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Peter E. Shaw,2 Harald Mischak,3 and Walter Kolch1,4*
The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD,1 School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH,2 Institute for Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom,4 Department of Nephrology, Medizinische Hochschule Hannover, 30625 Hannover, Germany3
Received 27 July 2001/ Returned for modification 19 September 2001/ Accepted 8 February 2002
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ABSTRACT |
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INTRODUCTION |
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In resting cells Raf-1 is phosphorylated on serines 43, 259, and 621 (27). Serines 43 and 621 have been previously described as target sites for the cAMP-regulated protein kinase, PKA, which inhibits Raf-1 (13, 25, 38). Phosphorylation of serine 43 was reported to diminish the affinity of Raf-1 for Ras (38) and thereby to interfere with Raf-1 activation (30, 38), although recent results dispute this (34). The role of serine 621 phosphorylation is more difficult to study, because mutation of this residue is incompatible with kinase function (25, 27). This observation was taken as an indication that serine 621 phosphorylation is essential for Raf-1 function. Biochemical experiments confirmed that the integrity of serine 621 was indeed required but also correlated its phosphorylation with the inhibition of catalytic activity (25). The latter experiments were done with the isolated Raf-1 kinase domain, and the role of serine 621 phosphorylation in the context of full-length Raf-1 is less clear. Recent experiments have shown that in macrophages the dephosphorylation of serines 621 and especially 259 is required for efficient Raf-1 activation (1). Blocking this dephosphorylation with concentrations of okadaic acid which selectively inhibited the serine/threonine phosphatase PP2A severely blunted the CSF-1-induced Raf-1 activation. Mutation of serine 259 rendered Raf-1 largely resistant to inhibition by okadaic acid. PP2A was found to associate with Raf-1 in the membrane compartment, suggesting that in macrophages Raf-1 membrane translocation is accompanied by PP2A binding and that PP2A enables Raf-1 activation by dephosphorylating serines 259 and 621 (1). Dephosphorylation of serine 259 was recently shown to be a key step in Raf-1 activation, regulating its binding to upstream activators as well as to its substrate, MEK (8). Serine 259 was also described as a target site for Raf-1 inhibition mediated by Akt/protein kinase B (PKB) (40), although this cross talk appears to occur only in certain cell types (31).
The present report highlights the importance of serine 259 for the regulation of Raf-1. It shows that serine 259 is a target site for phosphorylation by PKA and that it is the main site mediating PKA-induced Raf-1 inhibition. Using RafS259 mutants we also present evidence that PKA uses Raf-1 inhibition as main interface for the negative control of ERK activity and ERK-induced gene transcription.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression vector for the catalytic subunit of PKA was kindly provided by Stanley McKnight. All other expression vectors and baculoviruses have been described previously (13, 25). For Raf-1 immunoprecipitation and Western blotting crafVI, an antiserum raised against the 12 C-terminal amino acids of Raf-1, was used (13). The Raf-1 anti-phosphoserine 338 and anti-phosphoserine 259 antibodies were purchased from UBI and New England Biolabs, respectively. The Raf-1 anti-phosphoserine 43 antibody was a generous gift from Manuela Baccarini. The 9E10 myc and anti-hemagglutinin (HA) tag antibodies were from Roche Diagnostics; the anti-FLAG antibody was from Sigma. Cell culture and transfection. NIH 3T3 and COS-1 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine and 10% fetal calf serum. COS-1 cells were transfected in six-well plates with 1.5 µg of each DNA/well using DEAE dextrane and chloroquine as described previously (25) or Effectene according to the manufacturer's instructions. NIH 3T3 cells were transfected as described previously (19). Cells were serum starved overnight prior to treatment with mitogens. Protein expression in Sf-9 insect cells was done as described previously (25).
