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(Circulation Research. 1996;79:1007-1014.)
© 1996 American Heart Association, Inc.

Articles

Angiotensin II Stimulates MAP Kinase Kinase Kinase Activity in Vascular Smooth Muscle Cells

Role of Raf

Duan-Fang Liao, Jennifer L. Duff, Guenter Daum, Steven L. Pelech, Bradford C. Berk

the Departments of Medicine, Division of Cardiology (D.-F.L., J.L.D., B.C.B.), and Surgery (G.D.), University of Washington, Seattle; the Department of Medicine (S.L.P.), University of British Columbia and Kinetek Biotechnology Corp, Vancouver, Canada.

Correspondence to Bradford C. Berk, MD, PhD, Division of Cardiology, Box 357710, University of Washington, Seattle, WA 98195-7710. E-mail bcberk@u.washington.edu.

	Abstract

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Both angiotensin II (Ang II) and platelet-derived growth factor (PDGF) rapidly increase intracellular Ca²⁺ and activate protein kinase C (PKC) and MAP kinase in vascular smooth muscle cells (VSMCs). However, Ang II causes cell hypertrophy, whereas PDGF causes hyperplasia. These findings indicate that VSMCs are a good model for studying the relationship between cell growth and the MAP kinase pathway. In this study, we investigated the role of Raf in activation of 42- and 44-kD MAP kinases. Western blot analysis showed that c-Raf-1 was the predominant Raf isozyme in cultured rat aortic VSMCs. In response to Ang II, there was translocation of Raf to the membrane, which occurred significantly earlier than MAP kinase activation, suggesting that Raf activation precedes MAP kinase activation. Translocation of Raf to the membrane resulted in association with H-Ras as shown by c-Raf-1 coprecipitation with anti-Ras antibodies. Western blot analysis of H-Ras immunoprecipitates revealed c-Raf-1, but c-mos, MEK (MAP kinase/extracellular signal-regulated kinase) kinase-1 (MEKK-1), and Raf-B were not present. MAP kinase kinase kinase (MAPKKK) activity was assayed in c-Raf-1 and H-Ras immunoprecipitates by MAP kinase kinase–dependent phosphorylation of catalytically inactive 42-kD MAP kinase. In Ras immunoprecipitates, MAPKKK activity was stimulated approximately threefold by both Ang II and PDGF, with a peak at 5 minutes. Downregulation of PKC by 24-hour exposure to phorbol ester significantly inhibited Ang II–stimulated and PDGF-stimulated MAPKKK activity (80% decrease) and Raf translocation (90% decrease), suggesting that a phorbol-responsive PKC is upstream from MAPKKK and Raf. In contrast, Ang II (but not PDGF) stimulation of MAP kinase was unaffected by PKC downregulation or pharmacological PKC inhibition. These findings demonstrate for the first time that Ang II stimulation of MAP kinase may occur via a pathway independent of c-Raf-1 and of the phorbol-responsive PKC isozymes. The differing effects of Ang II and PDGF on VSMC growth may be a consequence of specific signal transduction events, as demonstrated here for activation of MAP kinase.

Key Words: angiotensin II • Raf • protein kinase C • mitogen-activated protein kinase • signal transduction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both Ang II and PDGF rapidly increase intracellular Ca²⁺ and activate PKC and MAP kinase in VSMCs. However, Ang II causes cell hypertrophy, whereas PDGF causes hyperplasia. Differences in signal transduction events activated by Ang II and PDGF may provide insight into their differing effects on VSMC growth. Ang II has been shown to be a powerful activator of MAP kinase in VSMCs in vivo¹ and in vitro.² It is well established that MAP kinase is activated in response to PDGF and other tyrosine kinase–coupled receptors by a signal transduction pathway dependent on Ras and Raf.³ It has previously been shown that Raf phosphorylation is increased in response to Ang II in mesangial cells.⁴ However, the activity of Raf and its role in Ang II–mediated activation of MAP kinase have not been delineated. Many of the effects of Ang II on gene expression, such as the induction of c-fos and c-myc, are mediated by PKC-dependent pathways that likely involve MAP kinase.⁵ PKC has been suggested to be both "upstream" and "downstream" from MAP kinase in signal transduction cascades.^{3 6} In addition, it has been suggested that Raf is potentially regulated by PKC.⁷ However, Ang II has been shown to stimulate many PKC-independent events in VSMCs as well. Because binding of Ang II to the Ang II type I receptor in VSMCs has been shown to stimulate PKC, Raf, and MAP kinase,⁸ this cell culture system is an excellent model for studying the roles of PKC and Raf in the regulation of MAP kinase.

