(Received for publication, July 3, 1996, and in revised form, October 3, 1996)
From the Friedrich Miescher Institute, P. O. Box
2543, CH-4002 Basel, Switzerland, and the
Department of Signal
Transduction, Parke Davis Research Division, Warner Lambert Company,
Ann Arbor, Michigan 48105
The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701).
Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.
Raf-1 is a ubiquitously expressed serine/threonine protein kinase that assumes a critical role in relaying proliferative and developmental signals initiated by cell-surface receptors to the nucleus (1-4). Genetic studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster as well as biochemical studies in vertebrate cells have elucidated that Raf-1 couples Ras to the MAP1 kinase cascade consisting of Raf-1 itself, the dual specificity MAP kinase kinases MKK1 and MKK2 (also termed MEK-1 and MEK-2), and the extracellular signal-regulated protein kinases (ERKs) or MAP kinases (5). The MAP kinases carry the signal to the nucleus, where they phosphorylate transcription factors capable of mediating changes in gene expression (6, 7).
While the regulation of MEK and MAP kinases by phosphorylation is well understood (8-10), the molecular mechanism(s) involved in Raf-1 regulation remain more obscure. The best understood aspects of Raf-1 kinase regulation are the initial events that precede its mitogen stimulation. Thus, growth factor receptor-induced activation of the mammalian nucleotide exchange factor mSOS, mediated by adapter proteins such as Shc and Grb2, stimulates the conversion of Ras from the inactive, GDP-bound state to the active, GTP-bound state. Activated Ras in turn directly interacts with Raf-1, resulting in the translocation of Raf from the cytoplasm to the plasma membrane. These conclusions are based on the findings that Raf-1 physically interacts with Ras (11-14) and that Raf-1 is associated with the plasma membrane in cells expressing oncogenic Ras (15-17). Importantly, Raf and Ras transiently interact in mammalian cells upon extracellular stimulation (18), providing a potential molecular basis for the transient membrane translocation of Raf observed in serum-stimulated cells (17). The physical interaction of Raf-1 with activated Ras in vitro, however, is insufficient for stimulation of Raf-1 kinase activity (Ref. 19; data not shown). Artificial plasma membrane localization, on the other hand, is sufficient for Raf kinase activation in a Ras-independent manner (15, 20). Taken together, these observations suggest that membrane localization of Raf-1 is necessary for its activation and that the role of Ras is to recruit Raf-1 to the membrane for activation by an as yet elusive mechanism.
Hyperphosphorylation of Raf-1 is a cellular response common to a wide range of physiological stimuli that activate the Raf-1/MEK/MAP kinase pathway and may be relevant to the process of Raf activity regulation (1, 3, 4). Phosphorylation of tyrosine residues 340 and 341 has been reported to be involved in the stimulation of Raf kinase activity in some cellular systems (21-23). However, in many cellular systems, Raf kinase activation occurs in the apparent absence of Raf-1 tyrosine phosphorylation. In fact, even under circumstances when tyrosine phosphorylation is observed, the majority of phosphorylation events occur on serine residues (1). Thus, Raf-1 is phosphorylated in vivo at Ser-43, Ser-259, Ser-499, and Ser-621 (24-26). While constitutive phosphorylation at Ser-621 might be necessary for Raf functionality, phosphorylation at Ser-259 and Ser-499 has been implicated in the protein kinase C-mediated activation of Raf (24, 26, 27). Raf phosphorylated at Ser-43 by cAMP-dependent protein kinase displays a decreased affinity for Ras in vitro. This could contribute to the negative regulation of the Raf-1/MEK-1/MAP kinase by cAMP-elevating agents observed in some cellular systems (28). Furthermore, phosphorylation of Raf on Thr-269 mediated by a ceramide-activated protein kinase has been implicated in tumor necrosis factor-induced Raf kinase activation (29).
The exact molecular relationship between these phosphorylation events and those that are associated with the characteristic retardation of the electrophoretic mobility of Raf-1 following stimuli that activate the Ras/Raf-1/MEK/MAP kinase pathway is not clear. Initial experiments employing serine/threonine-specific phosphatases suggested a causal relationship between serine/threonine phosphorylation and Raf mobility shift as well as Raf kinase activation (30). However, recent evidence argues against such a positive relationship between these two events (17, 31-34). We have previously observed that the kinetics of mitogen-stimulated Raf kinase activation and Raf mobility shift are poorly correlated events. Thus, while mitogen-stimulated Raf-1 kinase activation occurs rapidly and is transient in nature, the decrease in Raf-1 protein mobility only becomes apparent at later times and coincides with a marked attenuation of mitogen-stimulated Raf-1 kinase activity (17).
Here we show that the mobility shift-associated hyperphosphorylation of Raf is associated with a decreased affinity of this form of Raf for the plasma membrane. Since plasma membrane localization is a positive determinant for Raf kinase activity, this post-translational modification might represent a molecular mechanism accounting, at least in part, for the attenuation of Raf kinase activity following mitogen stimulation. We further demonstrate that hyperphosphorylation of Raf can be blocked by a specific inhibitor of MEK activation and that this correlates with restoration of plasma membrane-bound Raf to control levels. Thus, the mobility shift-associated hyperphosphorylation of Raf is likely a consequence of activating the downstream components in the MAP kinase cascade and might represent a negative feedback mechanism contributing to the desensitization of this signaling pathway.
