Both Gs and Gi Proteins Are Critically Involved in Isoproterenol ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 274, No. 14, Issue of April 2, pp. 9760 –9770, 1999 Printed in U.S.A.

Both Gs and Gi Proteins Are Critically Involved in Isoproterenol-induced Cardiomyocyte Hypertrophy* (Received for publication, November 9, 1998, and in revised form, December 21, 1998)

Yunzeng Zou‡§, Issei Komuro‡¶, Tsutomu Yamazaki‡i, Sumiyo Kudoh‡, Hiroki Uozumi‡, Takashi Kadowaki‡, and Yoshio Yazaki‡ From the ‡Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655, and the iHealth Service Center, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Activation of b-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked intracellular signal transduction pathways leading to activation of extracellular signalregulated kinases (ERKs) and cardiomyocyte hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that Gs protein is involved in the activation. Inhibition of Gi protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the Gbg subunit binding domain of the b-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of Gbg and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of dominant-negative mutants of Ras and Raf-1 kinase and of the b-adrenoreceptor mutant that lacks phosphorylation sites by PKA abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed by inhibitors for PKA, Gi, tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy through two different G proteins, Gs and Gi. cAMP-dependent PKA activation through Gs may phosphorylate the b-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Gi activates ERKs through Gbg, Src family tyrosine kinases, Ras, and Raf-1 kinase.

Cardiac hypertrophy is often associated with an increase in intracardiac sympathetic nerve activity and with elevated plasma catecholamines (1). Treatment of cardiac myocytes with catecholamines not only changes their functions such as beating rates and contractile activity but also induces typical hypertrophic responses (2–7). There are two major subtypes, a and b, in adrenoreceptors (ARs).1 AR agonists such as norepi* This work was supported in part by a grant-in-aid for scientific research, developmental scientific research, and scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan and a by grant from Japan Heart Foundation/Pfizer Pharmaceuticals for research on coronary artery diseases, Tanabe Medical Frontier Conference (to I. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a postdoctoral fellowship from the Japan Society for the Promotion of Science. ¶ To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3127); Fax: 81-3-3818-6673; E-mail: [email protected]. 1 The abbreviations used are: AR(s), adrenoreceptor(s); NE, norepinephrine; PHE, phenylephrine; ISO, isoproterenol; a- and b-AR, a- and b-adrenergic receptor, respectively; PKC, protein kinase C; ERK, extracellular signal-regulated protein kinase; PKA, cAMP-dependent protein

nephrine (NE) (a and b), phenylephrine (PHE) (a), and isoproterenol (ISO) (b) have been reported to induce cardiomyocyte hypertrophy (2–7). Prolonged infusion of subpressor doses of NE increases the mass of the myocardium and the thickness of the left ventricular wall, suggesting that NE has direct hypertrophic effects on cardiac myocytes without affecting afterload (2). We have reported that NE induces cardiomyocyte hypertrophy through both a- and b-ARs (7). It has been reported that PHE evokes hypertrophic responses in the cardiac myocytes of neonatal rats (5) and that expression of constitutively active a-AR induces cardiac hypertrophy in adult mice (8). Both in vivo and in vitro studies demonstrate that ISO also stimulates expression of proto-oncogenes in cardiomyocytes and induces cardiac hypertrophy (6, 9, 10). Activation of each AR evokes specific intracellular signals (4, 11). It has been shown that stimulation of a1-AR activates phosphoinositide-specific phospholipase C via Gq protein and hydrolyzes phosphoinositide 4,5-bisphosphate into inositol 1,4,5-triphosphate and diacylglycerol. Diacylglycerol activates protein kinase C (PKC) leading to activation of the Raf-1 kinase/extracellular signal-regulated protein kinase (ERK) cascade (12, 13). There was a report indicating that PHE activates ERKs and induces cardiomyocyte hypertrophy through the Rasdependent pathway (5). Regarding b-AR-induced signaling pathways, stimulation of b-AR activates adenylyl cyclase through a different G protein, Gs. Activation of adenylyl cyclase produces a second messenger cyclic adenosine monophosphate (cAMP), leading to activation of cAMP-dependent protein kinase (PKA) (14). PKA activation is important for the control of cell growth and differentiation. cAMP/PKA has been reported to have an inhibitory effect on the activation of ERKs stimulated by growth factors in many cell types such as Rat-1 cells, smooth muscle cells, Chinese hamster ovary cells, COS-7, and adipocytes (15–19). On the contrary, in some cell types such as PC12 cells, Swiss-3T3 cells, and S49 mouse lymphoma cells, cAMP activates ERKs and potentiates the effects of growth factors on differentiation and gene expression (20 –24). In human endothelial cells, down-regulation of the a subunit of Gs (Gsa) abolishes b-AR-mediated ERK activation by ISO (25), suggesting that Gsa-dependent cAMP elevation and PKA activation are responsible for the activation of ERKs. We and others have also demonstrated that b-AR agonists including ISO significantly activate ERKs and increase protein synthesis through cAMP/PKA in cardiac myocytes (6, 7). However, the

kinase; PTX, pertussis toxin; HA, hemagglutinin; HA-ERK2, HAtagged version of the wild type ERK2; bARK1495– 689, b-AR kinase 1 residues Gly495 to Leu689; Csk, COOH-terminal Src kinase; D.N.Raf-1, dominant negative mutant of Raf-1 kinase; D.N.Ras, dominant negative mutant of Ras; b2-ARwt, wild-type b2-AR; b2-ARmut, b2-AR mutant; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis.