Raf phosphorylation and two-dimensional phosphopeptide mapping. The phosphorylation of recombinant Raf-1 proteins with PKA in vitro was done as described previously (13) except that the Raf proteins were produced in COS-1 cells. Metabolic labeling with [32P]orthophosphoric acid and forskolin treatment of NIH 3T3 cells were done as previously described (13). Raf-1 was immunoprecipitated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiographed. Raf bands were cut out and subjected to tryptic digestion and two-dimensional peptide mapping as described earlier (14). A pH 8.9 buffer was used for the first dimension, and phosphochromatography buffer was used for the second dimension. Phosphopeptides were quantified with a Fuji phosphorimager.
Kinase assays and Western blotting.
Kinase assays and Western blotting were done as described earlier (25). Raf-1 activity was determined by a linked assay, where Raf-1 immunoprecipitates were incubated with recombinant MEK-1 and kinase-dead ERK-2 proteins. This assay measures the ability of Raf-1 to phosphorylate and activate MEK. MEK activation is detected by the phosphorylation of kinase-negative ERK. ERK activation was assayed with myelin basic protein as a substrate. In some experiments kinase activity was measured in a more sensitive two-step kinase assay exactly as described earlier (3). In this assay Raf proteins were immunoprecipitated with the crafVI antibody and used in the first step to activate recombinant MEK and ERK. In the second step an aliquot of the reaction is incubated with myelin basic protein in the presence of [
-32P]ATP to measure the activity of ERK. Western blots were developed by using the enhanced chemiluminescence kit from Roche Diagnostics according to the manufacturer's instructions. Densitometric evaluation was done with NIH Image.
Reporter gene assays. Reporter gene assays were done as described previously (19).
Ras pulldown assays. Ras pulldown assays were done as described previously (28), except that the Ha-Ras protein was bound to beads before loading with guanylimido diphosphate.
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RESULTS |
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Serine 43 does not mediate Raf-1 inhibition in COS-1 cells. The phosphorylation of serine 43 has been reported to prevent Raf-1 activation by Ras, because it lowers the affinity of Raf-1 towards activated Ras/GTP (5, 35, 38). However, a recent study challenges this conclusion by showing that the mutation of serine 43 to alanine does not prevent the inhibition of Raf-1 by cAMP (34). Using an antibody specific for phosphoserine 43, we first confirmed that treatment with cAMP agonists (forskolin plus IBMX) induced serine 43 phosphorylation (Fig. 2A). Phosphorylation of serine 43 was also induced by the potent Raf-1 activator TPA, which argues against a purely negative role of serine 43 phosphorylation. We then tested Raf-1 and the RafS43A mutant for Ras binding and kinase activity in response to stimulation with cAMP agonists and mitogens (Fig. 2). In serum-starved COS-1 cells 8Cl-cAMP treatment almost completely abolished the binding of Raf-1 to recombinant Ras/GTP immobilized on beads (Fig. 2B). In contrast, the Ras association of a RafS43A mutant was not affected. Surprisingly, when cells were treated simultaneously with 8Cl-cAMP plus epidermal growth factor (EGF) or 8Cl-cAMP plus tetradecanoylphorbol-13-acetate (TPA), the binding of Raf-1 to Ras/GTP was fully rescued. TPA or EGF alone did not influence the binding of Raf-1 or RafS43A to Ras/GTP, indicating that the mitogens selectively counteract the loss of binding caused by serine 43 phosphorylation rather than enhancing the affinity of Raf-1 towards Ras/GTP in general. Aliquots of the same cell lysates were used to immunoprecipitate and measure the kinase activities of Raf-1 and RafS43A (Fig. 2C). Here, no differences could be detected. RafS43A was consistently activated and inhibited to extents similar to those of wild-type Raf-1. These results confirm the initial observations that serine 43 phosphorylation diminishes the binding of Raf-1 to Ras (5, 38) but further suggest that this effect is counteracted by mitogen stimulation. Importantly, RafS43A was still fully susceptible to inhibition by PKA, demonstrating that phosphorylation on this residue is not required for the inhibitory effect.
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(data not shown). No kinase activity was observed in the kinase-dead RafK375W mutant, which was used as a control to exclude the possibility that any of the coexpressed kinases phosphorylates MEK.