[32P]orthophosphate and
[-32P]ATP were purchased from Amersham. FCS and Ham's
F-12 medium were from Life Technologies, Inc. Restriction enzymes were
from Boehringer Mannheim. Polyvinylidene difluoride membranes
(Immobilon-P) were obtained from Millipore Corp. Protein A-Sepharose
and Protein G-Sepharose were from Sigma. Recombinant kinase-inactive human MKK1 carrying an N-terminal hexahistidine tag
(His6-MKK(K97M)) was prepared by
Ni2+-NTA-agarose (Pharmacia Biotech Inc.) affinity
chromatography followed by DEAE-Sepharose (Pharmacia) chromatography as
described previously (35). The monoclonal
-Flag antibody M2 was
obtained from Kodak Scientific Imaging Systems, while the rabbit
-Raf (C-12) antiserum sc-133 was from Santa Cruz Biotechnologies,
Inc. Okadaic acid was purchased from LC Laboratories (Woburn, MA). D609
was from Kamiya Biomedical Co., and PD98059 was obtained from
Parke-Davis Research Division, Warner-Lambert Co. The
-MEK antiserum
2880 recognizing murine MEK1 (17) and the ERK2-specific antiserum (36)
have been previously described.
The plasmid pCMV-Flag-Raf-1
encoding human c-Raf-1 fitted with the Flag epitope tag (Kodak) at the
N terminus has been described previously (17). The retroviral vector
pBabe-Puro/Flag-Raf-1 was constructed by ligating the
EcoRI/XbaI-insert from pCMV-Flag-Raf-1 into
EcoRI/SalI-digested pBabe-Puro (37) followed by
blunt-ending and repeated ligation. CHO cells were maintained in Ham's
F-12 medium supplemented with 8% (v/v) FCS. The ecotropic helper-free packaging cell line E (37) was maintained in Dulbecco's modified Eagle's medium containing 8% FCS. CHO cells stably expressing Flag-Raf-1 were established by retroviral gene transfer. Briefly,
E
cells were transfected with pBabe-Puro/Flag-Raf-1, or pBabe-Puro (Vector Control) by lipofection using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocol. After 24 h, the transfected cells were placed in medium containing 2 µg/ml puromycin (Fluka). Recipient CHO cells were incubated for 20 h with
tunicamycin (0.3 µg/ml) to abrogate the resistance of these cells to
retroviral infection (38). Virus-containing medium collected from pools of puromycin-resistant
E cells was then used to infect the
tunicamycin-treated CHO cells in the presence of 8 µg/ml polybrene
(Sigma). Two days after infection, the infected CHO
cells were subjected to selection in medium containing 20 µg/ml
puromycin. In contrast to the previously described CHO/pCMV/Flag-Raf
cell line (17), the expression of the different Flag-Raf-1 proteins was
maintained at a constant level in the continuous presence of selective
pressure, as judged by Western blot analysis using the rabbit
-Raf-1
(C-12) antiserum sc-133. All experiments reported here were performed
with pools of CHO cells either stably transfected with pBabe-Puro (CHO)
or pBabe-Puro/Flag-Raf-1 (CHO/Flag-Raf-1)
CHO cells grown in
100-mm dishes were preincubated for 60 min in phosphate- and serum-free
Ham's F-12 medium at 37 °C. The cells were then incubated for
12 h in 10 ml of phosphate- and serum-free Ham's F-12 medium
containing 0.2 mCi/ml [32P]orthophosphate. At the end of
the labeling period, the cells were treated with 20% FCS or 10 µg/ml
D609 for defined times. The cells were then washed quickly in
phosphate-buffered saline and harvested in 500 µl of lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 2 mM
EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 25 mM -glycerophosphate) at 4 °C. Clarified supernatant
was prepared by sedimenting insoluble material by centrifugation at
14,000 × g for 15 min at 4 °C. The supernatant was
precleared by incubation with 20 µl of protein G-Sepharose for 30 min
at 4 °C. The precleared supernatant was then incubated for 1 h
at 4 °C with 2 µg of
-Flag monoclonal antibody M2 immobilized
on 20 µl of protein G-Sepharose. The immunoprecipitates were washed
once with lysis buffer, twice with 0.5 M LiCl, 0.1 M Tris (pH 7.4), 25 mM
-glycerophosphate,
and once with 10 mM Tris (pH 7.4), 25 mM
-glycerophosphate. The samples were boiled for 5 min supplemented
with 50 µl of 2 × sample buffer containing 100 mM
dithiothreitol and analyzed by SDS-PAGE (7% gel).
