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Molecular and Cellular Biology, June 2002, p. 3717-3728, Vol. 22, No. 11
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.11.3717-3728.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cyclic AMP Blocks Cell Growth through Raf-1-Dependent and Raf-1-Independent Mechanisms

Nicolas Dumaz, Yvonne Light, and Richard Marais*

Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, London SW3 6JB, United Kingdom

Received 1 November 2001/ Returned for modification 13 December 2001/ Accepted 2 March 2002


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ABSTRACT
 
It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.


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INTRODUCTION
 
A number of highly conserved intracellular signaling pathways have been described that control the responses of cells to extracellular signals. One such pathway is the Raf/extracellular signal-regulated protein kinase (ERK) signaling pathway, which controls complex cellular functions such as growth, survival, and differentiation (30, 36). The ERKs are highly conserved mitogen-activated protein (MAP) kinases that form part of a three-tiered protein kinase cascade involving the MAP kinase kinases MEK1 and MEK2 and the Raf family of MAP kinase kinase kinases (for reviews, see references 3, 15, and 22). The Rafs phosphorylate and activate the MEKs, which in turn phosphorylate and activate the ERKS. In mammals, there are three Raf genes (Raf-1, A-Raf, and B-Raf), and the proteins they express are cytosolic in resting cells. Raf proteins activation is initiated when they are recruited to the plasma membrane by Ras family small G proteins. Raf-1 is the most extensively studied isotype, and its activation is complex, requiring phosphorylation, interactions with other proteins, interactions with lipids, and possibly dimerization. Negative regulation of Raf-1 occurs though inhibitory phosphorylation events, through dephosphorylation of essential sites, and through interactions with other proteins (see references 3 and 22).

Another highly conserved signaling pathway that controls complex cellular responses is based on the second messenger cyclic AMP (cAMP) (for a review, see reference 19). Extracellular signals stimulate membrane-associated adenylate cyclases to convert ATP into cAMP, which regulates a large number and variety of cell-type-specific responses. A major class of cAMP targets is the cAMP-dependent protein kinase or protein kinase A (PKA) family. These heterotetrameric protein kinases, consisting of two kinase domains bound to two regulatory domains, are activated when cAMP binds to the regulatory domains and releases the kinase domains. A second family of cAMP targets, the cAMP-activated GTPase exchange factors, have recently been described. These proteins activate small G proteins of the Rap family, but their role in cell signaling is still unclear (see reference 4). cAMP signaling is terminated by phosphodiesterases, which degrade cAMP to AMP. The complexity of this pathway is underscored by the observation that there are at least 10 adenylate cyclases, as many as 30 phosphodiesterases, multiple PKA catalytic and regulatory domains, and at least two cAMP-activated GTPase exchange factors (see references 4 and 19).

It is well known that cAMP can suppress the growth of some cells, whereas it stimulates the growth of others (see reference 18). It has also been shown that there is significant cross talk between the cAMP and ERK signaling pathways. This cross talk appears to be cell type specific, since cAMP stimulates ERK in some cells but suppresses it in others. Even within the same cell type, confusion can occur. In some PC12 cell clones, cAMP appears to activate B-Raf in a Rap-dependent manner, whereas in other PC12 clones, this does not occur (see reference 4). It has also been shown that overexpressed Rap can sequester Raf-1 in a cAMP-dependent manner, thus preventing its activation by Ras, but endogenous Raps appear unable to mediate this antagonism (see reference 4). cAMP also counteracts some of the biological effects of oncogenes that activate ERKs. The cell-permeative cAMP analogue, 8-chloro-cAMP, can inhibit growth of cells transformed by Ki-Ras (41), and PKA activation reverses the transformation phenotype of v-Raf- and v-Abl-transformed cells (14, 42). Finally, 8-chloro-cAMP infusion induced regression of the LX-1 lung carcinoma in athymic mice in a dose-dependent manner (2).

cAMP also blocks Raf-1 activation in a number of cell types (6, 8, 14, 38, 43). Agents that elevate cellular cAMP stimulate Raf-1 phosphorylation on serine 43 (S43), and PKA can phosphorylate Raf-1 on S43 phosphorylation in vitro. S43 phosphorylation reduces binding of Raf-1 to Ras-GTP in vitro (43), and overexpression of the PKA kinase domain suppresses Raf-1 activity in vivo (34). A widely accepted model is that PKA phosphorylates S43 of Raf-1 and disrupts its binding to Ras-GTP, thereby suppressing ERK activation and cell growth (see references 3, 18, and 22). However, some data suggest this model is incomplete. For example, substitution of S43 for alanine does not prevent elevated cAMP from suppressing Raf-1 activity in vivo in some cells (39), and in CCL39 cells, cAMP appears to block cell growth without suppressing ERK activation (32). It has also been suggested that PKA can suppress Raf-1 by phosphorylating serine 621 (S621) (34). However, this may be an in vitro artifact because elevated cAMP does not stimulate S621 phosphorylation or suppress the activity of the isolated catalytic domain in vivo (39).

Here we describe a systematic analysis of the mechanism by which cAMP suppresses Raf-1 activation and the role played by Raf-1/ERK signaling in mediating cell growth suppression when cytosolic cAMP is elevated. We demonstrate that agents that elevate cAMP block Raf-1 activity but that the mechanism is considerably more complex than previously reported. Three sites on Raf-1 are phosphorylated. These are S43, serine 259 (S259), which has not been implicated in cAMP-mediated suppression of activity, and a previously unidentified site, serine 233 (S233). All three phosphorylation sites function independently, and cAMP inhibits Raf-1 by interfering with both Ras-dependent and Ras-independent mechanisms. In cells expressing Raf-1 with all three amino acids substituted for alanine to prevent their phosphorylation, Raf-1 and ERK activities were fully restored but cell growth was still blocked by elevated cAMP. Thus, although Raf-1 appears to be an important cAMP target, other pathways that regulate cell growth are also targeted.


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MATERIALS AND METHODS
 
Expression vectors. The mRaf-1 expression vector (pEFm/Raf-1) has been described (7). mL89Raf-1CAAX and m{Delta}RafCAAX have been described (27). Raf-1 point mutants were generated by the PCR (17) using standard techniques (37) and verifying vector sequences by automated dideoxy sequencing. For stable cell lines, mRaf-1 or its derivatives were cloned into the vector pMCEF-, which incorporates a Neor gene to allow selection of stable colonies in G418 (29).

