–induced increase in intracellular Ca²⁺ level (data not shown) or PMA-induced ERK activation (Figure 3C). These results suggest that Ang II stimulates ERK activity through a Ca²⁺/calmodulin-dependent mechanism.

View larger version (28K): [in this window] [in a new window]   Figure 3. Effects of Ca2+ signal inhibitors on intracellular Ca2+ levels and Ang II– or A23187-induced ERK activation. A, Fibroblasts were incubated for 30 minutes at 37°C with or without BAPTA-AM (10 µmol/L). Cells were trypsinized and resuspended in the buffer for measurement of intracellular Ca2+ levels using fura 2-AM and stimulated with Ang II (100 nmol/L). Traces are typical of those from 3 separate experiments. B and C, Fibroblasts were pretreated with or without the intracellular Ca2+ chelator BAPTA-AM (10 µmol/L) for 30 minutes, the intracellular Ca2+ chelator TMB-8 (100 µmol/L) for 30 minutes, the extracellular Ca2+ chelator EGTA (5 mmol/L) for 3 minutes, the L-type Ca2+ channel blocker nifedipine (1 µmol/L) for 3 minutes, the calmodulin inhibitors W7 (10 µmol/L) and calmidazolium (10 µmol/L) for 30 minutes, and then stimulated with Ang II (100 nmol/L), A23187 (10 µmol/L), or PMA (1 µmol/L) for 8 minutes. Results are mean±SE of 3 to 4 separate experiments.

AT₁-R–Mediated ERK Pathway Is Activated by Ca²⁺/Calmodulin-Dependent Protein Tyrosine Kinases
To determine whether tyrosine kinase activity is required for Ca²⁺-dependent ERK activation in response to Ang II, cells were pretreated with genistein (100 µmol/L) and ST638 (100 µmol/L), protein kinase inhibitors with a strong preference for tyrosine-specific kinases,^{25 26} and then stimulated with either Ang II or A23187. These inhibitors completely abolished both Ang II– and A23187-induced ERK activation with no effects on basal ERK activity (Figure 4A). Similar inhibitory effects were observed in Ang II–induced tyrosine phosphorylation of p42 ERK (Figure 4B). On the other hand, these tyrosine kinase inhibitors did not have any effect on Ang II–induced increase in intracellular Ca²⁺ level (data not shown) or PMA-induced ERK activation (Figure 4A and 4B). These findings suggest that protein tyrosine kinases activated downstream of the Ca²⁺/calmodulin pathway are closely involved in Ang II–induced ERK activation.

View larger version (41K): [in this window] [in a new window]   Figure 4. Effects of tyrosine kinase inhibitors on Ang II– and A23187-induced ERK activation. A and B, Fibroblasts were pretreated with or without tyrosine kinase inhibitors genistein (100 µmol/L) or ST638 (100 µmol/L) for 45 minutes and then stimulated with either Ang II (100 nmol/L), A23187 (10 µmol/L), or PMA (1 µmol/L) for 8 minutes. Arrows indicate tyrosine-phosphorylated p42ERK. Results are mean±SE of 3 to 4 separate experiments. Blots are representative of 3 separate experiments.

Ang II Increases GTP-Bound Ras and Ang II–Induced ERK Activation is Dependent on Ras
Ang II (100 nmol/L) induced a rapid accumulation of GTP-bound p21^Ras that reached a maximum in 4 to 5 minutes (1.8±0.3-fold increase relative to control, n=4; Figure 5A), gradually declined, and returned to the basal level within 20 minutes. Treatment with A23187 also elicited an increase in GTP-bound p21^Ras to a similar extent (1.7±0.3-fold increase relative to control, n=4; Figure 5A). To examine whether activation of p21^Ras and ERK by Ang II requires similar upstream signaling, the effects of several signal transduction inhibitors were tested on Ang II–induced p21^Ras activation. Pretreatment with genistein (100 µmol/L) and calmidazolium (10 µmol/L) (Figure 5A), but not with PTX (1 µg/mL for 24 hours) or calphostin C (50 nmol/L) (data not shown), completely prevented Ang II–induced p21^Ras activation with no effect on basal activity.