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These results pinpoint serine 259 as the major site for PKA-mediated Raf-1 inhibition. In order to test whether serine 43 can further modulate the activity and response to PKA of a RafS259 mutant, we generated a double mutant, RafS43A/S259A (Fig. 3C). In serum-starved cells the kinase activity of the double mutant behaved in a manner very similar to that of the RafS259A single mutant: it was constitutively elevated and refractory to inhibition by the cAMP agonists forskolin plus IBMX. Similarly, forskolin plus IBMX failed to suppress the TPA stimulation of both RafS259A and the RafS43A/S259A double mutant.
We used the phosphoserine 259-specific antiserum to estimate the stoichiometry of phosphorylation in response to cAMP agonists (Fig. 3D). Raf-1 was immunoprecipitated by using the phosphoserine 259-specific antiserum, and supernatants were subsequently immunoprecipitated with a non-phosphorylation-sensitive Raf-1 antiserum. For ease of comparison the gels were adjusted to equal loadings of total Raf-1. The induction of serine 259 phosphorylation was more than sevenfold in two independent experiments. This probably is an underestimation, since the immunodepletion of serine 259-phosphorylated Raf-1 was not complete. Importantly, the fraction of Raf-1 that is phosphorylated on serine 259 rises to almost 60% in response to cAMP agonists. This stoichiometry is consistent with the substantial inhibition of Raf-1 activity, which is of a similar extent (Fig. 3B).
In summary, these results indicate that in the context of full-length Raf-1 serine 259 represents the main target for inhibitory phosphorylation by PKA. They, however, do not strictly rule out a modulatory role for serine 43 which may fine-tune the exact extent of Raf-1 activation. The mutation of serine 259 enhances the affinity of Raf-1 towards Ras (8) and could override small modulatory effects of serine 43 phosphorylation in the RafS43A/S259A double mutant. The results further imply that the phosphorylation of serine 259 downregulates Raf-1 by preventing the transition from the inactive into a partially activated state.
Serine 259 modulates the phosphorylation of serine 338. In order to test the above hypotheses, the phosphorylation of serine 338 was examined. Serine 338 phosphorylation is required but not fully sufficient for Raf-1 activation (17, 23). Therefore, we assayed the phosphorylation status of serine 338 in Raf-1 and RafS259A (Fig. 4). Both mutants were poorly phosphorylated on serine 338 in starved cells. TPA increased serine 338 phosphorylation of both Raf-1 and RafS259A. In wild-type Raf-1 there appeared to be an inverse relationship between serine 259 and serine 338 phosphorylations: PKA activation decreased the phosphorylation of serine 338 in wild-type Raf-1, but not in RafS259A. These data suggest that serine 259 can at least in part control the phosphorylation of serine 338 and thus the transition from a state primed for activation into full activation.
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In order to test this hypothesis we examined the phosphorylation of Raf-1 on serine 259 and the phosphorylation of ERK on the activating sites over a time course of stimulation with forskolin plus IBMX (Fig. 5B). Forskolin plus IBMX treatment of growing cells induced the phosphorylation of Raf-1 on serine 259 within 10 min. In parallel, ERK activity declined steadily, although it took up to 1 h to see a marked inhibition. The delayed kinetics of ERK inactivation is consistent with the downregulation of an upstream activator, such as Raf-1, rather than with a direct inhibition of ERK by cAMP signaling. The induction of serine 259 phosphorylation and the inhibition of ERK were efficiently antagonized by H89, a chemical inhibitor of PKA.