In the present study, we compared the effects of Ang II and PDGF on Raf translocation and MAPKKK activity and characterized the role of PKC in the activation of MAPKKK. These findings were then correlated with the activation of MAP kinase. The results indicate that both Ang II and PDGF activate c-Raf-1, increase MAPKKK activity, and stimulate MAP kinase. Activation of c-Raf-1 is associated with membrane translocation and binding to H-Ras. Stimulation of H-Ras–associated MAPKKK activity by both PDGF and Ang II was predominantly PKC dependent. However, Ang II activated MAP kinase in a PKC-independent manner, whereas PDGF activated MAP kinase in a PKC-dependent manner. These findings demonstrate for the first time that Ang II stimulation of MAP kinase is via a pathway independent of c-Raf-1 and of phorbol-responsive PKC isozymes.

	Materials and Methods

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Cell Culture
VSMCs were isolated from 200- to 250-g male Sprague-Dawley rats and maintained in 10% calf serum/DMEM as described previously.⁹ VSMCs at passages 5 to 13 at 70% to 80% confluence in 100-mm dishes were growth-arrested by incubation in 0.1% calf serum/DMEM for 48 hours before use.

Western Blot Analysis
After treatment, the cells were washed with phosphate-buffered saline, and 0.5 mL TME lysis buffer (10 mmol/L Tris, pH 7.5, 5 mmol/L MgCl₂, 1 mmol/L EDTA, 25 mmol/L NaF, fresh 100 µmol/L Na₃VO₄, and 1 mmol/L dithiothreitol) was added.¹⁰ Cell lysates were prepared by freezing, thawing on ice, scraping, and Dounce homogenization (30 strokes). After centrifugation for 10 minutes at 1000g,⁴ the supernatant was centrifuged for 90 minutes at 100 000g (4°C). We will refer to the high-speed supernatant and pellet as cytosol and membrane fractions, respectively. Membrane proteins were solubilized with 1% Triton X-100, protein concentration was determined with the BioRad Bradford assay, and the samples were stored at -80°C. For Western blot analysis, 50 µg protein was subjected to SDS-PAGE under reducing conditions, and proteins were then transferred to nitrocellulose (Hybond-ECL, Amersham) as previously described.¹¹ The membrane was blocked for 2 hours at room temperature with a commercial blocking buffer from GIBCO. The blots were incubated for 1 hour at room temperature with the primary antibodies: anti-Raf and anti–c-mos antibodies from Santa Cruz Biotechnology, anti-MEKK and anti–H-Ras from Upstate Biotechnology Inc, and anti–Raf 5062 prepared against a 12–amino acid COOH terminal Raf peptide (raised by Dr G. Daum, University of Washington), followed by incubation for 1 hour with secondary antibody (horseradish peroxidase conjugated). Immunoreactive bands were visualized by ECL (Amersham International plc).

Immunoprecipitation and Immunoblot Analysis of Raf
VSMCs were lysed with HEB lysis buffer (25 mmol/L HEPES, pH 7.5, 5 mmol/L EGTA, 5 mmol/L EDTA, 150 mmol/L NaCl, 50 mmol/L NaF, 100 mmol/L Na₄P₂O₇, 10% glycerol, 1% Triton X-100, 1 mmol/L benzamidine, 1 mmol/L Na₃VO₄, 0.1% mercaptoethanol, 20 µg/mL leupeptin, 1 µg/mL pepstatin A, and 4 µg/mL aprotinin [50 kallikrein inhibiting units/mL]). The lysates were subjected to immunoprecipitation with anti–Raf 5062 antibody or anti–H-ras antibody. Immune complexes were recovered by the addition of protein A–agarose (GIBCO BRL), incubation overnight at 4°C, and centrifugation. The beads were washed once with HEB, twice with TTBS buffer (20 mmol/L Tris, pH 7.5, 500 mmol/L NaCl, 1% Triton X-100, and 0.1% ß-mercaptoethanol), and once with Raf buffer (50 mmol/L HEPES, pH 7.5, 0.1% Triton X-100, and 0.1% ß-mercaptoethanol). Immunoprecipitated proteins were then electrophoresed on a 9% SDS–polyacrylamide gel and transferred to nitrocellulose, and proteins were identified by ECL.

