Both Gs and Gi Proteins Are Critically
Involved in Isoproterenol-induced Cardiomyocyte Hypertrophy*
Yunzeng
Zou
§,
Issei
Komuro
¶,
Tsutomu
Yamazaki
,
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
Health Service Center, the
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
 |
ABSTRACT |
Activation of
-adrenoreceptors induces
cardiomyocyte hypertrophy. In the present study, we examined
isoproterenol-evoked intracellular signal transduction pathways leading
to activation of extracellular signal-regulated 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
G
subunit binding domain of the
-adrenoreceptor
kinase 1 and of COOH-terminal Src kinase, which inhibit functions of
G
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
-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
-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Gi
activates ERKs through G
, Src family tyrosine
kinases, Ras, and Raf-1 kinase.
 |
INTRODUCTION |
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,
and
, in adrenoreceptors
(ARs).1 AR agonists such as
norepinephrine (NE) (
and
), phenylephrine (PHE) (
), and
isoproterenol (ISO) (
) 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
- and
-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
-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
1-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 Ras-dependent pathway (5).
Regarding
-AR-induced signaling pathways, stimulation of
-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
subunit of Gs
(Gs
) abolishes
-AR-mediated ERK activation by ISO
(25), suggesting that Gs
-dependent cAMP
elevation and PKA activation are responsible for the activation of
ERKs. We and others have also demonstrated that
-AR agonists including ISO significantly activate ERKs and increase protein synthesis through cAMP/PKA in cardiac myocytes (6, 7). However, the
molecular mechanisms of
-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
-AR phosphorylates agonist-coupled
-AR, leading
to a change of the receptor coupling from Gs to
Gi (26). We examined whether phosphorylation of
-AR is
critical to ISO-induced activation of ERKs also in cardiac myocytes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[
-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
1- or
2-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 G
subunit binding domain of the bovine
-AR kinase 1 residues
Gly495 to Leu689 (
ARK1495-689),
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
2-AR
(pRK5-
2-ARwt) and
2-AR mutant lacking
phosphorylation sites for PKA (pcDNAI-
2-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 µg of HA-ERK2 plasmid
DNA was transfected with or without 7.5 µg of other relevant plasmids such as
2-ARwt,
2-ARmut,
ARK1495-689, 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 LacZ-containing 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 µCi/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 [
-32P]ATP.
Phosphorylation activities of ERKs were assayed by subjecting to
autoradiography. The 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 [
-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
-ARs in Cardiac Myocytes--
The amount of
-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 × 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 µg/ml aprotinin). Whole lysate was
first centrifuged at 200 × g to remove nuclei, and
then the supernatant was centrifuged at 15,000 × 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-
1-AR or an
anti-
2-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
-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 µM), ISO-evoked ERK activation was completely
suppressed (see Fig. 1A).
Pretreatment with a PKA inhibitor, H89 (10 µM), also
abolished ISO-induced activation of ERKs, whereas inhibition of PKC by
calphostin C (1 µM) 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.

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Fig. 1.
Involvement of PKA and PKC in ISO- or
PHE-induced activation of ERKs in cardiac myocytes. Cardiac
myocytes were preincubated with 100 µM RpcAMP for 10 min,
10 µM H89 or 1 µM calphostin C
(Calp.C) for 30 min and then treated with 10 µM ISO (panel A) or 10 µM 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 [ -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 ± S.E. of
three independent experiments. *p < 0.01 versus control.
|
|
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
-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.

