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INTRODUCTION |
The mitogen-activated protein
(MAP)1 kinase family
regulates diverse physiological processes including cell growth,
differentiation, and death. Activation of one of these MAP kinases (the
extracellular signal-regulated kinase or
ERK) is initiated by the recruitment of
the MAP kinase kinase kinase Raf-1 to the small G protein Ras, a
resident plasma membrane protein. The Ras family consists of three
members (Ha-Ras, Ki-Ras, and N-Ras) (1) that display overlapping but
distinct patterns of expression and function (2). Ras is tethered to
the membrane via a carboxyl CAAX motif containing a
cysteine followed by two aliphatic amino acids (A) and a
carboxyl-terminal amino acid (X) that directs the attachment
of a farnesyl moiety (3, 4). In addition to farnesylation, a second
signal assists in correct membrane targeting. For Ha-Ras and N-Ras,
this second signal is a palmitoyl moiety that is introduced on a
neighboring cysteine. In Ki-Ras, this second site is a polybasic
domain. The requirement of Ras membrane localization for Raf-1
activation has been confirmed by mutating the terminal cysteine in a
constitutively active Ras mutant, RasV12, resulting in a mutant that
cannot activate Raf-1 (5).
Membrane regions rich in cholesterol and sphingolipids, termed
"rafts" or detergent-insoluble glycolipid-enriched complexes have
been proposed to participate in signaling events by organizing additional molecules, such as c-Src, G protein subunits, and
phospholipases, into discrete membrane domains (6). Recent attention
has focused on the role of these specialized microdomains in Ras
signaling (7-10). A number of groups have shown that both Ras isoforms
Ki-Ras (11) and Ha-Ras are targeted to rafts (7-9, 12-14). Others
have suggested that Ha-Ras, but not Ki-Ras, can be targeted to raft microdomains (10, 15). Targeting of Ras isoforms to specific membrane
domains may be determined by the characteristics of the lipid
modifications on Ras, as well as other sequences found within the
hypervariable region (hvr) (10). Localization of Ras isoforms to
distinct membrane microdomains may influence selectivity of signaling
among the Ras isoforms (2). For example, Ki-Ras is thought to couple
well to Raf-1 but unlike Ha-Ras, couples poorly to phosphatidylinositol
3-kinase (16, 17). Differences between Ha-Ras and Ki-Ras in their
promotion of cell survival have also been noted (18), suggesting that
distinct localization of Ras isoforms dictate signaling pathways, as
recently proposed (19). Ras isoforms have been reported to display
distinct dependences on caveolin in their coupling to Raf-1 (9,
15).
Raf-1 recruitment to the membrane can be achieved independently of Ras
by the addition of Ras carboxyl-terminal sequences to the carboxyl
terminus of Raf-1. The addition of twenty amino acids from the carboxyl
terminus of Ki-Ras onto Raf-1 (Raf-KiCAAX) is sufficient to
redirect Raf-1 to the membrane where it is constitutively active (20,
21). Maximal activity of this chimera, however, requires additional
phosphorylation events (22, 23), consistent with the requirement of
specific kinases for full activation for wild type Raf-1 (24-26). In
particular, two phosphorylations on Ser-338 and Tyr-341 have been shown
to be required for full activity (27). Recently, two additional sites
within the kinase activation loop have also been shown to be required
(28). Phosphorylation of serine 338 may be mediated by the
serine/threonine kinase PAK (p21-associated protein) (29) and
phosphorylation of tyrosine 341 can be carried out by Src family
tyrosine kinases (23, 27, 30). The participation of specific membrane
microdomains in these modifications is not known.
In this study, we examined the membrane requirements for the
post-translational modification of Raf-1. In addition, we took advantage of chimeric Raf-1 molecules that are targeted to specific membrane domains to determine the specificity of these domains for raft
localization, phosphorylation, and constitutive activation of Raf-1.
Understanding the molecular basis for Raf-1 activation by Ras may also
help explain the actions of the related small G protein Rap1, which
recruits Raf-1, but unlike Ras, cannot activate it. In the present
study we address whether membrane localization also plays a role in
Rap1-mediated inhibition of Raf-1 activation by Ras. We found that
Raf-1 phosphorylation was intimately linked to proper membrane
targeting and that the ability of Ras and Rap1 to support Raf-1
phosphorylation dictated the biochemical actions of both Ras and Rap1
on Raf-1.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfections, and Stimulations--
COS-7 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. Cells were transfected using
LipofectAMINE 2000 according to the manufacturer's recommendations. Unless otherwise noted, cells were transfected with a total of 10 µg
of plasmid DNA, with pcDNA3.1 (vector) used to adjust DNA amounts
where necessary. After 24 h, transfected cells were switched to
low serum-containing medium and incubated for a further 12 h.
Cells were stimulated with 50 ng/ml epidermal growth factor (EGF) for
the indicated times. For cholesterol depletion experiments, serum-starved cells were preincubated for 1 h with 2% methyl
-cyclodextrin (CD, Sigma) in Dulbecco's modified Eagle's medium
prior to stimulation with EGF.
Plasmids--
The cDNA encoding Ha-Ras and Ha-RasV12 were
tagged at their amino termini with a FLAG epitope. The cDNA
encoding Ki-Ras and Ki-RasV12 were tagged at their amino termini with a
Myc epitope. The cDNA encoding N-Ras and N-RasV12 were tagged at
their amino termini with a hemagglutinin (HA) epitope and were
purchased from the Guthrie Institute, Sayre, PA. All other cDNAs
were tagged at the amino termini with a 2× FLAG epitope (Kodak) by PCR
and introduced into the BamH1 and XbaI sites of
pcDNA3.1 vector (Invitrogen), unless otherwise indicated. Raf-1 and
B-Raf mutations were introduced by PCR using the QuickChange
site-directed mutagenesis kit (Stratagene). The minimal membrane
targeting domain of Ha-Ras(
hvr) (31) was added to the 3'-end of
FLAGRaf-1 by PCR using a primer with Raf-1 sequences along with an
in-frame XhoI site (adding amino acids Leu and Glu) and the
last nine amino acids of Ha-Ras (CMSCKCVLS), a stop codon, and an
XbaI site. This construct is designated
Raf-HaCAAX(
hvr). Raf-Ha-RasCAAX(+hvr),
containing both the minimal membrane targeting domain and the
hypervariable domain, was constructed using a primer with an in-frame
XhoI site, the carboxyl-terminal 27 amino acids of Ha-Ras
(IRQHKLRKLNPPDESGPGCMSCKCVLS), a stop codon, and an XbaI
site by PCR. Raf-Rap1CAAX was constructed using the same strategy with the carboxyl-terminal 24 amino acids of Rap1b
(LVRQINRKTPVPGKARKKSSCQLL) introduced into the
XhoI-XbaI site of
Raf-Ha-RasCAAX(+hvr). B-Raf-Ha-RasCAAX, B-Raf-Rap1CAAX chimeras were constructed using a similar
strategy. RapE63-Ha-RasCAAX(+hvr) was constructed by
adding an in-frame XhoI site by PCR into RapE63 at
base pair 480 and cloning into the EcoR1-XhoI
sites of FLAGRaf-Ha-RasCAAX, thereby replacing the Rap
membrane targeting motif with the Ha-Ras hypervariable domain and
CAAX motif. The full-length coding sequences of human Ha-Ras, bovine Rap1b, RapE63-Ha-RasCAAX,
Raf-Rap1CAAX, and Raf(Y341D)-Rap1CAAX were
introduced in-frame into the pEGFP-C1 cloning vector
(Clontech). Wild type (CAV/WT) and mutant
caveolin-3 (CAV/DGV) plasmids were constructed as per Watson et
al. (32).