9760

This paper is available on line at http://www.jbc.org

Isoproterenol-induced Cardiomyocyte Hypertrophy molecular mechanisms of b-AR agonist-induced ERK activation remain largely unknown. In the present study, we examined the molecular mechanism of ISO-evoked activation of ERKs in the cardiomyocytes of neonatal rats. We observed that ISO-induced activation of ERKs is dependent on both Gs/cAMP/ PKA and Gi/Src/Ras signaling pathways. Quite recently, it has been reported that PKA activated through b-AR phosphorylates agonist-coupled b-AR, leading to a change of the receptor coupling from Gs to Gi (26). We examined whether phosphorylation of b-AR is critical to ISO-induced activation of ERKs also in cardiac myocytes. EXPERIMENTAL PROCEDURES

Materials—[g-32P]ATP and [3H]phenylalanine were purchased from NEN Life Science Products. Dulbecco’s modified Eagle’s medium, fetal bovine serum, and genistein were from Life Technologies, Inc. Pertussis toxin (PTX) was from List Biological Laboratories, Inc. Calphostin C was from BIOMOL. RpcAMP and H89 were from Biolog. Polyclonal antibodies against b1- or b2-AR, Shc, and Grb2 were from Santa Cruz Biotechnology Inc. Anti-hemagglutinin (HA) polyclonal antibody was from Mitsubishi Biochemical Laboratories (Japan). Other reagents were purchased from Sigma. cDNA Plasmids—HA-tagged ERK2 (HA-ERK2) in the SV40 promoter, cDNA encoding the COOH-terminal Gbg subunit binding domain of the bovine b-AR kinase 1 residues Gly495 to Leu689 (bARK1495– 689), wild-type COOH-terminal Src kinase (Csk), dominant-negative mutant of Ras (Asn-17) (D.N.Ras), and D.N.Raf-1 kinase (Ala-375), both of which are driven by the cytomegalovirus promoter, were provided and prepared as described previously (27, 28). Wild-type b2-AR (pRK5-b2ARwt) and b2-AR mutant lacking phosphorylation sites for PKA (pcDNAI-b2-ARmut; point mutations of Ser residues at 261, 262, 345, and 346 to Ala) were kind gifts from R. J. Lefkowitz and Y. Daaka (26). All plasmid DNA was prepared using QIAGEN plasmid DNA preparation kits (Hilden, Germany). Cell Culture—Cardiac myocytes from ventricles of 1-day-old Wistar rats were isolated as described previously (27). Cardiac myocytes were plated at a field density of 105 cells/cm2 on culture dishes with culture medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum). The culture medium was changed to serum-free Dulbecco’s modified Eagle’s medium at 48 h before treatment. Transfection—After 24 h of plating cardiac myocytes on culture dishes, DNA was transfected by the calcium phosphate method as described previously (27). For each dish, 2.5 mg of HA-ERK2 plasmid DNA was transfected with or without 7.5 mg of other relevant plasmids such as b2-ARwt, b2-ARmut, bARK1495– 689, Csk, D.N.Ras, or D.N.Raf-1. After transfection, the cells were maintained in serum-free Dulbecco’s modified Eagle’s medium for 48 h before stimulation with ISO. The transfection efficiency of each experiment was ;1% in cardiac myocytes as assessed by LacZ staining after transfection of a LacZcontaining expression plasmid. [3H]Phenylalanine Incorporation—Protein synthesis was determined by assessing the incorporation of labeled phenylalanine from the extracellular medium into the total trichloroacetic acid-precipitable cell protein (7, 29 –32). Cardiac myocytes were serum deprived for 2 days, pretreated with or without a variety of inhibitory agents, and then incubated for 24 h with ISO. [3H]Phenylalanine (1 mCi/ml) was added 2 h before the harvest. It has been reported that continuous long term (.24 h) labeling of cultured cardiomyocytes is intrinsically nonlinear, such that labeled amino acid accumulates asymptotically into protein (33). Because it has been reported that the specific radioactivities of the aminoacyl-tRNA pool may be equilibrated after 5 min and remain so over 2 h of labeling (29 –32, 34), pulse labeling for 2 h with [3H]phenylalanine is sufficient to achieve adequate equilibration of the specific activity of phenylalanyl-tRNA. At the end of the labeling incubation, the plates were placed on ice, quickly washed twice with ice-cold phosphate-buffered saline, incubated for 30 min with 10% trichloroacetic acid, and washed. Precipitates were solubilized for 30 min in 1 M NaOH and neutralized, and total radioactivity was measured by liquid scintillation spectroscopy. Assay of ERK Activity—ERK activities were measured using myelin basic protein (MBP)-containing gel as described previously (27). In brief, cell lysates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/ml MBP. ERKs in the gel were denatured in guanidine HCl, renatured in Tris-HCl containing Triton X-100 and 2-mercaptoethanol, and incubated with [g-32P]ATP. Phosphorylation activities of ERKs were assayed by subjecting to autoradiography. The