Cell culture and biochemical techniques. COS and NIH 3T3 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum or 5% donor calf serum, respectively. Both lines were transfected with lipofectamine in Optimem I reduced serum medium according to the manufacturer's instructions (Gibco-BRL Life Technologies Inc). COS and NIH 3T3 cells were serum starved in 0% fetal calf serum or 0.5% donor calf serum, respectively. Cells were treated with epidermal growth factor (EGF) (10 ng/ml) or platelet-derived growth factor (PDGF) (50 ng/ml) for the times indicated. Forskolin and isobutylmethylxanthine (IBMX) (Calbiochem) were dissolved in dimethylsulfoxide. 8-Bromo-cAMP (no. 203800; Calbiochem) was dissolved in water. For stable cell lines, NIH 3T3 cells were cultured in medium containing 1 mg of G418/ml, and individual clones were selected.

Cell extracts and Raf-1 kinase assays were handled as described previously (7, 28), using the monoclonal antibody M40091. G (Anogen, Mississauga, Canada) or the polyclonal antibody C20 (Santa Cruz Inc., Santa Cruz, Calif.) to immunoprecipitate endogenous Raf-1, or 9E10 to immunoprecipitate myc-tagged Raf-1 proteins. Ras activation assays and Ras-Raf-1 coimmunoprecipitation analysis were performed as described previously (26), except that Raf-1 was revealed using the antibody M40091G. ERK kinase assays were carried out as described previously (20). Briefly, ERK was immunoprecipitated with antibody 122, and [{gamma}-32P]ATP and myelin basic protein (MBP) were used as substrates. The samples were resolved on sodium dodecyl sulfate (SDS) gels, and [32P]orthophosphate incorporation into MBP was determined by a phosphorimager.

The following antibodies were used: phospho-ERK (clone MAPK-YT; M8159; Sigma), ERK2 (polyclonal serum 122 [24]), Raf-1 (R19120; Transduction Laboratories) (R5773; Sigma) (M40091.G; Anogen), Raf-1 phosphorylated on serine 259 (94215; New England Biolabs), Raf-1 phosphorylated on serine 338 (31), Ras (PanRas: R02120; Transduction Laboratories; Y13-238 [13]).

Generation of pS43-specific antibodies. The following peptides were synthesized by standard techniques (single-amino-acid code, where pS is phosphorylated serine): Raf39-47, QRRASDDGK; pRaf39-47, QRRApSDDGK; pRaf617-625, NRSApSEPSL.

The peptide pRaf39-47 was coupled to keyhole limpet hemocyanin (374817; Calbiochem) with glutaraldehyde (G5882; Sigma), and antibodies were raised in rabbits following standard protocols (16). The antisera were purified by affinity chromatography on pRaf39-47 coupled to affi-Gel10/15 (1536099/1536051; Bio-Rad). For competition analysis, the purified antibodies were preincubated with peptides (10 µM, 2 h at room temperature).

DNA synthesis analysis. NIH 3T3 cells were serum starved (48 h) and treated with growth factors. Where appropriate, they were pretreated (10 min) with forskolin and IBMX. Eight hours after treatment with growth factors, the cells were incubated with [3H]thymidine (0.4 µCi/ml, 24 h), washed in phosphate-buffered saline, fixed in 5% (wt/vol) trichloroacetic acid, and solubilized in 0.1 M NaOH-1% (wt/vol) SDS. The soluble fraction was added to 6 ml of scintillation fluid, and the samples were counted.

Phosphopeptide mapping and phosphoamino acid analysis. COS cells were transfected with expression plasmid as indicated. Approximately 32 h after the transfection, the cells were incubated in 1 ml of phosphate-free medium without serum and 1 mCi of [32P]orthophosphate (PBS-11; Amersham). The cells were incubated for a further 12 h and treated for 10 min with forskolin and IBMX, and protein was extracted as described above. The myc-tagged proteins were immunoprecipitated with 9E10, resolved in SDS-7% acrylamide gels, and transferred to a polyvinylidene difluoride (Millipore) membrane for analysis by two-dimensional thin-layer chromatography as described previously (5). The phosphorylated Raf-1 bands were excised and incubated with chymotrypsin (103314; Boehringer Mannheim) (10 µg, 37°C, overnight, followed by a second digestion of 10 µg at 37°C for 2 h). The released peptides were loaded onto crystalline cellulose thin-layer plates (1.005577; Merck), and the peptides were subjected to electrophoresis at pH 1.9 (45 min, 1,000 V) followed by ascending chromatography in phospho-chromatography buffer (5). For phosphoamino acid analysis, the phosphorylated peptides were eluted from the plates in pH 1.9 buffer and, following hydrolysis, were subjected to electrophoresis in the same crystalline cellulose plates at pH 1.9 (45 min, 1,000 V) to separate phosphoserine from phosphothreonine and phosphotyrosine (5).

Phosphorylation of peptides by PKA. The following peptides were used (single-amino-acid code where pS is phosphorylated serine): Raf228-238, SQHRYSTPHAF; pRaf228-238, SQHRYpSTPHAF.

For phosphorylation, the peptides (1 µg) were incubated with 30 U of PKA (V5161; Promega) in PKA buffer (50 mM Tris [pH 7.5], 10 mM MgCl2, 0.2 mg of bovine serum albumin/ml, 0.1 mM ATP, 1 µCi of [{gamma}-32P]ATP) (10 min, 30°C), and the reaction was terminated by addition of EDTA to 3 mM. The phosphorylated peptides were purified on a G25 column (17.0031-02; Amersham Biosciences). For digestion with chymotrypsin, peptides were incubated with 10 µg of chymotrypsin and analyzed by thin-layer chromatography as described above.


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RESULTS
 
S43 on Raf-1 does not account for its inhibition by agents that elevated cellular cAMP. In order to test whether S43 phosphorylation was the major target that mediated cAMP-dependent suppression of Raf-1 activity, we examined PDGF-stimulated Raf-1 activation in NIH 3T3 cells. For these studies a myc epitope-tagged version of Raf-1 (mRaf-1) in which S43 was mutated to alanine (mA43Raf-1) was transiently expressed in NIH 3T3 cells. The cells were pretreated with 8-bromo-cAMP, a cell-permeative analogue of cAMP (10 min), to increase intracellular cAMP. Raf-1 kinase activity was measured using an immunoprecipitation kinase assay with glutathione S-transferase-MEK, glutathione S-transferase-ERK, and MBP as sequential substrates, the mRaf-1 proteins being immunoprecipitated with the monoclonal antibody 9E10. PDGF strongly stimulated the activity of both mRaf-1 and mA43Raf-1, but the activity of both was completely abolished when the cells were pretreated with 8-bromo-cAMP (Fig. 1A).



