INTRODUCTION

One of the early obligatory responses elicited by mitogenic and hypertrophic factors is the stimulation of protein synthesis which results from changes at both the transcriptional and translational levels (1, 2, 3). Although the signaling mechanisms involved in this response remain poorly defined, it is known that phosphorylation/dephosphorylation reactions play a critical role in controlling the overall rate of protein synthesis (1, 4, 5). Signals initiated by growth factors interacting with receptor tyrosine kinases or G protein-coupled receptors are integrated and propagated through an elaborated network of cytoplasmic serine/threonine kinases (6, 7, 8). The best understood of these protein kinase cascades is the mitogen-activated protein (MAP)¹ kinase module leading to activation of the ERK subfamily of MAP kinases (9, 10, 11, 12). Two isoforms of ERKs, referred to as p44^mapk (ERK1) and p42^mapk (ERK2), have been described and found ubiquitously expressed in tissues (13, 14). ERK isoforms are activated by phosphorylation on both threonine and tyrosine residues by two dual-specificity MAP kinase kinases termed MEK1 and MEK2 (9, 12, 15, 16). MEKs are in turn activated by serine phosphorylation catalyzed by a number of MAP kinase kinase kinases which include Raf-1 (17, 18, 19, 20), B-Raf (21, 22, 23, 24), Mos (25), and MEK kinase-1 (26).

While the mechanisms of ERKs regulation are relatively well understood, the precise physiological roles of these enzymes remain to be established. The p44^mapk and p42^mapk isoforms are rapidly phosphorylated and activated in response to virtually all growth factors (14, 27). However, the observation that a MAP kinase is activated in a specific process does not demonstrate that this enzyme is functionally essential in vivo. Strong evidence for the critical involvement of ERKs in the regulation of cell proliferation were obtained from studies showing a close correlation between ERKs activation and DNA synthesis (28, 29) and from the demonstration that inhibition of cellular ERKs activity blocks cell cycle progression (30, 31). Studies using constitutively active and dominant-negative mutants of MEK1 or thiophosphorylated MAP kinase (32, 33), together with pharmacological blockade experiments (34), also demonstrated the absolute requirement of the ERK pathway for neuronal differentiation. The role of the ERK pathway in the regulation of protein synthesis and in many other growth-related processes remains to be clarified.

The peptide hormone AII provides a good model system to study the signaling pathways by which growth factors regulate the rate of protein synthesis. In vascular SMC, AII induces cellular hypertrophy as a result of increased protein synthesis, but has no effect on cell division (35, 36, 37, 38). The trophic action of AII is initiated by its interaction with the G protein-coupled AT₁ receptor, which stimulates the activity of phospholipase C to produce the second messengers IP₃ and diacylglycerol, and inhibits the activity of adenylyl cyclase (39, 40). These early signals ultimately result in the activation of ERKs (38, 41, 42) and of the 70/85-kDa S6 protein kinases (38).

The aim of this study was to evaluate the involvement of the ERK pathway in the stimulatory effect of growth factors on protein synthesis in vascular SMC. To this end, we used a selective inhibitor of this pathway, PD 98059, which has been shown to inhibit MEK activity in PC-12 (34) and Swiss 3T3 cells (43). We show that PD 98059 blocks AII-induced phosphorylation and activation of p44^mapk and p42^mapk in rat aortic SMC. Most importantly, we demonstrate that the drug inhibitor prevents the increased rate of protein synthesis by AII and other growth factors acting through distinct types of receptors. These results provide the first direct evidence that the ERK pathway plays a critical role in the regulation of global protein synthesis in mammalian cells.

EXPERIMENTAL PROCEDURES

AII was purchased from Hukabel Scientific. [³H]IP₃, [-³²P]ATP, [³²P]phosphoric acid, and [³H]leucine were from Amersham Corp. Protein A-Sepharose was obtained from Pharmacia Biotech Inc. Protease inhibitors and bovine MBP were from Sigma. IP₃ and phorbol 12-myristate 13-acetate were from LC Services. PD 98059 was a gift of Parke-Davis Pharmaceutical Research Division and was dissolved in dimethyl sulfoxide at a concentration of 30 mM. Rapamycin was a gift of Wyeth-Ayerst Research. The p70^S6K antiserum was generously provided by Dr. Frederick Hall (Children's Hospital of Los Angeles). Antiserum SM1 has been described previously and specifically immunoprecipitates p44^mapk protein (44). The antipeptide serum IIcp42 was kindly provided by Drs. Yizheng Wang and Michael Dunn (Case Western Reverse University) and specifically recognizes the native p42^mapk isoform (44, 45). The anti-MAPKK serum was provided by Drs. Gilles L'Allemain and Jacques Pouysségur (Université de Nice). This polyclonal antibody specifically immunoprecipitates the MEK1 isoform of MAP kinase kinases. The anti-MEK2 monoclonal antibody was purchased from Transduction Laboratories and specifically immunoprecipitates the MEK2 isoform. Anti-phosphotyrosine mAbs 4G10 and PY-20 were purchased from Upstate Biotechnology and ICN, respectively.