9761

activity of transfected HA-ERK2 was assayed as described previously (27). In brief, after transfection of HA-ERK2, cell lysates were incubated with an anti-HA polyclonal antibody. The immune complex was incubated with MBP and [g-32P]ATP for 10 min at 30 °C. The sample was subjected to SDS-PAGE, and the phosphorylated MBP band was visualized by autoradiography. Phosphorylation of Shc and Its Association with Grb2—Tyrosine phosphorylation of Shc and the association of Shc with Grb2 were examined by Western blot analysis as described previously (28). In brief, cell lysates were incubated with an anti-Shc or an anti-Grb2 antibody, and the immune complexes were subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The membranes were immunoblotted with an anti-phosphotyrosine antibody (PY20) or an anti-Shc antibody, and the immunoreactivity was detected using the enhanced chemiluminescence reaction (ECL) system (Amersham Pharmacia Biotech) according to the manufacturer’s directions. Examination of b-ARs in Cardiac Myocytes—The amount of b-ARs on the cell surface was examined by Western blotting after dividing the membrane fraction and the cytoplasmic fraction. Briefly, cells were lysed by RIPA buffer (1 3 phosphate-buffered saline, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 30 mg/ml aprotinin). Whole lysate was first centrifuged at 200 3 g to remove nuclei, and then the supernatant was centrifuged at 15,000 3 g for 30 min at 4 °C to pellet cell membrane. The pelleted membrane fractions were washed, subjected to SDS-PAGE, and then transferred to Immobilon-P membranes. After blocking with 30% non-fat dry milk, the membranes were incubated with an anti-b1-AR or an anti-b2-AR polyclonal antibody. The immunoreactivity was detected using the ECL system. Analysis of Statistics—Statistical comparison was carried out within three or more groups using one-way analysis of variance and Dunnett’s t test. Values of p , 0.05 were considered statistically significant. RESULTS

cAMP-dependent PKA Mediates ISO-induced Activation of ERKs—We have demonstrated recently that b-AR stimulation by NE activates ERKs through cAMP/PKA-dependent pathways in cardiac myocytes (7). We therefore defined the role of cAMP/PKA in ISO-induced ERK activation. When cAMP was inhibited by a cAMP analog, RpcAMP (100 mM), ISO-evoked ERK activation was completely suppressed (see Fig. 1A). Pretreatment with a PKA inhibitor, H89 (10 mM), also abolished ISO-induced activation of ERKs, whereas inhibition of PKC by calphostin C (1 mM) did not affect the ERK activation by ISO (Fig. 1A). On the other hand, PHE-induced activation of ERKs was suppressed by calphostin C but not by RpcAMP or H89 (Fig. 1B). These results suggest that cAMP-dependent PKA activation probably through Gs is required for ISO-induced activation of ERKs in cardiac myocytes. Gi Protein Is Involved in ISO-induced Activation of ERKs—We next examined the possibility that Gi protein is involved in ISO-induced ERK activation because b-AR was reported to bind Gi (35, 36), and activation of Gi protein can activate ERKs in many cell types (37, 38). We preincubated cardiac myocytes with 100 ng/ml PTX and then treated them with ISO or PHE. Activation of ERKs by ISO was abolished by the PTX treatment for 24 h, whereas PTX had no effect on PHE-induced ERK activation (Fig. 2A). These results suggest that PTX-sensitive Gi protein is responsible for ISO- but not PHE-induced activation of ERKs in cardiac myocytes. It has been reported recently that stimulation of Gi proteincoupled receptors activates ERKs by bg subunits (Gbg) but not by the a subunit (39 – 41). Therefore, the role of Gbg in ISOinduced ERK activation was examined by introducing HAERK2 and a minigene construct encoding bARK1495– 689 polypeptides, which inhibit Gbg-dependent activation of a wide variety of cell regulatory processes (39, 40) into cultured cardiac myocytes. ISO increased the activity of the transfected ERK2, and overexpression of bARK1495– 689 completely suppressed the ISO-induced activation of ERK2 (Fig. 2B). These findings suggest that ISO activates ERKs via the Gbg-depend-

9762

Isoproterenol-induced Cardiomyocyte Hypertrophy

FIG. 1. Involvement of PKA and PKC in ISO- or PHE-induced activation of ERKs in cardiac myocytes. Cardiac myocytes were preincubated with 100 mM RpcAMP for 10 min, 10 mM H89 or 1 mM calphostin C (Calp.C) for 30 min and then treated with 10 mM ISO (panel A) or 10 mM PHE (panel B) for 8 min. ERK activities were measured by the in-gel method. In brief, cell lysates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/ml MBP. ERKs in the gel were denatured in guanidine HCl and renatured in Tris-HCl containing Triton X-100 and 2-mercaptoethanol. Phosphorylation activities of ERKs were assayed by incubating the gel with [g-32P]ATP. After incubation, the gel was washed extensively and subjected to autoradiography. Representative autoradiograms of MBP phosphorylation are shown. The intensities of 42-kDa ERK band were measured by densitometric scanning of the autoradiograms. The activity was expressed relative to that of 42-kDa ERK obtained from unstimulated cardiomyocytes. The data are indicated as the mean 6 S.E. of three independent experiments. *p , 0.01 versus control.

ent pathway in cardiac myocytes. Inhibition of Gi by PTX Does Not Affect Abundance of b-ARs—There is a possibility that blockade of Gi may lead to

sustained stimulation of adenylyl cyclase and may induce sustained phosphorylation and desensitization (and perhaps down-regulation) of b-ARs (35). It has also been reported that


9763

FIG. 2. Effect of PTX or overexpression of bARK1495– 689 on ISO-induced ERK activation in cardiac myocytes. Panel A, cardiac myocytes were preincubated with 100 ng/ml PTX for 24 h. The cells were then treated with 10 mM ISO or 10 mM PHE for 8 min. Activities of ERKs were assayed as described in the legend of Fig. 1. Panel B, cDNA encoding bARK1495– 689 polypeptide was cotransfected with cDNA encoding HA-ERK2 into cardiac myocytes. 8 min after the addition of 10 mM ISO, HA-ERK2 was immunoprecipitated with an anti-HA polyclonal antibody, and the immune complex was incubated with 25 mg of MBP and 2 mCi of [g-32P]ATP for 10 min. Aliquots of the reaction mixture were subjected to SDS-PAGE, and the gel was washed, dried, and subjected to autoradiography. A representative autoradiogram is shown. Relative kinase activities of 42-kDa ERK were determined by scanning each band with a densitometer. Activities were expressed relative to those of 42-kDa ERK obtained from unstimulated cardiomyocytes. The results are presented as the mean 6 S.E. from three independent experiments. *p , 0.01 versus control.