View larger version (42K): [in this window] [in a new window]   Figure 5. Role of Ras in Ang II–induced ERK activation. A, Cardiac fibroblasts were labeled with 32P-orthophosphate for 18 hours and treated with Ang II (100 nmol/L) or A23187 (10 µmol/L) for 5 minutes in the presence or absence of pretreatment (30 minutes) with genistein (100 µmol/L) and calmidazolium (10 µmol/L). Cell lysates were treated with anti–Ha-Ras antibody, and [32P]GTP/GDP in the immunoprecipitate was separated. A representative autoradiogram from 3 separate experiments is shown. B, Fibroblasts were pretreated with or without the Ras inhibitor manumycin (10 µmol/L) for 60 minutes and stimulated with Ang II (100 nmol/L) or A23187 (10 µmol/L) for 8 minutes. Results are mean±SE of 3 separate experiments. C, Epitope-tagged ERK was transiently transfected with pRS vector alone or dominant negative Ras (RasN17) into cardiac fibroblasts. Cells were stimulated with Ang II (100 nmol/L), A23187 (10 µmol/L), epidermal growth factor (EGF) (10 ng/mL), or PMA (1 µmol/L) for 8 minutes. The activity of transfected ERK was assessed by measuring myelin basic protein (MBP) phosphorylation. A representative autoradiogram from 3 separate experiments is shown.

We further examined the role of Ras in the AT₁-R/ERK cascade pharmacologically using manumycin, which is a Ras farnesyl-transferase inhibitor and effectively suppresses Ras biological functions.²⁷ Although the basal ERK activity was slightly increased by pretreatment with manumycin (10 µmol/L), both AT₁-R– and A23187-induced ERK activation were abolished by manumycin to a similar extent (Figure 5B). Furthermore, we investigated the effect of overexpression of the dominant negative Ras (RasN17). We transfected an epitope-tagged ERK with or without RasN17 into fibroblasts and treated cells with Ang II. It was very likely that both epitope-tagged ERK and RasN17 DNAs were transfected into the same cells, and the ERK immunoprecipitated with anti–epitope-tag antibody was able to reflect the effect of RasN17. In preliminary experiments, by examining the cotransfected ß-galactosidase transgene activity, we found that the transfection efficiency of epitope-tagged ERK was not changed with or without the RasN17. As shown in Figure 5C, both Ang II– and A23187-induced ERK activation were completely abolished by cotransfection of RasN17, and EGF-induced ERK activation (well known to be dominantly mediated through Ras) was also inhibited by RasN17, whereas PMA-induced ERK activation was not affected in cells overexpressing RasN17.

Ang II–Dependent ERK Activation and GTP Loading of Ras Is Sensitive to Pyk2
We next examined the role of a Ca²⁺-sensitive tyrosine kinase, Pyk2,²⁸ in Ang II–mediated ERK signaling. Although it has been reported that Pyk2, also termed CAKß²⁹ or RAFTK,³⁰ was expressed at high levels mainly in cells of neuronal origin^{28 29 30} but not in cardiac muscle,³¹ we found for the first time that Pyk2 was abundantly present in cardiac fibroblasts (Figure 6A) and that both Ang II and A23187 markedly stimulated tyrosine phosphorylation of Pyk 2 (Figure 6B). The Ang II–induced Pyk2 activation was nearly abolished by Ca²⁺/calmodulin kinase inhibitor (W7) and chelation of intracellular Ca²⁺ with BAPTA-AM (Figure 6B). W7 or BAPTA-AM alone had no effect on the basal phosphorylation level of Pyk2, and tyrosine phosphorylation of Pyk2 was very rapid and occurred before maximal activation of ERK by Ang II (5 minutes) (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 6. Involvement of Pyk2 in Ang II–induced ERK activation and GTP loading of Ras. A, Pyk2 immunoblot (116 kDa) of whole-cell lysates from untransfected cardiac fibroblasts, untransfected PC12 cells, and cardiac fibroblasts stably transfected with cDNA for a dominant negative mutant of Pyk2 (PKM). B, Phosphorylation of Pyk2 in response to Ang II. Serum-starved cardiac fibroblasts treated with A23187 (10 µmol/L) or Ang II (100 nmol/L) for 2 minutes in the presence and absence of pretreatment with W7 (10 µmol/L for 30 minutes) or BAPTA-AM (10 µmol/L for 30 minutes) were immunoprecipitated with anti-Pyk2 antibody and blotted with anti-phospho tyrosine antibody (upper panel). Same membranes were reprobed and blotted with anti-Pyk2 antibody (lower panel). C, Cells stably transfected with pRK5 (mock-transfected control) or PKM were stimulated with Ang II (100 nmol/L) or A23187 (1 µmol/L) for 8 minutes, and ERK activities and tyrosine phosphorylation levels were determined. As a control experiment, these cells were stimulated with PMA (1 µmol/L for 8 minutes). Results are mean±SE of 6 separate experiments. *P<0.01 vs data of mock-transfected control. D, GTP loading of Ras was examined using cells overexpressing PKM with the same experimental method described in Figure 5.