Western Blotting and Immunoblotting--
COS-7 cells were
stimulated and lysates prepared as described. Protein concentrations
were determined by using the Bio-Rad protein assay dye reagent
according to manufacturer's recommendations. Equal amounts of lysate
were immunoprecipitated with either FLAG M2 antibody coupled to agarose
(Sigma) or anti-Myc antibody (9E10) coupled to agarose (Santa Cruz
Biotechnology) where indicated and examined by Western blot as
previously described (33). Samples were separated by SDS-PAGE and
transferred to PVDF membrane. Expression of FLAG and Myc-tagged
proteins was detected using monoclonal FLAG M2 antibody or monoclonal
anti-Myc 9E10 antibody. Rabbit polyclonal anti-phospho-MEK1/2 antibody
(Cell Signaling Technology) was used to detect activated GST-MEK1
(Upstate Biotechnology). Polyclonal anti-phospho-ERK1/2 (Cell Signaling
Technology) was used to detect activated MycERK2 (pMycERK2).
Phosphorylation of Raf-1 at Ser-338 or at Tyr-341 was detected using
anti-phospho-Raf-1 Ser-338 or -phosphotyrosine (pTyr, 4G10) (Upstate
Biotechnology). All experiments were repeated at least three times, and
representative blots are shown. The expression of endogenous N-Ras,
Ki-Ras, and Ha-Ras was examined using subtype-specific Ras antisera
from Santa Cruz Biotechnology.
Cell Fractionation--
COS-7 cells were washed twice in
phosphate-buffered saline (PBS) before scraping into 0.5 ml of
hypotonic lysis buffer (10 mM Tris, pH 7.5, 5 mM Mg2Cl, 25 mM NaF, 25 mM
-glycerophosphate, 1 mM dithiothreitol,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 mM sodium
orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). After 10 min., cells were homogenized at 4 °C by 50 strokes in a
tight fitting Dounce homogenizer. Nuclei and unbroken cells were
pelleted by centrifugation at 1500 × g. Supernatants
were then centrifuged at 100,000 × g in a Beckman TLA
45 rotor at 4 °C for 30 min. The supernatant was collected and
designated the cytosolic fraction (S100), and the pellet was
resuspended in 250 µl of hypotonic lysis buffer and designated the
membrane (P100) fraction.
Sucrose Gradients--
Membrane microdomains were isolated based
on their buoyant density using isopycnic equilibrium sucrose density
gradient centrifugation. A non-detergent method for lipid raft
isolation was used based on the method of Smart et al. (34)
but modified for equilibrium centrifugation, and sucrose was used
instead of Optiprep. Briefly, transfected COS-7 cells (2 × 106 cells) were rinsed twice in PBS and scraped into 0.5 ml
of MES buffer (25 mM MES, pH 6.5, 10 mM NaCl, 5 mM Mg2Cl, 10 µg/ml aprotinin, 10 mg/ml
leupeptin, 25 mM NaF, 25 mM
-glycerophosphate, 5 mM sodium orthovanadate). Cells
were homogenized at 4 °C by 30 passes through a 23-gauge syringe and
sonicated on ice for 30 s at setting 2, 30 s at setting 3, and 30 s at setting 4 (Sonic Dismembrater, Fisher). The lysate was
mixed with 0.5 ml of 90% sucrose in MES buffer and placed at the
bottom of a 2.4-ml Beckman ultracentrifuge tube. The gradient was
constructed by overlaying the 45% sucrose/lysate mixture with 1.2 ml
of 35% sucrose, 1 ml of 30% sucrose, 1 ml of 25% sucrose, and
ultimately 1 ml of 5% sucrose. The tubes were centrifuged at 4 °C
in a Beckman SW 55 rotor for 16 h at 48,000 rpm. A visible band
3-4 mm from the top was observed after centrifugation and corresponded
to the lipid raft/caveolae fraction. Twelve 0.43-ml fractions were
collected from the top of the gradient. From each fraction, 40 µl was
removed for refractometry and protein determination using the Bradford
method (BioRad). The remainder of each fraction was diluted with 1 ml
of MES buffer to dilute out the sucrose, and membranes and proteins
were pelleted at 100,000 × g for 45 min in a TLA 45 rotor. Pellets were resuspended in Laemmli buffer, separated by
SDS-PAGE, and transferred to a PVDF membrane for analysis by
immunoblotting. Only the first 10 fractions are shown for each
gradient. Similar results were obtained using the sodium carbonate
method of raft preparation (10).
Immunofluorescence--
COS-7 cells were grown on glass
coverslips and transfected with 100 ng of FLAGRaf cDNAs using
LipofectAMINE 2000. Cells were fixed in PBS containing 4%
formaldehyde, permeablized in PBS containing 0.1% Triton X-100, and
blocked with PBS, 5% horse serum. Localization of transfected proteins
was detected with a 1:2000 dilution of FLAG M2 antibody (Sigma) in 5%
horse serum, 0.01% Tween followed by a 1:10,000 dilution of an
anti-mouse-fluorescein isothiocyanate conjugate in 5% horse serum,
0.01% Tween. Cells were visualized using a Zeiss Axioplan 2 microscope. To examine the expression patterns of the Myc-tagged wild
type caveolin (CAV/WT) or DGV mutant (CAV/DGV) constructs, cells
were transfected with 5 µg of plasmid cDNAs as described above.
The expressed caveolin constructs were detected by mouse anti-Myc
monoclonal antibody (Santa Cruz Biotechnology) at 1:100 dilution,
followed by Texas-Red anti-mouse secondary antibody at 1:100 (Jackson
Immunoresearch Laboratories). Endogenous caveolin was detected by
rabbit anti-caveolin polyclonal antibody (Transduction Laboratories) at
1:200 dilution, followed by Alexa-488 anti-rabbit secondary antibody at
1:100 dilution (Molecular Probes). Cells were imaged with a Zeiss 510 scanning laser confocal microscope.
Raf-1 Kinase Assays--
COS-7 cells were transfected, lysed,
and lysates prepared as described (28). FLAGRaf proteins were
immunoprecipitated from 500 µg of cell lysate with 30 µl of FLAG M2
agarose at 4 °C for 6 h. Immune complexes were washed twice
with lysis buffer and once with kinase assay buffer (20 mM
MOPS, pH 7.2, 25 mM
-glycerophosphate, 5 mM
EGTA, 1 mM sodium orthovanadate, 1 mM
dithiothreitol, 5 µg/ml aprotinin). Pellets were resuspended in 40 µl of kinase assay buffer with 1.5 mM Mg2Cl
and 7.5 µM ATP along with 0.4 µg of GST-MEK1 (Upstate
Biotechnology), and the reaction was incubated for 30 min at 30 °C.
The kinase reaction was terminated by adding 45 µl of 2× Laemmli
buffer, boiled for 5 min, resolved by SDS-PAGE, and transferred to PVDF
membrane. Raf-1 activity was evaluated by immunoblotting with
anti-phospho-MEK1/2 antibody.
Phospho-MycERK2 Assay--
For MycERK2 assays, treated and
untreated cells were lysed in ERK assay buffer and activation of
MycErk2 was detected as described previously (33).
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RESULTS |
Raf-1 Is Relocalized to Raft Microdomains upon EGF
Stimulation--
Activation of Raf-1 by Ras-dependent
signals induced by growth factors is associated with a redistribution
of Raf-1 from the cytoplasm to the cell membrane, where it associates
with Ras (35). Recent studies suggest that Ras may be localized to
specific cholesterol-rich membrane microdomains called rafts (11), and
upon Ras activation, Raf-1 may also be recruited to rafts (12).
Proteins that localize to cholesterol-rich microdomains (rafts) can be
detected within low density fractions of sucrose density gradients (10,
14, 36). The method is illustrated in Fig. 1A. The density gradient
achieved following equilibrium centrifugation is shown in Fig.