PTX increases the sensitivity of b-AR to ISO (42). To test whether pretreatment of cardiac myocytes with PTX might affect the density of b-ARs on the cell membrane, we examined the amount of cell surface b-ARs, b1-AR and b2-AR, by Western blot analysis after long term treatment with PTX. As shown in Fig. 3, PTX did not affect the protein levels of b1-AR and b2-AR

in membrane fractions. These results suggest that the inhibition of ISO-induced ERK activation by PTX is not caused by down-regulation of b-ARs. Tyrosine Kinases Including Src Family Tyrosine Kinases Modulate ISO-induced Activation of ERKs—Gbg has been reported to activate ERKs through non-receptor-type tyrosine

9764


FIG. 3. b-ARs in PTX-treated or untreated cardiac myocytes. Cardiac myocytes were treated with 100 ng/ml PTX for 24 h and 10 mM ISO for 8 min. Membrane fractions of the cell extracts were subjected to SDS-PAGE. The blotted membrane was incubated with an anti-b1-AR or an anti-b2-AR polyclonal antibody for 1 h at 37 °C. The immunoreactivity was detected using ECL system. PTX did not affect the protein levels of b1-AR and b2-AR in membrane fractions. Representative autoradiograms are shown.

kinases including Src family tyrosine kinases in many types of cells (28, 43). We examined whether tyrosine kinases are responsible for activation of ERKs induced by ISO in cardiac myocytes. Activation of ERKs by ISO was suppressed completely when cardiomyocytes were pretreated with a broad spectrum tyrosine kinase inhibitor, genistein, whereas the activation of ERKs by PHE was not affected by the same pretreatment (Fig. 4A). These suggest that tyrosine kinases are involved in ISO-induced activation of ERKs in cardiac myocytes, whereas PHE-induced activation of ERKs is not dependent on the tyrosine kinase pathway. We examined further the role of Src family tyrosine kinases in ISO-induced ERK activation in cardiac myocytes. Csk has been reported to phosphorylate the tyrosine residue in the carboxyl terminus of Src family protein kinases and thereby inactivate their function (44, 45). We cotransfected Csk with ERK2 into cardiomyocytes and examined the activity of the transfected ERK2 after stimulation with ISO or PHE. Although cotransfection of Csk did not affect PHE-induced ERK2 activation, overexpression of Csk completely inhibited activation of ERK2 by ISO (Fig. 4B), suggesting that Src family tyrosine kinases are also involved in ISO-induced ERK activation in cardiac myocytes. ISO Enhances Tyrosine Phosphorylation of Shc and Association of Shc with Grb2—Adapter proteins containing Src homology 2 domains such as Shc and Grb2 transduce activation of tyrosine kinases to the Ras/ERK pathway via the guanine nucleotide exchange factor Sos (46 – 48). We therefore examined whether Shc is activated by ISO in cardiac myocytes. ISO rapidly (within 30 s) increased levels of tyrosine phosphorylation of 52-kDa Shc (Fig. 5A). Phosphorylation levels decreased from 5 min and returned to the basal levels by 15 min. A faint band corresponding to 46-kDa Shc was also observed with long exposure (data not shown). Next, association of Grb2 with Shc was examined by immunoprecipitation with anti-Grb2 antibody and immunoblotting with an anti-Shc antibody. The intensities of the bands around 52 kDa and 46 kDa corresponding to Shc were enhanced by ISO stimulation (Fig. 5B), suggesting that the 52- and 46-kDa Shc form a complex with Grb2 after ISO stimulation. Ras and Raf-1 Kinase Activation Is Essential to ISO-induced ERK Activation—Association of Grb2 with Shc usually results in the recruitment of a Ras activator Sos to the membrane fraction, leading to activation of Ras (46 – 48). The role of Ras was next analyzed in ISO-stimulated cardiomyocytes. ISOinduced activation of ERKs was suppressed by overexpression of D.N.Ras (Fig. 6A), suggesting that activation of Ras is re-