Raf Assays
Raf activity was assayed by MAPKK-dependent phosphorylation of recombinant catalytically inactive 42-kD MAP kinase as previously described.¹² In brief, cell lysates were prepared in HEB buffer, which was centrifuged at 1000g for 5 minutes. The supernatant was immunoprecipitated with anti–c-Raf-1 or anti–H-Ras antibodies, and the immunoprecipitates were washed twice with TTBS and once with Raf buffer. The kinase assay was performed in 30 µL containing 10 µL 3x Raf buffer, 10 µL histidine-42 kD MAP kinase (400 ng per assay),¹³ 5 µL glutathione-S-transferase (GST)-MEK (200 ng per assay), and 5 µL Mg²⁺-ATP (60 mmol/L MgCl₂, 0.6 mmol/L ATP, and 5 µCi per assay) for 30 minutes at room temperature. The assay was stopped by addition of 4x Laemmli buffer, and proteins were separated on 10% SDS-PAGE. Raf activity was measured by densitometry of 42-kD MAP kinase phosphorylation on autoradiograms (in the linear range of film exposure) using NIH Image 1.59 software.

MAP Kinase Assays
A myelin basic protein in-gel kinase assay to measure MAP kinase phosphotransferase activity was performed exactly as previously described.¹¹ MAP kinase activity was measured by densitometry of autoradiograms (in the linear range of film exposure) using NIH Image 1.59.

Materials
All materials were from Sigma Chemical Co except where indicated. Recombinant PDGF-BB was from Collaborative Research, and GF-109203 was from LC Service Inc.

Statistical Analysis
All experiments were performed at least three times, and data are presented as mean±SE. Significant differences were determined by Student's t test (P<.05).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MAP Kinase Activation by Ang II and PDGF Follows a Similar Time Course
The goal of the present study was to determine the role of Raf in the activation of MAP kinase by Ang II and PDGF. To begin this analysis, we determined the time course for MAP kinase activation by 100 nmol/L Ang II and 10 ng/mL PDGF. As shown in Fig 1, both Ang II and PDGF rapidly activated MAP kinase, with onset at 2 minutes, peak at 5 minutes, and sustained activation for 120 minutes.

View larger version (38K): [in this window] [in a new window]   Figure 1. Time course for MAP kinase activation by Ang II and PDGF in VSMCs. Cells were exposed to 100 nmol/L Ang II and 10 ng/mL PDGF for the indicated times, and lysates were prepared. In-gel kinase analysis was then performed using 10 µg cell protein per lane. Arrows indicate the position of the 42-kD and 44-kD bands identified as MAP kinases. Results are representative of four experiments.

Ang II and PDGF Stimulate Rapid Increases in MAPKKK Activity
To correlate Raf activity with activation of MAP kinase, we measured Raf activity in c-Raf-1 immunoprecipitates from VSMCs stimulated for varying times with Ang II and PDGF. The Raf activity assay is based on recombinant MAPKK-dependent phosphorylation of catalytically inactive 42-kD MAP kinase. Both Ang II and PDGF stimulated rapid increases in presumptive Raf activity measured in c-Raf-1 immunoprecipitates with peaks between 2 and 5 minutes for Ang II and PDGF (Fig 2A, top blot). There was a return to baseline by 20 minutes.

View larger version (31K): [in this window] [in a new window]   Figure 2. Time course for Raf and MAPKKK activation in VSMCs. A, Cells were exposed to 100 nmol/L Ang II and 10 ng/mL PDGF for the indicated times and harvested. c-Raf-1 was then immunoprecipitated from whole-cell lysates with anti–Raf-5062 antibody (top blot) or H-Ras was immunoprecipitated with anti–H-Ras antibody (bottom blot). Equal amounts of protein were then used to assay MAPKKK activity by MAPKK-dependent phosphorylation of 42-kD MAP kinase. Results are representative of five experiments. IP indicates immunoprecipitation. B, The relative MAPKKK activity in the Ras immunoprecipitates was measured by densitometry of autoradiograms in the linear range of film development. The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. The time-dependent response to Ang II and PDGF was then determined on a relative basis. Results are the mean±SE of three to five determinations.

Activation of Raf has been shown to require interaction with membrane-bound Ras.¹⁴ Thus, activated Raf should coprecipitate with Ras from agonist-treated cells. Therefore, we measured the ability of proteins that immunoprecipitated with H-Ras to activate MAPKK and phosphorylate 42-kD MAP kinase. We refer to this kinase activity as MAPKKK activity because we believe that Raf is responsible for only a portion of this activity, as discussed below. Peak MAPKKK activity in H-Ras immunoprecipitates occurred at 5 minutes with Ang II and at 2 to 5 minutes with PDGF (Fig 2A, bottom blot), similar to results in c-Raf-1 immunoprecipitates. MAPKKK activity in H-Ras immunoprecipitates was of greater magnitude than in c-Raf-1 immunoprecipitates, so subsequent experiments were performed using H-Ras immunoprecipitates. c-Raf-1 protein levels were the same in both c-Raf-1 and H-Ras immunoprecipitates as shown below (Fig 3C and 3D), so this difference was not due to differences in precipitation. Analysis of five experiments (Fig 2B) indicated that peak MAPKKK activity (3.5-fold increase) occurred at 5 minutes in response to both Ang II and PDGF.

