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J Biol Chem, Vol. 274, Issue 28, 19762-19770, July 9, 1999

Regulation of Ras·GTP Loading and Ras-Raf Association in Neonatal Rat Ventricular Myocytes by G Protein-coupled Receptor Agonists and Phorbol Ester
ACTIVATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE CASCADE BY PHORBOL ESTER IS MEDIATED BY Ras^*

Antonio Chiloeches, Hugh F. Paterson^§, Richard Marais^§, Angela Clerk^¶, Christopher J. Marshall^§, and Peter H. Sugden^**

From the  National Heart and Lung Institute Division (Cardiac Medicine), Imperial College School of Medicine, London SW3 6LY, United Kingdom, the ^§ Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, United Kingdom, and the ^¶ Division of Biomedical Sciences (Molecular Pathology), Imperial College School of Medicine, London SW7 2AZ, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras·GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras·GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and G_i-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of the membrane-associated small (21-kDa) G protein Ras family (Ha-Ras, K-Ras, N-Ras) are important in eukaryotic signal transduction (reviewed in Ref. 1). In its GDP-ligated state, Ras is inactive. By increasing its GTP loading in a regulated manner, Ras acts as a "molecular switch" and transmits signals from a variety of cell surface receptors to downstream effector proteins. The best characterized effector is c-Raf, one of the mitogen-activated protein kinase (MAPK)¹ kinase kinases of the extracellular signal-regulated kinase (ERK) cascade. Ras·GTP (but not Ras·GDP) has a high affinity for c-Raf, causing it to translocate to the membrane (reviewed in Ref. 2), where, in a process that possibly involves its phosphorylation (3-5), c-Raf becomes fully activated. Several other potential Ras effectors have been identified. These include phosphatidylinositol 3'-kinase, other small G proteins, the Ral-GDS family, and other protein kinases (reviewed in Ref. 1). Ras is converted back to its inactive state by its innate GTPase activity, which may be enhanced by GTPase-activating proteins.

In the terminally differentiated, cell cycle-arrested ventricular myocyte (in contrast to transformed cell lines and other dividing cells), G protein-coupled receptor (GPCR) agonists such as endothelin-1 (ET-1), bradykinin (BK), and ₁-adrenergic agonists promote a stronger activation of the ERK cascade members (c-Raf/A-Raf, MAPK kinase 1/2 (MKK1/2), and ERK1/ERK2) than peptide growth factors (6-9). This activation is mediated at least in part through the G_q/G₁₁ group of GPCRs (recently also demonstrated in vivo (10)), stimulation of membrane phospholipid hydrolysis, and activation of the diacylglycerol-sensitive isoforms of protein kinase C (PKC) (reviewed in Ref. 11). Thus, phorbol esters such as phorbol 12-myristate 13-acetate (PMA) also powerfully activate the ERK cascade in myocytes (6, 12). In terms of biology, ET-1, ₁-adrenergic agonists such as phenylephrine (PE), and phorbol esters cause myocytes to hypertrophy, a response that manifests itself with increases in myocyte size, increased myofibrillogenesis, and alterations in gene and protein expression (reviewed in Refs. 13 and 14). Myocyte hypertrophy is important in pathophysiological conditions in vivo, since it allows the heart to maintain or increase its contractile power following partial loss of viable myocytes (as occurs following myocardial infarction) or when faced with a demand for increased pressure-volume work. There is evidence that GPCR- and G_q-mediated activation of Ras and the ERK cascade brings about at least some of these changes (reviewed in Ref. 14). Although regulation of Ras activity has been examined extensively in cell lines, relatively little is known about this topic in primary cells (such as the ventricular myocyte). In this study, we have used a nonradioactive method (15, 16) to assess the activation of Ras by PMA and GPCR agonists in myocytes. We have examined the stimulation of the association between Ras·GTP and c-Raf and show that Ras is essential for the PMA-stimulated phosphorylation of ERKs. These results are relevant to the understanding of myocyte hypertrophy and of GPCR-linked signaling pathways generally.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Mouse monoclonal antibodies to pan-Ras (catalog no. R02120) and c-Raf (catalog no. R19120) were from Transduction Laboratories. The antibody to Ha-Ras was from Quality Biotech Inc. (catalog no. LA069), and those to K-Ras (catalog nos. sc-030, sc-521, and sc-522) and N-Ras (catalog no. sc-031) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Y13-238 and Y13-259 monoclonal anti-Ras antibodies were purified from rat lymphocyte-myeloma hybridoma cultures (17) and were kindly provided by M. Valeri and J. Cordell (The Hybridoma Unit, Institute of Cancer Research, Sutton, Surrey, United Kingdom). Anti-dually phosphorylated ERKs (monoclonal MAPK-YT) were from Sigma. "ChromPure" normal rat IgG, fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (preadsorbed against rat IgG), and Texas Red-conjugated donkey anti-rat IgG (preadsorbed against mouse IgG) were from Jackson Immunoresearch. The recombinant Ras binding domain (RBD) of c-Raf (residues 51-131 (18-20)), MKK1, and ERK2 were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST-MKK1 and GST-ERK2 were purified by glutathione-Sepharose (Amersham Pharmacia Biotech) chromatography (21). Microcystin LR was from Alexis, and a 1 mM stock solution was prepared in Me₂SO. [-³²P]ATP was from NEN Life Science Products. Prestained molecular mass markers, ECL Western blotting reagents, and Hyperfilm MP were from Amersham Pharmacia Biotech. The antibodies used for ECL (rabbit anti-mouse and horseradish peroxidase-linked goat anti-rabbit immunoglobulins) were from Dako. SDS-polyacrylamide gel electrophoresis reagents and Bradford protein assay reagent were from Bio-Rad. Nitrocellulose (0.45 µm; Schleicher & Schuell) was supplied by Anderman. ET-1 was from Bachem. GF109203X and Ro318220 were from Calbiochem and were prepared as 10 mM stocks in Me₂SO. PMA (prepared as a 1 mM stock in Me₂SO) and other agonists, pertussis toxin (PTX), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) were from Sigma. Myelin basic protein was from Upstate Biotechnology, Inc. (Lake Placid, NY). General laboratory reagents were from Sigma or Merck.