quired for ISO-induced activation of ERKs in cardiac myocytes. Activated Ras usually induces activation of ERKs through Raf-1 kinase and mitogen-activated protein kinase/ERK kinase (49). Activation of the transfected ERK2 by ISO or PHE was abolished by the overexpression of D.N.Raf-1 kinase (Fig. 6B), indicating that Raf-1 kinase is crucial for activation of ERKs by ISO and PHE in cardiac myocytes. Activation of ERKs by ISO Requires PKA-dependent Phosphorylation of b-AR—We next examined how both Gs/PKA- and Gi/Ras-dependent pathways are involved in ISO-induced activation of ERKs. Quite recently Daaka et al. (26) have reported that phosphorylation of b2-AR by PKA changes coupling of the receptor from Gs to Gi protein. We therefore examined this possibility by introducing the gene of b2-ARmut, which lacks phosphorylation sites for PKA, into cardiomyocytes. ISO-induced activation of ERK2 was completely suppressed by overexpression of b2-ARmut (Fig. 7). In contrast, overexpression of b2-ARwt enhanced the activation of ERK2 by ISO. These results suggest that PKA-dependent phosphorylation of b-AR is necessary to evoke signals from b-AR to ERKs in cardiac myocytes. ISO-induced Protein Synthesis Also Occurs through Both Gsand Gi-dependent Signal Transduction Pathways—ISO activates ERKs through both Gs/PKA- and Gi/Ras-dependent pathways. To determine whether cardiomyocyte hypertrophy is also induced through the same signal transduction pathways, we examined the effects of various inhibitory agents for signaling molecules on the ISO-induced increase in protein synthesis. We pretreated cardiac myocytes with PTX, genistein, manumycin (a Ras farnesyltransferase inhibitor), H89, or RpcAMP and stimulated the cells with ISO for 24 h. Although the pretreatment with these inhibitors alone did not affect the basal level of protein incorporation significantly (Fig. 8A), an ISO-induced increase in protein synthesis was suppressed significantly by all of these inhibitors (Fig. 8B), indicating that ISO enhanced protein synthesis through cAMP/PKA, Gi, tyrosine kinases, and Ras. These results suggest that the signal transduction pathway leading to activation of ERKs is also important for ISO-induced protein synthesis in cardiac myocytes. DISCUSSION

Stimulation of b-AR usually activates an effector enzyme adenylyl cyclase through Gs. The activation of adenylyl cyclase induces an increase in cAMP levels, which in turn activates PKA (14, 19, 50, 51). In addition, a previous report indicated that down-regulation of Gsa abolishes ISO-induced ERK activation in human endothelial cells (25), suggesting that Gsa is required for activation of ERKs by b-AR. Our previous (7) and present studies also showed that ISO activates ERKs through cAMP/PKA in cardiac myocytes, supporting that Gs protein plays an essential role in the activation of ERKs. Many laboratories have reported that activation of cAMP-dependent PKA inhibits activation of the Raf-1 kinase/ERK cascade in various cell types such as Rat-1 cells, smooth muscle cells, Chinese hamster ovary cells, COS-7 cells, and adipocytes (16 –19). In other cell types such PC12 cells, S49 mouse lymphoma cells, and Swiss-3T3 cells, however, cAMP activates ERKs and potentiates the effects of growth factors on differentiation and gene expression (20 –24). Taken together, cAMP-dependent PKA activation may have different effects on ERKs among cell types, and PKA may activate ERKs in cultured cardiac myocytes. By examining the signal transduction pathway of ISO-induced activation of ERKs, we found that the Gi protein/Src/Ras pathway is also required for ISO-induced ERK activation. How do two different pathways, cAMP/PKA pathway and Gi/Src/Ras pathway, converge at the ERK cascade? Gs-coupled b2-AR was reported to activate simultaneously the pathway that leads to


FIG. 4. Role of tyrosine kinases including Src family tyrosine kinases in ISO-induced activation of ERKs in cardiac myocytes. Panel A, cardiac myocytes were pretreated with 30 mM genistein for 30 min and stimulated with 10 mM ISO or 10 mM PHE for 8 min. ERK activities were assayed as described under legend of Fig. 1. Panel B, HA-ERK2 was cotransfected into cardiac myocytes with Csk gene. 8 min after the addition of 10 mM ISO or 10 mM PHE, the transfected ERK2 activity was measured using MBP as described in the legend of Fig. 2B. A representative autoradiogram is shown. Relative kinase activities of 42-kDa ERK were determined by scanning each band with a densitometer. Activities were expressed relative to that obtained from unstimulated cardiomyocytes. Data are presented as the mean 6 S.E. from three independent experiments. *p , 0.01 versus control.

9765

9766


FIG. 5. Phosphorylation and association of Shc with Grb2 by ISO. Cardiac myocytes were stimulated with 10 mM ISO for the indicated periods of time. The cell extracts were incubated with an anti-Shc (panel A) or an anti-Grb2 (panel B) polyclonal antibody, and the immune complex was subjected to SDS-PAGE. The blotted membrane was incubated with an antiphosphotyrosine monoclonal antibody (PY20) (panel A) or an anti-Shc antibody (panel B). The immunoreactivity was detected using the ECL system. Representative autoradiograms are shown. Relative phosphorylation of 52-kDa Shc was determined by scanning each band with a densitometer. The intensities were expressed relative to that of 52-kDa Shc obtained from unstimulated cardiomyocytes. Results are indicated as the mean 6 S.E. of three independent experiments. *p , 0.05 versus control.

functional inhibition of cAMP/PKA pathway via Gi protein in cardiac cells (36). Moreover, it has been reported recently that PKA-induced phosphorylation of b-AR changes the coupling of the receptor from Gs to Gi and activates ERKs through the Src/Ras pathway in HEK293 cells (26). We therefore examined the role of Gi protein in ISO-evoked ERK activation and found that Gi protein is also essential to ISO-induced activation of

ERKs in cardiac myocytes. Although it has been reported that inhibition of Gi protein may lead to sustained stimulation of adenylyl cyclase and down-regulation of b-ARs in some types of cell (35), Western blot analysis revealed that blocking of Gi by PTX did not affect the abundance of b-ARs in the membrane fraction of cardiac myocytes, suggesting that in cardiac myocytes, the inhibitory effect of PTX is attributable to suppression


FIG. 6. Role of Ras and Raf-1 in ISOinduced activation of ERKs in cardiac myocytes. HA-ERK2 was cotransfected with D.N.Ras (panel A) or D.N. Raf-1 (panel B) into cardiac myocytes, and the cells were stimulated with 10 mM ISO or 10 mM PHE for 8 min. The activities of ERKs were assessed by measuring MBP phosphorylation as described in the legend of Fig. 2B. Representative autoradiograms are shown. Relative kinase activities of MBP were determined by scanning each band with a densitometer. Results are presented as the mean 6 S.E. from three independent experiments. *p , 0.01 versus control.