To investigate the involvement of Pyk2 in the Ang II–induced Ras/ERK pathway, cells were stably transfected with a dominant negative mutant of Pyk2 (PKM) lacking its kinase domain³² (Figure 6A). We obtained several cloned cells expressing PKM and selected the clone that most abundantly expressed the PKM (Figure 6A). AT₁-R numbers in these clones were examined by the ligand binding assay using the membrane fraction, and its expression level was found to be comparable with that in the control cells (Table). Ang II–induced elevation of intracellular Ca²⁺ level in this cloned cell was comparable with that in the control cells (data not shown), and PMA-induced ERK activation that utilizes upstream pathways different from AT₁-R–mediated ERK signaling was preserved in the transfectants (Figure 6C). Interestingly, overexpression of this mutant significantly attenuated Ang II– or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, P<0.01, respectively) and ERK tyrosine phosphorylation levels. We also examined the effect of PKM overexpression on GTP loading of Ras. Ang II–induced accumulation of GTP-bound Ras was significantly inhibited (47±3%, P<0.001, n=3) in cells overexpressing PKM compared with that in the mock-transfected cells (Figure 6D). These data suggest that Pyk2 plays a critical role in the efficient activation of the Ang II–induced Ras/ERK pathway in a Ca²⁺/calmodulin-sensitive manner.

View this table: [in this window] [in a new window]   Table 1. AT1-R Expression in Cells Expressing Pyk2 Dominant Negative Mutant

	Discussion

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The present study demonstrates that in cardiac fibroblasts, Ang II–induced Ras/ERK activation is dominantly mediated by a G_q-coupled Ca²⁺/calmodulin-dependent mechanism and that activation of Pyk2 that lies downstream of Ca²⁺/calmodulin signals plays an important role in this signaling pathway. Earlier studies using VSMCs¹³ reported an involvement of PKC or Ca²⁺-insensitive tyrosine kinase in the AT₁-R/ERK signaling pathway, whereas the critical role of intracellular Ca²⁺ was also reported in VSMCs¹² or cardiac myocytes.^{10 11} Booz et al⁶ previously reported that in cardiac fibroblasts, Ang II induces ERK activity by both PKC-independent and -dependent pathways, with increases in intracellular Ca²⁺ playing an important role in the PKC-independent pathway; this contrasts with our present data indicating the dominant role of a Ca²⁺-mediated pathway in Ang II–induced ERK activation. However, we also found that PKC depletion by prolonged PMA treatment had a minor effect on Ang II–induced ERK activation, whereas the PKC inhibitors GF109203X and calphostin C, known to inhibit PKC-, -ßI, -ßII, and -,³³ had no effect on ERK activation by Ang II (Figure 2). Thus, it might be possible that the pathway by which GF109203X- and calphostin C– or phorbol ester–insensitive isoforms of PKC such as PKC- transmit Ang II signaling to ERK is at least partially involved in Ang II–induced ERK activation, and that this PKC isoform is relatively downregulated in cells used in this study compared with that of Booz et al⁶ because of the influence of passage numbers or the difference in experimental conditions. In the present study, we further extended the study by Booz et al⁶ by examining the downstream pathway of Ca²⁺ signaling and found for the first time that Pyk2 activated downstream of G_q-coupled Ca²⁺/calmodulin signals plays an important role in the efficient activation of the AT₁-R–mediated Ras/ERK signaling pathway.