Rat aortic SMC were cultured and synchronized in the quiescent state as described previously (38). For experiments with PD 98059 and rapamycin, the cells were treated with vehicle alone or with the indicated concentrations of agents for 30 min before addition of growth factors.

Quiescent aortic SMC were stimulated with 100 nM AII for 5 (ERK assays), 3 (MEK assays), or 15 min (p70^S6K assays). The enzymatic activity of ERK isoforms was measured by a specific immune complex kinase assay using MBP as substrate as described (38, 44). The phosphotransferase activity of p70^S6K was measured by an immune complex kinase assay using the S6 peptide RRRLSSLRA (Upstate Biotechnology) as substrate (38). The enzymatic activity of MEK1 and MEK2 was assayed by measuring their ability to increase the MBP kinase activity of recombinant p44^mapk in vitro. Details of the procedure will be described elsewhere.² Briefly, cell lysates were prepared as described (38) and 100 µg (MEK1 assays) or 600 µg (MEK2 assays) of lysate proteins were incubated for 4 h at 4 °C with 1 µl of anti-MAPKK serum or 4 µl of anti-MEK2 mAb preadsorbed to protein A-Sepharose beads. The immune complexes were washed 3 times with lysis buffer, once with kinase assay buffer (20 mM Hepes, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.4), and then resuspended in kinase assay buffer containing 50 µM ATP, 5 µCi of [-³²P]ATP, and 300 ng of recombinant p44^mapk. After incubation at 30 °C for 30 min, bovine MBP (0.25 mg/ml) was added and the incubation was continued for an additional 10 min. The reaction was stopped by addition of 2 × Laemmli's sample buffer. The samples were analyzed by SDS-gel electrophoresis on 12% acrylamide gels and the band corresponding to MBP was excised and counted in a liquid scintillation counter.

Quiescent rat aortic SMC in 100-mm Petri dishes were metabolically labeled for 5 h at 37 °C in bicarbonate and phosphate-free Hepes-buffered Dulbecco's modified Eagle's medium containing 0.75 mCi/ml [³²P]phosphoric acid. The cells were then stimulated by addition of 100 nM AII to the medium for 5 min and quickly washed with ice-cold phosphate-buffered saline. Cell lysates were prepared as described above. The lysates were then precleared for 1 h with 10 µl of normal rabbit serum and incubated for 4 h at 4 °C with either 10 µl of SM1 antiserum, 3 µl of IIcp42 antiserum, 4 µl of MAPKK antiserum, or 10 µl of anti-MEK2 mAb preadsorbed to protein A-Sepharose beads. Immune complexes were washed six times with lysis buffer. Protein complexes were eluted by heating at 95 °C for 5 min in denaturating sample buffer and analyzed by SDS-gel electrophoresis on 10% acrylamide gels.

The intracellular mass of IP₃ was measured by a specific radioreceptor assay (46). Quiescent aortic SMC in 35-mm Petri dishes were stimulated with AII for 15 s at 37 °C. The incubation was terminated by addition of 100 µl of cold 40% trichloroacetic acid (final concentration of 10%). After extraction for 10-20 min on ice, the cells were scraped and centrifuged at 13,000 × g for 5 min at 4 °C. The resulting supernatant was extracted five times with 2 ml of water-saturated diethyl ether and neutralized with 30 µl of 1 M NaHCO₃ (pH 8.5). An aliquot of 100 µl of cell extract was then assayed for IP₃ mass. For IP₃ binding assays, 750 µg of adrenocortical membranes (46) were incubated with 1 nM [³H]IP₃ and an aliquot of cell extract or varying concentrations of unlabeled IP₃ for 30 min at 4 °C in a total volume of 500 µl of IP₃ binding buffer (25 mM Tris-HCl, pH 8.5, 100 mM KCl, 20 mM NaCl, 5 mM KH₂PO₄, 1 mM EDTA, 0.1% bovine serum albumin). Bound [³H]IP₃ was separated from free ligand by centrifugation at 13,000 × g for 5 min at 4 °C. The supernatant was removed by aspiration and the radioactivity in the pellet was determined by liquid scintillation counting. Averages of duplicate determinations of bound [³H]IP₃ were used for data analysis. The mass of IP₃ is expressed as picomoles of IP₃ produced per mg of protein.