View larger version (47K): [in this window] [in a new window]   Figure 3. Time course for Raf translocation in VSMCs. Cells were exposed to 100 nmol/L Ang II and 10 ng/mL PDGF for the indicated times, and cell membrane and cytosol fractions were prepared. A, Western blot analysis was performed with anti–c-Raf-1 antibody on cell membranes. Results are representative of three experiments. B, The relative amounts of c-Raf-1 in the membrane fractions were measured by densitometry in the linear range of film development. The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. The time-dependent response to Ang II and PDGF was then determined on a relative basis. Results are the mean±SE of three or four experiments. C, Cells were treated with 10 ng/mL PDGF and 100 nmol/L Ang II for 5 minutes, and cell lysates were prepared. H-Ras was immunoprecipitated, and Western blot analysis was performed with anti–c-Raf-1 antibody. Preimmune serum (PIS) was substituted for H-Ras antibody. IP indicates immunoprecipitation; IB, immunoblot. D, Cells were treated with 10 ng/mL PDGF and 100 nmol/L Ang II for 5 minutes, and cell lysates were prepared. c-Raf-1 was immunoprecipitated, and Western blot analysis was performed with anti–c-Raf-1 antibody. PIS was substituted for c-Raf-1 antibody. E, Lysates from cells treated for 5 minutes with vehicle only (control), 100 nmol/L Ang II, or 10 ng/mL PDGF were size-fractionated by SDS-PAGE, and Western blot analysis was performed with anti–c-Raf-1 antibody. F, Lysates from control cells were immunoprecipitated with anti–H-Ras antibody or PIS, and Western blot analysis was performed with anti–H-Ras antibody. G, The distribution of H-Ras in membrane and cytosol fractions was determined by Western blot analysis. Cells were treated with 10 ng/mL PDGF and 100 nmol/L Ang II for 5 minutes or exposed to 1 µmol/L PDBU for 24 hours before treatment with Ang II. Results are representative of three experiments.

Raf Translocates to the Membrane and Associates With Ras in Response to Ang II and PDGF
We next studied the time course for c-Raf-1 membrane translocation and association with H-Ras, because Raf translocation has been correlated with Raf activity.¹⁴ Growth-arrested VSMCs were stimulated with Ang II and PDGF, cell lysates were fractionated into membrane and cytosol, and c-Raf-1 was identified by Western blot. Both Ang II and PDGF stimulated increases in immunoreactive c-Raf-1 in the membrane fraction (Fig 3A). The increase in response to Ang II peaked at 2 to 5 minutes and returned to near baseline at 20 minutes (Fig 3A). In response to PDGF, Raf translocation peaked at 5 minutes and remained elevated for at least 20 minutes (Fig 3A). Retardation of Raf electrophoretic mobility ("band shift") compared with control was present (although to a small extent) in all agonist-stimulated membrane fractions (Fig 3A). The retardation of Raf mobility has been previously reported to correlate with increased phosphorylation of Raf.¹⁵ Thus, increases in Raf membrane association and phosphorylation occurred within 2 minutes in response to both Ang II and PDGF. There was a corresponding decrease in cytosolic c-Raf-1 (not shown). The results of several experiments are summarized in Fig 3B and show that peak Raf translocation occurred at 2 minutes in response to Ang II and at 5 minutes in response to PDGF. Thus, the time course for MAPKKK activity (Fig 2B) correlates well with Raf translocation (Fig 3B).

To demonstrate that translocated Raf associated with Ras, we repeated the Raf translocation experiment using anti–H-Ras antibody to coimmunoprecipitate Raf. Ang II and PDGF stimulated increases in immunoreactive Raf associated with H-Ras (Fig 3C). Analysis of three such experiments showed that Ang II and PDGF stimulated approximately fourfold increases in c-Raf-1 associated with H-Ras. Of importance, the time for peak Raf association with H-Ras was at 2 minutes, earlier than the time for peak MAP kinase activation, suggesting that Raf activation precedes MAP kinase activation (data not shown). The amount of c-Raf-1 that coprecipitated with H-Ras was similar to that immunoprecipitated by c-Raf-1 antibodies as assayed by Western blot under identical conditions (Fig 3D). Although Western blot analysis is semiquantitative, there appeared to be increases in c-Raf-1 in H-Ras immunoprecipitates (compare panels C and D of Fig 3) and in immunoprecipitates from cells stimulated with Ang II and PDGF (compare lanes 2 and 3 versus lane 1 in Fig 3D). The apparent increase in c-Raf-1 after Ang II and PDGF stimulation is likely due to enhanced recognition of phosphorylated c-Raf-1 by the antibody. However, this is a property of the antibody only when used to immunoprecipitate c-Raf-1. When used for Western blot analysis on total cell lysates (Fig 3E), the anti–c-Raf-1 antibody recognized equally both unphosphorylated (lower band) and phosphorylated (upper band) forms of c-Raf-1 from control and stimulated cells.