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  		FIG. 1. Activity and phosphorylation of Raf-1 in NIH 3T3 cells. (A) mRaf-1 kinase activity. mRaf-1 (Raf) or mA43Raf-1 (A43) was transiently expressed in NIH 3T3 cells as indicated. The cells were serum starved (24 h) and left untreated or treated with 8-bromo-cAMP (cAMP) (1 mM) as indicated. Ten minutes later, the cells were treated with PDGF (10 min) as indicated, cell extracts were prepared, and Raf kinase activity was measured using 9E10 to capture the myc-epitope-tagged proteins. Background counts (~5,800 cpm) were subtracted. The results are for one experiment assayed in triplicate, with error bars representing standard deviations. Similar results were obtained in two independent experiments. (B) Phosphorylation of endogenous Raf-1. NIH 3T3 cells were serum starved (24 h) and left untreated (Control) or pretreated with forskolin and IBMX (F/I) as indicated. Ten minutes later, the cells were treated with PDGF for the indicated times and cell extracts were prepared. Endogenous Raf-1 was immunoprecipitated with polyclonal antibody C20 for blotting with phospho-specific antibodies to S43 (p43; upper panel), S338 (p338; second panel) and S259 (p259; third panel). Blots were reprobed with monoclonal antibody M40091.G to reveal total Raf-1, and one representative reprobed blot is shown (lower panel). Similar results were obtained in three independent experiments. WB, Western blot.

Thus, S43 phosphorylation cannot account for cAMP-mediated suppression of Raf-1 in NIH 3T3 cells. Therefore, we examined if this was because cAMP failed to stimulate S43 phosphorylation in these cells. For these studies, we developed a phospho-specific antibody for Raf-1 phosphorylated on S43. Rabbits were immunized with a synthetic peptide corresponding to amino acids 39 to 47 of Raf-1, in which S43 was phosphorylated (pRaf39-47). Following purification of the antiserum over an immobilized pRaf39-47 column, the sera from one of these rabbits bound to Raf-1 from NIH 3T3 cells that had been treated with forskolin (25 µM) and IBMX (500 µM) for 10 min, but not to Raf-1 from untreated cells (Fig. 1B, lanes 1 and 7). Forskolin activates adenylate cyclase and IBMX inhibits phosphodiesterases, respectively, thus working in concert to increase intracellular cAMP levels. The binding of this antibody was blocked by the immunizing, phosphorylated peptide but not by the corresponding unphosphorylated peptide (Raf39-47) or by an unrelated peptide containing a phosphorylated serine (pRaf617-625; data not shown). The antibody also bound to mRaf-1 transiently expressed in COS cells that had been treated with forskolin and IBMX but not to untreated cells or to mA43Raf-1 from cells treated with forskolin and IBMX (data not shown). Thus, the antibody is specific and binds to mRaf-1 only when S43 is phosphorylated. PDGF stimulated very low-level, transient phosphorylation on S43 that was detectable at 2 and 5 min (Fig. 1B). Forskolin-IBMX-stimulated strong S43 phosphorylation on endogenous Raf-1, which was maximal within 10 min (Fig. 1B and data not shown). Subsequent treatment with PDGF did not have any immediate effects on S43 phosphorylation, but it was reduced 20 min after PDGF treatment and returned to basal levels after 60 min (Fig. 1B).

S259 phosphorylation is stimulated by forskolin and IBMX. Thus, S43 is clearly phosphorylated in NIH 3T3 cells treated with agents that stimulate elevated levels of cAMP, but it appears not to account for the suppression of activity. We therefore examined the phosphorylation of Raf-1 on other sites for which phospho-specific antibodies are available. Previously, we described a phospho-specific antibody that binds to S338 on Raf-1 (31). This site is phosphorylated when Raf-1 is recruited to the plasma membrane in a Ras-dependent manner, and its phosphorylation, although not indicative of Raf-1 activation, is required for activation of Raf-1 in growth factor-stimulated cells (11, 31). PDGF stimulates rapid and sustained phosphorylation of this site on endogenous Raf-1 in NIH 3T3 cells, and this is strongly suppressed by pretreatment with forskolin and IBMX (Fig. 1B). Finally, we examined the phosphorylation of S259, for which a phospho-specific antibody is available. This site was weakly phosphorylated on endogenous Raf-1 from resting cells, and its phosphorylation was not stimulated by PDGF but at later times appears to be weakly dephosphorylated. Forskolin-IBMX-stimulated strong phosphorylation of this site which was maximal within 10 min and remained elevated for the entire time course studied (Fig. 1B).

The effects of cAMP are cell type specific, so we examined Raf-1 phosphorylation in forskolin- and IBMX-treated COS cells and obtained results that were similar to those for NIH 3T3 cells. S43 was not phosphorylated in serum-starved COS cells, and EGF stimulated only weak phosphorylation at 5 and 10 min (Fig. 2A). Forskolin and IBMX stimulated rapid and sustained phosphorylation of this site, and subsequent EGF treatment did not affect the levels of phosphorylation. S338 phosphorylation, which is rapidly stimulated by EGF in COS cells, was also found to be strongly suppressed when the cells were pretreated with forskolin and IBMX (Fig. 2A). Finally, S259 was weakly phosphorylated in COS cells but was not strongly affected by EGF treatment (Fig. 2A). However, forskolin and IBMX stimulated rapid and sustained phosphorylation on S259 (Fig. 2A).