Quiescent aortic SMC in 100-mm Petri dishes were washed once and stimulated with 100 nM AII at 37 °C for 5 min. The cells were then washed twice with ice-cold phosphate-buffered saline and lysed in 0.8 ml of Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM sodium fluoride, 5 mM EDTA, 40 mM -glycerophosphate, 1 mM sodium orthovanadate, 10⁴ M phenylmethylsulfonyl fluoride, 10⁶ M leupeptin, 10⁶ M pepstatin A, 1% Triton X-100) for 20 min at 4 °C. Lysates were clarified by centrifugation at 13,000 × g for 10 min and equal amounts of lysate proteins (100 µg) were subjected to immunoprecipitation with 20 µl of agarose-coupled PY-20 mAb for 2 h at 4 °C. Immune complexes were washed three times with lysis buffer prior to electrophoresis on 7.5% acrylamide gels. After electrophoresis, proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham) in 25 mM Tris, 192 mM glycine. Membranes were blocked in Tris-buffered saline containing 5% non-fat dry milk for 1 h at 37 °C prior to incubation for 1 h at 25 °C with mAb 4G10 (1:5,000) in blocking solution. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham).

Quiescent aortic SMC in triplicate wells of 24-well plates were stimulated with the indicated concentrations of growth factors in serum-free quiescence medium containing 0.5 µCi/ml [³H]leucine. After 24 h of stimulation, the medium was aspirated and the cells were incubated for a minimum of 30 min in cold 5% trichloroacetic acid. The wells were then washed once with trichloroacetic acid and three times with tap water. The radioactivity incorporated into trichloroacetic acid-precipitable material was measured by liquid scintillation counting after solubilization in 0.1 M NaOH. For experiments with drug inhibitors, quiescent cells were pretreated for 30 min with the indicated concentrations of agents and stimulated for 24 h in the continuous presence of the inhibitor.

Protein concentrations were measured using the BCA protein assay kit (Pierce) with bovine serum albumin as standard. Dose-response curves were analyzed according to a four-parameter logistic equation using the ALLFIT computer program (47).

RESULTS

PD 98059 is a synthetic drug inhibitor that selectively blocks the activity of the ERK pathway at the level of MEK (34, 43, 48). To explore the role of the ERK pathway in the hypertrophic action of AII, we first examined the effect of PD 98059 on AII-dependent phosphorylation and activation of MEK and ERK isoforms in rat aortic SMC. Growth-arrested cells were labeled with ³²P_i, stimulated with AII for 5 min, and MEK1, MEK2, p44^mapk, and p42^mapk were immunoprecipitated from cell lysates prior to analysis by gel electrophoresis. Little phosphorylation of MEK1 and MEK2 was detected in resting cells and treatment with AII significantly increased the phosphate content of the two proteins (Fig. 1A). Pretreatment of the cells with PD 98059 completely suppressed the phosphorylation of MEK1 and MEK2, thereby suggesting that the inhibitor interferes with the upstream activation of these enzymes. In parallel to these experiments, extracts from similarly-treated cells were used to test the enzymatic activity of MEKs in a reconstitution assay using recombinant p44^mapk and MBP as substrate. Addition of AII to quiescent SMC caused a 9-fold increase in both MEK1 and MEK2 activity, which was inhibited approximately 80% by preincubation of the cells with 30 µM PD 98059 (Fig. 1B). These results confirm that the compound exerts inhibitory effects on both isoforms of MEK in intact cells. As previously reported (38, 41, 42), AII strongly stimulated the phosphorylation and enzymatic activation of p44^mapk and p42^mapk isoforms in quiescent aortic SMC. Pretreatment of cells with PD 98059 prevented the phosphorylation of the two enzymes and, as a consequence, blocked their activation by the growth factor (Fig. 2). Thus, these results confirm that PD 98059 is a valuable tool to inhibit the cellular activity of the ERK pathway in rat aortic SMC.