Phosphorylated proteins were detected by autoradiography, and the proteins of interest were consecutively excised from the dried gel. Tryptic digestion and two-dimensional phosphopeptide analysis of Raf phosphoproteins were performed basically as described by Stover et al. (39). Briefly, dried gel slices were rehydrated in 50% acetonitrile, air-dried for 4 h, and then soaked in 100 µl of 100 mM NaHCO3 containing 5 µg sequencing grade trypsin (Boehringer Mannheim). The digestion was left to proceed overnight at 30 °C, and the phosphopeptides were then eluted by rocking in 50% acetonitrile for 4 h. After lyophilization, phosphopeptides were mapped using the HTLE-7000 peptide mapping system according to the manufacturer's protocol (CBS Scientific Co.). Briefly, lyophilized peptides were resuspended in 10 µl of pH 1.9 buffer (2.2% formic acid and 7.8% acetic acid). 2 µl (~1000-1500 cpm) of the sample was spotted on cellulose TLC plates (Merck), and electrophoresis at pH 1.9 and 1000 V proceeded for 45 min. After drying, the plates were placed in a chromatography tank containing phosphochromatography buffer (37.5% n-butanol, 25% pyridine, and 7.5% acetic acid) for 12-16 h for separation in the second dimension. Phosphopeptides were detected by autoradiography. Phosphoamino acid analysis was performed by partial acid hydrolysis (1 h at 110 °C in 6 M HCl) and thin layer electrophoresis as described (40).
In Vitro Protein Kinase AssaysCHO cells were grown in 100-mm dishes, starved for 18 h in serum-free Ham's F-12 medium prior to the experiment, and then treated as indicated in the figure legends. The cells were then washed quickly with ice-cold phosphate-buffered saline and harvested in 500 µl of lysis buffer. Clarified supernatant was prepared by sedimenting insoluble material by centrifugation at 14,000 × g for 10 min at 4 °C.
Raf protein kinase activity was measured using an immunocomplex protein
kinase assay with recombinant kinase-inactive human MKK1,
His6-MKK(K97M), as an exogenous substrate basically as
described previously (17). Clarified supernatant was incubated for
1 h with 2 µg of the -Flag monoclonal antibody M2 immobilized
on 20 µl of protein G-Sepharose (4 °C). The immunoprecipitates
were then washed three times with lysis buffer and twice with kinase buffer (25 mM Hepes (pH 7.5), 25 mM
-glycerophosphate, 1 mM dithiothreitol, 5 mM
MnCl2, 15 mM MgCl2). The washed
Flag-Raf-1 immunoprecipitates were incubated with 50 µM
[-32P]ATP (10 Ci/mmol) and 200 ng of
His6-MKK(K97M) (~70-80% pure) in a final volume of 50 µl for 30 min at 30 °C. The reactions were terminated by the
addition of 50 µl of 2 × sample buffer and then boiled for 5 min prior to analysis by SDS-PAGE (7% gel). The gel was dried, and the
phosphorylation of MKK was quantitated using a PhosphorImager and
ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA).
MAP kinase (ERK2) activity was measured in an immunocomplex kinase
assay using myelin basic protein as an exogenous substrate as described
previously (36) with minor modifications. ERK2 was immunoprecipitated
from clarified cell lysates obtained as described above using 2 µl of
specific antiserum (36) adsorbed to Protein A-Sepharose.
Immunocomplexes were washed three times with lysis buffer and once with
kinase buffer (30 mM Tris-HCl (pH 8.0), 20 mM
MnCl2, and 2 mM MgCl2) and then
incubated in a final reaction volume of 30 µl together with 15 µg
of myelin basic protein, 10 µM unlabeled ATP, and 0.1 µM [-32P]ATP (1200 Ci/mmol) for 30 min
at 37 °C. Reactions were terminated by the addition of sample
buffer, proteins were subjected to SDS-PAGE (15% gel), and
phosphorylation of myelin basic protein was quantified with a
PhosphorImager and ImageQuant software (Molecular Dynamics Inc.,
Sunnyvale, CA).
PKA activity in crude cell extracts was assayed using kemptide as a substrate basically as described (41).
In Vitro Dephosphorylation of Raf and Determination of Phosphatase ActivitiesPP1 and PP2A were partially purified from
rabbit skeletal muscle as described previously (42). 15 min prior to
use, PP1 and PP2A were diluted 10-fold into dephosphorylation buffer
(50 mM Tris-HCl (pH 7.5), 0.1% -mercaptoethanol, 3 mM MnCl2, 0.1 mM EDTA, 50 mM NaCl). In some assays, dephosphorylation buffer was
supplemented with 1 µM okadaic acid to inactivate
phosphatases. Flag-Raf-1 was immunoprecipitated from CHO cells treated
for 60 min with 10 µg/ml D609 as described above. Immunoprecipitates were washed twice with lysis buffer and twice with phosphatase buffer
(50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, and 50 mM NaCl). The washed immunoprecipitates were incubated for
60 min at 30 °C in a final volume of 50 µl (50 mM
Tris-HCl (pH 7.5), 0.1%
-mercaptoethanol, 1 mM
MnCl2, 0.1 mM EDTA, 50 mM NaCl)
with naive or okadaic acid-pretreated, diluted PP1 and PP2A.
The effects of D609 on the catalytic activity of PP1 and PP2A in vivo were determined using clarified cell lysates prepared in the absence of phosphatase inhibitors, while the in vitro effects of D609 were measured using partially purified PP1 and PP2A catalytic subunit. Assays were performed with phosphorylase a as a substrate as described (42).