To examine signaling downstream of ERK, we used a transcriptional readout that relies on the phosphorylation of Elk-1, a member of the ternary complex transcription factor family. Elk-1 is a well-characterized substrate for ERK, which enhances Elk-1-mediated transcription by phosphorylating the transactivation domain (12). If the Elk-1 transactivation domain is fused to a GAL4 DNA binding domain, the transcription of a reporter gene under the control of GAL4 binding sites becomes strongly dependent on the activity of the ERK pathway. The expression of the reporter gene is induced by TPA or growth factors that stimulate Raf-1 and can be suppressed by dominant negative Raf-1 mutants (data not shown). Transfection of Raf-1 enhanced the TPA-induced activation of the GAL4-Elk-1 fusion protein (Fig. 5C). This increase was thoroughly suppressed when cAMP degradation was inhibited by IBMX. The expression of RafS259D stimulated the basal activity to levels comparable to those obtained with TPA induction. This activation was completely insensitive to IBMX, demonstrating that cAMP blocks the pathway at the level of Raf-1 and does not affect downstream components. Likewise, RafS259A or RafS259E induced the constitutive activation of the GAL4-Elk-1 reporter, whereas RafS43A behaved like wild-type Raf-1 and RafS621A behaved like a dominant negative mutant (data not shown).
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DISCUSSION |
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Serine 43 can be phosphorylated by PKA (5, 30, 38) and the cyclic GMP-dependent kinase, PKG (35), and was reported to prevent Raf-1 activation in these studies by interfering with the binding of Raf-1 to Ras. These observations have been challenged by a recent report that dissociated Raf-1 inhibition by PKA from the phosphorylation of serine 43 (34). Our data suggest that the effects of serine 43 phosphorylation are context dependent as the inhibition of Ras binding can be abolished by TPA or EGF. These agents induce serine 43 phosphorylation on their own (Fig. 2A) (8), making it unlikely that they rescue Ras binding by preventing serine 43 phosphorylation. However, serine 43 phosphorylation could serve as a timing device which determines the duration of the Ras-Raf interaction. Given that Ras interacts with multiple effectors (reviewed in reference 4), which all compete for binding to a single shared effector domain, there must be mechanisms to coordinate the timely interaction with the different effectors.
Serine 621 also appears to have a complex role. Unfortunately, this residue is not readily amenable to mutational analysis. Changing it to a number of other amino acids resulted in the loss of catalytic function (25, 27). This observation demonstrates that serine 621 is essential for kinase activity and has further been used to argue that the phosphorylation of this residue is necessary for kinase activity (36). Biochemical experiments have confirmed that serine 621 is essential but also indicated that the phosphorylation of this residue inhibits catalytic activity (25). These experiments were performed with the isolated Raf-1 kinase domain expressed in Sf-9 insect cells, but it has also been previously shown that the activity of v-Raf, which consists of the Raf-1 kinase domain fused to retroviral Gag sequences, is also downregulated by PKA (13). It has been shown that the Raf-1 regulatory domain restrains catalytic activity through physical interactions with the kinase domain (6). Thus, it is possible that serine 621 becomes fully accessible for phosphorylation only once the regulatory domain is removed. This could explain why the inhibitory effects of serine 621 are not evident in full-length Raf-1 but readily become apparent in the context of the isolated kinase domain. This hypothesis is supported by the observation that in vitro serine 621 in the isolated Raf-1 kinase domain is a much better substrate for PKA than serine 621 in full-length Raf-1 (13).
A possible explanation for the dual role of serine 621 may be related to 14-3-3 binding. 14-3-3 proteins are ubiquitously expressed adapter proteins that require phosphoserine for binding (11, 24, 29). Both serines 259 and 621 in Raf-1 are docking sites for 14-3-3 proteins, and 14-3-3 proteins have been implicated in the activation of Raf-1 and the regulation of serine 621 phosphorylation (36, 39) as well as in stabilizing both active and inactive Raf-1 conformations (37). The displacement of 14-3-3 from Raf-1 by a synthetic peptide encompassing phosphoserine 621 in vitro led to a complete loss of Raf-1 catalytic activity, which could be restored by the addition of recombinant 14-3-3 protein. Interestingly, a RafS259A mutant also was susceptible to inhibition by this phosphopeptide (37). Since the RafS259A mutant still has an intact 14-3-3 docking site on phosphoserine 621, this experiment suggests that the inhibitory effect of serine 621 phosphorylation can be reversed by 14-3-3 binding. This hypothesis is currently being investigated.