Primary Culture of Ventricular Myocytes-- Myocytes were prepared from the ventricles of neonatal Harlan Sprague Dawley rat hearts by an adaptation of the method of Iwaki et al. (22) as described previously (23). The cells were preplated to remove fibroblasts. For measurement of Ras·GTP loading and Ras-Raf association etc., myocytes were plated at a density of 1.4 × 10³ cells/mm² onto 60-mm gelatin-coated dishes in Dulbecco's modified Eagle's medium/medium 199 (4:1, v/v) containing 10% (v/v) horse serum, 5% (v/v) fetal calf serum and 100 units/ml each of penicillin and streptomycin. After 18 h, the cells were confluent and beating. The cells were incubated for 24 h in 4 ml of serum-free medium and before exposure to agonists. For immunofluorescence and microinjection experiments, cells were plated in 60-mm dishes at a density of 700 cells/mm².

Western Blot Analysis-- Proteins were separated by SDS-polyacrylamide gel electrophoresis on 10 or 12% (w/v) polyacrylamide gels and transferred electrophoretically to nitrocellulose as described earlier (24). Nonspecific binding sites were blocked with 5% (w/v) nonfat milk powder in 20 mM NaH₂PO₄, 80 mM Na₂HPO₄, 100 mM NaCl, 0.05% (v/v) Tween 20 (pH 7.5) (PBST) for 30 min, and the blots were incubated with Ras or c-Raf antibodies (1:1000 dilution in blocking solution, overnight, 4 °C). For monoclonal antibodies, the blots were washed with PBST (three times for 5 min, room temperature), incubated with a polyclonal rabbit anti-mouse secondary antibody (1:5000 dilution in PBST containing 1% (w/v) nonfat milk powder, 1 h, room temperature) and then washed again in PBST (three times for 5 min, room temperature). Blots were then incubated with a horseradish peroxidase-linked goat anti-rabbit IgG tertiary antibody (1:5000 dilution in PBST containing 1% (w/v) nonfat milk powder, 1 h, room temperature), and, after washing in PBST (three times for 5 min, room temperature), bands were detected using ECL with exposure to Hyperfilm MP. When polyclonal rabbit antibodies were used, the rabbit anti-mouse antibody was omitted. Blots were quantified by laser scanning densitometry.

Activated Ras Affinity Precipitation Assay-- The assay was performed essentially as described by de Rooij and Bos (16). Ventricular myocytes were exposed to agonists in serum-free medium. The medium was aspirated, and the cells were washed three times with ice-cold PBS and extracted by scraping into 300 µl of buffer A (20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 100 mM KCl, 5 mM MgCl₂, 5 mM NaF, 0.2 mM Na₃VO₄, 2 µM microcystin, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 0.5% (v/v) 2-mercaptoethanol, 10 mM benzamidine, 0.2 mM leupeptin, 0.01 mM trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane, 0.3 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged (10,000 × g, 5 min, 4 °C), and the supernatants were assayed for protein by the Bradford method (25). Supernatants (equalized for protein) were incubated with mixing at 4 °C for 2 h with GST-RBD that had been previously bound to glutathione-Sepharose beads resuspended in Buffer A. Beads were washed four times with 1 ml of buffer A and boiled with SDS-polyacrylamide gel electrophoresis sample buffer (0.33 M Tris-HCl pH 6.8, 10% (w/v) SDS, 13% (v/v) glycerol, 133 mM dithiothreitol, 0.2 mg/ml bromphenol blue). The eluted proteins were resolved on 12% polyacrylamide gels and transferred to nitrocellulose. Ras was visualized with the monoclonal pan-Ras antibody (1:1000). In some cases, membranes were probed with antibodies to Ha-Ras or N-Ras (1:1000).

Formation of Complexes Between Ras and c-Raf or MKK1 Activating Activity-- Ras was immunoprecipitated from 2 mg of supernatant protein with 10 µg of Y13-238 antibody prebound to protein G-Sepharose beads by rocking at 4 °C for 2 h. Myocyte supernatants were prepared as for the Ras·GTP loading assays described above (four dishes pooled). The beads were washed four times in 1 ml of buffer A, and immunocomplexes were immunoblotted for c-Raf or Ras.