Western Blot AnalysisTotal cellular proteins or
immunoprecipitates were resolved by SDS-PAGE and electrophoretically
transferred to polyvinylidene difluoride membranes (Immobilon-P) and
analyzed by Western blotting using the polyclonal -Raf-1(C-12)
antiserum sc-133 (Santa Cruz Biotechnology, Inc.). Immunocomplexes were
visualized with corresponding secondary antibodies using the enhanced
chemiluminescence procedure (Amersham International PLC).
Subcellular
fractionation was performed essentially as described (43) with minor
modifications. Cells were washed with ice-cold phosphate-buffered
saline, disrupted by sonification in hypotonic buffer (25 mM Hepes, pH 7.5, 25 mM -glycerophosphate, 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride,
and 10 µg/ml leupeptin), and centrifuged for 30 min at 100,000 × g yielding the cytosol (S100) and the membrane pellet
(P100). The latter was resuspended in hypotonic buffer containing 1%
Triton X-100, sonicated, and subsequently recentrifuged for 30 min at
100,000 × g in order to obtain a clarified Triton
X-100-extractable membrane fraction.
When analyzing the subcellular distribution of Raf in vitro, the membrane pellet obtained from parental CHO cells was resuspended in hypotonic buffer lacking Triton X-100. These resuspended membranes were then incubated for 30 min at 4 °C with the cytosolic (S100) fraction obtained from Flag-Raf-expressing CHO cells. Subsequently, the "reconstituted hypotonic extract" was subjected to subcellular fractionation as described above to obtain the cytosolic and clarified Triton X-100-extractable membrane fractions.
Raf-1 shares general structural features with members of the protein kinase C family, including the presence of a zinc-binding cysteine-rich domain termed the "zinc finger" (44). The corresponding motifs in protein kinase C mediate the binding of diacylglycerols and phorbol esters necessary for its activation (45). This structural similarity has led to the hypothesis of allosteric regulation of Raf-1 by a lipid co-factor (44).
In order to investigate a potential role for lipid second messengers in
mitogen-stimulated Raf-1 kinase activation, we tested the effect of
inhibitors of different phospholipases on serum-stimulated Raf-1 kinase
activity. Ligand-induced activation of the Ras/Raf-1/MEK/MAP kinase
pathway is tightly associated with a hyperphosphorylation-mediated decrease in the electrophoretic mobility of Raf-1 upon
SDS-polyacrylamide gel electrophoresis. Thus, in our initial
experiments, we examined the effect of the various inhibitors on the
electrophoretic mobility of Raf-1 in a CHO cell line overexpressing an
epitope-tagged version of Raf-1 (CHO/Flag-Raf-1). While none of the
tested inhibitors influenced the serum-induced mobility shift of Raf-1
(data not shown), control experiments performed in the absence of serum stimulation revealed that treatment with D609, a xanthate derivative that inhibits phosphatidylcholine-specific phospholipase C (PC-PLC) (46) caused a mobility shift of Raf-1 in a dose- and
time-dependent manner (Fig. 1, A
and B, respectively). The kinetics of appearance of Raf
species with retarded mobility lagged slightly behind those observed in
response to serum treatment of CHO/Flag-Raf-1 cells (Fig.
1C). We next compared the Raf kinase activity of cells
treated with serum or D609 for 15 and 30 min, respectively. While serum treatment consistently enhanced Raf protein kinase activity, incubation of cells with D609 failed to do so, despite the induction of a comparable mobility shift in Raf. On the contrary, in D609-treated cells we generally observed a reduction of Raf kinase activity below
the basal level observed in serum-starved cells (Fig. 1D). However, the extent of reduction varied among the experiments (0-89%;
see "Discussion"). Potassium ethyl xanthate (PEX), a
biologically inactive structural homologue of D609, affected neither
Raf-1 mobility (Fig. 1B) nor Raf-1 protein kinase activity
when applied at equimolar concentration (data not shown).
These results clearly demonstrate that the marked electrophoretic mobility shift of Raf-1 observed in response to mitogens and other physiological stimuli is not required for Raf-1 kinase activation and, hence, that the Raf mobility shift is an inadequate monitor for the activation state of Raf.
D609- and Serum-induced Raf-1 Mobility Shift Is Due to Hyperphosphorylation and Occurs on Identical SitesThe molecular
relationship between the multiple Raf phosphorylation events and the
the Raf mobility shift is not known. Thus, it is possible that
differences in the phosphorylation pattern, which do not translate into
an altered electrophoretic mobility of Raf, account for the
differential modulation of Raf kinase activity by serum and D609. We
therefore analyzed the phosphorylation state of Raf following serum or
D609 treatment. To this end, Flag-Raf-1 was immunoaffinity-purified
from CHO/Flag-Raf-1 cells metabolically labeled with
[32P]orthophosphate. Raf-1 phosphoproteins were then
analyzed by gel electrophoresis and autoradiography. As shown in Fig.
2A, serum as well as D609 elicited an
increase in the total phosphate content of Raf-1 as compared with the
untreated control (1.5- and 1.4-fold, respectively). Phosphoamino acid
analysis revealed that both basal phosphorylation as well as D609- and
serum-induced hyperphosphorylation occurred predominantly on serine and
to a minor extent on threonine, while tyrosine phosphorylation was not
detected (data not shown). Raf-1 phosphoproteins were further analyzed
by tryptic two-dimensional phosphopeptide mapping (Fig. 2B).