1B with the corresponding protein concentrations. One of
these raft proteins, caveolin-1, was used to identify the density of
these cholesterol-rich raft domains (Fig. 1C, top
panel) (6). Using this technique, we show that endogenous Raf-1
was excluded from raft microdomains in untreated cells (Fig.
1C, second panel), and EGF treatment induced the
redistribution of endogenous Raf-1 into raft domains (Fig.
1C, third panel). A significant fraction of Raf-1
protein was also detected at higher densities within the gradient.

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Fig. 1.
EGF stimulation relocalizes Rap-1 to raft
microdomains. A, schematic illustration of the sucrose
density gradient assay. Isopycnic centrigugation is used to achieve an
equiibrium gradient. The lipid-associated proteins migrate to buoyant
densities, where they can be detected within the top fractions of the
gradient. The localization of Raf-1 in EGF-treated cells is shown as an
example. B, sucrose density gradient profiles of COS-7
cells. Cells were lysed and proteins prepared for sucrose equilibrium
density gradient centrifugation as described under "Experimental
Procedures." The top twelve 0.43-ml fractions are shown (open
squares). The protein content of each fraction is shown as
black circles. C, growth factor stimulation
recruits Raf-1 to lipid rafts. Cells were treated with or without EGF
as indicated, and proteins separated into the indicated fractions by
equilibrium centrifugation on a sucrose density gradient
(fraction 1 is top). Fractions 1-10 were
pelleted and proteins separated by SDS-PAGE. The position of caveolin-1 within the gradient is shown
in the upper panel using caveolin-1 antisera. The presence
of endogenous Raf-1 within fractions (1-10) of each gradient is shown
in the middle and lower panels, using antisera to
Raf-1. D, sucrose density gradients of protein fractions
containing Raf-1 and Raf mutants. Cells were transfected with wild type
FLAG-tagged Raf-1 (Raf-1 WT), treated with or without EGF or
CD, as indicated, and proteins separated into the indicated fractions
by sucrose density gradients. The presence of FLAGRaf-1 constructs
within fractions 1-10 of each gradient is shown in the lower two
panels, using FLAG antibody. E, activated Ras proteins
recruit Raf-1 into raft microdomains. COS-7 cells were transfected with
FLAGRaf-1 and either vector, Ha-RasV12 (top panel),
Ki-RasV12 (second panel), N-RasV12 (third panel),
or vector (bottom panel) as indicated, and proteins were
separated into the indicated fractions by sucrose density
centrifugation. The presence of FLAGRaf-1 within fractions (1-10) of
each gradient is shown using the FLAG antibody.
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Similar results were seen using transfected cDNAs encoding wild
type Raf-1 tagged with the FLAG epitope. We show that wild type Raf-1
was excluded from raft microdomains in untreated cells (Fig.
1D, top panel), and EGF treatment induced the
redistribution of wild type Raf-1 into raft domains (Fig.
1D, middle panel). Raft domains can
be disrupted by the cholesterol-depleting agent CD (11). In the
presence of CD, EGF recruitment of Raf-1 to low density fractions was
inhibited (Fig. 1D, bottom panel), confirming that these low density fractions represented cholesterol-rich membrane
microdomains. This provides strong evidence that the buoyant fractions
containing Raf-1 represented cholesterol-rich membrane microdomains
consistent with rafts. Raf-1 could also be redistributed to rafts in
cells transfected with constitutively active mutants of Ha-Ras,
Ki-Ras, and N-Ras (Fig. 1E).
The localization of Ras proteins to rafts is not completely understood,
and some controversies remain (37) with some groups showing that
Ha-Ras, but not Ki-Ras, requires raft localization for full activity
(9, 15). COS-7 cells express detectable levels of Ki-Ras and N-Ras
(Fig. 2A; middle
and right panels, untr.). The expression of
Ha-Ras was not detected (Fig. 2A; left panel,
untr.). As a control, cells were transfected with each Ras
isoform and lysates probed with isoform-specific antisera. Endogenous
Ki-Ras and N-Ras were localized to lipid rafts in resting cells (Fig.
2B). Similar results were seen in cells transfected with
wild type as well as constitutively active (V12) Ha-Ras, Ki-Ras, and
N-Ras mutants, although transfected N-Ras wild type appeared in higher
density fractions as well (Fig. 2, C and D).

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Fig. 2.
Ras isoforms are localized to raft domains.
A, Western blot of Ras isoforms expressed in COS-7 cells.
Cells were left untransfected or transfected with FLAG-HaRas,
Myc-KiRas, or HA-NRas. Proteins were assayed by Western blot for
endogenous and transfected Ras proteins, using isoform-specific Ras
antibodies directed against Ha-Ras, Ki-Ras, and N-Ras as indicated.
B, sucrose density gradients of membrane fractions from
unstimulated COS-7 cells. The presence of endogenous Ki-Ras (top
panel) and N-Ras (bottom panel) are shown using Ki-Ras
or N-Ras antisera, respectively. C, sucrose density
gradients of membrane fractions containing transfected Ras proteins.
Cells were transfected with WT, FLAG-HaRas, Myc-KiRas, or HA-NRas as
indicated, and proteins were separated into the indicated fractions by
sucrose density gradients The presence of Ha-Ras (top
panel), Ki-Ras (middle panel), and N-Ras (lower
panel) within fractions 1-10 of each gradient were analyzed using
FLAG, Myc, and HA antibodies respectively. D, sucrose
density gradients of membrane fractions containing transfected Ras
proteins. Cells were transfected with constitutively active
FLAG-HaRasV12, Myc-KiRasV12, or HA-NRasV12, as indicated. The presence
of Ha-RasV12 (top panel), Ki-RasV12 (middle
panel), and N-RasV12 (lower panel) within fractions
1-10 of each gradient were analyzed using FLAG, Myc, and HA antibodies
respectively.
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Raf-1 Activation and Ser-338 Phosphorylation Require Intact Raft
Microdomains--
It has long been appreciated that Raf-1 recruitment
to Ras is insufficient by itself to trigger Raf-1 activation and that additional post-translational modifications are required (27). The two
best studied modifications are serine phosphorylations on serine 338 and tyrosine 341. Fig. 3A
demonstrates this requirement. Constitutively active Ras (RasV12)
activated wild type Raf-1 (Raf-1 WT), as measured by in
vitro Raf-1 assay. However, Raf-1 that was mutated at either
Ser-338 to alanine (RafS338A) or Tyr-341 to alanine (RafY341A) could no
longer be activated by Ha-RasV12 (Fig. 3A).

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Fig. 3.
Ser-338 phosphorylation and activation
requires intact raft domains. A, Raf-1 kinase assays.
HaRasV12 and either FLAG-tagged Raf-1, RafS338, RafY341A, or pcDNA3
vector was transfected into COS-7 cells. Equivalent amounts of
Raf protein were immunoprecipitated using the FLAG antibody and assayed
for the ability to phosphorylate MEK (pMEK) in
vitro (top panel). The levels of FLAGRaf (middle
panel) and MycRasV12 (lower panel) are shown.
B, cells were transfected with FLAG Raf-1 WT and treated
with EGF and/or CD as indicated. Lysates were subjected to FLAG
immunoprecipitation and assayed for p338 (upper panel) and
the ability to phosphorylate MEK (pMEK) in vitro
(middle panel). The levels of FLAGRaf-1 expression are shown
in the lower panel, using FLAG antibody. C, cells
were transfected with FLAGRaf-1WT or FLAGRafY341A and treated with EGF
and/or CD as indicated. Lysates were subjected to FLAG
immunoprecipitation and assayed for tyrosine phosphorylation with
phospho-Tyr antibody (pTyr) (upper panel).