Immunoprecipitated Ras was assayed for associated MKK1 activating activity using a coupled assay with GST-MKK1, GST-ERK2, and myelin basic protein in the presence of [-³²P]ATP (7). The protein G-Sepharose beads were washed four times with buffer B (30 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 20 mM MgCl₂, 5 mM NaF, 0.2 mM Na₃VO₄, 1 µM microcystin, 0.1% (v/v) Triton X-100, 0.3% (v/v) 2-mercaptoethanol). Immunoprecipitates were resuspended in 20 µl of buffer B containing unlabeled 0.16 mM ATP, 6.5 µg/ml GST-MKK1, and 100 µg/ml GST-ERK2. The reactions were incubated at 30 °C for 30 min with intermittent mixing and were stopped by 20 µl of 30 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM NaF, 0.2 mM Na₃VO₄, 1 µM microcystin, 0.1% (v/v) Triton X-100, 0.3% (v/v) 2-mercaptoethanol, 20 mM EGTA and then were cooled on ice. Supernatants were mixed with 40 µl of 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 12.5 mM MgCl₂, 0.4 mg/ml myelin basic protein, and 1 µCi of [-³²P]ATP. After 15 min at 30 °C, the reactions were terminated by spotting 40 µl of the reaction mixture onto P81 papers, and the papers were washed four times in 75 mM H₃PO₄. Radioactivity was counted by Cerenkov counting. Radioactivity incorporated into myelin basic protein in the absence of Y13-238 was similar to blanks performed in the absence of extract.

Assay of cAMP-dependent Protein Kinase Activity-- After incubation with agonists and washing three times in ice-cold PBS, myocytes were extracted by scraping into ice-cold 10 mM Na₂HPO₄, 10 mM NaH₂PO₄, 1 mM EDTA, 0.2 mM 1-methyl-3-isobutylxanthine, 1 mM dithiothreitol, 100 mM NaCl (pH 6.8). Extracts were centrifuged (10,000 × g, 5 min, 4 °C), and the supernatants were assayed for cAMP-dependent protein kinase (PKA) by phosphorylation of 50 µM Kemptide in the absence or presence of 2 µM cAMP as described previously (26). Results were expressed as activity ratios (activity in the absence of cAMP relative to that in the presence of 2 µM cAMP).

Microinjection Experiments-- Myocytes in serum-containing medium were microinjected (150-200 cells in marked areas of the dishes) intracytoplasmically at 24 h after plating with anti-Ras (Y13-259) (6 mg/ml in PBS) or normal rat IgG (6 mg/ml in PBS) (27). After 20 h, the cells were placed in 4 ml of serum-free medium. After a further 9 h, PMA in Me₂SO was added to a final concentration of 1 µM. An equivalent volume of Me₂SO was added to the control (0.1% (v/v) final concentration). At 4 min, cells were rinsed in PBS and fixed for 15 min in 4% formaldehyde in PBS at room temperature.

Immunofluorescence of Phospho-ERKs-- All washes and dilutions were performed in PBS. Fixed cells were washed for 30 min and permeabilized in 0.2% (v/v) Triton X-100 for 10 min, and nonspecific binding sites were blocked with 10% fetal calf serum. Cells were incubated with mouse phospho-ERK antibody (1:50 dilution, 2 h, 37 °C) and washed for 30 min. Cells were incubated (1 h, 37 °C) with a mixture of fluorescein isothiocyanate-anti-mouse IgG (1:250 dilution) to reveal phospho-ERKs and Texas Red-conjugated anti-rat IgG (1:250 dilution) to reveal microinjected rat IgG. Following a final 30-min wash, areas of dishes containing microinjected cells were mounted in Moviol aqueous mountant (28) under glass coverslips. In immunohistochemical experiments that did not involve microinjection, the Texas Red anti-IgG was omitted. Immunofluorescence analysis was performed using a Bio-Rad MRC 1024 confocal imaging system in conjunction with a Nikon Eclipse 600 fluorescence microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hypertrophic Agonists Increase Ras·GTP Loading in Ventricular Myocytes-- Using concentrations of agonists that we have shown elsewhere to be maximally effective in activating the ERK cascade, we examined the stimulation of Ras·GTP loading by PMA, ET-1, and PE using the pan-Ras antibody (Fig. 1). Small amounts of Ras·GTP were detected in control myocytes. PMA (1 µM) induced a rapid 12-15-fold increase in Ras·GTP loading, which was maximal within 1-2 min and was sustained for at least 10 min (Fig. 1, A, top panel, and B). Both ET-1 (100 nM) and PE (100 µM) stimulated Ras·GTP loading by approximately 12- and 10-fold, respectively, with a similar time course to PMA (Fig. 1, A, center panels, and B). Although Ras·GTP loading declined subsequently, it remained above control values for at least 10 min (Fig. 1, A, center panels, and B). In all cases, we confirmed that the total amounts of Ras in myocyte extracts before affinity precipitation were similar, and a typical result for a PMA time course is shown in Fig. 1A, bottom panel. There was no change in Ras·GTP loading with time in cells not exposed to agonists (results not shown). We next examined whether there were differences in GTP loading of Ras isoforms by probing with antibodies selective for N-Ras and Ha-Ras. Both isoforms were activated in myocytes exposed to PMA, ET-1, or PE for 1 min (Fig. 1C). We also attempted to determine GTP loading of K-Ras. Although some experiments indicated that the stimulation of K-Ras·GTP loading by PE was less than that with PMA or ET-1, the results were equivocal (results not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Time courses and specificity of Ras·GTP loading. Ras·GTP loading was determined by its ability to bind to GST-RBD and subsequent immunoblotting as described under "Experimental Procedures." A, typical immunoblots of Ras·GTP in myocytes exposed to 1 µM PMA (top panel), 100 nM ET-1 (upper center panel), or 100 µM PE (lower center panel) for the times indicated. An immunoblot of total Ras in a 5% extract of myocytes exposed to 1 µM PMA is shown as a loading control (bottom panel). B, quantification of time courses of Ras·GTP loading as determined by laser densitometry of immunoblots for myocytes exposed to PMA (, solid line), ET-1 (, dashed line), or PE (, solid line). Results are means ± S.E. expressed as -fold stimulation relative to control for four separate myocyte preparations. C, immunoblots for N-Ras·GTP (top panel) and Ha-Ras·GTP (bottom panel) in myocytes under control (C) conditions, or exposed to agonists for 1 min.