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  		FIG. 2. Activity and phosphorylation of Raf-1 in COS cells. (A) Phosphorylation of endogenous Raf-1. COS cells were serum starved (24 h) and left untreated (Control) or pretreated with forskolin and IBMX (F/I) as indicated. Ten minutes later, the cells were treated with EGF for the indicated times and cell extracts were prepared. Endogenous Raf-1 was immunoprecipitated with polyclonal antibody C20 for blotting with phospho-specific antibodies to S43 (p43; upper panel), S338 (p338; second panel) and S259 (p259; third panel). Western blots (WB) were reprobed with monoclonal antibody M40091.G to reveal total Raf-1, and one representative reprobed blot is shown (lower panel). Similar results were obtained in three independent experiments. (B) mRaf-1 kinase activity. COS cells were transiently transfected with mRaf-1 (Raf), mA43Raf-1 (A43), mA259Raf-1 (A259), and mA43, A259Raf-1 (A43, A259). The cells were serum starved (for 24 h) and left untreated or pretreated with forskolin and IBMX (F/I). Ten minutes later, the cells were treated with EGF (20 min) as indicated and extracts were prepared. The kinase activity of the mRaf-1 proteins was measured using 9E10 monoclonal antibody to capture the myc-tagged proteins. Background counts (~5,000 cpm) were subtracted; the results are for one experiment assayed in triplicate, with error bars to indicate standard deviations from the mean. Similar results were obtained in two independent assays.

S259 phosphorylation does not account for the suppression of Raf-1 activity by forskolin and IBMX. The suppression of S338 phosphorylation in both cell lines is consistent with suppression of Raf-1 activity, and the above data also suggest that the forskolin-IBMX effect may be due to the phosphorylation of S43 and S259. To test this directly, we measured the activation of transiently expressed mRaf-1, mA43Raf-1, and mA259Raf-1 (in which S259 is replaced with alanine) in COS cells. In COS cells, mRaf-1 and mA43Raf-1 were robustly activated by EGF and both were strongly suppressed by forskolin-IBMX pretreatment (Fig. 2B). Although mA259Raf-1 had elevated basal activity compared to mRaf-1, it was further activated by EGF but was still suppressed by forskolin-IBMX pretreatment, albeit not completely (Fig. 2B). Finally, we tested a double mutant in which both S43 and S259 were mutated to alanine (mA43,A259Raf-1). This mutant also had elevated basal activity but was activated by EGF and strongly suppressed by forskolin and IBMX (Fig. 2B).

cAMP targets both Ras-dependent and Ras-independent mechanisms of Raf-1 activation. The above data demonstrated that phosphorylation of Raf-1 on S43 and S259 is not sufficient to mediate its suppression by cAMP in vivo. The data also demonstrate that S338 phosphorylation is blocked by forskolin and IBMX, and since this requires Ras-mediated membrane recruitment (31), the data suggest that the effects of cAMP may be directed against Ras. We therefore examined Ras activation in these cells, using a nonradioactive capture assay in which active Ras is detected by Western blotting (9). PDGF stimulated rapid Ras activation in NIH 3T3 cells (Fig. 3A), and EGF stimulated rapid Ras activation in COS cells (Fig. 3B). However, forskolin and IBMX did not suppress Ras activation in either cell line (Fig. 3A and B), so we tested whether forskolin and IBMX affected the binding of Raf-1 to Ras in vivo, since this has previously been tested only in vitro (43).



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  		FIG. 3. Role of Ras in cAMP-mediated suppression of Raf-1. (A and B) Activation of endogenous Ras. NIH 3T3 cells (A) or COS cells (B) were serum starved (for 24 h) and left untreated (Control) or pretreated with forskolin and IBMX (F/I). Ten minutes later, the cells were treated with PDGF (A) or EGF (B) as indicated, and cell extracts were prepared at the indicated times for Ras activation assays. Total Ras protein in 10% of the extracts is shown in the upper panels, and activated Ras (Ras.GTP) is shown in the lower panels. Similar results were obtained in two independent assays. (C) Ras-Raf-1 binding. Serum-starved COS cells were treated as described for panels A and B, and extracts were prepared for Ras-Raf-1 binding assays. The levels of Raf-1 and Ras in 10% of the cell extracts are shown in the upper two panels and the immunoprecipitated Ras is shown in the lower panel. Raf-1 coprecipitated with Ras.GTP is shown in the third panel. Raf-1 was revealed with antibody M40091.G, and Ras was revealed with the pan-Ras antibody. Similar results were obtained in two independent experiments. WB, Western blot.

The only antibodies known to immunoprecipitate endogenous Ras-Raf-1 complexes are the monoclonal antibodies Y13-238 and LA069 (12), which unfortunately do not bind to Ki-Ras or N-Ras in murine cells (Y. Light and R. Marais, unpublished data), and curiously, in our NIH 3T3 cells Ha-Ras was not activated by PDGF (data not shown). However, Y13-238 binds to both Ha-Ras and Ki-Ras in COS cells (Light and Marais, unpublished), and we have previously shown that EGF induces rapid binding of Raf-1 to Ha- and/or Ki-Ras in these cells (26). Endogenous Ha- and Ki-Ras were immunoprecipitated with the monoclonal antibody Y13-238, and the complexes were examined for bound endogenous Raf-1 by Western blotting. EGF stimulated rapid binding of Raf-1 to Ras in COS cells, complexes being observed within 2 min and persisting out to 10 min (Fig. 3C). Pretreatment of the cells with forskolin and IBMX severely suppressed the formation of these complexes, and at 10 min, no complexes were observed (Fig. 3C). These data show for the first time that Raf-1 binding to Ras is suppressed in vivo, and although we were unable to perform these experiments with NIH 3T3 cells, the suppression of S338 phosphorylation in these cells (Fig. 1B) is consistent with suppression of the formation of Ras-Raf-1 complexes in NIH 3T3 cells too.

Since these data imply that cAMP targets recruitment of Raf-1 to the plasma membrane by blocking the interaction between Ras and Raf-1 in vivo, we tested whether Ras-independent Raf-1 kinase activity was insensitive to elevated cAMP levels. For this experiment, we used mL89RafCAAX. In this version of mRaf-1, the Ras binding domain is disrupted by substitution of R89 for leucine, and the protein is targeted to the plasma membrane by fusion to its C terminus of the membrane localization signal from Ki-Ras (25, 27, 40). EGF stimulated mL89RafCAAX activity in COS cells, but this activity was still partly (~30%) suppressed by pretreatment with forskolin and IBMX (Fig. 4). However, when the N terminus of mL89RafCAAX was deleted, leaving just the kinase domain (amino acids 325 to 648) targeted to the plasma membrane (m{Delta}RafCAAX), the EGF-stimulated kinase activity was insensitive to forskolin-IBMX pretreatment (Fig. 4).