The levels of FLAGRaf-1 expression are shown in the lower
panel, using FLAG antibody.
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Raft microdomains were required for phosphorylation on Ser-338, since
CD inhibited EGF-induced phosphorylation of FLAGRaf-1 on Ser-338 (p338)
and Raf-1 activation (Fig. 3B). These data suggest that
targeting to raft domains was required for full Raf-1 activation and
phosphorylation at Ser-338. In contrast, tyrosine phosphorylation of
FLAGRaf-1 was not affected by CD (Fig. 3C, upper
panel, lanes 1-4). The absence of phosphorylation of
Tyr-341 in FLAGRafY341A is provided as a negative control (Fig.
3C, upper panel, lanes 5-8).
Recent studies have shown that Ras proteins are localized to
specialized raft domains called caveolae (11). These microdomains are
enriched for caveolin, and a requirement for caveolin can be assessed
using interfering mutants such as CAV/DGV, a truncated form of
caveolin-3 (9, 10, 32). In COS-7 cells, expression of CAV/DGV was
detected within cytoplasmic vesicles, as previously reported (9, 10)
and interferes with the localization of caveolin-1 (Fig.
4A). CAV/DGV did not interfere
with the ability of either EGF (Fig. 4B) or constitutively
active Ras mutants (Fig. 4C) to activate Raf-1 or stimulate
Ser-338 phosphorylation.

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Fig. 4.
Interfering mutants of caveolin do not
disrupt Ras function. A, epifluorescent micrographs of
CAV/WT and CAV/DGV. COS-7 cells were transfected with Myc-CAV/WT (WT;
panels 2, 4, and 6) or Myc-CAV3/DGV
(DGV; panels 1, 3, 5, and
7). The expression of Myc-CAV/DGV is shown in panels
1 and 2 (anti-Myc; red). The expression of
endogenous caveolin-1 is shown in panels 3 and 4 (anti-caveolin; green). Merges of the red and green
images are shown in panels 5 and 6. Panel
7 shows a magnification of the boxed area in
panel 5. Bar is 10 µM. The zoom
field is 10 × 10 µm. B, cells were transfected with
FLAGRaf-1 and either vector ( ) or Myc-CAV/DGV (+), and treated with
EGF or left untreated, as indicated. Lysates were subjected to FLAG
immunoprecipitation and assayed for p338 (top panel) and the
ability to phosphorylate MEK (pMEK) in vitro
(second panel). The levels of FLAGRaf-1 expression are shown
in the third panel, using FLAG antibody. The levels of
Myc-CAV/DGV expression are shown in the fourth panel, using
Myc antibody. C, cells were transfected with FLAGRaf-1 along
with Ha-RasV12 (Ha), Ki-RasV12 (Ki), or N-RasV12
(N), and vector (V), with CAV/DGV; (+) or without
CAV/DGV; ( ). Lysates were subjected to FLAG immunoprecipitation and
assayed for p338 (top panel) and the ability to
phosphorylate MEK (pMEK) in vitro (second
panel). The levels of FLAGRaf-1 expression are shown in the
third panel, using FLAG antibody. The levels of Myc-CAV/DGV
expression are shown in the fourth panel, using Myc
antibody.
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Tyr-341 Is Required for Targeting to Raft Microdomains and
Phosphorylation of Ser-338--
We next examined the requirement of
Ser-338 and Tyr-341 in Raf-1 localization. EGF was able to direct
RafS338A into rafts (Fig. 5A,
upper panel), but was not able to direct RafY341A into rafts
(Fig. 5A, lower panel). Neither Raf mutant
RafS338A nor RafY341A entered rafts in the absence of EGF stimulation
(data not shown). This suggests that phosphorylation of Tyr-341
participates in localization of Raf-1 to lipid rafts, while Ser-338
phosphorylation occurs once proper localization has been achieved. In
Fig. 5B, we show that the tyrosine at residue 341 was
essential for EGF and Ha-RasV12 to phosphorylate Ser-338 (Fig.
5B, upper panel) and activate Raf-1 (Fig.
5B, middle panel). Replacing tyrosine with
aspartate in the mutant Raf Y341D did not affect EGF actions (Fig.
5B).

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Fig. 5.
The requirement for Tyr-341 can be overcome
by targeting Raf-1 to raft domains. A, sucrose density
gradients of membrane fractions containing Raf-1 mutants. Cells were
transfected with FLAG-tagged RafY341A, and RafS338A, treated with EGF
as indicated, and proteins separated into the indicated fractions by
sucrose density gradients, as described in the legend to Fig. 1. The
presence of FLAGRaf constructs within each gradient is shown in both
panels, using FLAG antibody. B, phosphorylation and
activation of Raf-1 mutants by EGF. FLAG-tagged Raf-1, RafY341A, or
RafY341D were transfected into COS-7 cells, and cells were either
treated with EGF or left untreated, as indicated. Lysates were
subjected to FLAG immunoprecipitation and assayed for p338 (upper
panel) and the ability to phosphorylate MEK (pMEK)
in vitro (middle panel). The levels of FLAGRaf
proteins are shown in the lower panel, using FLAG antibody.
C, immunofluorescence of Raf-Ras chimeras. COS-7 cells were
transfected with FLAG-tagged Raf-1 WT, Raf-HaCAAX(+hvr), and
Raf-HaCAAX( hvr), as indicated. Cells were prepared for
epifluorescent microscopy as described under "Experimental
Procedures," and representative cells are shown. D,
sucrose density gradients of Raf-Ras chimeras. Cells were transfected
with FLAG-tagged Raf-HaCAAX(+hvr), Raf
Y341AHaCAAX(+hvr), and Raf S338AHaCAAX(+hvr), and
left untreated. Proteins were separated as in Fig. 1 and the presence
of the chimera within each fraction is shown, using FLAG antibody.
E, lack of requirement of Tyr-341 for Ser-338
phosphorylation in targeted chimeras. Cells were transfected with Raf-1
WT, Raf-HaCAAX(+hvr), RafS338A-HaCAAX(+hvr), or
RafY341AHaCAAX(+hvr), and treated with or without EGF, as
indicated, and assayed for p338 (upper panel), or Raf-1
kinase activity (pMEK, middle panel). The levels
of FLAG-containing proteins are shown in the lower
panel.
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The Requirement of Tyr-341 Phosphorylation for Raf-1 Activation Can
Be Overcome by Targeting to Raft Domains--
Raf-1 can be
constitutively targeted to the membrane following the attachment of a
Kirsten Ras carboxyl-terminal domain (Raf-KiCAAX) (20, 21).
Here, we examined the localization of a related chimera created by
fusing the Raf-1 protein to the carboxyl 27 amino acids of Ha-Ras
including both the CAAX domain and the hvr; Raf-HaCAAX(+hvr) (16). As expected, wild type Raf-1 was
located within the cytoplasm of resting cells (Fig. 5C,
left panel), and the chimera Raf-HaCAAX(+hvr) was
present on the plasma membrane (Fig. 5C, middle
panel). The chimera Raf-HaCAAX(
hvr) that lacked hvr
sequences was also present on the plasma membrane (Fig. 5C, right panel).
Raf-HaCAAX(+hvr) is constitutively localized within raft
domains (Fig. 5D, upper panel). Unlike Raf Y341A,
the introduction of Y341A into Raf-HaCAAX(+hvr)
[RafY341A-HaCAAX(+hvr)] did not prevent raft localization
(Fig. 5D, middle panel), constitutive phosphorylation of Ser-338 or activation of Raf-1 (Fig. 5E).