In serum-starved cells, two major phosphopeptides were present (peptides 3 and 4). Serum treatment of cells resulted in the enhanced phosphorylation or de novo appearance of several peptides
(peptides 5 and 6, and peptides 1, 2, and 7, respectively). The pattern of phosphopeptides induced by D609 was virtually identical to that
induced by serum. Taken together, the absence of detectable differences
in the hyperphosphorylation pattern of Raf induced by serum and D609,
in contrast to the differential effect of these treatments on Raf
kinase activity (Fig. 1), strongly implies that these phosphorylation
events are not per se involved in the regulation of the
enzymatic activity of Raf.
D609-induced Hyperphosphorylation of Raf-1 Correlates with Decreased Membrane Localization of Raf-1 in Vivo and in Vitro
Plasma membrane localization represents an important
determinant in Raf-1 kinase activation. It is thus conceivable that
mobility shift-associated hyperphosphorylation could be involved in Raf kinase activity attenuation by negatively modulating plasma membrane localization of Raf. In order to test this hypothesis, we first investigated the effect of D609 on the subcellular localization of
Raf-1 in vivo. As shown in Fig.
3A, incubation of cells with D609 for times
that lead to a Raf mobility shift (see Fig. 1B) induced a
decrease in plasma membrane-associated Raf-1. Although this correlation
implies a direct mechanistic relationship between these two phenomena,
one cannot rule out from these experiments the possibility that the
decrease in plasma membrane localization of Raf-1 is due to effects of
D609 on plasma membrane components regulating the association of Raf
with this subcellular compartment rather than being a direct
consequence of Raf hyperphosphorylation.
In order to discount a contribution of modulated plasma membrane components in the observed phenomenon, we analyzed the membrane association of Raf-1 in vitro by employing a cell-free reconstitution system. Serum-starved parental CHO cells were subjected to subcellular fractionation as described above, except that the initial P100 pellet was resuspended in hypotonic buffer devoid of detergents. Portions of these "naive" membranes were then mixed with cytosolic fractions obtained from either quiescent or D609-treated CHO/Flag-Raf-1 cells. After incubation on ice for 30 min, these "reconstituted" hypotonic extracts were subjected to standard subcellular fractionation, and distribution of Raf-1 between the cytosolic and the membrane fractions was analyzed as described above. However, in order to facilitate visual quantification, proteins were separated on 12% gels to prevent resolution of individual mobility-shifted Raf species observed on 7% gels. In addition, exposure times during Western blot analysis using the enhanced chemiluminescence detection method were adjusted to give similar intensities for immunodetected Raf-1 recovered from the cytosolic fraction and the particulate fraction. Judging from the difference in exposure times during enhanced chemiluminescence-based immunodetection of Raf-1, about 5% of Flag-Raf-1 was recovered from the Triton X-100-extractable membrane fraction in this assay. This ratio is in good agreement with that estimated for the in vivo distribution of Raf-1 based on subcellular fractionation of whole cells (Ref. 18; Fig. 3A). As shown in Fig. 3B, the amount of Flag-Raf-1 recovered in the membrane fraction was reduced when cytosolic extracts from D609-treated cells were compared with those of control cells. This was not due to differences in the amount of Raf-1 present in the S100 fractions of serum-starved and D609-treated cells (Input). Importantly, as shown in Fig. 3C, a similar reduction of Raf recovered from the particulate fraction was observed when cytosolic extracts obtained from serum-treated cells were analyzed in this reconstitution assay. Thus, these data strongly suggest that mobility shift-associated hyperphosphorylation in response to mitogen stimulation of cells negatively regulates plasma membrane localization of Raf-1. Since the kinetics of appearance of mobility-shifted Raf correlate with a reduction in Raf-1 kinase activity following an initial maximal stimulation in response to serum treatment (17), the proposed mechanism may account, at least in part, for the attenuation of Raf kinase activity following mitogen stimulation.
Inhibition of D609-induced MAP Kinase Activation by PD98059 Restores Membrane-associated Raf Protein to Control LevelsThe
molecular events that lead to hyperphosphorylation of Raf-1 in response
to serum- or D609-treatment of CHO cells are enigmatic. It is
conceivable, however, that these treatments result either in the
activation of a latent Raf-1 kinase kinase or inhibition of a
constitutive Raf-1 kinase phosphatase. We therefore initially investigated the effect of D609 on the cellular activity of several protein kinases that had been reported to phosphorylate Raf in vitro. While PKA activity was not altered in response to D609 (data not shown), surprisingly, ERK2 activity was strongly stimulated with kinetics and to an extent similar to that induced by FCS (Fig.
4). Incubation of cells with potassium ethyl xanthate, a biologically inactive D609 homologue, did not result in ERK2
activation. The kinetics of stimulation of ERK2 activity in response to
D609 and FCS preceded those of the appearance of the Raf mobility
shift, which ensued with slightly different lag times (Fig.
1B; see also "Discussion"). Furthermore, Western blot
analysis of lysates prepared from parallel dishes using a MEK-specific
antiserum revealed a time-dependent retardation of the
electrophoretic mobility of MEK in response to D609 (data not shown).