To demonstrate that PD 98059 selectively blocks the activation of ERKs, we then examined the effects of the compound on AII-stimulated IP₃ production, p70^S6K activity, and tyrosine phosphorylation in rat aortic SMC. AII binding to the AT₁ receptor rapidly stimulates the activity of phospholipase C in vascular SMC, leading to the formation of IP₃ (39, 40, 49). As shown in Fig. 3A, pretreatment of quiescent aortic SMC with 30 µM PD 98059 did not prevent the rapid increase in the production of IP₃ induced by AII. We have previously demonstrated that AII potently stimulates the phosphotransferase activity of p70^S6K in aortic SMC (38). To determine if PD 98059 interferes with p70^S6K activation, cells were treated with the MEK inhibitor prior to AII stimulation and the activity of p70^S6K was measured by immune complex kinase assay. Fig. 3B shows that treatment with 30 µM PD 98059 had no effect on AII-dependent activation of p70^S6K in aortic SMC. Stimulation of vascular SMC with AII also leads to increased tyrosine phosphorylation of several proteins, including two major bands of apparent molecular mass 65-75 and 120 kDa (49, 50, 51). The 65-75-kDa band has been recently identified as the focal adhesion-associated protein paxillin, whereas the 120-kDa band may correspond to p125 focal adhesion kinase (52, 53). To further test the selectivity of PD 98059, rat aortic SMC were treated with or without PD 98059, followed by exposure to AII for 5 min. Cells lysates were prepared and subjected to immunoprecipitation with agarose-linked PY-20 antiphosphotyrosine mAb, prior to analysis by immunoblotting with 4G10 antiphosphotyrosine mAb. Fig. 3C shows that PD 98059 did not affect AII-stimulated tyrosine phosphorylation of the M_r 65,000-75,000 and 120,000 protein bands. Together, these results indicate that PD 98059 selectively inhibits the activation of ERK isoforms in intact SMC.

We next examined the effect of PD 98059 on growth factor-stimulated protein synthesis. For these experiments, quiescent rat aortic SMC were preincubated for 30 min with PD 98059 prior to stimulation with growth factors for 24 h in the continuous presence of the drug. PD 98059 was found to potently inhibit AII-induced protein synthesis in these cells, with 70% inhibition observed at a concentration of 30 µM (Fig. 4A). Half-maximal inhibition was observed in the presence of 4.3 ± 1.6 µM PD 98059 (n = 2), which is similar to the concentration required for 50% inhibition of [³H]thymidine incorporation in platelet-derived growth factor-stimulated Swiss 3T3 cells (43). To verify the general involvement of the ERK pathway in the regulation of protein synthesis by growth factors, aortic SMC were treated as above and stimulated with different growth factors acting through distinct types of receptors. PD 98059 was found to also inhibit the increased rate of protein synthesis induced by thrombin, insulin, basic fibroblast growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate (Fig. 4B). The extent of inhibition by PD 98059 varied for the different growth factors studied, suggesting that the ERK pathway may be of greater importance to the action of certain agonists in these cells.

We have recently reported that the immunosuppressant drug rapamycin, which selectively blocks p70^S6K activation, strongly inhibits AII-stimulated protein synthesis in aortic SMC (38). In view of the above findings, it was of interest to examine the relative contribution of the ERK and p70^S6K pathways to the overall regulation of protein synthesis. Simultaneous treatment of aortic SMC with 30 µM PD 98059 and 10 ng/ml rapamycin, concentrations which maximally inhibit the AII response (Fig. 4A and Ref. 38), had a significant additive effect on the inhibition of AII-induced protein synthesis when compared to the effect of each drug alone (Fig. 5). These results suggest that p44^mapk/p42^mapk and p70^S6K operate via distinct signaling pathways to increase the rate of protein synthesis in rat aortic SMC.

DISCUSSION

ERK isoforms are coordinately activated in response to a wide range of mitogenic and non-mitogenic stimuli (14, 27). Evidence for a physiologically relevant role of these enzymes in growth factor-dependent cell proliferation and cell differentiation have been obtained from a combination of pharmacological, biochemical, and genetic approaches (54, 55, 56). Biochemical studies have shown that ERKs can phosphorylate a large number of proteins, including transcription factors, protein kinases, cytosolic enzymes, and others (27, 57). Despite these observations, the precise role of the ERK pathway in several growth-related processes remains largely unknown. In this study, we have used a synthetic MEK inhibitor to investigate the involvement of ERK isoforms in the stimulation of global protein synthesis by growth factors in rat aortic SMC. This compound, PD 98059, was found to selectively block the phosphorylation and activity of MEK1 and MEK2 and, as a consequence, of p44^mapk and p42^mapk isoforms in intact cells. We report that PD 98059 potently inhibits growth factor-stimulated leucine incorporation, demonstrating for the first time, a direct role of the ERK pathway in the overall regulation of protein synthesis.