These data suggest that phosphorylation of Tyr-341 is required for raft localization, Ser-338 phosphorylation, and activation of wild type
Raf-1 but that Tyr-341 phosphorylation is not required if Raf-1 is
constitutively targeted to rafts. In contrast, mutating Ser-338 to
alanine, in the chimera RafS338A-HaCAAX(+hvr), completely abolished activation of Raf-1 (Fig. 5E), without affecting
raft localization (Fig. 5D, lower panel),
demonstrating the requirement of phospho-Ser-338 for kinase activation,
but not raft localization.
In addition to the CAAX domain, Ha-Ras contains a
hypervariable region that influences specific membrane localization
(10). Here we examined chimeras either containing the Ha-Ras
hypervariable region (+hvr) or lacking these sequences (
hvr). Like
Raf-HaCAAX(+hvr), Raf-HaCAAX(
hvr) was present
within the particulate (P100) fraction (Fig.
6A) and was detected on the
plasma membrane (Fig. 5C, right panel). Unlike
Raf-HaCAAX(+hvr), Raf-HaCAAX(
hvr) was excluded from low density gradient fractions (Fig. 6B, upper
panel), suggesting that despite its membrane localization, the
chimera was targeted differently than Raf-HaCAAX(+hvr).
Raf-HaCAAX(+hvr) gradients were included as a control (Fig.
6B, lower panel).

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Fig. 6.
Ectopic targeting of Raf-1 to raft domains is
required for constitutive Ser-338 phosphorylation, activation of Raf-1,
and activation of ERKs. A, localization of Raf-Ras
chimeras. Cells were transfected with FLAG-tagged Raf-1 WT and the
Raf-Ras chimeras as indicated and fractionated into S100 and P100
fractions, and FLAG-containing proteins detected by Western blot.
B, sucrose density gradients of
Raf-HaCAAX( hvr). Cells were transfected with FLAG-tagged
Raf-HaCAAX( hvr), and left untreated. Proteins were separated as in
the legend to Fig. 1, and the presence of the chimera within each
fraction is shown (upper panel), using FLAG antibody.
Gradients of lysates expressing Raf-HaCAAX(+hvr) are shown
as a control (lower panel). C, raft-targeted
chimeras show constitutive activity. Cells were transfected with
MycERK2 and individual Raf-1 chimeras. Cells transfected with wild type
Raf-1 were also treated with EGF as indicated. FLAG-containing proteins
were recovered by immunoprecipitation and examined for Ser-338
phosphorylation (p338, first panel), and
phosphorylation of MEK in vitro, as in Fig. 1 (pMEK,
second panel). The position of FLAGRaf proteins is shown in the
third panel (FlagRaf). In the lower two
panels, the lysates were subjected to Myc immunoprecipitation and
the recovered MycERK2 examined for phosphorylation
(pMycERK2) or total MycERK levels
(MycERK2).
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Only Raf-HaCAAX(+hvr) (lane 4), but not
Raf-HaCAAX(
hvr) (lane 3) displayed
constitutive phosphorylation of Ser-338 (Fig. 6C,
p338, first panel). Raf-HaCAAX(+hvr),
but not Raf-HaCAAX(
hvr), was active in Raf-1 kinase assays
in vitro (Fig. 3C, pMEK, second panel) and ERK activation assays in vivo (Fig.
6C, pMycERK2, fourth panel). As a
control, EGF stimulation of wild type Raf-1 was included, which
resulted in phosphorylation of Ser-338 and activation of both Raf-1 and
ERK (lanes 1 and 2). High levels of activation of
Raf-HaCAAX(+hvr), but not Raf-HaCAAX(
hvr), were
also seen in coupled in vitro kinase assays (data not
shown). Therefore, both Ser-338 phosphorylation and biochemical
activity of these chimeras paralleled raft localization.
Y341D Restored Raft Localization and Activity to
Raf-HaCAAX(
hvr)--
For Raf-HaCAAX(
hvr), membrane
targeting was not sufficient to trigger Ser-338 phosphorylation and
Raf-1 activity. Since Tyr-341 phosphorylation potentiates the
localization of Raf-1 within rafts, which may be required for
subsequent Ser-338 phosphorylation, we tested whether the Y341D
mutation could restore Ser-338 phosphorylation in the
HaCAAX(
hvr) chimera. Indeed, this mutant,
RafY341D-HaCAAX(
hvr), but not
RafS338D-HaCAAX(
hvr), was capable of entering raft domains (Fig. 7A). Moreover,
RafY341D-HaCAAX(
hvr) was phosphorylated on Ser-338 and
showed constitutive activity to levels similar to those seen with
Raf-HaCAAX(+hvr) (Fig. 7B). Furthermore, the introduction of Y341D into Raf-HaCAAX(+hvr) did not
significantly increase Raf-1 activity (Fig. 7, B and
C). These data suggest that one of the functions of Tyr-341
is to localize Raf-1 to specific membrane microdomains permitting
efficient phosphorylation of Ser-338 and coupling to downstream
effectors.

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Fig. 7.
Mutation of Y341D restores phosphorylation of
Ser-338, activation, and raft localization of inactive Raf/Ras( hvr)
chimeras. A, raft localization of
RafY341DHaCAAX( hvr). Cells were transfected with
RafY341DHaCAAX( hvr) and proteins fractionated as described
in the legend to Fig. 1. RafS338D-HaCAAX( hvr) is shown as
a control (lower panel). The presence of FLAG-containing
proteins within each fraction is shown. B, Ser-338
phosphorylation and Raf-1 activity assays. Cells were transfected with
either FLAG-tagged Raf-HaCAAX( hvr),
RafY341DHaCAAX( hvr), Raf-HaCAAX(+hvr), or
RafY341DHaCAAX(+hvr) and immunoprecipitated using FLAG
antibody and assayed for Ser-338 phosphorylation (upper
panel) and the ability to phosphorylate MEK (pMEK)
in vitro (middle panel). The levels of FLAGRaf-1
expression are shown in the lower panel, using FLAG antibody.
C, Raf-1 assays from three independent experiments, as in
Fig. 4. The data are shown as fold activation above that seen for Raf-1
WT, with S.E.
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Rap1 Is Unable to Activate Raf-1 Because it Cannot Induce Tyr-341
Phosphorylation--
The ability of small G proteins to recruit Raf-1
to the membrane is not sufficient for full activation of Raf-1 (22).
For example, Rap1 is a small G protein within the Ras family that can
associate with Raf-1 but cannot activate it (38). Unlike Ras, which is
located at the plasma membrane, Rap1 is located in vesicular membranes
(39-41). This localization is directed by carboxyl-terminal sequences
of Rap1 that contain a distinct CAAX motif that regulate the
attachment of geranyl modifications that direct Rap1 to vesicular
membranes (42, 43). This could be shown using GFP fusions to the RapE63
protein (GFP-Rap), which, unlike GFP alone (Fig.
8A, a), was
localized to perinuclear vesicles within the cytoplasm (Fig.
8A, b). In contrast, GFP-Ha-RasV12 was detected
at the plasma membrane, consistent with recent reports (19) (Fig.
8A, c). The chimera
GFP-RapE63-Ha-RasCAAX was also present on the plasma
membrane, confirming that the carboxyl-terminal sequences of Ha-Ras
could redirect ectopic proteins (Fig. 8A, d).

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Fig. 8.
The inability of Rap1 to activate Raf-1 is
due to the inability of Rap1 to induce Ser-338 phosphorylation.
A, GFP epifluorescence. Cells were transfected with GFP
(a), GFP-RapE63 (b), GFP-HaRasV12 (c),
and GFP-RapE63-HaRas-CAAX (d). The locations of
the transfected proteins were examined by epifluoresent microscopy, and
representative cells are shown. B, Rap1 supports neither
Raf-1 activation nor Ser-338 phosphorylation. Cells were transfected
with Raf-1 WT along with pcDNA3, RapE63,
RapE63-HaCAAX(+hvr) or HaRasV12 and examined for Ser-338
phosphorylation (p338) and Raf-1 activity (pMEK).