This mobility shift is believed to correlate with MEK activation (47).
These observations thus raise the intriguing possibility that
hyperphosphorylation of Raf-1 in response to D609 is the consequence of
activation of the MAP kinase cascade at a step distal to Raf, probably
at the level of MEK.
We therefore investigated the effect of blocking activation of the MAP
kinase pathway on the D609-induced mobility shift and subcellular
relocalization of Raf. Pretreatment of CHO/Flag-Raf-1 cells with the
MEK activation inhibitor PD98059 (48) reduced the basal activity of
ERK2 and completely blocked D609-induced ERK2 activation (Fig.
5C). Analysis of the subcellular localization of Raf revealed that preincubation of cells with PD98059 completely prevented the D609-induced reduction of membrane-associated Raf (Fig.
5A). Significantly, this correlated with a reversion of the
D609-induced mobility shift of Raf (Fig. 5B). Furthermore, analysis of the relative levels of the different forms of
hyperphosphorylated Raf species revealed an inverse correlation between
the degree of mobility shift-associated hyperphosphorylation and
membrane association of Raf (Fig. 5B, lane
3).
In summary, the results presented here strongly suggest that mobility shift-associated hyperphosphorylation of Raf (a) is a consequence of activation of components in the MAP kinase pathway downstream from Raf and (b) reduces its association with the plasma membrane. Since membrane localization represents a crucial role in Raf kinase activation and since D609- and serum-induced hyperphosphorylation of Raf-1 occur on identical sites, hyperphosphorylation-mediated reduction of plasma membrane association of Raf may represent a molecular mechanism accounting for the "down-regulation" of Raf-1 kinase activity that follows its initial mitogen stimulation.
The molecular mechanism(s) involved in the regulation of Raf-1 protein kinase activity are complex and still incompletely understood. The initial events leading to activation of Raf-1 seem to involve Ras-mediated membrane translocation and phosphorylation on tyrosine residues (21-23). Despite the obvious physiological importance, little is known about the mechanism(s) involved in turning Raf-1 kinase "off." Treatment of cells with a wide range of physiological stimuli results in hyperphosphorylation of Raf. While phosphorylation and activation of Raf by protein kinase C (24, 27) and/or tyrosine kinases such as Src and Lck (22, 23) might represent early receptor-stimulated events, these post-translational modifications are unlikely to account for the characteristic retardation of the mobility of Raf upon SDS-PAGE. The nature and significance of these mobility shift-associated hyperphosphorylation events have been controversial. Although experiments with serine/threonine-specific phosphatases initially suggested a stimulatory role for these post-translational modifications of Raf-1 (30), recent evidence has challenged this hypothesis (17, 31-34). First, the kinase activity negatively affected by phosphatase treatment in the experiments of Kovacina et al. (30) is unlikely to reflect Raf-1, since the peptide used, Syntide-2, is not a Raf-1 substrate (49). While the more recent demonstration of Raf-1 kinase inactivation following phosphatase treatment was performed using the physiological Raf-1 substrate MEK, it was not demonstrated whether this correlated with corresponding changes in the electrophoretic mobility of Raf-1 (50). Furthermore, only highly purified Raf-1 devoid of the putative chaperone proteins hsp90 and 14-3-3, which form a complex with Raf-1 in vivo (17, 51-54), was susceptible to inactivation by phosphatase treatment. Second, a comparison of the time course of activation and mobility shift of Raf in response to various agonists has shown that these processes are poorly correlated (17, 33, 55). Thus, we have previously demonstrated that the transient activation of Raf protein kinase in response to serum stimulation of cells correlated with a transient membrane translocation of Raf and occurred in the absence of a Raf mobility shift. On the contrary, in accordance with the the kinetics of serum-stimulated Raf hyperphosphorylation presented here, the appearance of the Raf mobility shift correlated with an attenuation of Raf kinase activity and was paralleled by a reduction in the amount of membrane-associated Raf (17).
Here we show that mobility shift-associated hyperphosphorylation of Raf induced by treatment of cells with D609, a PC-PLC inhibitor, correlates with a decrease in membrane-localized Raf below that observed in untreated cells. The results obtained in the cell-free reconstitution experiments demonstrated that this phenomenon is not due to effects of D609 on the plasma membrane. Importantly, hyperphosphorylated Raf from serum-treated cells displayed a similar decreased tendency to localize to the membrane fraction in this in vitro assay. Furthermore, comparison of the phosphopeptide maps revealed no significant differences in the phosphorylation state of Raf in D609- and mitogen-stimulated cells. Taken together, these results suggest that negative modulation of membrane localization mediated by mobility shift-associated hyperphosphorylation of Raf represents an important molecular mechanism that could account, at least in part, for the attenuation of Raf kinase activity that follows its initial mitogen activation.