Numerous agonists (epinephrine, norepinephrine, isoproterenol, angiotensin II (ANGII), carbachol (CCH), BK, fibroblast growth factors) activate ERKs to varying degrees in myocytes (6, 8, 12, 29-31). Some of these are strongly hypertrophic (e.g. norepinephrine (32)), whereas others (e.g. CCH and BK) are much less so (8, 33). We examined the stimulation of Ras·GTP loading at 1 min by a variety of GPCR agonists and by acidic fibroblast growth factor (aFGF). This time point was chosen because Ras·GTP was maximal at 1-3 min for PMA, ET-1, and PE (Fig. 1B), as it was for aFGF, ANGII, and isoproterenol (results not shown). All agonists tested increased Ras·GTP loading, although the extent of stimulation differed (Fig. 2). Activation of Ras·GTP loading by PE was 77 ± 9% of the level achieved by ET-1 (the most potent agonist), whereas activation by other catecholamines such as epinephrine or norepinephrine was 65 ± 13 or 60 ± 14%, respectively (Fig. 2). BK was also very effective, stimulating Ras to 85 ± 12%, whereas that for aFGF was 50 ± 13% (all relative to ET-1). The poorest activators were isoproterenol, ANGII, and CCH, with a relative potency of 35-45% (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Comparison of Ras·GTP loading induced by different agonists. Ras·GTP loading was determined by its ability to bind to GST-RBD and subsequent immunoblotting and laser densitometry as described under "Experimental Procedures" in myocytes exposed to 1 µM PMA, 100 nM ET-1, 100 µM PE, 100 µM epinephrine (EPI), 100 µM norepinephrine (NE), 50 µM isoproterenol (ISO), 1 µM ANGII, 100 µM CCH, 1 µM BK, or 25 ng/ml aFGF for 1 min. Results are means ± S.E. expressed as -fold stimulation relative to control for 4-8 separate myocyte preparations.

Effects of PKC Inhibition and PTX on Ras·GTP Loading-- PMA pretreatment (1 µM for 24 h) essentially completely down-regulates the "classical" and "novel" PKCs (cPKCs and nPKCs) present in ventricular myocytes (34). The inhibition of the stimulation of Ras·GTP loading by PMA, ET-1, or PE in PMA-pretreated cells was 75-90% (Table I). However, interpretation of the data is complicated by the fact that PMA pretreatment increased basal Ras·GTP loading by about 4-fold (Fig. 3A). We have encountered a similar problem when examining activation of c-Raf in myocytes (7). Despite this problem, the stimulation of Ras·GTP loading elicited by acute exposure of PMA-pretreated myocytes to agonists was always less than that in myocytes that had not been pretreated (Fig. 3A). These data suggest that cPKCs/nPKCs are involved in the stimulation of Ras·GTP loading by PMA, ET-1, and PE. We also assessed the effects of the PKC-selective inhibitors GF109203X and Ro318220. Both had minimal effects on basal Ras·GTP loading (results not shown). Although GF109203X inhibited agonist-stimulated Ras·GTP loading by 45-55%, Ro318220 significantly inhibited only PMA-stimulated Ras·GTP loading (Table I). Stimulation of Ras·GTP loading by ET-1 or PE was decreased by 70-80% by 150 ng/ml PTX (Table I), suggesting that G_i/G_o proteins are involved in addition to PKC. Although PMA-stimulated Ras·GTP loading was decreased by PTX (Table I), this result did not attain statistical significance (p > 0.02). In a second series of experiments, we examined the effects of PKC inhibition and PTX on aFGF-induced Ras·GTP loading (Fig. 3B and Table I). In contrast to Fig. 3A, the stimulation of Ras·GTP loading elicited by acute exposure of PMA-pretreated myocytes to aFGF was always greater than that in myocytes exposed to aFGF alone (Fig. 3B), and any inhibition (after subtraction of the appropriate controls) was relatively slight (Table I). GF109203X inhibited aFGF-induced Ras·GTP loading (Table I), but this could reflect inhibition of the "atypical" PKC species that are not down-regulated by PMA (34). PTX did not inhibit aFGF-induced Ras·GTP loading (Table I).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of PKC down-regulation on agonist-induced Ras·GTP loading. Myocytes were either not pre-exposed to PMA (open bars) or preincubated with 1 µM PMA for 24 h (solid bars). Ras·GTP loading was determined as described under "Experimental Procedures." A, immunoblot (top panel) and quantification (bottom panel) of Ras·GTP loading in myocytes exposed to 100 nM ET-1, 1 µM PMA, or 100 µM PE for 1 min. Results are means ± S.E. for 4-7 separate myocyte preparations expressed as stimulation relative to that by 1 µM PMA or 100 nM ET-1 (as appropriate) in cells not pre-exposed to PMA. B, a similar experiment for myocytes exposed to 25 ng/ml aFGF for 1 min. Results are means ± S.E. for three separate myocyte preparations expressed as stimulation relative to cells pre-exposed to PMA and then to aFGF.