C, Ser-338 phosphorylation and activation of RafY341D by
Rap1. Cells were transfected with RafY341D along with either
pcDNA3, RapE63, RapE63-HaCAAX(+hvr) or HaRasV12 and
examined for Ser-338 phosphorylation (p338) and kinase activity of
Raf-1 (pMEK). D, Raf-Rap1 chimeras are not
constitutively active. Cells were transfected with either FLAG Raf-1
wild type (WT), Raf-Rap1CAAX, RafY341D-Rap1CAAX, or
Raf-HaCAAX(+hvr). Cells were treated with EGF (+) or left
untreated ( ) and lysates immunoprecipitated using FLAG antibody and
assayed for both Ser-338 phosphorylation (upper panel) and
Raf-1 activity in vitro (middle panel). The
levels of FLAGRaf expression are shown in the lower panel,
using FLAG antibody. E, GFP epifluorescence. Cells were
transfected with GFP-Raf-Rap1CAAX (a) or
GFP-RafY341-Rap1CAAX (b). The locations of the
transfected proteins were examined by epifluoresent microscopy, and
representative cells are shown.
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Activated Rap1 does not activate Raf1 (44). Rap1's inability to permit
Ser-338 phosphorylation and activation of Raf-1 could be partially
overcome by swapping the Rap1 CAAX domain with that of Ras
(RapE63/Ha-RasCAAX) (Fig. 8B). Although RapE63
could not activate wild type Raf-1, it could activate RafY341D, as
measured by Ser-338 phosphorylation and kinase activation (Fig.
8C). These data suggest that the inability of RapE63 to
activate Raf-1 was due to the inability of Raf-1 to be correctly
phosphorylated when recruited by Rap1, since Rap1 was capable of
supporting Ser-338 phosphorylation in the Y341D mutation. These data
also suggest that the inability of Rap1 to activate Raf-1 is not just a
consequence of the interaction between Rap1 and Raf-1, as has been
proposed (44) but may also be dictated by the localization of Rap1.
However, Ha-RasV12 was better than RapE63-HaCAAX(+hvr) in
activating both Raf-1 and RafY341D (Fig. 8, B and
C), suggesting that sequences within Ras distinct from the
carboxyl-terminal membrane-targeting domain are critical for maximal
activation of Raf-1.
To examine the effect of relocalizing Raf-1 to Rap1-containing
membranes, we generated chimeras of Raf-1 fused to the Rap1 carboxyl-terminal CAAX motif (Raf-Rap1CAAX) (Fig.
8D). Raf-Rap1CAAX was not constitutively active and could
not be activated (Fig. 8D, middle panel) or
phosphorylated on Ser-338 by EGF (Fig. 8D, upper
panel). These data suggest that Raf-1 needs to be targeted to
specific membranes in order to be activated and that Raf-1 targeting to
Rap1-specific membrane domains does not support Ser-338 phosphorylation
or activation. The inability of Rap1 to direct the proper
phosphorylation of Raf-1/Rap chimeras could be overcome by introducing
negative charges into Raf-1 at Tyr-341. Mutation of Tyr-341 to
aspartate to generate RafY341D-RapCAAX increased the basal
levels of both phosphorylation of Ser-338 and Raf-1 activation compared
with Raf-Rap1CAAX (Fig. 8D), which were not further increased by EGF. In Fig. 8E, we show the
subcellular localization of GFP-fusion proteins,
GFP-Raf-Rap1CAAX (Fig. 8E, a),
GFP-RafY341-Rap1CAAX (Fig. 8E, b). Both chimeras
are largely localized to perinuclear regions, with little or no
staining detected at the cell surface.
Rap1 Activation of B-Raf Requires Aspartic Acid at Residues
Asp-447/Asp-448--
The Y341D mutation in Raf-1 resembles
the naturally occurring sequence within the Raf isoform, B-Raf. B-Raf
lacks tyrosines at the site corresponding to Tyr-341 in Raf-1 (448 in
B-Raf). Instead, it contains aspartic acids at residues 447 and 448, that appear to mimic phosphorylation at these sites (27). Moreover, B-Raf was phosphorylated at the serine corresponding to Ser-338 in
Raf-1 (Ser-445 in B-Raf) in resting cells (Fig.
9A, lane 1, upper panel), and remained phosphorylated at this residue in
the presence of the raft-disrupting agent, CD (data not shown).

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Fig. 9.
The ability of B-Raf to be activated by Rap1
requires aspartic acid residues at positions Asp-447/Asp-448 in
B-Raf. A, p445 phosphorylation and B-Raf activity
assays. Cells were transfected with FLAGB-Raf wild type (B-Raf
WT) or FLAGB-RafYY (B-RafYY) along with either
pcDNA3, HaRasV12, or RapE63, as indicated. Lysates were subjected
to FLAG immunoprecipitation and assayed for both p338 (upper
panel) and Raf-1 activity in vitro (pMEK,
middle panel). The levels of FLAGB-Raf expression are shown
in the lower panel, using FLAG antibody. B, ERK
activation by B-Raf requires targeting to either Rap1- or
Ras-containing membranes. Cells were transfected with MycERK2 and
either FLAG B-Raf WT, B-Raf-HaCAAX(+hvr), or
B-Raf-Rap1CAAX and immunoprecipitated using FLAG antibody
and assayed for Ser-445 phosphorylation (p445, upper panel).
In the middle panel (pMycERK2), the total lysates were
assayed for phosphorylation of MycERK2. The levels of FLAGB-Raf
expression are shown in the lower panel, using FLAG
antibody.
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Unlike Raf-1, B-Raf can be activated by the small G protein Rap1 (44).
Because of this, Rap1 can activate MEK and ERK in B-Raf-expressing
cells (45-47). The ability of Rap1 to activate B-Raf is shown in Fig.
9A (lanes 1-3). Both constitutively active mutants of Ras (RasV12) and Rap1 (RapE63) could activate wild type
B-Raf (B-RafWT) (Fig. 9A, lanes 2 and
3). In contrast to B-RafWT, expression of a mutant B-Raf in
which the aspartic acid residues were mutated to the corresponding
tyrosines residues in Raf-1 (B-RafYY) showed no basal phosphorylation
on Ser-445 (Fig. 9A, lane 4). B-RafYY was no longer
activated by constitutively active Rap1 (RapE63), as measured by both
Ser-445 phosphorylation and kinase activity (lane 6).
Ha-RasV12 stimulated Ser-445 phosphorylation and activity of the
B-RafYY mutant (lane 5). When Rap1-CAAX sequences were coupled to B-Raf, the resulting chimera,
B-Raf-Rap1CAAX, was phosphorylated on p445 and activated
ERKs to a similar degree as B-Raf-HaCAAX(+hvr) (Fig.
9B). Although wild type B-Raf was constitutively
phosphorylated on Ser-445 and displayed detectable constitutive kinase
activity against MEK in vitro (Fig. 9A,
lane 1), it could not activate ERKs unless it was targeted
to Ras or Rap1 (Fig. 9B), reflecting the requirement of
specific membrane targeting for the B-Raf activation of MEK/ERK
in vivo. Therefore, we propose that B-Raf's ability to
mimic phosphorylation at residues 447 and 448 is critical for its
ability to be activated by Rap1.