Several studies have observed that the mutation of serine 259 modestly increases the catalytic activity of Raf-1 (8, 24, 27, 32, 37). The same activated phenotype is produced by replacing serine 259 with either alanine, aspartic acid, or glutamic acid. Due to their negative charge, aspartic and glutamic acids often act as phosphomimetic substitutes. But our results indicate that the physical presence of a phosphate group at serine 259 is necessary for inhibition. It has been suggested that 14-3-3 binding to phosphoserine 259 (32, 37) inhibits Raf-1. As 14-3-3 docking requires a phosphoserine (24, 29), the activated phenotype of RafS259 mutants could be due to the loss of 14-3-3 binding. To test the contribution of 14-3-3 binding, we have mutated both 14-3-3 docking sites, but unfortunately the serine 259/621 double mutant is devoid of kinase activity (data not shown), precluding a conclusive investigation. The present study demonstrates that both the basal and the stimulated kinase activities are enhanced by mutation of serine 259. Raf S259 mutants are largely refractory to the inhibitory effects of PKA, demonstrating that serine 259 phosphorylation is sufficient for the inhibition of full-length Raf-1 by PKA.
It is important to note that this conclusion does not discount a role for the other PKA phosphorylation sites serines 43 and 621. They could serve as conditional modulators. As outlined above, serine 43 phosphorylation may be part of an intricate regulatory mechanism that modulates Raf-1 activation by Ras, while serine 621 phosphorylation may regulate the Raf-1 catalytic activity in a 14-3-3-dependent manner. This regulation may apply to different pools of Raf-1. Compartmentalization of signaling molecules is increasingly being recognized as an important regulatory mechanism, in particular in the ERK and cAMP signaling systems (reviewed in references 15 and 18). It has been recently shown that serine 259 phosphorylation affects the subcellular localization of Raf-1 (8, 20). In a broader sense compartmentalization can also be perceived in terms of functional separation because of differential modifications. As the phosphopeptide maps show, the cAMP-induced increase in serine 43, 259, and 621 phosphorylation is not uniform, implicating that subpopulations of Raf-1 which bear different combinations of phosphorylations exist. It will be a challenge to sort out whether PKA inhibits Raf-1 solely via the serine 259 master site, as this study implies, or whether there is a subtle interplay between the three PKA target sites.
In addition to inhibiting Raf-1 by direct phosphorylation, PKA may also use an indirect route to prevent Raf-1 activation. The suppression of Raf-1 activation in detached cells caused by the loss of integrin engagement was also recently shown to result from the PKA-mediated inhibition of PAK (16). PAK has been described as the kinase which phosphorylates Raf-1 on serine 338 (17). Work with mutants has suggested that serine 338 phosphorylation is essential but not sufficient for the full activation of Raf-1 (9, 17, 23) and that S338 phosphorylation is indicative of, but does not quantitatively correlate with, Raf-1 activity (23). As shown in Fig. 4, we found that cAMP elevation reduced Raf-1 phosphorylation on serine 338, which conceivably could contribute to Raf-1 inhibition. Mutation of serine 259 completely prevented the downregulation of serine 338 phosphorylation by cAMP. These observations suggest that, rather than suppressing a serine 338 kinase or inducing a serine 338 phosphatase, the effect of PKA on serine 338 phosphorylation is regulated by the phosphorylation of serine 259. Since serine 259 regulates Raf-1 membrane localization and its interaction with Ras (8, 20), a plausible mechanism whereby phosphorylation of serine 259 reduces serine 338 phosphorylation is by diminishing the accessibility of Raf-1 to membrane-bound serine 338 kinases. These results also raise the possibility that in adherent cells PKA influences serine 338 phosphorylation through the modulation of serine 259 phosphorylation rather than through the inhibition of PAK. In summary, the inhibition of Raf-1 by PKA seems to utilize multiple layers of regulation and begins to unfold a complexity similar to that of Raf-1 activation itself.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Cancer Research Campaign and the DFG.
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FOOTNOTES |
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Present address: BioChip Technologies GmbH, GeneScan Europe AG, D-79108 Freiburg, Germany.
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