c-Raf and MKK1 Kinase Activating Activity Forms Complexes with Ras in Ventricular Myocytes-- Ras was immunoprecipitated (antibody Y13-238) from myocytes exposed to PMA, ET-1, or PE and was immunoblotted for associated c-Raf (Fig. 4). Activation of Ras resulted in the formation of Ras·c-Raf complexes following stimulation by all agonists. We also attempted to determine whether A-Raf, the other Raf isoform detectable in ventricular myocytes by immunoblotting (7), associated with Ras·GTP. We were not able to demonstrate an association, but, in the absence of a suitable positive control, these results are equivocal.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Association of c-Raf with immunoprecipitated Ras. c-Raf associated with immunoprecipitated Ras was immunoblotted as described under "Experimental Procedures." Myocytes were incubated in the absence of agonists or exposed to 1 µM PMA, 100 nM ET-1, or 100 µM PE for 1 min. c-Raf was immunoblotted in the 5% total extract as a loading control (top left panel) or in the Ras immunoprecipitates (top right panel). As controls, Ras was immunoblotted in the 5% total extract (bottom left panel) and in the Ras immunoprecipitates (IP) (bottom right panel). The results of a typical experiment, which was repeated, are shown.

c-Raf, A-Raf, and B-Raf (and possibly other MAPK kinase kinases) activate MKK1. Ras immunoprecipitates (antibody Y13-238) from myocytes exposed to PMA or ET-1 clearly contained a MKK1 activating activity (Fig. 5), for which c-Raf is likely to be at least partly responsible (Fig. 4). The stimulation of MKK1 activating activity (about 10-fold) was maximal within 1 min and was sustained for at least 3 min (Fig. 5). Only small amounts of MKK1 activating activity could be detected in Ras immunoprecipitates from PE-treated myocytes (Fig. 5), although PE clearly increased Ras·GTP loading to a degree comparable with that with PMA or ET-1 (Figs. 1 and 2), and also promoted association of Ras and c-Raf (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Association of MKK1 activating activity with immunoprecipitated Ras. Ras was immunoprecipitated, and associated MKK1 activating activity was assayed as described under "Experimental Procedures." Myocytes were exposed to 1 µM PMA (, solid line), 100 nM ET-1 (, dashed line), or 100 µM PE (, solid line). Results are means ± S.E. expressed as -fold stimulation relative to control for at least three separate myocyte preparations.

PMA pretreatment for 24 h essentially abolished the activation of Ras-associated MKK1 activating activity by 1 µM PMA or 100 nM ET-1 (Fig. 6). PMA pretreatment did not significantly affect the basal activities of Ras-associated MKK1 activating activity (Fig. 6), although it increased basal Ras·GTP loading (Fig. 3). PTX pretreatment also completely abolished the stimulation of Ras-associated MKK1 activating activity by ET-1. Somewhat surprisingly, it also caused a 60% decrease in the PMA response (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of PKC down-regulation or PTX on agonist-induced stimulation of MKK1 activating activity associated with Ras. Myocytes were incubated in the absence of inhibitory treatments (open bars), in the presence of 1 µM PMA for 24 h (solid bars), or in the presence of 150 ng/ml PTX for 24 h (stippled bars). They were then incubated in the absence of agonists (control) or in the presence of 100 nM ET-1 or 1 µM PMA for 3 min. Ras was immunoprecipitated, and the associated MKK1 activating activity was assayed as described under "Experimental Procedures." Results are means ± S.E. expressed as -fold stimulation relative to myocytes incubated in the absence of inhibitors and agonists for three separate myocyte preparations.