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DISCUSSION |
Multiple Ras Isoforms Localize Raf-1 to Raft Microdomains--
Both endogenous Ki-Ras and N-Ras were readily detected in COS-7
cells and localized to raft domains. In contrast, Ha-Ras was not
detected in COS-7 cells, using isoform-specific antisera. This is
consistent with the results of Kranenburg et al. (11), who
also detected little or no Ha-Ras in COS-7 cells. In that study,
Ki-Ras, largely, and N-Ras, partially, colocalized with caveolin.
Colocalization with caveolin did not appear essential for activation as
EGF activated N-Ras but not Ki-Ras in COS-7 cells (11). We show that
the cholesterol-depleting agent CD blocked EGF activation of Raf-1
kinase activity and Ser-338 phosphorylation, consistent with
CD's ability to block EGF activation of MEK/ERK in these cells (11).
Although, it has been shown that CD potentiated activation of Ki-Ras
(but not N-Ras), CD completely blocked the coupling of Ras to
Raf-1/MEK/ERK (11).
Ser-338 Phosphorylation of Raf-1 Requires Targeting to Raft
Microdomains--
Recent studies suggest that the ability of small G
proteins to regulate signaling cascades is dictated not only by the
specificity of effector utilization, but also by their subcellular
localization (2). Differences in the localization of specific Ras
isoforms within rafts has been reported by some (2) but not others
(11). Paradoxically, disruption of rafts by CD could completely inhibit coupling to downstream effectors, while actually increasing the GTP
loading of selected Ras isoforms (11). This may reflect the need for
selected Ras isoforms to shuttle in and out of the raft (10, 48). In
this study, we focused our attention on the requirement of raft
localization not on Ras activation but activation of the proximal
downstream effector Raf-1. We show that one of the functions of raft
localization is that it permits phosphorylation of Raf-1 on
Ser-338.
Localization of Raf-1 to rafts appears to be required for full
activation of ERKs (12). Using the cholesterol-depleting agent CD, this
group and others (11, 13) have shown that disruption of raft
microdomains interferes with signaling of Raf-1 to ERKs. Raf-1
activation also requires phosphorylation at serine 338. This activating
phosphorylation occurs within the plasma membrane for Raf-1, subsequent
to Raf-1 recruitment to Ras (27). Using CD to disrupt rafts, we show
that intact rafts are required for proper phosphorylation at 338. Therefore, the requirement of raft localization for full Raf-1 activity
is coupled to Ser-338 phosphorylation, extending previous studies
showing that the membrane-localized Ser-338 kinase was required for
Raf-1 activation by oncogenic Ras (29). A candidate kinase, PAK, has
been proposed (49-51); however, its role has been challenged (52).
CAV/DRG Does Not Disrupt Ras Activation of Raf-1 in
COS-7 Cells--
A number of studies have demonstrated that at least
two types of rafts exist; those that contain caveolin and those that do not (6, 11, 32). Caveolins are integral components of caveolae, 50-100-nm vesicular invaginations of the plasma membrane involved in
vesicular trafficking and cell signaling (53). The role of caveolin in
ERK signaling has received much recent attention, and both positive and
negative affects on ERKs have been reported (9, 11, 54-57). Ras
isoforms appear heterogeneous in their ability to couple to caveolins
and to localize to caveolin-containing membranes. For example, in one
study, Ki-Ras largely colocalized with caveolin in COS-7 cells, whereas
N-Ras only partially colocalized with caveolin (11). In BHK cells,
Ki-Ras was largely excluded from caveolin-containing membrane fractions
(10).
The caveolin-3 mutant DGV (CAV/DGV) acts as an interfering mutant of
caveolin function (10, 32). In BHK cells, this mutant inhibits Raf-1
activation by Ha-RasV12 but not by Ki-RasV12 (N-Ras was not examined)
(9, 15). In these cells, CAV/DGV expression increased the buoyant
density of Ras-containing membranes. The effect of this shift was
reflected in the redistribution of caveolin and to a lesser extent of
Ha-Ras, with no redistribution of Ki-Ras detected (9). This suggests
that Ki-Ras, and to a lesser extent H-Ras, remains localized to low
density raft domains even in the presence of CAV/DGV. This is likely
true for N-Ras as well, since N-Ras only partially colocalizes with
caveolin-containing membranes.
We have shown that disrupting caveolin function by overexpressing
CAV3/DGV did not block the activation of Raf-1 by EGF, Ha-RasV12, Ki-RasV12, or N-RasV12 in these cells. Our finding that CAV3/DGV did
not block EGF activation of Raf-1 supports the results of Kranenburg
et al. (11), and may reflect the prominent role of endogenous Ki-Ras and N-Ras in EGF action in COS-7 cells. However, the
inability of CAV/DGV to block the action of Ha-RasV12 on Raf-1 appears
to conflict with the results of Roy et al. (9). This may
reflect differences in the membrane compositions of the BHK cells (9,
10) and the COS-7 cells used in this study. For example, Ki-Ras, which
this group and others (11) identified in raft microdomains in COS-7
cells, has been localized to non-raft domains in BHK cells (10).
In this study we compared the ability of distinct CAAX
motifs to potentiate the phosphorylation and activation of a variety of
chimeric Raf-1/CAAX proteins whose carboxyl-terminal domains were derived from Ha-Ras or Rap1. We show that a Raf-1 chimera that
included the complete carboxyl-terminal membrane targeting domains from
Ha-Ras was localized to rafts, showed both constitutive activity and
phosphorylation of Ser-338, and activated ERKs. Chimeras containing
only the minimal membrane-targeting motif
[Raf-HaCAAX(
hvr)], however, had no basal activity. We
suggest that the lack of activity of this chimera was a direct
consequence of its inability to be phosphorylated on serine 338. The
ability of Y341D to restore the raft localization and Ser-338
phosphorylation of Raf-1-HaCAAX(
hvr) and kinase activity
argues that localization to specific raft microdomains may be necessary
and sufficient for Ser-338 phosphorylation and activation of Raf-1.
Recent studies have proposed that hvr sequences help shuttle Ha-Ras out
of the rafts in a GTP-dependent fashion (10) and cooperate
in effector utilization (16). Differences in the localization of mutant
Ha-Ras proteins and Raf/Ras chimeras may be due to the influence of
sequences in Ras mutants that are absent from the Raf/Ras chimeras. It
is also possible that activated Ras shuttles Raf-1 into the raft where
it is phosphorylated on Ser-338 and subsequently exits the raft, as
suggested by recent studies (10, 48).
Phosphorylation of Tyr-341 Is Required for Proper Raft Localization
and Subsequent Phosphorylation of Ser-338--
Upon EGF stimulation,
RafS338A was localized to a raft microdomain. Moreover,
RafS338A-HaCAAX(+hvr) was constitutively localized to a raft
domain. These data demonstrate that Ser-338 phosphorylation was not
required for raft localization, but likely occurs subsequently. This is
consistent with a model that Raf-1 activation by Ha-Ras requires
post-translational modifications, including Ser-338 phosphorylation, which occurs within specialized microdomains.
We show here that Tyr-341 phosphorylation of Raf-1 was a prerequisite
for Ser-338 phosphorylation, consistent with previous results (27).
Marais and co-workers (27) also showed that tyrosine phosphorylation by
Src enhanced Ser-338 phosphorylation of Raf-1. The data presented here
suggest that one of the consequences of Tyr-341 phosphorylation may be
the repositioning of Raf-1 near potential Ser-338 kinases. The
requirement of Tyr-341 in Raf-1 activation and Ser-338
phosphorylation, however, could be overcome by membrane targeting,
suggesting that one of the functions of Tyr-341 phosphorylation is to
facilitate proper membrane localization. Indeed, RafY341A mutants were
unable to enter rafts upon EGF stimulation, unless linked to ectopic
raft-targeting domains.