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  		FIG. 4. Membrane-targeted Raf-1 is still subject to cAMP-mediated suppression. mRaf-1 (Raf), m89LRafCAAX (89L-CX), and m{Delta}RafCAAX ({Delta}-CX) were transiently transfected into COS cells and treated as described for Fig. 2B, for the Raf-1 kinase assay. The results are for one experiment assayed in triplicate, with error bars indicating standard deviations from the mean. Similar results were obtained in two independent experiments.

Forskolin and IBMX stimulate Raf-1 phosphorylation on S233. The above-described data showed that cAMP targets both Ras-dependent and Ras-independent components of Raf-1 activation. The difference in response between mL89RafCAAX and m{Delta}RafCAAX suggests that the Ras-independent suppression occurs through the N-terminal domain, but it does not appear to be due to S43 and/or S259 phosphorylation. However, during the course of these studies, we observed that the binding of a commercial monoclonal antibody (R19120; Transduction Laboratories) to endogenous Raf-1 from NIH 3T3 cells was strongly decreased when the cells were pretreated with forskolin and IBMX (Fig. 5A). However, this reduction was not seen with another monoclonal antibody, M40091.G (Fig. 5A), or a third one (R5773; data not shown); similar results were obtained in COS cells (data not shown). We therefore considered the possibility that the antibody binding was hampered because cAMP stimulated a phosphorylation event that disrupted the R19120 epitope, so we mapped the epitope. The minimal epitope was encoded by amino acids 234 to 239 of Raf-1 (Fig. 5B). Mutation of these amino acids in mRaf-1 to those present in A-Raf ablated R19120 binding to mRaf-1 (Light and Marais, unpublished). Intriguingly, the epitope is adjacent to a weak consensus sequence for a PKA phosphorylation site centred on serine 223 (S223) (Fig. 5B). Mutation of S233 to alanine (creating mA233Raf-1) did not the affect binding of R19120 to mRaf-1 from untreated COS cells (Fig. 5C), indicating that S233 is not part of the antibody epitope. Importantly, however, the binding of R19120 to mRaf-1 from COS cells treated with forskolin and IBMX was suppressed, whereas the binding to mA233Raf-1 was unaffected (Fig. 5C), suggesting that S233 phosphorylation is induced by cAMP and reduces binding of R19120 to Raf-1.



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  		FIG. 5. Binding of antibody R19120 to Raf-1. (A) Binding to endogenous Raf-1 protein. NIH 3T3 cells were treated as described in Fig. 1B, and Western blots (WB) were performed using monoclonal antibodies R19120 (upper panel) or M40091.G (lower panel). Similar results were obtained in at least three independent experiments. (B) Details of the R19120 epitope. The sequence of human Raf-1, amino acids 228 to 241, is shown. The R19120 minimal epitope is indicated by the open box, and the PKA consensus and the sequence of synthetic peptide Raf228-238 are indicated by the short and long lines, respectively. S233 is highlighted in the oblong, and the potential chymotrypsin cleavage sites are indicated by the arrowheads. (C) Effects of mutants on R19120 binding. COS cells transiently expressing mRaf-1 (Raf) or mA233Raf-1 (A233) were serum starved (for 24 h) and left untreated (C) or treated with forskolin and IBMX (F/I) (10 min). Myc-epitope-tagged proteins were immunoprecipitated with 9E10 and immunoblotted with R19120 (upper panel) or M40091.G (lower panel). Vector, vector control transfection. Similar results were obtained in two independent experiments.

To determine whether S233 was a site of phosphorylation on Raf-1, we performed in vivo labeling experiments. mRaf-1 and mA233Raf-1 were immunoprecipitated from cells that had been metabolically labeled with [32P]orthophosphate and subjected to phosphopeptide mapping following digestion with chymotrypsin. In untreated cells, three major (C, D, and F) and five minor (A, B, E, H, and G) phosphopeptides were observed (Fig. 6A). Treatment of the cells with forskolin and IBMX strongly stimulated the relative amount of phosphorylation on peptides A, B, and G and also stimulated phosphorylation on E and H, but to a lesser degree (Fig. 6A). In mA233 Raf-1, the three major peptides (C, D, and F) were present, and minor peptides G and H were also present, but minor peptides A, B, and E were absent (Fig. 6A). Following forskolin and IBMX treatment, only peptide G displayed increased relative levels of phosphorylation in mA233Raf-1, and peptides A, B, and E were still absent. The mixing experiment demonstrates that peptides C, D, F, G, and H comigrate from mRaf-1 and mA233Raf-1, indicating that they are derived from the same sites on these two proteins (Fig. 6A).




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  		FIG. 6. S233 is phosphorylated in vitro by PKA and in vivo following F/I treatment. (A) Two-dimensional phosphopeptide mapping. COS cells were transfected with mRaf-1 (Raf-1) or mA233Raf-1 (A233) and labeled with [32P]orthophosphate. Cells were either untreated or treated for 10 min with forskolin and IBMX (F/I), and mRaf-1 proteins were immunoprecipitated for two-dimensional phosphopeptide mapping analysis. For each sample, ~1,000 cpm (Cerenkov counting) were loaded, except for the mix, where ~2,000 cpm were loaded. The peptides whose phosphorylation was enhanced by F/I treatment are indicated with arrows, and the peptides absent in the map from mA233Raf-1 are indicated with open arrows. A scheme of the peptide identity is presented, with peptides being labeled from A to G. O, origin. Similar results were obtained in two experiments. (B) Phosphorylation of Raf228-238 by PKA. The peptides Raf228-238 and pRaf228-238 were incubated with PKA for in vitro phosphorylation, followed by digestion with chymotrypsin as indicated, and separated by electrophoresis at pH 1.9 on phospho-cellulose plates. O, origin. Similar results were obtained in two experiments. (C) Two-dimensional phosphopeptide mapping. Upper panel, phosphopeptide map of Raf-1 treated with forskolin and IBMX as described for panel A (~2,000 cpm). Middle panel, the PKA phosphorylated chymotryptic fragment of peptide Raf228-238 from panel B (peptide; ~100 cpm). Lower panel, mixture of Raf-1 treated with forskolin and IBMX (~2,000 cpm) and the fragment from Raf228-238 (~100 cpm). The peptides which comigrate are indicated by an arrow. (D) Phosphoamino acid analysis. The peptides indicated by the arrows in Fig. 6C above were subjected to phosphoamino acid analysis. The circles indicate the positions of migration of phosphoserine (P-S) and phosphothreonine plus phosphotyrosine (P-T/P-Y) standards. Pi, inorganic phosphate.