9767

9768


FIG. 7. Role of PKA-dependent b-AR phosphorylation in ISO-induced ERK activation. b2-ARmut or b2-ARwt was cotransfected into cardiomyocytes with HA-ERK2. The cells were stimulated by ISO (10 mM) for 8 min. The transfected ERK2 activity was measured using MBP as described in the legend of Fig. 2B. Representative autoradiograms are shown. The intensities of MBP band were measured by densitometric scanning of the autoradiogram. Values represent the mean 6 S.E. of four independent experiments. *p , 0.01 versus control.

of signaling from b-ARs to downstream but is not caused by down-regulation of b-ARs. It is necessary to define how G proteins are involved in the ISO-induced ERK activation in cardiac myocytes. It has been reported in COS-7 cells that activation of b-AR activates ERKs through Gsbg but not Gsa. Gsbg activates ERKs through a Rasdependent pathway, whereas Gsa inhibits activation of ERKs via cAMP/PKA. The balance between these two opposite mechanisms of regulation may control the ERK response to b-AR agonists (19). However, this model may not be applied to cardiac myocytes because ISO-stimulated ERK activation is dependent on cAMP/PKA, suggesting the importance of Gsa protein. It is also possible that ISO activates ERKs through both Gs and Gi proteins independently. In the present study, however, because inhibition of cAMP/PKA or of Gi completely suppressed ISO-induced ERK activation, it may be unlikely that Gs and Gi proteins are involved independently. We introduced b2-ARmut lacking phosphorylation sites for PKA with HAERK2 into cardiac myocytes and showed that inhibition of b2-AR phosphorylation inhibits ISO-induced activation of ERKs. These results are consistent with the hypothesis that activation of cAMP-dependent PKA phosphorylates the activated receptor, leading to the change in coupling of the receptor from Gs to Gi protein. Gbg subunit derived from PTX-sensitive Gi protein regulates many effectors within the cell (22, 41, 52, 53). Stimulation of various receptors such as a2A-adrenergic, M2 muscarinic acetylcholine, D2 dopamine, A1 adenosine, or angiotensin II type 1

receptors induces Ras-dependent ERK activation via the Gbg subunit in COS-7 cells (53). By introducing the bARK1495– 689 polypeptide minigene, we showed in the present study that the Gbg subunit is required for ISO-induced ERK activation in cardiac myocytes. There is a report showing that the bg subunit of Gs protein activates ERKs in COS-7 cells (19). Because activation of adenylyl cyclase is usually mediated by the a subunit rather than the bg subunit of Gs (14, 24, 25, 50, 51) and ISO activated ERKs through a cAMP/PKA-dependent pathway in this study, ISO-induced activation of ERKs might be dependent on the a subunit of Gs and on the bg subunit of Gi in cardiac myocytes. The bg subunit of Gi protein activates ERKs through tyrosine kinases including Src family tyrosine kinases (43, 54). Because b-AR itself does not possess tyrosine kinase activity, non-receptor-type tyrosine kinases may be responsible for ISOinduced ERK activation. We therefore examined the involvement of tyrosine kinases including Src family tyrosine kinases in ISO-induced ERK activation in cardiac myocytes. Pretreatment with a tyrosine kinase inhibitor or overexpression of Csk strongly inhibited ISO-induced activation of ERKs in cardiac myocytes. In contrast, PHE-induced activation of ERKs was not dependent on the tyrosine kinase pathway. We have reported that angiotensin II activates ERKs through PKC but not through tyrosine kinases in cardiac myocytes (27). These results collectively suggest that tyrosine kinases including Src family tyrosine kinases mediated Gi-coupled receptor-induced ERK activation, whereas PKC but not tyrosine kinases play a


FIG. 8. Effects of various inhibitors for protein kinases on protein synthesis in cardiac myocytes. Serum-deprived (48 h) cardiac myocytes were pretreated with 100 mM RpcAMP for 10 min, 10 mM H89, 30 mM genistein (Geni), 1 mM manumycin (Manu) for 30 min, or 100 ng/ml PTX for 24 h. After incubation with vehicle (panel A) or 10 mM ISO for 24 h (panel B), cardiomyocytes were pulse labeled with [3H]phenylalanine (1 mCi/ml) for the final 2 h. Incorporation of [3H]phenylalanine into the acid-precipitable cellular fraction was determined by liquid scintillation counting. The data are presented as the means 6 S.E. from three independent experiments (control 5 100%). *p , 0.05 versus control. † p , 0.05 versus ISO stimulation alone.