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Fig. 2.
Effect of PTX or overexpression of
ARK1495-689 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 µM ISO or 10 µM
PHE for 8 min. Activities of ERKs were assayed as described in the
legend of Fig. 1. Panel B, cDNA encoding
ARK1495-689 polypeptide was cotransfected with
cDNA encoding HA-ERK2 into cardiac myocytes. 8 min after the
addition of 10 µM ISO, HA-ERK2 was immunoprecipitated
with an anti-HA polyclonal antibody, and the immune complex was
incubated with 25 µg of MBP and 2 µCi of [ -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 ± S.E. from three
independent experiments. *p < 0.01 versus
control.
|
|
It has been reported recently that stimulation of Gi
protein-coupled receptors activates ERKs by 
subunits
(G
) but not by the
subunit (39-41). Therefore,
the role of G
in ISO-induced ERK activation was
examined by introducing HA-ERK2 and a minigene construct encoding
ARK1495-689 polypeptides, which inhibit
G
-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
ARK1495-689 completely suppressed the ISO-induced
activation of ERK2 (Fig. 2B). These findings suggest that
ISO activates ERKs via the G
-dependent pathway in cardiac myocytes.
Inhibition of Gi by PTX Does Not Affect Abundance of
-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
-ARs (35). It has also been reported that PTX
increases the sensitivity of
-AR to ISO (42). To test whether
pretreatment of cardiac myocytes with PTX might affect the density of
-ARs on the cell membrane, we examined the amount of cell surface
-ARs,
1-AR and
2-AR, by Western blot
analysis after long term treatment with PTX. As shown in Fig.
3, PTX did not affect the protein levels of
1-AR and
2-AR in membrane fractions.
These results suggest that the inhibition of ISO-induced ERK activation
by PTX is not caused by down-regulation of
-ARs.

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Fig. 3.
-ARs in PTX-treated or
untreated cardiac myocytes. Cardiac myocytes were treated with 100 ng/ml PTX for 24 h and 10 µM ISO for 8 min. Membrane
fractions of the cell extracts were subjected to SDS-PAGE. The blotted
membrane was incubated with an anti- 1-AR or an
anti- 2-AR polyclonal antibody for 1 h at 37 °C.
The immunoreactivity was detected using ECL system. PTX did not affect
the protein levels of 1-AR and 2-AR in
membrane fractions. Representative autoradiograms are shown.
|
|
Tyrosine Kinases Including Src Family Tyrosine Kinases Modulate
ISO-induced Activation of ERKs--
G
has been
reported to activate ERKs through non-receptor-type tyrosine 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.

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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 µM genistein for 30 min and stimulated with 10 µM ISO or 10 µM 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 µM ISO or
10 µM 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 ± S.E. from three
independent experiments. *p < 0.01 versus
control.
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|
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.

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Fig. 5.
Phosphorylation and association of Shc with
Grb2 by ISO. Cardiac myocytes were stimulated with 10 µM 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 anti-phosphotyrosine 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 ± S.E. of three independent experiments. *p < 0.05 versus control.
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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. ISO-induced activation of ERKs was
suppressed by overexpression of D.N.Ras (Fig.
6A), suggesting that
activation of Ras is required 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.

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Fig. 6.
Role of Ras and Raf-1 in ISO-induced
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 µM ISO or 10 µM 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 ± S.E. from three independent experiments.
*p < 0.01 versus control.
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Activation of ERKs by ISO Requires PKA-dependent
Phosphorylation of
-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
2-AR by PKA changes coupling of the receptor from
Gs to Gi protein. We therefore examined this
possibility by introducing the gene of
2-ARmut, which
lacks phosphorylation sites for PKA, into cardiomyocytes. ISO-induced
activation of ERK2 was completely suppressed by overexpression of
2-ARmut (Fig. 7). In
contrast, overexpression of
2-ARwt enhanced the
activation of ERK2 by ISO. These results suggest that
PKA-dependent phosphorylation of
-AR is necessary to
evoke signals from
-AR to ERKs in cardiac myocytes.

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Fig. 7.
Role of PKA-dependent
-AR phosphorylation in ISO-induced ERK
activation. 2-ARmut or 2-ARwt was
cotransfected into cardiomyocytes with HA-ERK2. The cells were
stimulated by ISO (10 µM) 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 ± S.E. of four
independent experiments. *p < 0.01 versus
control.
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ISO-induced Protein Synthesis Also Occurs through Both
Gs- and 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.