The specificity of the H-Ras antibody was determined and showed that the dominant protein immunoprecipitated and recognized on Western blot was 21-kD Ras (Fig 3F). Preimmune serum failed to immunoprecipitate either H-Ras (Fig 3F) or c-Raf-1 (Fig 3C). To verify that immunoreactive Ras was found only in the membrane fraction, cell lysates were fractionated into membrane and cytosol, and H-Ras was identified by Western blot (Fig 3G). Immunoreactive H-Ras was present as a single band of 21 kD only in the membrane fractions. As a control for subsequent experiments, there was no change in H-Ras immunoreactivity in cells pretreated with 1 µmol/L PDBU for 24 hours to downregulate PKC. In summary, the increase in c-Raf-1 shown by Western blots of H-Ras immunoprecipitates (Fig 3C) demonstrates that c-Raf-1 translocation to the membrane had occurred in response to Ang II and PDGF because H-Ras is found only in the membrane (Fig 3G).

c-Raf-1 Is the Predominant Raf Isozyme in VSMCs and Associates With H-Ras
To determine the extent to which MAPKKKs other than c-Raf-1 were present in VSMCs and coprecipitated with H-Ras, Western blot analysis was performed with antibodies to Raf-A, Raf-B, c-mos, and MEKK. Growth-arrested VSMCs were prepared, and total cell lysates were analyzed for expression of Raf isozymes. The predominant Raf isoform in VSMC lysates was c-Raf-1, although small amounts of Raf-B were also present (Fig 4A). No Raf-A was present (Fig 4A). We next determined the association of c-Raf-1 and Raf-B with H-Ras (Fig 4B). c-Raf-1 showed an increase in immunoreactive protein associated with H-Ras in response to both Ang II and PDGF. However, there was no Raf-B associated with H-Ras (Fig 4B). Small amounts of c-mos and MEKK immunoreactive proteins were present in VSMC lysates (data not shown), but neither c-mos nor MEKK was detected in H-Ras immunoprecipitates (data not shown). However, differences in antibody affinity and the effects of posttranslational modifications such as phosphorylation make these results qualitative. Thus, the only MAPKKK that we could identify in association with H-Ras in VSMCs after stimulation by Ang II and PDGF was c-Raf-1.

View larger version (43K): [in this window] [in a new window]   Figure 4. Only c-Raf-1 associates with H-Ras in VSMCs. A, Growth-arrested VSMCs were harvested, and Western blot analysis was performed on whole-cell lysates using Raf isoform–specific antibodies. Care was taken to ensure equal loading of cell protein, antibody dilutions, and ECL exposure. B, Cells were exposed to 100 nmol/L Ang II and 10 ng/mL PDGF for 5 minutes and harvested. H-Ras was then immunoprecipitated from whole-cell lysates, and the immunoprecipitated proteins were subjected to SDS-PAGE and transferred. Western blot analysis was performed with anti–c-Raf-1 and anti–Raf-B antibodies. IP indicates immunoprecipitation; IB, immunoblot.

Ang II and PDGF Stimulation of MAPKKK Activity Are PKC Dependent in VSMCs
PKC has been reported to be an upstream activator of Raf in several cell types.⁶ Both Ang II and PDGF have previously been demonstrated to activate PKC in VSMCs, and Ang II–mediated and PDGF-mediated events, such as c-fos mRNA induction, are PKC dependent.¹⁶ To investigate the role of PKC in Raf activation, PKC was downregulated by incubation with PDBU (1 µmol/L for 24 hours). Previous studies have shown that PKC downregulation blocks Ang II–mediated induction of proto-oncogene expression,¹⁶ MAP kinase phosphatase-1 expression,⁹ and myristoylated alanine–rich-C-kinase substrate phosphorylation¹⁷ and partially inhibits activation of the Na⁺-H⁺ exchanger.¹⁸ PKC downregulation significantly inhibited MAPKKK activity associated with H-Ras after stimulation by 200 nmol/L PMA, Ang II, and PDGF (Fig 5A). Analysis of five experiments indicated that PKC downregulation caused 81%, 80%, and 82% inhibition of H-Ras–associated MAPKKK activity (at 5 minutes) stimulated by PMA, Ang II, and PDGF, respectively (Fig 5B).