Mutation of S233 appears to cause the loss of three peptides from mRaf-1. In order to determine whether one or more of these peptides was derived from the phosphorylation of S233, we performed synthetic peptide phosphorylation studies using PKA. Chymotrypsin digests proteins after large hydrophobic residues (Phe, Trp, and Tyr), and so it would be expected to cleave Raf-1 after Y232 and after F238 (Fig. 5B. However, a peptide encompassing these amino acids may not be phosphorylated by PKA, since the basic residues H230 and R231 would be absent. Therefore, we generated a synthetic peptide corresponding to amino acids 228 to 238 of Raf-1 (Raf228-238) (Fig. 5B). PKA phosphorylated Raf228-238 on S233, because a similar peptide that was phosphorylated on S233 (pRaf228-238) during synthesis was not a substrate for PKA (Fig. 6B. Phosphorylated Raf228-238 was cleaved by chymotrypsin as demonstrated by its increase in electrophoretic mobility that was seen following incubation with the protease (Fig. 6B. The only potential chymotrypsin cleavage site in Raf228-238 is Y232, which would yield fragments of amino acids 228 to 233 to 238.

The inability of PKA to phosphorylate pRAF228-238 suggests that the 233-238 fragment is phosphorylated only on S233 and this fragment comirated with peptide A from Raf-1 treated with forskolin and IBMX (Fig. 6C). However, these peptides also contain a threonine at position 234, and to ensure that this threonine was not phosphorylated, we performed phosphoamino acid analysis on peptide A and the 233-238 fragment of PKA-phosphorylated Raf228-238 and found only phosphoserine (Fig. 6D). We conclude that peptide A is derived from amino acids 233 to 238 of Raf-1 and contains phosphorylated S233, providing direct evidence that this amino acid is phosphorylated on Raf-1 in vivo. The identify of peptides B and E has not been established, but they may arise due to incomplete digestion of this region of Raf-1 by chymotrypsin or from other as yet unidentified phosphorylation events.

cAMP regulation of Raf-1 occurs through multiple phosphorylation events. Thus, forskolin and IBMX stimulate phosphorylation of three amino acids on Raf-1: S43, S233, and S259. We therefore tested whether S233 phosphorylation could account for forskolin-IBMX-mediated suppression of Raf-1. EGF-stimulated activation of mA233Raf-1 was still suppressed by pretreatment with forskolin and IBMX (Fig. 7A), so we tested combinations of mutations. When S43A was combined with S233A (mA43,A233Raf-1), EGF-stimulated kinase activity was still suppressed by forskolin-IBMX pretreatment (Fig. 7A), but when S233 and S259 were combined (mA233,A259Raf-1), EGF-stimulated kinase activity was almost completely resistant to forskolin-IBMX pretreatment (Fig. 7A). This result suggests that the suppression of Raf-1 activity by cAMP can be fully accounted for by phosphorylation on S233 and S259 and that S43 phosphorylation plays only a minor role. However, S43 was only poorly phosphorylated on mA259Raf-1 and mA233,A259Raf-1 (Fig. 7B), and when all three mutants were combined (mA43,A233,A259Raf-1), the basal kinase activity of the mutant was strongly increased and EGF-stimulated activity was completely resistant to forskolin-IBMX pretreatment (Fig. 7A). These data demonstrate that Raf-1 is fully resistant to elevated cAMP only when all three sites are mutated.



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  		FIG. 7. Raf-1 regulation by cAMP involves multiple phosphorylation events. (A) mRaf-1 kinase activity. mRaf-1 (Raf), mA233Raf-1 (A233), mA43,A233Raf-1 (A43, A233), mA233,A259Raf-1 (A233, A259) or mA43,A233,A259Raf-1 (A43,A233,A259) were transiently expressed in COS cells and treated as for Fig. 2B for the Raf-1 kinase assay. The results are for one experiment assayed in triplicate, with error bars indicating standard deviations from the mean. Similar results were obtained in two independent experiments. (B) S43 phosphorylation. mRaf-1 (Raf), mA233Raf-1 (A233), mA259Raf-1 (A259), mA233,A259Raf-1 (A233,A259) or mA43,A233,A259Raf-1 (A43,A233,A259) were transiently expressed in COS cells which were either untreated or treated for 10 min with forskolin and IBMX as indicated. An equal amount of myc-tagged Raf-1 protein was immunoprecipitated and probed with a phospho-specific antibody to S43. Similar results were obtained in two independent experiments.

ERK activation but not DNA synthesis is rescued by mA43,A233,A259Raf-1. Finally, we explored the role played by Raf-1/ERK signaling in mediating suppression of proliferation when cAMP levels are elevated. For these studies, we used NIH 3T3 cells, since their PDGF-stimulated proliferation (measured by incorporation of [3H]thymidine into DNA) is completely blocked by a 10-min pretreatment with forskolin and IBMX (Fig. 8A). Forskolin and IBMX also completely block the PDGF-stimulated activation of endogenous Raf-1 in NIH 3T3 cells (Fig. 8B). However, the effects of forskolin and IBMX on ERK activation are more modest. Endogenous ERK activation was monitored using a phospho-specific antibody that only binds to the dually phosphorylated, active forms of ERK1 and ERK2. When the cells were pretreated with forskolin and IBMX, ERK activity was fully blocked at 2 min, but at later times ERK was still active, albeit to lower levels (Fig. 8C). This result was verified by use of an immunoprecipitation kinase assay that measures ERK2 activity directly, using MBP as a substrate. ERK activity was almost completely blocked at 2 min and was suppressed at all other times (Fig. 8D).