The results presented here imply that mobility shift-associated hyperphosphorylation does not directly regulate the catalytic activity of Raf, but rather exerts its effect indirectly, by modulating the ability of Raf to associate with the plasma membrane, the residence of the elusive Raf-"activating principle." Consistent with this hypothesis, dephosphorylation of mobility-shifted Raf, isolated from D609 or serum-treated cells, by incubation with either of the serine/threonine-specific protein phosphatases PP1 or PP2A, did not significantly alter the in vitro protein kinase activity of Raf (data not shown). Thus, total cellular Raf kinase activity might be the result of the stimulus-independent intrinsic catalytic activity of Raf and a component determined by the stimulus-dependent levels of a membrane-localized "activating principle," such as a putative lipid co-factor. Following serum starvation, the levels of this "activating principle" are likely low, but not necessarily absent. Fluctuations in the levels of the Raf activator in serum-starved cells might explain the varying degrees of reduction (0-89%) below basal Raf kinase activity observed in D609-treated cells. In serum-stimulated cells, on the other hand, the level of the Raf activator is likely significantly increased and might account for the intermediate, rather than basal or reduced, Raf kinase activity of mobility-shifted Raf observed at later times following mitogen stimulation.
It has previously been demonstrated in NIH3T3 cells that inhibition of endogenous PC-PLC by D609 blocks activation of Raf-1 in response to mitogenic growth factors (56). However, in contrast to our results obtained with CHO cells, Cai et al. (56) failed to observe a Raf-1 mobility shift in response to treatment of serum-starved NIH3T3 cells with D609. This is probably due to the different cell system or the higher concentration of D609 (35 µg/ml) used in their experiments. While we observed a pronounced Raf protein mobility shift at low concentrations of D609, the shift was less pronounced at higher concentrations (>35 µg/ml; Fig. 1A). Thus, the inhibition of serum-stimulated Raf-1 kinase activation at concentrations of D609 higher than 35 µg/ml might not rely on the effect of hyperphosphorylation of Raf-1 proposed here but might be the consequence of inhibiting mitogen-stimulated PC-PLC activity. Furthermore, it remains to be established whether the D609-induced hyperphosphorylation of Raf-1 reported here is a consequence of inhibition of PC-PLC or whether D609 modulates targets distinct from PC-PLC.
Several recent reports support the hypothesis that mobility shift-associated hyperphosphorylation of Raf is a feedback consequence of activation of the MAP kinase pathway. Hormone-induced activation of an estrogen-dependent form of oncogenic Raf-1, stably expressed in quiescent 3T3 cells, resulted in increased activity of MEK and, to a lesser extent, MAP kinase. Under these conditions, a mobility shift-associated hyperphosphorylation of the endogenous pool of Raf-1 was observed without a concomitant stimulation of Raf-1 kinase activity (32). Ueki et al. (33) reported that overexpression of MAP kinase in CHO cells overexpressing human insulin receptor (CHO/IR) led to hyperphosphorylation of Raf-1, which, significantly, was accompanied by a decrease in the MEK kinase activity of Raf-1. The clearest evidence arguing for an involvement of MEK or a downstream component in the hyperphosphorylation of Raf, however, has been provided by the demonstration that blocking activation of MEK by PD98059, or by overexpression of a dominant negative MEK mutant, abolishes the insulin-stimulated mobility shift of Raf in CHO/IR cells (57). Furthermore, Alessi et al. (48) demonstrated that PD98059 blocks the platelet-derived growth factor-stimulated hyperphosphorylation of Raf and, interestingly, prevents the attenuation of Raf kinase activity that occurred following its initial stimulation. Here, by the use of an agent that activates MAP kinase pathway components downstream of Raf in a PD968059-sensitive manner, we present further evidence in favor of the hypothesis that mobility shift-associated hyperphosphorylation of Raf in response to mitogens, and probably other stimuli, occurs in a negative feedback manner. First, time course analyses demonstrated that D609- and FCS-stimulated activation of ERK2 precedes, rather than ensues, the appearance of mobility-shifted Raf. The fact that D609-induced activation of ERK2 and hyperphosphorylation of Raf is PD98059-sensitive strongly suggests the involvement of a component of the MAP kinase pathway at a point distal to Raf in this phenomenon. While MEK-1 is unable to phosphorylate recombinant Raf-1 in vitro (data not shown) and thus is an unlikely Raf-1 kinase kinase, the classical MAP kinase isoforms are able to phosphorylate Raf-1 in vitro (Refs. 58 and 59; data not shown). While this is consistent with MAP kinase being a potential mediator of Raf-1 hyperphosphorylation in vivo, phosphorylation of Raf-1 by the classical MAP kinases in vitro fails to cause the characteristic mobility shift of Raf-1 (data not shown). Thus, it is likely that the putative Raf kinase kinase responsible for mobility shift-associated hyperphosphorylation of Raf lies downstream of ERK2. The molecular mechanism of how D609 leads to the activation of ERK2 (and MEK; data not shown) and whether D609 affects additional components in this pathway remains to be established. Differential modulation of component(s) downstream of ERK2 by D609 and FCS might explain the observation that, while D609 and FCS stimulated ERK2 activity with similar kinetics, the time course of appearance of mobility-shifted Raf elicited by D609 was somewhat slower than that induced by FCS.