critical role in activation of ERKs by Gq-coupled receptors in cardiac myocytes. Activation of tyrosine kinases leads to Ras activation through adaptor proteins such as Shc and Grb2 and the guanine exchange factor Sos (46 – 48). It has been demonstrated that Shc is tyrosine phosphorylated in response to angiotensin II in cardiac myocytes (55) and that Shc serves as a converging target in the growth factor- and G protein-coupled receptorstimulated signal transduction events resulting in activation of Ras protein (56). The present results indicated that ISO rapidly induces phosphorylation of Shc and association of Shc with Grb2, which might result in the translocation of the Ras guanine nucleotide exchange factor Sos to the membrane fraction. It has been recognized that Ras plays a key role in a variety of cell functions through the sequential activation of Raf-1 kinase and ERKs (57, 58). On the other hand, it has been reported that cAMP activates ERKs through a B-Raf- and a PKA-activated Rap1-dependent pathway, but not through Ras/Raf-1 kinase-

9769

dependent pathway in PC12 cells (24, 59). It has also been shown that cAMP activates ERKs independently of Raf-1 kinase (60). By contrast, we observed in the present study that Ras and Raf-1 kinase are necessary for ISO-induced ERK activation. Taken collectively, these findings suggest that ISO may activate ERKs through the signaling pathway consisting of Src, Shc, Grb2, Sos, Ras, and Raf-1 kinase in cardiac myocytes. As reported previously, ERK activation is required for cell growth including cardiac hypertrophy (6, 7, 34, 61, 62). We also showed in the present study that the signal transduction pathway leading to activation of ERKs is important for ISO-induced protein synthesis in cardiac myocytes. The effect of isoproterenol on the index of net protein synthesis is small compared with severalfold increases in the activity of each signaling steps. Although there are great differences between signaling steps and protein synthesis in many aspects such as the basal levels, the responsive ability to stimulation, and the methods of examination among them, it is also possible that the initiation of protein synthesis needs a much greater fold-increase in the activity of signaling molecules. Many previous studies also showed that signaling steps such as ERKs are activated more than severalfold by stimulation, whereas an increase in the protein synthesis rate is less than 50% (6, 7, 62, 63). Although it is unknown at present why there are big differences in the time course for protein synthesis and activation of signaling molecules, there are several possibilities. Protein synthesis may need activation of many signaling steps, or the initiation step of protein synthesis needs some time period after activation of molecules responsible to protein synthesis. Further studies are necessary to determine these possibilities. In summary, the b-AR-evoked signal transduction pathways to activation of ERKs are different among cell types. In cardiac myocytes, ISO activates ERKs through the signal transduction pathway consisting of b-AR phosphorylation by Gs/cAMP-dependent PKA, Gbg subunits derived from Gi, Src family tyrosine kinases, the formation of the Shc-Grb2-Sos complex, Ras, and Raf-1 kinase. We showed in this study that the phosphorylation of b-AR by PKA and the change of coupling of the receptor from Gs to Gi play an important role in ISO-induced cardiomyocyte hypertrophy. Acknowledgments—We thank Drs. M. Karin, R. J. Lefkowitz, Y. Daaka, Y. Takai, H. Sabe, and K. Touhara for plasmids. REFERENCES 1. 2. 3. 4. 5. 6. 7.

8.

9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19.

Siri, F. M. (1988) Am. J. Physiol. 255, H452–H457 Laks, M. M., Morady, F., and Swan, H. J. C. (1973) Chest 64, 75–78 Simpson, P. (1983) J. Clin. Invest. 72, 732–738 Zierhut, W., and Zimmer, H. G. (1989) Circ. Res. 65, 1417–1425 Thorburn, A. (1994) Biochem. Biophys. Res. Commun. 205, 1417–1422 Bogoyevitch, M. A., Andersson, M. B., Gillespie, B. J., Clerk, A., Glennon, P. E., Fuller, S. J., and Sugden, P. H. (1996) Biochem. J. 314, 115–121 Yamazaki, T., Komuro, I., Zou, Y., Kudoh, S., Shiojima, I., Hiroi, Y., Mizuno, T., Aikawa, R., Takano, H., and Yazaki, Y. (1997) Circulation 95, 1260 –1268 Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable, M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109 –10113 Brand, T., Sharma, H. S., and Schaper, W. (1993) J. Mol. Cell. Cardiol. 25, 1325–1337 Slotkin, T. A., Lappi, S.E., and Seidler, F. J. (1995) Toxicol. Appl. Pharmacol. 133, 188 –195 Graham, R. M. (1990) Cleve. Clin. J. Med. 57, 481– 491 Terzic, A., Puceat, M., Vassort, G., and Vogel, S. M. (1993) Pharmacol. Rev. 45, 147–175 Karliner, J. S., Kagiya, T., and Simpson, P. C. (1990) Experientia (Basel) 46, 81– 84 Morgan, H. E., and Baker, K. M. (1991) Circulation 83, 13–25 Cook, S. J., and McCormick, F. (1993) Science 262, 1069 –1072 Graves, L. M., Bornfeldt, K. E., Raines, E. W., Potts, B. C., Macdonald, S. G., Ross, R., and Krebs, E. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10300 –10304 Burgering, B. M., Pronk, G. J., Weeren, P. C., Chardin, P., and Bos, J. L. (1993) EMBO J. 12, 4211– 4220 Hordijik, P. L., Verlaan, I., Jalink, K., van Corven, E. J., and Moolenaar, W. H. (1994) J. Biol. Chem. 269, 35334 –35338 Crespo, P., Cachero, T. G., Xu, N., and Gutkind, J. S. (1995) J. Biol. Chem. 270,