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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 µM
RpcAMP for 10 min, 10 µM H89, 30 µM
genistein (Geni), 1 µM manumycin
(Manu) for 30 min, or 100 ng/ml PTX for 24 h. After
incubation with vehicle (panel A) or 10 µM ISO
for 24 h (panel B), cardiomyocytes were pulse labeled
with [3H]phenylalanine (1 µCi/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 ± S.E. from three independent experiments (control = 100%).
*p < 0.05 versus control. p < 0.05 versus ISO stimulation
alone.
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|
 |
DISCUSSION |
Stimulation of
-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 Gs
abolishes ISO-induced ERK
activation in human endothelial cells (25), suggesting that
Gs
is required for activation of ERKs by
-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
2-AR was
reported to activate simultaneously the pathway that leads to
functional inhibition of cAMP/PKA pathway via Gi protein in
cardiac cells (36). Moreover, it has been reported recently that
PKA-induced phosphorylation of
-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
-ARs in some
types of cell (35), Western blot analysis revealed that blocking of
Gi by PTX did not affect the abundance of
-ARs in the
membrane fraction of cardiac myocytes, suggesting that in cardiac
myocytes, the inhibitory effect of PTX is attributable to suppression
of signaling from
-ARs to downstream but is not caused by
down-regulation of
-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
-AR activates ERKs through Gs
but not Gs
. Gs
activates ERKs through a Ras-dependent pathway, whereas
Gs
inhibits activation of ERKs via cAMP/PKA. The balance
between these two opposite mechanisms of regulation may control the ERK
response to
-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 Gs
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
2-ARmut lacking
phosphorylation sites for PKA with HA-ERK2 into cardiac myocytes and
showed that inhibition of
2-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.
G
subunit derived from PTX-sensitive Gi
protein regulates many effectors within the cell (22, 41, 52, 53). Stimulation of various receptors such as
2A-adrenergic,
M2 muscarinic acetylcholine, D2 dopamine,
A1 adenosine, or angiotensin II type 1 receptors induces
Ras-dependent ERK activation via the G
subunit in COS-7 cells (53). By introducing the
ARK1495-689 polypeptide minigene, we showed in the
present study that the G
subunit is required for
ISO-induced ERK activation in cardiac myocytes. There is a report
showing that the 
subunit of Gs protein activates
ERKs in COS-7 cells (19). Because activation of adenylyl cyclase is
usually mediated by the
subunit rather than the 
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
subunit of
Gs and on the 
subunit of Gi in cardiac myocytes.
The 
subunit of Gi protein activates ERKs through
tyrosine kinases including Src family tyrosine kinases (43, 54).
Because
-AR itself does not possess tyrosine kinase activity,
non-receptor-type tyrosine kinases may be responsible for ISO-induced
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 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 receptor-stimulated 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-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
-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
-AR phosphorylation by Gs/cAMP-dependent
PKA, G
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
-AR by PKA and the change of coupling of the receptor from
Gs to Gi play an important role in ISO-induced
cardiomyocyte hypertrophy.
 |
ACKNOWLEDGEMENTS |
We thank Drs. M. Karin, R. J. Lefkowitz, Y. Daaka, Y. Takai, H. Sabe, and K. Touhara for plasmids.
 |
FOOTNOTES |
*
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. The 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:
komuro-tky{at}umin.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
AR(s), adrenoreceptor(s);
NE, norepinephrine;
PHE, phenylephrine;
ISO, isoproterenol;
- and
-AR,
- and
-adrenergic receptor,
respectively;
PKC, protein kinase C;
ERK, extracellular
signal-regulated protein kinase;
PKA, cAMP-dependent
protein kinase;
PTX, pertussis toxin;
HA, hemagglutinin;
HA-ERK2, HA-tagged version of the wild type ERK2;
ARK1495-689,
-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;
2-ARwt, wild-type
2-AR;
2-ARmut,
2-AR mutant;
MBP, myelin basic protein;
PAGE, polyacrylamide gel electrophoresis.
 |
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