cAMP Decreases Ras-associated MKK1 Activating Activity and c-Raf, but Does Not Affect Ras·GTP Loading-- Exposure of myocytes to 100 µM CPT-cAMP caused a complete activation of PKA within 3 min (Table II) but did not significantly affect either the basal Ras·GTP loading or the increases in Ras·GTP loading induced by PMA, ET-1, or PE at 3 min (Fig. 7A). This result was independent of whether the agonist and CPT-cAMP were added simultaneously or whether cells were preincubated with CPT-cAMP for 3 min before the addition of agonist. Because the effect of CPT-cAMP alone on Ras·GTP loading is minimal, these results suggest that the stimulation of Ras·GTP loading by the -adrenoreceptor agonist isoproterenol (Fig. 2) is independent of its established ability to activate adenylyl cyclase and PKA. Ras-associated MKK1 activating activity in myocytes exposed to PMA or ET-1 for 3 min was almost completely inhibited by 100 µM CPT-cAMP (Fig. 7B), as was the ET-1-induced association of c-Raf and Ras (Fig. 7C).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effects of PKA activation on agonist-stimulated Ras·GTP loading and association of Ras with MKK1 activating activity or c-Raf. Myocytes were incubated with 100 nM ET-1, 1 µM PMA, or 100 µM PE for 3 min in the absence (open bars) or presence (cross-hatched bars) of 100 µM CPT-cAMP. A, Ras·GTP loading was determined by its ability to bind to GST-RBD and subsequent immunoblotting as described under "Experimental Procedures." B, MKK1 activating activity associated with immunoprecipitated Ras was assayed as described under "Experimental Procedures." Results in A and B are means ± S.E. expressed as -fold stimulation relative to myocytes incubated in the absence of CPT-cAMP and agonists, for three separate myocyte preparations. C, c-Raf associated with immunoprecipitated (IP) Ras was immunoblotted as described under "Experimental Procedures." Where applicable, myocytes were preincubated with 100 µM CPT-cAMP for 3 min (lanes 2 and 4). Myocytes were incubated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 100 nM ET-1 for 1 min. c-Raf was immunoblotted in the 5% total extract as a loading control (top panel, four left hand lanes) or in the Ras immunoprecipitates (top panel, four right hand lanes). As controls, Ras was immunoblotted in the immunoprecipitates (bottom panel). The results of a typical experiment, which was repeated, are shown.

PE stimulated Ras·GTP loading (Figs. 1 and 2) and the association of c-Raf with Ras·GTP (Fig. 4) but induced only a very slight increase in Ras-associated MKK1 activating activity (Fig. 5). We therefore considered the possibility that PE, although principally an -adrenergic agonist, might stimulate PKA activity at the high concentrations used (100 µM). Myocytes were incubated with 100 µM PE for 3 min in the presence or absence of -adrenergic antagonist, propranolol. The PKA activity ratios were not significantly different from control values (Table II). Furthermore, propranolol did not increase the activation of Ras-associated MKK1 activating activity by PE (results not shown).

Ras Is Necessary for the Stimulation of ERK Phosphorylation by PMA-- Using an anti-phospho-ERK antibody, we examined the subcellular distribution of activated ERKs in myocytes exposed to PMA. Phospho-ERKs were initially barely detectable (Fig. 8A). After 2 min, there was an increase in cytoplasmic staining (Fig. 8B), followed by intense nuclear staining at 4 min (Fig. 8C), which was maintained at 8 min (Fig. 8D). By 16 min, nuclear staining was declining (Fig. 8E) but was still clearly detectable in the cytoplasm (Fig. 8E). After 32 min, diffuse staining was still detectable (Fig. 8F). These results show that PMA transiently activates ERKs in both the nuclear and cytoplasmic compartments of myocytes.

View larger version (105K): [in this window] [in a new window]   Fig. 8.   Stimulation of ERK phosphorylation by PMA. Myocytes were exposed to 1 µM PMA for 0 min (A), 2 min (B), 4 min (C), 8 min (D), 16 min (E), or 32 min (F). Phospho-ERK was immunostained as described under "Experimental Procedures." A typical field from a typical experiment (from three independent experiments) is shown (540× magnification).

We examined whether Ras was necessary for PMA-induced ERK phosphorylation by microinjecting the neutralizing Y13-259 anti-Ras antibody (Fig. 9). In control myocytes microinjected with normal rat IgG (Fig. 9A) or Y13-259 (Fig. 9B), phospho-ERK staining was essentially undetectable. After exposure to 1 µM PMA for 4 min, phospho-ERK was clearly detectable in nuclei of myocytes microinjected with normal rat IgG and in uninjected cells in the same field (Fig. 9C). In contrast, Y13-259 completely prevented the appearance of phospho-ERK staining, which, however, was still clearly visible in uninjected myocytes (Fig. 9D). Thus, active Ras is essential for the coupling of PMA stimulation to the activation of the ERK cascade in myocytes.

View larger version (47K): [in this window] [in a new window]   Fig. 9.   Ras is necessary for the stimulation of ERK phosphorylation by PMA. Myocytes were microinjected with rat IgG (A and C) or rat anti-Ras (B and D) and incubated under control conditions (A and B) or in the presence of 1 µM PMA for 4 min (C and D). Microinjected cells were detected with Texas Red-conjugated donkey anti-rat IgG, and phospho-ERK was detected with mouse monoclonal anti-phospho-ERK and fluorescein isothiocyanate-donkey anti-mouse IgG as described under "Experimental Procedures." A typical field from a typical experiment (from three independent experiments) is shown (540× magnification).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ventricular myocyte withdraws from the cell cycle during the perinatal period, and any subsequent adaptive growth is brought about by hypertrophy (reviewed in Refs. 13 and 14). The intracellular signaling pathways responsible are still not clear. Although it has recently been suggested that calcineurin may be involved (35), a large body of earlier work has implicated PKC, Ras, and the MAPK superfamily (reviewed in Ref. (14)). The fact that PMA strongly stimulates phosphorylation (activation) of ERKs in the nucleus (Fig. 8) is consistent with a role for these kinases in the transcriptional changes associated with hypertrophy. When some cells are exposed to suitable agonists, activated ERKs may translocate from the cytoplasm into the nucleus (36, 37), but we have been unable to detect this in myocytes.² Evidence for an involvement of Ras in hypertrophy derives from transient transfection of constitutively activated or dominant negative ras (38-41), from microinjection of constitutively active Ras protein (38), and from transgenic mice that cardiospecifically express constitutively activated Ras (42). The overall hypertrophic effects of Ras may depend on the activation of a number of Ras effectors, including c-Raf, Ral-GDS, and possibly phosphatidylinositol 3'-kinase (43).