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  		FIG. 8. cAMP regulates of Raf-1/ERK signaling and cell growth in NIH 3T3 cells. (A) DNA synthesis. NIH 3T3 cells were serum starved and left untreated or treated with forskolin and IBMX (F/I) as indicated. Ten minutes later, the cells were treated with PDGF as indicated and DNA synthesis was measured. The results presented are for one experiment assayed in triplicate with error bars representing standard deviations. Similar results were obtained in three independent experiments. (B) Endogenous Raf-1 activity. NIH 3T3 cells were serum starved (for 24 h) and left untreated (Control) or pretreated with forskolin and IBMX (F/I). Ten minutes later, the cells were treated with PDGF as indicated, and cell extracts were prepared at the indicated times. Endogenous Raf-1 was immunoprecipitated with M40091.G, and kinase activity was measured. The results presented are for one experiment assayed in triplicate with error bars representing standard deviations. Background counts (~5,000 cpm) were subtracted, and similar results were obtained in three independent assays. (C) Phosphorylation of endogenous ERK. NIH 3T3 cells were serum starved (for 24 h) and left untreated (Control) or pretreated with forskolin and IBMX (F/I). Ten minutes later, the cells were stimulated with PDGF for the indicated times. ERK phosphorylation is shown in the upper blot, and total ERK is shown in the lower blot. Results are for one assay, and similar results were obtained in two independent experiments. WB, Western blot. (D) Endogenous ERK activity. The kinase activity of ERK2 was measured from the same samples as in panel C. Results are for one assay, and similar results were obtained in two independent experiments.

Thus, endogenous Raf-1 activity is completely blocked, whereas endogenous ERK activity is only partially blocked, when cAMP levels are elevated, so we tested whether this was sufficient to account for the suppression of cell growth. We generated stable NIH 3T3 clones expressing mRaf-1 or mA43,A233,A259Raf-1. PDGF stimulated rapid activation of both of these exogenously expressed proteins, with kinetics that were similar to those seen for endogenous Raf-1 (compare Fig. 8B and 9A ). However, whereas mRaf-1 activity was completely blocked by forskolin-IBMX pretreatment, mA43,A233,A259Raf-1 activity was completely resistant to these agents (Fig. 9A). Similar results were obtained with two independent clones expressing mRaf-1 and with three clones expressing mA43,A233,A259Raf-1 (data not shown). We next examined the activity of endogenous ERK in these clones. In the clone expressing mRaf-1, forskolin and IBMX still almost completely blocked PDGF-stimulated ERK activation at 2 min, whereas in the clone expressing mA43,A233,A259Raf-1, PDGF-dependent ERK activation at 2 min was completely insensitive to forskolin-IBMX pretreatment (Fig. 9B). Similar results were obtained with two independently isolated mRaf-1 clones and two mA43,A233,A259Raf-1 clones. Thus, mRaf-1 expression did not restore activation of endogenous ERK in forskolin-IBMX-pretreated NIH 3T3 cells, but mA43,A233,A259Raf-1 did restore the activity of endogenous ERK.



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  		FIG. 9. mA43,A233,A259Raf-1 rescues ERK activation but not cell proliferation. (A) Raf-1 kinase activity. NIH 3T3 cells stably expressing mRaf-1 (Raf; upper panel) or mA43,A233,A259Raf-1 (A43,A233,A259; lower panel) were treated as described for Fig. 8B, and extracts were prepared. Myc-tagged Raf proteins were immunoprecipitated using 9E10 for Raf kinase assay determination. The results are for one experiment assayed in triplicate, with error bars representing standard deviations from the mean. Similar results were obtained with two independently derived clones for each line, each assayed on two separate occasions. (B) Endogenous ERK activation. NIH 3T3 cells stably expressing mRaf-1 (Raf) or mA43,A233,A259Raf-1 (A43,A233,A259) were serum starved and treated with forskolin and IBMX (F/I) for 10 min followed by PDGF for a further 2 min as indicated. Extracts were prepared, and immunoblots were performed for ppERK (upper blot) or total ERK (lower blot). Similar results were obtained in two independent assays. WB, Western blot. (C) DNA synthesis. NIH 3T3 cells stably expressing mRaf-1 (Raf) or mA43,A233,A259Raf-1 (A43,A233,A259) were treated as described for Fig. 8A and analyzed for DNA synthesis. The results presented are for one experiment assayed in triplicate with error bars representing standard deviations. Similar results were seen with two individual clones of each line, each assayed at least twice.

Finally, we examined DNA synthesis in these clones. PDGF-stimulated DNA synthesis in the lines overexpressing mRaf-1 was completely blocked by forskolin-IBMX pretreatment (Fig. 9C). The clone expressing mA43,A233,A259Raf-1 had elevated DNA synthesis under resting conditions compared to mRaf-1 expressing controls, but DNA synthesis was still stimulated by PDGF (Fig. 9C). Surprisingly, however, forskolin and IBMX still strongly suppressed both the PDGF-independent and PDGF-stimulated DNA synthesis (Fig. 9C). However, whereas the suppression of DNA synthesis in the mRaf-1-overexpressing clones was complete, residual DNA synthesis was still seen in the clones expressing mA43,A233,A259Raf-1. Approximately 21% of growth factor-independent DNA synthesis was resistant to forskolin and IBMX, and ~24% of the DNA synthesis seen in the presence of PDGF escaped forskolin-IBMX treatment (Fig. 9C). Similar results were obtained with two independently derived mRaf-1 clones and two mA43,A233,A259Raf-1 clones.


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DISCUSSION
 
In this paper, the widely accepted model suggesting that Raf-1 activity is suppressed because S43 is phosphorylated in cells when cAMP levels are elevated is shown to be incomplete (see, for example, references 3 and 22). We show for the first time that elevating cAMP does suppress the binding of Raf-1 to Ras.GTP in vivo and show that membrane-dependent events, such as S338 phosphorylation, are blocked. However, we also show that when Raf-1 is localized to the plasma membrane and its activity is Ras independent, it is still partly suppressed by forskolin and IBMX, thereby demonstrating that cAMP targets both Ras-dependent and Ras-independent mechanisms to suppress Raf-1 activity.

We show that the regulation of Raf-1 by agents that elevate cAMP is considerably more complex than was previously accepted. We provide direct evidence that Raf-1 is phosphorylated on three sites, using phospho-specific antibodies and phosphopeptide mapping. These sites are the previously identified S43 and S259 and also a new site, not previously identified: S233. However, our data do not support a role for S621 phosphorylation in negative regulation of Raf-1 by cAMP in vivo, as has been suggested (34). S621 was intact in mA233,A259Raf-1, mA43,A233,A259Raf-1, and m{Delta}RafCAAX, and yet forskolin and IBMX did not suppress their activities. Together with the demonstration that elevated cAMP does not stimulate S621 phosphorylation or block the activity of the Raf-1 catalytic domain in vivo (39), our data suggest that the suppression of Raf-1 activity by S621 phosphorylation is an in vitro artifact.