View larger version (36K): [in this window] [in a new window]   Figure 5. PKC downregulation inhibits MAPKKK activity in VSMCs. A, Growth-arrested VSMCs were exposed to 1 µmol/L PDBU for 24 hours (+) to downregulate PKC. Cells were then exposed to 200 nmol/L PMA and 10 ng/mL PDGF for 5 minutes and to 100 nmol/L Ang II for the indicated times. Cell lysates were immunoprecipitated with anti–H-Ras antibody. Equal amounts of protein were then used to assay MAPKKK activity by MAPKK-dependent phosphorylation of 42-kD MAP kinase. Results are representative of five experiments. B, The relative MAPKKK activity in the Ras immunoprecipitates was measured by densitometry of autoradiograms in the linear range of film development. The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. The response to Ang II, PDGF, and PMA at 5 minutes was then determined on a relative basis. Results are the mean±SEM of five experiments. DMSO indicates dimethyl sulfoxide. C, Growth-arrested VSMCs were exposed to 100 nmol/L Ang II and 10 ng/mL PDGF for 5 minutes. PKC was downregulated by exposure to 1 µmol/L PDBU for 24 hours. c-Raf-1 translocation was assessed by H-Ras immunoprecipitation, followed by Western blot analysis with c-Raf-1 antibody.

The effect of PKC downregulation on c-Raf-1 translocation and association with H-Ras was also determined. PDBU treatment completely prevented the association of c-Raf-1 with H-Ras in cells stimulated by Ang II and PDGF (Fig 5C). These findings suggest that the inhibition of MAPKKK activity by PDBU was due in part to inhibition of c-Raf-1 membrane translocation and activation.

Ang II Stimulation of MAP Kinase Activity Is PKC Independent but PDGF Stimulation Is PKC Dependent in VSMCs
To determine whether PKC downregulation also inhibited MAP kinase activation, the same VSMC cell lysates prepared in Fig 5 were used to measure MAP kinase activity by an in-gel kinase assay. PMA, PDGF, and Ang II stimulated fourfold to sevenfold increases in MAP kinase activity at 5 minutes (Fig 6A, top, lanes 2 to 4). In PKC-downregulated VSMCs, there was nearly complete inhibition of PMA- and PDGF-stimulated MAP kinase activity (Fig 6A, compare lanes 2 and 3 with lanes 6 and 7). In contrast, there was only a minimal decrease in Ang II–stimulated MAP kinase activity (Fig 6A, compare lanes 4 and 8). Analysis of multiple experiments indicated that there was no significant inhibition of Ang II–stimulated MAP kinase activity in PKC-downregulated cells (Fig 6C, P>.05, n=11), whereas both PMA and PDGF were inhibited by >90% (Fig 6C, P<.001, n=5 and 7).

View larger version (33K): [in this window] [in a new window]   Figure 6. PKC inhibition blocks PDGF- and PMA-stimulated, but not Ang II–stimulated, MAP kinase activity in VSMCs. A, PKC was downregulated by exposure to 1 µmol/L PDBU for 24 hours (+). B, PKC was inhibited by treatment with 1 µmol/L GF-109203 for 10 minutes (+) before agonist exposure. Cells were then treated with 200 nmol/L PMA, 10 µg/mL PDGF, or 200 nmol/L Ang II for 5 minutes, and lysates were prepared. In-gel kinase analysis was then performed with 10 µg cell protein per lane. Arrows indicate the position of the 42-kD and 44-kD bands identified as MAP kinase. Results are representative of four experiments. C, The relative MAP kinase activity determined by in-gel kinase assay was measured by densitometry of autoradiograms in the linear range of film development. The density of 42-kD and 44-kD MAP kinases were measured individually and together. There were no significant differences using either method, and because of better visibility, the 42-kD MAP kinase density was analyzed. The results for each experiment were normalized to the density of the control sample, which was arbitrarily adjusted to 1.0. The responses to Ang II, PDGF, and PMA were then determined on a relative basis. Results are the mean±SEM of four determinations. *P<.01 vs 0.1% dimethyl sulfoxide (DMSO).