Previous studies from our laboratory have clearly shown that activation of the ERK pathway is not sufficient to mediate the increased rate of protein synthesis by AII in rat aortic SMC. We have demonstrated that treatment of aortic SMC with rapamycin, which totally blocks the activation of p70^S6K by AII, causes a major but incomplete inhibition of AII-stimulated protein synthesis (38). However, the activation of p44^mapk and p42^mapk is not affected by rapamycin under similar conditions. The observation that rapamycin never completely inhibit AII-stimulated protein synthesis suggested that additional signaling pathways, such as the ERK pathway, were recruited by AII to regulate the rate of protein synthesis. More recently, we have shown that inhibition of tyrosine phosphorylation by the tyrosine kinase inhibitors genistein and herbimycin A results in a complete inhibition of AII-stimulated protein synthesis in rat aortic SMC (49). Again, the two inhibitors do not interfere with the activation of p44^mapk and p42^mapk in these cells. Finally, we have recently observed that a variety of agents known to elevate the intracellular concentration of cyclic AMP potently inhibit the stimulatory effect of AII on protein synthesis, without affecting AII-dependent activation of ERK or MEK isoforms in rat aortic SMC.³

The site of action of ERK isoforms in the control of protein synthesis is not known. Activation of protein synthesis involves changes not only at the level of mRNA translation but also in transcriptional processes. While the role of ERKs has been well characterized in the latter (58, 59, 60), their involvement in the regulation of translational processes remains hypothetical. It has been recently proposed that ERKs mediate insulin-dependent phosphorylation of the translational repressor 4E-BP1 (also known as PHAS-I), thereby providing a direct link between these enzymes and the translational machinery (61). This hypothesis was based on the observation that ERK efficiently phosphorylates 4E-BP1 on a single serine residue (serine 64) in vitro and that phosphorylation markedly decreases its affinity for eIF-4E (61, 62). This serine site is also phosphorylated in vivo in adipocytes in response to insulin (62). However, more recent studies indicate (63)⁴ that p44^mapk or p42^mapk are unlikely to play a major role in the phosphorylation of 4E-BP1 in intact cells.⁵ We have found that 4E-BP1 is phosphorylated on several residues in addition to the single ERK site and that the latter is not phosphorylated at a time when ERKs activity is already high in aortic SMC.⁵ Another potential mechanism by which ERK isoforms could modulate the rate of translation initiation is by regulating the phosphorylation level of eIF-2B. eIF-2B is a guanine nucleotide-exchange factor which plays a critical role in translation initiation by mediating the recycling of eIF-2 (5). It has been recently reported that GSK3 can phosphorylate the largest subunit of eIF-2B in vitro (65), possibly leading to inhibition of eIF-2B activity (5). Interestingly, the  and  isoforms of GSK3 are rapidly inactivated by insulin treatment in vivo (65, 66) and by phosphorylation with p90^RSK in vitro (67, 68). Since the activity of p90^RSK is under control of ERKs (13), this could provide a link between the ERK pathway and eIF-2B. However, several important issues need to be resolved to establish the relevance of this pathway.

Interestingly, we report here that PD 98059 and rapamycin exert additive inhibitory effects on AII-stimulated leucine incorporation, providing strong evidence that the ERK pathway and p70^S6K regulate global protein synthesis by distinct mechanisms. It has been demonstrated that rapamycin selectively represses translation of mRNAs containing a polypyrimidine tract immediately after their cap structure (69, 70). This family of mRNAs includes transcripts for elongation factors and ribosomal proteins. More recently, rapamycin was shown to block growth factor-dependent phosphorylation of 4E-BP1 (63, 71, 72) and, most importantly, to reduce cap-dependent initiation of translation (72). In addition to its effects on translation, rapamycin also exert some actions at the level of transcription (reviewed in Ref. 73). For example, a recent study has shown that rapamycin inhibits serum-induced cAMP-responsive element modulator activation by preventing its phosphorylation by p70^S6K (64). An interesting challenge will be to determine how signals from ERKs and p70^S6K are integrated to increase the global rate of protein synthesis.

In conclusion, the results presented here indicate that activation of the ERK pathway is necessary but not sufficient for growth factor-induced protein synthesis in vascular SMC. Further work is required to address the exact role of ERK isoforms in the nuclear and cytoplasmic events controlling the rate of protein synthesis.