One explanation for the increased phosphorylation on Ser-338 seen in
Y341D mutants is that this reflects the strong cooperativity between
the phosphorylations of these sites (50). However, in studies examining
the ability of PAK to phosphorylate Raf-1 in vitro, this was
not the case (58). Another explanation is that Y341D mutants relocalize
Raf-1 to sites of Ser-338 phosphorylation. Phosphorylation of Tyr-341
has been proposed to function in concert with pS338 to provide a
negatively charged surface on the Raf-1 protein (27). We suggest that
one additional function of phosphorylation of Tyr-341 that is distinct
from that of Ser-338, is to target Raf-1 to specific membrane sites
that participate in subsequent phosphorylations.
Rap1 Association with Raf-1 Is Not Sufficient for the
Phosphorylation of Tyr-341--
The inability of some small G proteins
to activate Raf-1 despite recruiting Raf-1 to the membrane also
suggests that recruitment to the membrane is not sufficient for Raf-1
activation. One small G protein that binds Raf-1, without activating
it, is Rap1 (44). Chimeric Ras/Rap1 proteins that replace
membrane-targeting domains of Ras with those of Rap1 are growth
inhibitory (42), but this inhibition can be relieved by constitutively
active Raf-1, suggesting that the inhibitory effects of this chimera
were due to impaired Raf-1 activation.
One proposed function for Ras is the displacement of the 14-3-3 protein
from its binding site on residue 259 within Raf-1 (35). The inability
to displace 14-3-3 from Raf-1 may explain the inability of selected G
proteins to activate Raf-1 (59). However, for Rap1, such a model has
been ruled out (59). This suggests that other mechanisms account for
the inability of Rap1 to activate Raf-1. Studies have demonstrated that
activation of endogenous Rap1 limits Ras activation of Raf-1 (33, 38,
44). It has been proposed that Rap1 interferes with Ras by trapping the
Ras/Raf-1 complex in an inactive conformation (60, 61). However, recent
studies have demonstrated that Ras and Rap1 occupy distinct subcellular
regions (39, 41, 43), even following Rap1 activation (40).
In part because of its distinct location, Rap1 has been proposed to
inhibit Ras activation of Raf-1 by sequestering Raf-1 from Ras. This is
consistent with studies showing a loss of Ras/Raf-1 association (and a
parallel increase in Rap1/Raf-1 association) upon Rap1 activation (38).
Data presented here suggest a possible explanation for the inability of
Rap1 recruitment of Raf-1 to activate Raf-1-the inability of Rap1 to
support Raf-1 phosphorylations. First, Raf-1 chimeras that were
targeted to Rap1-containing membranes via Rap1CAAX motifs
were neither activated nor phosphorylated on Ser-338. Second,
retargeting Rap1 by swapping in Ha-RasCAAX sequences allowed
Rap1 to activate Raf-1 and to phosphorylate Ser-388. Mutation of Raf-1
to mimic Tyr-341 phosphorylation (Y341D) resulted in a Raf-1 protein
that could be activated and phosphorylated on Ser-338 following Rap1
activation, suggesting that the phosphorylation on Tyr-341 can
partially overcome Rap1's inability to activate Raf-1. This may be due
to the lack of specific Tyr-341 kinases within Rap1 domains. Therefore,
we propose that Rap1 prevents Raf-1 activation by positioning it away
from tyrosine kinases that are required for Tyr-341 phosphorylation.
One of the functions of Tyr-341 phosphorylation might be to provide a
regulatable interaction with proteins or lipids to participate in
proper targeting of Ras/Raf-1 (62, 63).
The Lack of Dependence of B-Raf on Tyrosine Phosphorylation
Accounts for Its Activation by Rap1--
The Y341D mutation in Raf-1
resembles the naturally occurring sequences within the Raf isoform,
B-Raf. B-Raf lacks a tyrosine at the site corresponding to Tyr-341 in
Raf-1 (448 in B-Raf). Like Raf-1Y341D, B-Raf is constitutively
phosphorylated on the nearby serine (Ser-338 in Raf-1, Ser-445 in
B-Raf). Although B-Raf was constitutively active in in vitro
kinase assays, we show that membrane recruitment was required to permit
B-Raf to activate MEK and ERKs in vivo. Moreover, targeting
of B-Raf chimeras via either Rap1-CAAX or
Ras-CAAX was sufficient. The ability of B-Raf-Rap1 chimeras
to activate ERKs confirms that the requirement for membrane localization for B-Raf activation by small G proteins is less stringent
than that of Raf-1. The mutant of B-Raf in which Asp-447/Asp-448 was
replaced by tyrosines (B-RafYY) behaved like Raf-1; it was no longer
activated by Rap1, but retained the ability to be activated by Ras. The
unique specificity of Rap1 for B-Raf activation, but not Raf-1
activation, can be largely explained by the distinct requirements of
each kinase for specific membrane targeting for phosphorylation and
activation. Future studies examining the ability of Rap1 to support
additional critical phosphorylations, including Thr-491 and Ser-494 in
Raf-1 (Thr-598 and Ser-601 in B-Raf) (28, 64) may be informative as well.
It has been proposed that sequences within the cysteine-rich domain
(CRD) of Raf-1 and B-Raf dictated the contrasting actions of Rap1 on
each Raf isoform (44). However, the ability of Rap1 to activate
RafY341D, as well as the ability of Rap/Ras chimeras to activate wild
type Raf-1, both argue strongly that the interactions between Rap1 and
Raf-1 are not the only determinants of Raf-1 inhibition. It should be
noted that Rap1/Ras chimeras were not as effective as Ras in
activating/phosphorylating Raf-1, suggesting that Ras also provides an
activation function that is distinct from localization (22, 59, 65).
Furthermore, the lack of activation of B-RafYY by Rap1 suggests that
interactions between the B-Raf CRD and Rap1 are also not sufficient to
promote activation, although they may be important (44). We propose
that the carboxyl-terminal domain of Rap1 provides specificity to Rap1
signaling in addition to that provided through the interaction between
Rap1's effector loop and the Raf CRDs.
In conclusion, we show that Raf-1 phosphorylation at Ser-338 requires
membrane targeting of Raf-1 to specific raft microdomains. We propose
that tyrosine phosphorylation of Tyr-341 potentiates Ser-338
phosphorylation by facilitating proper membrane localization. This
two-step mechanism is outlined in Fig.
10 and may explain the contrasting
actions of Ras and Rap1 on Raf isoforms.

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Fig. 10.
A model of sequential phosphorylation of
Raf-1 for full activation. EGF induces the recruitment of Raf-1 to
plasma membrane-bound Ras. Following this association of Ras with Raf-1
two sequential modifications occur. The first modification is the
phosphorylation of Tyr-341 by membrane-bound Tyr-341 kinases, whose
activities are induced by EGF and/or Ras activation. This
phosphorylation may relocalize the Ras/Raf-1 complex within specialized
plasma membrane microdomains where a second phosphorylation on Ser-338
can occur that renders the Raf-1 molecule competent in phosphorylating
downstream effectors like MEK/ERK. Rap1 signaling is depicted in the
lower portion of the figure. Rap1 activation does not lead
to phosphorylation of Raf-1 or activation of MEK/ERK, although Raf-1 is
recruited to Rap1-containing membranes. In contrast, B-Raf is
constitutively phosphorylated on Ser-445 (Ser-338 equivalent site), and
the adjacent tyrosines in Raf-1 are replaced with aspartate residues
(Y447D/Y448D), and therefore do not need to be phosphorylated upon
recruitment to Rap1. In this case, Rap1 is capable of coupling B-Raf to
MEK/ERK signaling. We suggest that the lack of Tyr-341 activity within
Rap1 domains is the limiting step in Rap1's inability to activate
Raf-1. Gray and white circles represent Tyr-341
kinases and Ser-338 kinases, respectively.
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