To investigate the role of PKC further, the specific PKC inhibitor GF-109203 was tested. This inhibitor has been reported to block agonist-mediated activation of Ca²⁺- and phospholipid-dependent PKC isozymes.^{19 20} Pretreatment with GF-109203 (1 µmol/L for 10 minutes) completely inhibited MAP kinase activation by PMA and PDGF (Fig 6B, compare lanes 2 and 3 with lanes 6 and 7) but had minimal effect on Ang II–mediated MAP kinase activity (Fig 6B, compare lanes 4 and 8). Analysis of multiple experiments indicated that there was no significant inhibition of Ang II–stimulated MAP kinase activity in cells treated with GF-109203 (Fig 6C, P>.05, n=4), whereas both PMA and PDGF were inhibited by >90% (Fig 6C, P<.001, n=4). These results indicate that stimulation of MAP kinase activity by PDGF and PMA is dependent on phorbol ester–binding PKC isozymes that are regulated by Ca²⁺ and phospholipid. In contrast, activation of MAP kinase by Ang II was independent of these PKC isozymes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of the present study is that Ang II and PDGF differ in the signal transduction pathways that lead to MAP kinase activation in VSMCs. Whereas both Ang II and PDGF increase MAPKKK activity, cause c-Raf-1 translocation, and stimulate MAP kinase in VSMCs, Ang II stimulation of MAP kinase occurs via a c-Raf-1–independent pathway. The presence of a c-Raf-1–independent pathway is supported by findings that on one hand Ang II stimulation of MAP kinase was not altered when PKC was inhibited pharmacologically (GF-109203) or by downregulation (PDBU for 24 hours). On the other hand, inhibition of PKC blocked c-Raf-1–mediated translocation and association with Ras. Thus, under conditions in which no Raf associated with the membrane, PDGF-mediated MAP kinase activity was inhibited, whereas Ang II–mediated MAP kinase was unaffected, defining a separate Ang II–activated pathway.

The present findings extend previous studies that reported Ang II stimulation of Raf.^{4 15 21} It should be noted that these studies showed Raf activation indirectly by measurement of Raf hyperphosphorylation and a band shift that occurred 10 to 20 minutes after Ang II stimulation. The fact that Raf phosphorylation peaked 10 to 20 minutes after Ang II stimulation in these studies made its role in the activation of MAP kinase, which peaks at 5 minutes,² dubious. However, these previous studies failed to investigate the association of Raf with H-Ras, translocation of Raf to the plasma membrane, or MAPKKK activity. Our results are the first to show that Ang II stimulates Raf translocation, Raf association with H-Ras, and Raf activity (as measured by MAPKKK activity), within 2 minutes. These data suggest that Raf plays an important role in the early signal events stimulated by Ang II.

However, two findings reported in the present study suggest that c-Raf-1 is not the predominant Ang II–stimulated MAPKKK in VSMCs. First, Ang II–mediated MAP kinase activation was PKC independent, as there was no significant inhibition of MAP kinase in PDBU-treated cells. However, Ang II stimulation of MAPKKK activity and c-Raf-1 translocation were predominantly PKC dependent, as shown by 80% inhibition in PDBU-treated cells. Thus, in the same cells in which MAPKKK activity associated with H-Ras was decreased by 80%, there was still activation of MAP kinase. These findings suggest that the MAPKKK stimulated by Ang II, which is responsible for activating MAP kinase, is not c-Raf-1. We could not demonstrate association of potential MAPKKKs, such as MEKK, c-mos, or B-Raf, with H-Ras, so future work will be required to identify other H-Ras–associated kinases with MAPKKK activity. Several other groups have reported examples in which MAP kinase activation occurs by Raf-dependent and Raf-independent pathways, as demonstrated by differential effects of agonists or antagonists (especially cAMP).^{22 23 24 25} Second, in contrast to Ang II, PDGF-mediated MAP kinase activity and MAPKKK activity were both completely inhibited by PDBU treatment. Thus, the PDGF-stimulated MAPKKK responsible for MAP kinase activation associates with H-Ras and is regulated by PKC, findings consistent with c-Raf-1 being the PDGF-stimulated MAPKKK.