In agreement with previous studies (39), we show that S43 phosphorylation did not account for cAMP-mediated suppression of Raf-1, because S233 and S259 function to override activation when phosphorylation of S43 is blocked. Indeed, our data imply that whereas S233 and S259 can function independently to suppress Raf-1 activity (A43,A233Raf-1 and A43,A259Raf-1 activities were efficiently suppressed by forskolin and IBMX [Fig. 2B and 7A]), S43 did not appear to function independently (A233,A259Raf-1 activity was only poorly suppressed [Fig. 7A]). However, this appears to be because S43 is not phosphorylated efficiently in the A233,A259Raf-1 double mutant (Fig. 7B), and in fact, our data suggest that all three of the sites mediate suppression of Raf-1 kinase activity even under resting conditions. For S233 and S259, this was evident from the fact that the single mutants had elevated basal kinase activity even in serum-starved cells, and when these mutations were combined, basal kinase activity was further elevated (Fig. 2B and 7A). We also show that both S259 (Fig. 1B and 2A) and S233 (Fig. 6A) are weakly phosphorylated even in serum-starved cells. S43 appears to have a more subtle effect. The basal kinase activity of A43Raf-1 was not elevated, and it did not appear to cooperate with A233 or A259 to elevate basal activity. We also did not find any evidence that this site was phosphorylated in resting cells. However, when the A43 mutant was combined with the A233,A259Raf-1 double mutant, the levels of basal kinase activity were further elevated.

Thus, it appears that all three sites may play a role in suppressing Raf-1 activity even in resting cells, but what are the biological consequences of this regulation? When the regulation was completely lost, as in the cell lines expressing A43,A233,A259Raf-1, the cells were driven into proliferation even in the absence of serum (Fig. 9C). Thus, it appears that loss of these biological controls drives cells into proliferation, suggesting that if somatic mutations occurred in these sites, Raf-1 may contribute to inappropriate cell growth. This may explain the apparent redundancy of these controls; it ensures that single somatic mutants are not sufficient to create oncogenic versions of Raf-1. Indeed, we have found that A259Raf-1, despite its elevated basal kinase activity, is not transforming to NIH 3T3 cells (Light and Marais, unpublished). Although we do not yet understand how these mutants suppress Raf-1 activity and therefore why mutating them is activating, there is an interesting parallel with B-Raf, which has recently been shown to have cryptic negative-regulatory phosphorylation sites (44).

Although S259 is a known negative-regulatory site on Raf-1 (see references 3 and 22) that is implicated in suppression of Raf-1 by PKB in differentiated myotubes (45), this site has not previously been implicated in the negative regulation of Raf-1 activity by PKA. Previously it has been shown that S259 phosphorylation does not change in growth factor-stimulated cells (35). We also did not find significant changes in the levels of S259 phosphorylation in growth factor-stimulated cells at times when Raf-1 activity was maximal, although some reduction in phosphorylation occurred at later times (Fig. 1 and 2). Since forskolin-IBMX stimulated strong S259 phosphorylation, this site cannot be fully occupied in serum-starved cells. Thus, there appear to be two populations of Raf-1, one that is phosphorylated on S259 and one that is not. Our data imply, in agreement with a negative role for this phosphorylation event, that when S259 is phosphorylated, Raf-1 cannot be activated. It has recently been argued that S259 needs to be dephosphorylated in order for Raf-1 to be activated (1, 10, 21), but our data show that some of the Raf-1 in cells is not phosphorylated on S259 under resting conditions, so dephosphorylation may not be a necessary prelude to activation. S259 phosphorylation is required for 14-3-3 binding to CR2 in Raf-1, which raises interesting questions about the dynamics of Raf-1-14-3-3 interactions and the role played by 14-3-3 in mediating suppression of Raf-1 by cAMP. We are currently examining these questions.

Finally, the ability to generate a version of Raf-1 that was no longer suppressed by elevated cAMP enabled us to directly test whether cAMP suppresses cell growth because of its action on Raf-1/ERK signaling. Raf-1 was completely blocked in our parental NIH 3T3 cells, but ERK activity was only weakly blocked. This result is in line with studies in which it was shown that ERK was activated normally in cells derived from animals that are null for the Raf-1 gene (20, 33). Clearly, redundant routes exist from growth factor receptors to ERK activation (possibly involving B-Raf or A-Raf), and the major signal from PDGF receptor to ERK in NIH 3T3 cells is Raf-1 independent. Why then is Raf-1 targeted and inhibited so comprehensively by cAMP? This is particularly curious in light of the observation that this only translates to a relatively small effect on ERK activity and implies that Raf-1 may be involved in other processes that are independent of MEK/ERK. However, even when endogenous ERK activity was fully restored in the clones expressing A43,A233,A259Raf-1, forskolin and IBMX still strongly suppressed cell growth, although some DNA synthesis (20 to 25%) was resistant to forskolin and IBMX. This observation has two implications. It demonstrates that strong Raf-1 signaling does stimulate some cAMP-independent cell growth, indicating that inhibition of this pathway is necessary for suppression of growth, both in the presence and absence of growth factors, and that the Raf-1/ERK pathway is an important target of cAMP. It also demonstrates however, that this is not the only pathway that is targeted and that cAMP also targets other proliferative pathways. Thus, the generally accepted model is incomplete. It is worth noting that in CCL39 cells, cyclin D1 expression but not ERK activity appears to be an important target of cAMP (23, 32). However, overexpression of cyclin D1 only partly restored the growth of these cells, demonstrating that in these cells cAMP also targets multiple pathways. We are currently examining the relationship between Raf-1/ERK signaling and cyclin D1 with respect to cAMP-mediated cell growth suppression in NIH 3T3 cells.


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ACKNOWLEDGMENTS
 
We thank A. Chiloeches for helpful comments and for generating pMCEF-mRaf-1. We thank members of the Signal Transduction Laboratory for stimulating discussions and C. Marshall for critical reading of the manuscript. We thank J. Metcalf for peptide synthesis, H. King for DNA sequencing, and J. Cordell for technical expertise.

This work was funded by grants from the Medical Research Council (component grant: G9900391), Cancer Research UK, and The Institute of Cancer Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Rd., London SW3 6JB, United Kingdom. Phone: 020 7878 3856. Fax: 020 7352 3299. E-mail: rmarais{at}icr.ac.uk. Back


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Molecular and Cellular Biology, June 2002, p. 3717-3728, Vol. 22, No. 11
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.11.3717-3728.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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