Our interests in the hypertrophic response and GPCR signaling to the ERK cascade led us to examine the regulation of endogenous Ras activity in myocytes. Using ³²P_i prelabeling of guanine nucleotide pools, others have demonstrated stimulation of Ras·GTP loading by ANGII (40) and PE (44). This is a technically demanding method, making a thorough survey impractical. Increased Ras·GTP loading has also been demonstrated in permeabilized cells exposed to PE and extracellular radiolabeled GTP (45). We used a novel method that uses the RBD of c-Raf as a probe (15, 16), and we show here that PMA and ET-1 cause a 10-15-fold increase in Ras·GTP loading within 1-2 min (Fig. 1, A and B). Maximal Ras·GTP loading precedes the activation of Raf (maximal at 3 min (7)) and ERKs (maximal at 5 min (6, 12, 46)), consistent with a precursor function. The rapid Ras·GTP loading induced by PMA in myocytes (Fig. 1B) contrasts with the situation in COS cells, where, using similar methodology, Marais et al. (47) found that Ras·GTP loading did not attain maximal values until 40 min. In 3T3 cells, activation of Ras by PMA was not detectable (48), and thus the response may be cell-specific.

Drawing mainly on our own data for the sake of consistency, we have correlated our results for Ras·GTP loading (Fig. 2) with activation of the ERK cascade and with the hypertrophic response in myocytes (Table III). PMA and ET-1 powerfully stimulate the ERK cascade as well as stimulating phosphatidylinositol hydrolysis (ET-1) and PKC translocation (ET-1 and PMA), and both are strongly hypertrophic. ANGII and CCH stimulate a lesser (4-6-fold) increase in Ras·GTP loading, are poor activators of the ERK cascade, and, in the hands of other investigators (33, 49), are weakly hypertrophic. For these agonists, the correlations are reasonably secure. However, there are exceptions. Thus, PE stimulates Ras·GTP loading by 9-10-fold (Figs. 1C and 2) and is strongly hypertrophic, yet is a poor activator of Raf and a moderate activator of ERKs. Essentially the converse is true for BK, which is at best weakly hypertrophic. We also examined the association of Ras with c-Raf (Fig. 4) and with MKK1 activating activity (Fig. 5). PMA and ET-1 increase these associations, and c-Raf is presumably at least partly responsible for the Ras-associated MKK1 activating activity. In contrast, PE only slightly stimulated Ras-associated MKK1 activating activity (Fig. 5), although it did increase the association between Ras and c-Raf (Fig. 4). We do not understand the reasons for the failure of PE to increase Ras-associated MKK1 activating activity, but the finding is in agreement with the fact that PE is a poor activator of c-Raf and A-Raf in comparison with PMA and ET-1 in myocytes (7). Our preliminary experiments have suggested that PE may not stimulate K-Ras·GTP loading to the same extent as PMA or ET-1, and it has been recently demonstrated that K-Ras translocates and activates c-Raf more efficiently than Ha-Ras (50). We suggest that there may be contributions from Ras-dependent Raf-independent signaling pathways to the stimulation of hypertrophy by PE.

Some GPCRs (e.g. the _2A-adrenoreceptor, the lysophosphatidic acid receptor) activate the ERK cascade in a Ras-dependent manner through a G_i-dependent -mediated mechanism (51, 52) that is independent of PKC. The data presented (Table I) are largely consistent with a requirement for both PKC and G_i/G_o in the stimulation of Ras·GTP loading by ET-1 and PE. Although we used much higher concentrations of PTX than necessary to cause complete ADP-ribosylation of G proteins in myocytes (22), this treatment did not affect aFGF-stimulated Ras·GTP loading (Table I), indicating that the inhibition by PTX of the Ras·GTP loading induced by other agonists is not caused by nonspecific toxic effects. Stimulation of Ras-associated MKK1 activating activity by ET-1 or PMA also required PKC and G_i/G_o (Fig. 6). The effects of PTX on the activation of the ERK cascade in myocytes are confusing. ET-1 couples to both G_q/G₁₁- and G_i/G_o-dependent pathways in ventricular myocytes (53) and other cells (54). Although the stimulation of c-Raf and A-Raf by ET-1 is decreased by PTX (7), the activation of ERKs by ET-1 is largely insensitive to inhibition by PTX (23, 55), possibly reflecting the amplifying capacity of the ERK cascade or the existence of alternative pathways. PTX raises cAMP concentrations in myocytes (56), and phosphorylation of Ser-43 in c-Raf by PKA inhibits its ability to bind to Ras·GTP (reviewed in Ref. 2). Thus, it was possible that the inhibition of the association between Ras and MKK1 activating activity by PTX could result from increased cAMP concentrations. Although cAMP does not inhibit agonist-stimulated Ras-GTP loading (Fig. 7A), it reduces the association of MKK1 activating activity (Fig. 7B) with Ras, suggesting that some of the effects of PTX may result from increases in cAMP concentrations.