Two caveats should be raised regarding the interpretation of experiments involving Ang II–stimulated and PDGF-stimulated MAPKKK activity in Ras immunoprecipitates. First, the magnitude of MAPKKK activity was significantly greater in Ras immunoprecipitates than in Raf immunoprecipitates. This difference could be due to several factors, including stabilization of Raf protein by Ras, increased levels of c-Raf-1 in the Ras immunoprecipitates, maintenance of Raf in a more active state by Ras, inhibition of Raf activity by the anti–c-Raf-1 antibody, or coprecipitation of another MAPKKK in the Ras immunoprecipitates. Second, determining MAPKKK activity by MAPKK-dependent phosphorylation of catalytically inactive MAP kinase will not measure all potential MAPKKK proteins present in the immunoprecipitates. In the experiments performed in the present study, either c-Raf-1 or H-Ras was first immunoprecipitated, and the coprecipitated proteins were assessed for activity under conditions that were optimized to detect Raf activity. However, kinases that are dependent on Ca²⁺, lipids, fatty acids, or other cofactors would not be expected to have significant kinase activity under the conditions used here. This may be important in VSMCs, where we have detected PKC in Ras immunoprecipitates (D.F. Liao, unpublished data, 1996). PKC is a Ca²⁺-independent and phorbol ester–nonresponsive enzyme that has recently been reported to have Raf-like qualities.^{26 27} In contrast, potential MAPKKK proteins other than Raf would be fully active in the intact cell. Finally, there may be MAPKK-independent mechanisms by which Ang II regulates MAP kinase activity, including inhibition of MAP kinase phosphatases¹¹ and/or inactivation of MAP kinase inhibitors.²⁸ We have shown that MAP kinase phosphatase-1 is the predominant mechanism that inactivates MAP kinase in response to Ang II,¹¹ and there is no difference between Ang II and PDGF with respect to this phosphatase (authors' unpublished data, 1996). However, alterations in MAP kinase inhibitors remain to be studied.

The finding that Raf translocation by Ang II and PDGF is PKC dependent in VSMCs provides insight into the nature of the PKC isozyme(s) that may be responsible for regulation of Raf. VSMCs are known to express the following PKC isozymes: , ß, , , and .²⁹ Among these isozymes, those that bind phorbol esters and hence are likely to be downregulated by PDBU include , ß, , and . These PKC isozymes are therefore likely candidates to mediate the PKC-dependent activation of MAP kinase stimulated by PDGF and PMA.

In summary, the present studies support the concept that early signal transduction events activated by the Ang II type I receptor resemble those events activated by tyrosine kinase–coupled receptors, such as the PDGF receptor. These events now include activation of phospholipase C-,²⁹ Janus kinase and TYK kinases,³⁰ src kinase,³¹ H-Ras,³² Raf,²¹ and MAP kinase.² As suggested by van Biesen et al,³³ pathways shared by G protein–coupled receptors and tyrosine kinase receptors are likely a consequence of ß activation of an upstream tyrosine kinase. This kinase then phosphorylates Shc and leads to activation of Ras. Ang II–mediated phosphorylation of Shc in VSMCs has recently been reported, lending support to this concept.³⁴ In the present study, we have defined another shared signal pathway, which is the PKC-dependent association of c-Raf-1 with H-Ras and activation of c-Raf-1. However, a novel finding of the present study is that although PDGF-mediated MAP kinase activation appears to be PKC dependent, Ang II–mediated MAP kinase activation is PKC independent (defined by PKC downregulation with PDBU). Our findings are supported by a recent report from Inagami's lab,³⁵ which also showed that Ang II–mediated MAPK activation was not inhibited by GF-109203 and was only partially decreased by phorbol ester pretreatment. In addition, these investigators suggested that a Ca²⁺-calmodulin–dependent tyrosine kinase was required for Ang II–mediated MAP kinase activation. The identity of this tyrosine kinase remains to be determined. A new candidate to mediate Raf-independent (and possibly PKC-independent) activation of the MAP kinase pathway is kinase suppressor of Ras (KSR-1).³⁶ Although there are few data regarding signal transduction by KSR-1 in mammalian cells, it has been proposed to act on a pathway parallel to Ras or on a pathway parallel to Raf. Such functions would explain the results we have observed. Alternatively, PKC has been reported to act in a fashion similar to Raf and activate MAP kinase–mediated signal transduction.^{27 37} The differing effects of Ang II and PDGF on VSMC growth may be a consequence of specific signal transduction events that have been demonstrated here for activation of MAP kinase. Future studies will be necessary to define the nature of this potentially novel signal transduction pathway.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ECL = enhanced chemiluminescence GST = glutathione-S-transferase MAP = mitogen-activated protein MAPKK = MAP kinase kinase MAPKKK = MAP kinase kinase kinase MEK = MAP kinase/extracellular signal-regulated kinase MEKK = MEK kinase PDBU = phorbol 12,13-dibutyrate PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by National Institutes of Health grants R01 HL-44721 to Dr Berk. Dr Berk is an Established Investigator of the American Heart Association. We thank Arnold Baas, Masatoshi Kusuhara, and members of the Berk laboratory for helpful discussions.

	Footnotes

 
This manuscript was sent to Robert J. Lefkowitz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received March 27, 1996; accepted August 12, 1996.

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