Our present data demonstrated that both Ang II and A23187 activate p21^Ras and that the Ras-specific inhibitor manumycin and overexpression of the dominant negative mutant of Ras nearly abolished Ang II– or A23187-induced ERK activation. Ang II–induced p21^Ras activation was also blocked by either chelation of intracellular Ca²⁺ or tyrosine kinase inhibitors, suggesting the presence of a common Ras/ERK pathway shared between Ang II and Ca²⁺ signaling. It was reported that the adaptor protein Shc activated by receptor or nonreceptor tyrosine kinases is involved in the Ras/ERK signaling pathway.³⁴ Tyrosine-phosphorylated Shc can activate p21^Ras by binding to the SH2 domain of adaptor protein Grb2, which is complexed to the guanine nucleotide exchange factor Sos through its SH3 domains. Recently, Schorb et al⁷ reported that Ang II phosphorylates p46^Shc and p56^Shc in cardiac fibroblasts. In addition, Ang II–induced Shc phosphorylation resulted in the subsequent formation of a complex between Shc and Grb2 in cardiac myocytes¹⁰ or VSMCs.²¹ Taken together, these data suggest that Ca²⁺-dependent ERK activation by Ang II is mediated by the signaling pathway initiated by tyrosine phosphorylation–mediated Shc-Grb2-Sos complex formation, resulting in the activation of p21^Ras.

Ca²⁺-dependent activation of a novel focal adhesion kinase family protein tyrosine kinase, Pyk2, also termed CAKß²⁹ or RAFTK,³⁰ has been shown to mediate G_q-coupled receptor–stimulated ERK activation in neuronal cells²⁸ via a direct interaction with c-Src or association with Grb2.³² In neuronal cells, association of p60^c-Src with Pyk2 mediates both Shc phosphorylation and ERK activation.³⁵ Although it has been reported that Pyk2 was expressed at high levels, mainly in cells of neuronal origin^{28 29 30} but not in cardiac muscle,³¹ we found for the first time that Pyk2 was abundantly present in cardiac fibroblasts and that Ang II markedly stimulated tyrosine phosphorylation of Pyk2 in a Ca²⁺/calmodulin-sensitive manner. Consistent with our observations, Brinson et al³⁶ also have very recently reported that Pyk2 is activated by Ang II in a nonneuronal cell type such as VSMCs. The detection of Pyk2 in cell lysates from cardiac fibroblasts, as well as the sensitivity of AT₁-R–mediated ERK activation in this cell to the dominant negative mutants of Pyk2, suggests that a Pyk2-mediated mechanism of Ras activation may represent a paradigm for mitogenic signaling in a variety of nonneuronal cell types. Considering that dominant negative mutants of Pyk2 moderately inhibited Ang II–induced ERK activity in contrast with complete inhibition by a dominant negative mutant of Ras, it is likely that an unidentified pathway other than Pyk2 that transmits Ca²⁺ signal to Ras activation exists in this signal transduction system. As indicated by the involvement of the platelet-derived growth factor receptor²¹ or epidermal growth factor receptor^{17 37} in Ang II signaling in VSMCs or cardiac fibroblasts, Ang II–induced transactivation of growth factor receptors is likely involved in this signal transduction system, and Pyk2 may contribute at least partially to such a transactivation mechanism. The mechanism of Ca²⁺/calmodulin-mediated Pyk2 activation remains unclear, since Pyk2 lacks a calmodulin binding motif and is not directly activated by Ca²⁺.^{28 29} However, it was shown that in HEK-293 cells, Pyk2 activated in a Ca²⁺/calmodulin-dependent manner is involved in G_i- and G_q-mediated ERK activation.³² Taken together with the fact that a Ca²⁺/calmodulin-activated tyrosine kinase has been purified from bovine uterus,³⁸ these findings suggest the presence of a Ca²⁺/calmodulin effector protein to activate Pyk2. Further studies are required to completely elucidate the AT₁-R–mediated Ca²⁺/calmodulin-dependent signaling pathway leading to activation of the Ras/ERK cascade.

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science, and Culture, Japan; the Study Group of Molecular Cardiology, Naito Foundation; the Clinical Pharmacology Foundation; and the Japan Medical Association; and the Japan Smoking Foundation, Japan Heart Foundation. Dr Satoshi Murasawa is a Research Fellow of the Japan Society for the Promotion of Science.

Received April 8, 1998; first decision May 4, 1998; accepted July 2, 1998.

	References

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