9770


25259 –25265 20. Heidemann, S. R., Joshi, H. C., Schechter, A., Fletcher, J. R., and Bothwell, M. (1985) J. Cell Biol. 100, 916 –927 21. Frodin, M., Peraldi, P., and Van Obberghen, E. (1994) J. Biol. Chem. 269, 6207– 6214 22. Faure, M., Voyno-Yasenetskaya, T. A., and Bourne, H. R. (1994) J. Biol. Chem. 269, 7851–7854 23. Yao, H., Labudda, K., Rim, C., Capodieci, P., Loda, M., and Stork, P. J. S. (1995) J. Biol. Chem. 270, 20748 –20753 24. Wan, Y., and Huang, X.-Y. (1998) J. Biol. Chem. 273, 14533–14537 25. Sexl, V., Mancusi, G., Holler, C., Gloria-Maercker, E., Schutz, W., and Freissmuth, M. (1997) J. Biol. Chem. 272, 5792–5799 26. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88 –91 27. Zou, Y., Komuro, I., Yamazaki, T., Aikawa, R., Kudoh, S., Shiojima, I., Hiroi, Y., Mizuno, T., and Yazaki, Y. (1996) J. Biol. Chem. 271, 33592–33597 28. Zou, Y., Komuro, I., Yamazaki, T., Kudoh, S., Aikawa, R., Zhu, W., Shiojima, I., Hiroi, Y., Tobe, K., Kadowaki, T., and Yazaki, Y. (1998) Circ. Res. 82, 337–345 29. McKee, E. E., Cheung, J. Y., Rannels, D. E., and Morgan, H. E. (1978) J. Biol. Chem. 253, 1030 –1040 30. Eckel, J., van, E. G., and Reinauer, H. (1985) Am. J. Physiol. 249, H212–H221 31. Kent, R. L., Hoober, J. K., and Cooper, G. (1989) Circ. Res. 64, 74 – 85 32. McDermott, P. J., and Morgan, H. E. (1989) Circ. Res. 64, 542–553 33. Wiesner, R. J., and Zak, R. (1991) Am. J. Physiol. 260, L179 –L188 34. Cobb, M. H., Boulton, T. G., and Robbins, D. J. (1991) Cell Regul. 2, 965–978 35. Abramson, S. N., Martin, M. W., Hughes, A. R., Harden, T. K., Neve, K. A., Barrett, D. A., and Molinoff, P. B. (1988) Biochem. Pharmacol. 37, 4289 – 4297 36. Xiao, R. P., Ji, X., and Lakatta, E. G. (1995) Mol. Pharmacol. 47, 322–329 37. Letterio, J. J., Coughlin, S. R., and Williams, L. T. (1986) Science 234, 1117–1119 38. van, C. E., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H. (1989) Cell 59, 45–54 39. Inglese, J., Luttrell, L. M., Iniguez, L. J., Touhara, K., Koch, W. J., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3637–3641 40. Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 6193– 6197 41. van, B. T., Hawes, B. E., Luttrell, D. K., Krueger, K. M., Touhara, K., Porfiri, E., Sakaue, M., Luttrell, L. M., and Lefkowitz, R. J. (1995) Nature. 376, 781–784 42. Lonnqvist, F., and Arner, P. (1989) Biochem. Biophys. Res. Commun. 161,

654 – 660 43. Luttrell, L. M., Hawes, B. E., van-Biesen, T., Luttrell, D. K., Lansing, T. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 19443–19450 44. Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T., and Nakagawa, H. (1991) J. Biol. Chem. 266, 24249 –24252 45. Hata, A., Sabe, H., Kurosaki, T., Takata, M., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 7306 –7313 46. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93–104 47. Rozakis, A. M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature 360, 689 – 692 48. Egan, S.E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993) Nature 363, 45–51 49. Kyriakis, J. M., App, H., Zhang, X., Banerjee, P., Brautigan, D. L., Rapp, U. R., and Avruch, J. (1992) Nature 358, 417– 421 50. Strulovici, B., Cerione, R. A., Kilpatrick, B. F., Caron, M. G., and Lefkowitz, R. J. (1984) Science 225, 837– 840 51. Hausdorff, W. P., Aguilera, G., and Catt, K. J. (1989) Cell. Signalling 1, 377–386 52. Clapham, D. E., and Neer, E. J. (1993) Nature 365, 403– 406 53. Koch, W. J., Hawes, B. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12706 –12710 54. Chen, Y. H., Pouyssegur, J., Courtneidge, S. A., and Van Obberghen-Schilling, E. (1994) J. Biol. Chem. 269, 27372–27377 55. Sadoshima, J., and Izumo, S. (1996) EMBO J. 15, 775–787 56. Schorb, W., Peeler, T. C., Madigan, N. N., Conrad, K. M., and Baker, K. M. (1994) J. Biol. Chem. 269, 19626 –19632 57. Nishida, E., and Gotoh, Y. (1993) Trends Biochem. Sci. 18, 128 –131 58. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267, 24149 –24152 59. Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. (1997) Cell 89, 73– 82 60. Faure, M., and Bourne, H. R. (1995) Mol. Biol. Cell 6, 1025–1035 61. Gillespie-Brown, J., Fuller, S. J., Bogoyevitch, M. A., Cowley, S., and Sugden, P. H. (1995) J. Biol. Chem. 270, 28092–28096 62. Jones, L. G., Gause, K. C., and Meier, K. E. (1996) Life Sci. 58, 617– 630 63. Kinugawa, K., Takahashi, T., Kohmoto, O., Yao, A., Ikenouchi, H., and Serizawa, T. (1995) Cardiovasc. Res. 30, 419 – 431