PKA phosphorylates Raf-1 on sites that either negatively affect the Raf-1/Ras interaction (i.e. Ser-43) or inhibit the catalytic activity of Raf (28). PKA is thus a potential Raf kinase kinase whose activation might account for the phenomenon reported here. Indeed, comparison of the phosphopeptide maps presented here with those reported by Morrison and co-workers (26) suggests that D609 as well as serum induce the phosphorylation of Raf on Ser-43 (Fig. 2, phosphopeptide 1 and/or 2). However the in vivo phosphorylation of this site is probably not mediated by PKA, since we did not observe activation of PKA in response to either stimulus (data not shown). Furthermore, it remains to be established whether mobility shift-associated hyperphosphorylation interferes with membrane localization of Raf at the level of interaction with Ras or other membrane components. Interestingly, it has been suggested that membrane anchoring of Raf, in contrast to its initial translocation, occurs in a Ras-independent manner (20).
An alternative mechanism that could account for Raf-1 hyperphosphorylation is the negative modulation of phosphatases involved in the regulation of MAP kinase signal transduction. PP2A has been implicated in the negative regulation of MEK and MAP kinases (60) and is able to dephosphorylate Raf-1 in vitro (data not shown). Cellular inactivation of PP2A might therefore not only result in enhanced phosphorylation and activation of MEK and MAP kinase, but might at the same time allow more efficient Raf-1 hyperphosphorylation. Interestingly, it has been reported that PP2A is inhibited in response to growth factor or insulin stimulation of cells, likely as a consequence of tyrosine and/or threonine phosphorylation of the catalytic subunit of PP2A (61, 62). While D609 failed to inhibit the catalytic activity of PP2A or PP1 in vivo as well as in in vitro (data not shown), it might alter the substrate specificity or subcellular localization of PP2A, two parameters that might be controlled by the association of the catalytic subunit with different regulatory subunits (63, 64). Furthermore, the involvement of other serine/threonine-specific phosphatases in the regulation of Raf hyperphosphorylation cannot be ruled out. The strong amplification potential of the MAP kinase cascade (48), together with the inhibition of a serine/threonine-specific phosphatase(s), might explain the apparent paradox of D609-mediated MEK and MAP kinase activation in the absence of Raf kinase stimulation. Alternatively, other MEK kinases, such as MEKK-1 (65) or Tpl-2 kinase (66), might be involved in the D609-induced response.
Resetting signaling cascades to "default values" following stimulation is crucial for any cellular system to be able to appropriately respond to changes in its environment. Attenuation of signal transduction via the MAP kinase cascade is achieved at various levels and by different mechanisms. Some of the desensitization mechanisms are built into the signal transducer itself. Thus, Ras can revert to its inactive state by virtue of its intrinsic GTPase activity, a function that is further enhanced in vivo by GTPase-activating proteins (67). Other inactivation processes are implemented as a consequence of signal transduction through the MAP kinase pathway in a feedback manner. For example, insulin-stimulated Ras activation is transient despite the continuous activation of the insulin receptor tyrosine kinase activity and prolonged Shc tyrosine phosphorylation (57). This phenomenon appears to be, at least in part, due to MAP kinase-mediated phosphorylation of the Ras nucleotide exchange factor, mSOS, which results in its dissociation from the adapter protein Grb2 and its functional inactivation (57, 68). Based on the results presented here, we suggest that mobility shift-associated hyperphosphorylation of Raf, by virtue of decreasing its ability to associate with the plasma membrane, represents an additional molecular feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade. This mechanism might be of particular importance, since the results obtained in the in vitro reconstitution experiments strongly suggest that this process is independent of the Ras activation state as well as potential plasma membrane-localized Raf modulators.
Hyperphosphorylation of Raf is invariably associated with activation of
the Ras/Raf/MEK/MAP kinase cascade. While some phosphorylation events
may directly modulate the catalytic activity of Raf, the data presented
here point to a novel mechanism of Raf kinase regulation that involves
hyperphosphorylation-mediated subcellular relocalization of Raf-1, as
schematically summarized in Fig. 6. In resting cells, hypophosphorylated Raf-1 resides in the cytoplasm in a latent, activation-competent state. Upon mitogen stimulation, a fraction of the
cytoplasmic Raf-1 pool is recruited by GTP-bound Ras to the plasma
membrane, where Raf-1 becomes activated in an as yet ill defined
fashion that might include stimulatory phosphorylation events, putative
lipid second-messengers, or both. At a later stage following serum
stimulation, the majority of Raf-1 becomes hyperphosphorylated on sites
that decrease its affinity for the plasma membrane as a consequence of
activation of downstream components in the MAP kinase signaling
cascade. This in turn reduces the amount of Raf-1 available for further
activation at the plasma membrane. Following this desensitization
period, a hypothetical phosphatase might be responsible for converting
Raf-1 to an activation-competent state, thus resetting the system to a
latent state ready to respond to new signals.
Dr. Roger J. Davis (Howard Hughes Medical
Institute, Program in Molecular Medicine, Worcester, MA) is thanked for
plasmid pCMV/Flag-Raf-1 as well as -MEK antiserum 2880. Escherichia coli strain BL21(DE3)pLysS transformed with
pKH-1 encoding human MKK1 fitted with an N-terminal hexahistidine tag
was generously provided by Dr. N. Ahn (University of Colorado, Boulder,
CO). Drs. Kurt Ballmer-Hofer, George Thomas and Xiu-Fen Ming (Friedrich
Miescher Institute, Basel) are thanked for helpful discussions and
critical reading of the manuscript.