We show a scheme (Fig. 10) to account for the various effects of inhibitors on the different stages in the activation of Ras and MKK1 activating activity based on differential activation of PKC isoforms. There is evidence that the different subgroups of PKCs perform divergent functions within the context of c-Raf activation (57). We have previously shown that PMA, ET-1, and PE differentially translocate (activate) cPKC, nPKC, and nPKC in ventricular myocytes (34, 46). nPKC is translocated by all three agonists, whereas nPKC is translocated only by PMA and ET-1, and cPKC is translocated only by PMA. We suggest that ET-1 or PE "primes" Ras·GDP for activation in a G_i-dependent manner, whereas PMA stimulates the process through activation of cPKC. Exchange of GDP for GTP on "primed" Ras·GDP requires activation of nPKC and can therefore be brought about by all three agonists. c-Raf then associates with Ras·GTP, but the c-Raf is catalytically inactive. However, assuming that nPKC is essential for full activation of c-Raf (or alternatively activation of K-Ras; see above), PMA and ET-1 would induce activation of c-Raf, whereas PE would be ineffective.

View larger version (10K): [in this window] [in a new window]   Fig. 10.   A possible mechanism of Ras and c-Raf activation by PMA, ET-1, and PE in ventricular myocytes.

There have been relatively few studies on the ability of PKC inhibition or PTX to prevent myocyte hypertrophy, largely for technical reasons (cytotoxicity of inhibitors over the 24-48 h necessary for the induction of this complex response, the fact that chronic exposure of myocytes to PMA induces hypertrophy). PTX does not inhibit the PE-induced expression of c-fos or egr-1 or the accumulation of myosin light chain protein, changes typical of hypertrophy (22). We have found that inhibition of the ERK cascade by limited exposure of myocytes to PD98059 reduces myofibrillogenesis in response to ET-1 or PE (58). However, PD98059 does not inhibit the increases in cell size induced by ET-1 (58), and prolonged exposure even potentiates the re-expression of the atrial natriuretic peptide gene (33, 59), re-expression of this gene being a characteristic of hypertrophy (reviewed in Refs. 13 and 14).

As discussed in Ref. 47 (and reviewed in Ref. 60), there is confusion about the role of Ras in the PMA/PKC- and the G_q PCR/PKC-mediated activation of the ERK cascade. Dominant negative N17Ras inhibits activation of the cascade by PMA or G_q PCR agonists in the hands of some workers (61, 62) yet fails to do so in the hands of others (47, 52, 63-67). Although little work has been carried out in cardiac myocytes, dominant negative A15Ras has been shown to inhibit the stimulation of ERKs by PE (68). One group has shown that PMA still activates transfected c-Raf(Arg-89  Leu), a mutant that is unable to interact with Ras·GTP, albeit to a lesser extent than wild type c-Raf (69). However, others have shown that mutations in c-Raf that interfere with its ability to interact with Ras·GTP render it insensitive to activation by PMA (47, 70) or G_q PCR agonists (47). Furthermore, the evidence that PKC directly phosphorylates and activates c-Raf (71-74) (thereby bypassing Ras) has recently been challenged (57). In an approach that does not involve transient transfection, microinjection of a neutralizing antibody against Ras (Y13-259) abolished the phosphorylation (activation) of overexpressed ERK2 by PMA (47). Consistent with this, we found that microinjection of Y13-259 abolished the increase in phosphorylation of endogenous ERK by PMA (Fig. 9). These experiments suggest an essential role of Ras in PKC-mediated activation of the ERK cascade. It is not clear how activation of PKC results in increased Ras·GTP loading, but it may inhibit the activity of the Ras GTPase activating protein, Ras·GAP (75), possibly by direct phosphorylation (76). The possibility that PMA increases Ras·GTP loading by a PKC-independent mechanism cannot be discarded. In this regard, a Ras guanyl nucleotide-releasing protein (Ras·GRP) that contains a diacylglycerol/phorbol ester binding domain has recently been identified (77, 78). Overexpression of Ras·GRP stimulates Ras·GTP loading in fibroblasts (77). However, Ras·GRP transcripts are not detectable in heart (78). Taken as a whole, our results are consistent with a role for Ras in myocyte hypertrophy, and in activation of the ERK cascade by PMA.

	FOOTNOTES

^* This work was supported by grants from the British Heart Foundation.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.

Gibb Life Research Fellow of the Cancer Research Campaign.

^** To whom correspondence should be addressed: NHLI Division (Cardiac Medicine), Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, United Kingdom. Tel.: 44-171-352-8121 (ext. 3306/3314); Fax: 44-171-823-3392; E-mail: p.sugden@ic.ac.uk.

² A. Clerk and P. H. Sugden, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; ANGII, angiotensin II; BK, bradykinin; CCH, carbachol; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; ERK, extracellular signal-regulated kinase; ET-1, endothelin-1; aFGF, acidic fibroblast growth factor; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; MKK, MAPK kinase; PBS, phosphate-buffered saline; PE, phenylephrine; PKA, protein kinase A; PKC, protein kinase C; PTX, pertussis toxin; RBD, Ras binding domain; PMA, phorbol 12-myristate 13-acetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES