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INTRODUCTION |
The small GTPase, Ras, is involved in numerous aspects of normal
cellular metabolism including proliferation, survival/apoptosis, differentiation, and adhesion/motility (1). Ras must be localized to
the inner leaflet of the plasma membrane where, in its GTP-bound active
form, it translocates cytosolic effector proteins such as Raf kinase,
Ral guanine nucleotide dissociation stimulator (RalGDS),1 or
phosphoinositide 3-kinase (PI3-K) to the membrane for subsequent activation. Ras is anchored at the membrane as the result of a series
of post-translational modifications beginning with the attachment of a
farnesyl isoprenoid to a conserved cysteine residue (cysteine 186 in H-
and N-Ras; cysteine 185 in K-Ras) located within the "CAAX
box," a motif consisting of the carboxyl-terminal four amino acids of
Ras. Mutation of the CAAX cysteine therefore prevents both
Ras farnesylation and membrane association, leaving the Ras protein
unprocessed in the cytosol, where the mutationally active, GTP-bound
form can bind effector proteins but without translocating them to the
membrane for activation. Thus, cytosolic GTP-bound H-Ras(C186S)
is thought to act as a dominant negative (DN) inhibitor of normal Ras
signaling (2, 3) by sequestering Ras effectors in nonproductive
complexes (2, 4).
This mode of DN action is in contrast to that of the conventional DN
Ras mutant (S17N) (5), a distinct class of DN Ras that is well
characterized (reviewed in Ref. 2). Ras(S17N) is membrane-localized but
GDP-bound. Therefore, Ras(S17N) fails to bind effector proteins but
instead binds tightly to guanine nucleotide exchange factors,
sequestering them in non-productive complexes and thereby preventing
them from activating Ras. The Ras(G15A) mutant is also of this
class of DN, although it is considered to be nucleotide-free and
therefore even more efficient at sequestering Ras guanine nucleotide
exchange factors. Ras(S17N) is widely used as a specific inhibitor of
Ras activation and has proven useful in defining the role of Ras in a
variety of cellular functions (2).
Despite their potential utility as biochemical tools to study Ras
signaling, cytosolic GTP-bound Ras proteins are not well characterized,
and literature regarding them is limited. Cytosolic GTP-bound Ras(G12V)
was originally shown to inhibit Ras-induced germinal vesicle breakdown
in Xenopus oocytes (6), implying the existence of a
cytosolic factor required for Ras function. Later, Ras(S17N) and
cytosolic GTP-bound Ras were shown to inhibit preferentially signaling
by wild-type and oncogenic Ras, respectively (3), which is consistent
with their different mechanisms of action. We have shown previously (4)
that cytosolic GTP-bound H-Ras(Q61L), generated pharmacologically by
treatment with an inhibitor of the Ras-prenylating enzyme
farnesyltransferase, forms nonproductive cytosolic complexes with Raf
and prevents Raf activation. Similar results were obtained using
H-Ras(G12V) rendered cytosolic by mutation of the farnesylated cysteine
(7). However, it is not known whether N- or K-Ras can also act as DN
inhibitors, whether DN activity is dependent on the Ras-activating
mutation (position 12 versus 61), or whether Ras
effectors other than Raf can be functionally blocked.
One potential advantage of cytosolic GTP-bound Ras is that additional
single mutations (e.g. at residues 35, 37, and 40) can be
introduced within the Ras effector binding domain (residues 32-40)
that selectively impair Ras association with specific effector proteins
(8), including the most well characterized Ras effectors, the
serine/threonine kinase Raf, PI3-K, and RalGDS. Ras effector domain
mutants (EDMs) are well characterized and, in their fully processed and
membrane-localized forms, have been used extensively to demonstrate
that individual Ras effector pathways are sufficient to induce
particular Ras-dependent phenotypes (see for example Refs.
8-15). We envisioned that, in a complementary way, the unprocessed and
therefore cytosolic GTP-bound Ras EDMs could be used to selectively impair Ras signaling through specific effectors and to show that particular pathways are necessary for Ras-dependent phenotypes.
To characterize the potential of cytosolic GTP-bound Ras EDM proteins
to act as DN inhibitors of Ras activity, we generated a series of
unprocessed, oncogenically activated H-, N-, and K-Ras4B mutants to
study various phenotypes that are altered during Ras-induced transformation, including activation of transcription factors and
signaling intermediates and changes in cell morphology and adhesion.
Together, our data characterize the function of cytosolic GTP-bound H-,
N-, and K-Ras4B proteins and demonstrate that they can be used
effectively as DN inhibitors of Ras-dependent signaling, thereby permitting the dissection of Ras signaling pathways in ways
different from those of the more conventional Ras(S17N) and Ras(G15A) DN mutants.
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MATERIALS AND METHODS |
Generation of Cytosolic GTP-bound Ras Constructs--
The
following unprenylated Ras mutants were generated by PCR by methods
described previously (16) using templates containing activating
mutations at either position 12 or position 61: H-Ras(G12V/C186S), H-Ras(G12V/T35S/C186S), H-Ras(G12V/E37G/C186S), H-Ras(G12V/Y40C/C186S), H-Ras(Q61L/C186S), H-Ras(Q61L/T35S/C186S), H-Ras(Q61L/E37G/C186S), H-Ras(Q61L/Y40C/C186S), N-Ras(G12D/C186S), N-Ras(G61K/C186S), and
K-Ras(G12V/C185S). Mutation of the prenylated cysteine (position 185/186) near the carboxyl terminus was accomplished using mutagenic 3'-PCR primers (University of North Carolina Lineberger Comprehensive Cancer Center, Nucleic Acids Core Facility). Effector domain
mutants (EDMs) were generated from templates that also contained
effector domain mutations at positions 35, 37, or 40. PCR primers were designed to introduce restriction sites for subcloning of the product
into the mammalian expression vectors pZIPneoSV(X)1 (17) or
the pZIP-related vector pZBE-HA (G. Clark, National Institutes of
Health). Both vectors drive protein expression from a Moloney murine leukemia virus 5'-long terminal repeat promoter and contain an
SV40 origin of replication (16). Ras coding sequences in pZBE-HA are
fused to the coding sequence for the hemagglutinin epitope
(MASSYPYDVPDYASLGGPSS-). The Ras coding sequence of each construct was
confirmed before use by automated DNA sequencing (Automated Sequencing
Facility, University of North Carolina).
Transient Transcriptional Transactivation Assay for Elk-1
Activation--
NIH 3T3 fibroblasts were maintained humidified at
37 °C in DMEM-H (Invitrogen) with 10% Colorado calf serum (Colorado
Serum Co., Denver, CO) and antibiotics. All luciferase assays were
performed in NIH 3T3 fibroblasts as described previously (18). Briefly, plasmid DNA was precipitated in the presence of high molecular weight
carrier DNA (calf-thymus DNA, Roche Molecular Biochemicals) with 125 mM calcium phosphate and layered onto cells for 3-5 h. Cells were washed, shocked in 15% glycerol for 3 min, and returned to
complete medium for 48 h, washed twice with phosphate-buffered saline (PBS), pH 7.2. Cells were then lysed and assayed for luciferase activity using reagents from the Enhanced Luciferase Assay Kit (PharMingen, San Diego, CA) according to the manufacturer's protocols.
To evaluate the ability of each cytosolic Ras protein to inhibit Elk-1
activation by each membrane-localized Ras protein, NIH 3T3 cells
(2 × 105 cells/35-mm dish plated the day before use)
were transiently transfected in duplicate with 100 ng of plasmid (pZIP)
containing the coding sequence for each membrane-localized GTP-bound
Ras protein (to drive Elk-1 activation) along with 1 µg of vector (pZIP or pZBE-HA) or vector containing the coding sequence of each
cytosolic Ras protein to inhibit Elk-1 activation. Each transfection also contained 250 ng of a Gal-Elk-1 reporter plasmid and 2.5 µg of
5×Gal-Luciferase plasmid (19). Raw data from duplicate samples were
averaged and expressed as percent luciferase activity compared with
vector co-transfected cells. All assays were performed at least three
times, and representative data are displayed ± S.D.
Generation and Characterization of Stable Cell Lines--
NIH
3T3 fibroblasts plated at 2.5 × 105/60-mm dish the
day before use were transfected as described above with 100-200 ng of each Ras-expressing construct by calcium phosphate precipitation. After
48 h, cells were passaged into complete medium with selection (750 µg/ml G418) and fed every other day with fresh selection medium.
Colonies appeared after 2 weeks, and 40-70 colonies of each cell line
were pooled for use. Protein expression was confirmed by SDS-PAGE and
Western analysis or by autoradiography following radiolabeling of cells
with [35S]cysteine/methionine and immunoprecipitation
using Ras-specific antibodies. Similar expression levels were observed
for each protein (data not shown). To confirm that apparent functional
differences were not because of unusually unstable proteins, we
performed pulse-chase assays on each of the stable cell lines. Our data (not shown) indicate that all cytosolic Ras constructs had a half-life comparable to that of membrane-localized H-Ras(Q61L) (i.e.
22 h (20)) and that our results were therefore unlikely to result from variations in protein stability. We also confirmed that all unprenylated Ras proteins remained cytosolic by cell fixation in
formaldehyde followed by immunofluorescent staining as described previously (21), using either anti-HA or anti-pan-Ras antibodies visualized with FITC-conjugated secondary antibody on a Zeiss Axioskop
fluorescence microscope.
Inhibition of PDGF- or Ras-induced Akt and Erk Activation by
Cytosolic GTP-bound Ras--
To evaluate the ability of cytosolic Ras
proteins to inhibit the phosphorylation of Akt and Erk by growth factor
stimulation, NIH 3T3 cells stably expressing cytosolic
H-Ras(Q61L/C186S), its corresponding effector domain mutants (T35S,
E37G, or Y40C), or vector were plated at 1 × 104
cells/35-mm dish and allowed to adhere overnight. Cells were serum-starved (0.5% serum) overnight and then stimulated with 50 ng/ml
platelet-derived growth factor (PDGF; Sigma) for 5 min. Alternatively,
adherent cells were transfected as described above with 200 ng of
oncogenic H-Ras(Q61L) and serum-starved overnight. In either case,
cells were washed with PBS and lysed in 1% Triton X-100, 10% glycerol
in 50 mM Tris, pH 7.4, 100 mM NaCl, plus
protease inhibitors (10 µg/ml aprotinin (Sigma), 5 µg/ml leupeptin,
0.5 mM Pefabloc (Roche Molecular Biochemicals), 20 mM 
-glycerophosphate (Sigma), and 10 mM
para-nitrophenyl phosphate (Sigma)) and a phosphatase inhibitor (1 mM sodium vanadate). Lysates were normalized
to total protein, and 5 µg of total protein/lane was subjected to
SDS-PAGE and Western analysis using either anti-phospho(serine 473)-Akt antibody, anti-total Akt antibody, anti-phospho-Erk antibody, or
anti-total Erk antibody (all antibodies from Cell Signaling, Beverly,
MA). To demonstrate expression of each cytosolic Ras protein, Western
analyses using anti-H-Ras antibody (146-3E4, Quality Biotech, Camden,
NJ) were also done to show relative expression levels of cytosolic Ras
proteins. Proteins were visualized by ECL (SuperSignal, Pierce).
Cell Adhesion Assays--
Confluent NIH 3T3 cells in 100-mm
dishes were harvested with 4 ml of pre-warmed non-enzymatic cell
dissociation solution (Specialty Media, Phillipsburg, NJ).
Alternatively, cells were removed from the dish using pre-warmed 0.25%
trypsin/EDTA (Invitrogen) and resuspended in 8 ml of DMEM-H containing
9% bovine calf serum. Adhesion results were similar with both methods.
Harvested cells were allowed to recover in complete medium for 40 min
at 37 °C, pelleted by low speed centrifugation, and resuspended in 8 ml of DMEM-H with 1% bovine serum albumin (BSA, Pentex) but no serum. Cells were washed twice in the same media and manually counted using a
hemocytometer. Cells (1 × 104 per well) were loaded
in a 96-well plate coated with 1% BSA or 10 ng/ml fibronectin (Enzyme
Research, South Bend, IN). Cells were incubated for 40 min at 37 °C
and then washed 5 times with 100 µl of the above media. Adherent
cells were quantitated using an assay that measures luciferase activity
as a function of intracellular ATP, a co-substrate for luciferase
(ATP-lite, Packard Instrument Co.). Cell numbers were determined by
comparing luminescence values of samples to a standard curve plotted as
luminescence versus known cell number. Number of cells
adhering to BSA was defined as nonspecific and subtracted from number
of cells adhering to fibronectin. Adhesion was expressed as the ratio
(%) of adherent cells to total cells loaded into the assay (1 × 104).
To determine whether integrin-specific antibodies could block cell
adhesion to fibronectin, subconfluent NIH 3T3 cells were harvested with
0.25% trypsin, 1 mM EDTA and allowed to recover at
37 °C for 30 min in serum-containing growth media. Cells were washed
three times in DMEM, 1% BSA to remove serum, counted, and incubated
with 40 µg/ml of 1 integrin antibody (CD29, clone
Ha2/5; BD PharMingen) or control antibody (clone G235-1; BD
PharMingen) at 37 °C for 30 min. After incubation with the blocking
antibody, 1 × 104 cells were loaded into individual
wells of a 96-well plate (Falcon) that had been previously coated with
10 ng/ml human fibronectin (Enzyme Research Laboratories, South Bend,
IN). Adhesion was allowed to take place for 25 min at 37 °C. Wells
were washed with DMEM, 1% BSA five times to remove unbound cells. The
remaining adherent cells were quantitated using the
ATP-dependent luminescent assay described above. Results
were displayed as the percentage of total cells bound.
Flow Cytometry Analysis of Integrin Expression--
Subconfluent
NIH 3T3 cells stably expressing each cytosolic Ras mutant or vector
were harvested with 0.25% trypsin, 1 mM EDTA, added to 10 ml of serum-containing media, and allowed to recover for 40 min at
37 °C. Cells were washed three times in PBS, 1% BSA, resuspended in
2 ml of PBS, 1% BSA, and incubated with 1 integrin
antibody (CD29, clone Ha2/5 BD PharMingen) in parallel with an
isotype-matched control antibody (clone G235-1; BD PharMingen) at 1 µg/ml for 60 min on ice. For detection of 1 integrin,
FITC-conjugated anti-mouse Ig Fab' antibody
(BIOSOURCE International, Camarillo, CA) was used
at 10 µg/ml for 30 min on ice in the dark. Cells were washed two
times in cold PBS, 1% BSA and resuspended in 500 µl of the same
buffer. Fluorescence was detected by a FACScan flow cytometer
(Becton-Dickinson, Franklin Lakes, NJ).
Focus Formation Assays--
To assess the ability of cytosolic
Ras proteins to inhibit the growth of oncogenic H-Ras-induced foci on a
monolayer of NIH 3T3 fibroblasts, cells stably expressing cytosolic Ras
proteins or vector were transiently transfected by calcium phosphate
precipitation (22) with 200 ng of pZIP H-Ras(G12V) or H-Ras(Q61L).
Cells were maintained at 37 °C and 10% CO2 in DMEM-H
(Invitrogen) containing 10% Colorado calf serum plus antibiotics and
fed every other day. After 2 weeks cells were washed twice in
phosphate-buffered saline, pH 7.4, fixed for 10 min in 75% methanol,
25% acetic acid, stained for 1 min with 0.4% crystal violet in 20%
ethanol, and washed extensively with water until excess stain was
removed. All transfections were done in duplicate.
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RESULTS |
Cytosolic GTP-bound H-, N-, and K-Ras4B Inhibit Elk-1
Signaling--
Cytosolic Ras proteins containing GTPase-inactivating
mutations have been shown to act as DN inhibitors of Ras signaling, whereas corresponding versions lacking a GTPase-inactivating
mutation do not (3, 4, 6, 7). However, these studies were somewhat limited. First, only H-Ras, but not N- or K-Ras, was evaluated for DN
activity. Second, direct comparisons between Ras(G12V) and Ras(Q61L) to
determine the respective effects of these activating mutations on the
potency of DN activity were not performed. Third, although it was
shown that Ras-dependent Raf signaling could be inhibited,
the potential of cytosolic GTP-bound Ras to inhibit other Ras
effectors, such as PI3-K, was not evaluated. To address these points,
we generated a series of H-, N-, and K-Ras mutants that were rendered
unprenylated and, therefore, cytosolic by cysteine to serine mutations
at position 186 (185 in K-Ras). Each protein also contained an
activating mutation at either position 12 or 61. These constructs were
H-Ras(G12V/C186S), H-Ras(Q61L/C186S), N-Ras(G12D/C186S),
N-Ras(Q61K/C186S), and K-Ras4B(G12V/C186S). In addition,
H-Ras(G12V/C186S) and H-Ras(Q61L/C186S) constructs also containing
single mutations in the Ras effector domain (residues 32-40) were
generated. Effector domain mutations (EDMs) at positions 35, 37, and 40 were originally identified by their abilities to bind preferentially to
and activate the Ras effector Raf, RalGDS, or PI3-K, respectively (8,
11, 23). Therefore, these Ras proteins H-Ras(G12V/T35S/C186S),
H-Ras(G12V/E37G/C186S), H-Ras(G12V/Y40C/C186S), H-Ras(Q61L/T35S/C186S), H-Ras(Q61L/E37G/C186S), and
H-Ras(Q61L/Y40C/C186S) were designed to preferentially block Ras
signaling through particular effector pathways. Although the H-Ras
constructs but not the N- or K-Ras constructs contained an
amino-terminal hemagglutinin (HA) tag, we have shown
previously that there is no difference between tagged and untagged Ras
proteins in either signaling or transformation (16).
To determine whether DN activity is specific to H-Ras or a general
feature of Ras proteins, we first assessed the ability of cytosolic,
activated H-, N-, and K-Ras to inhibit Elk-1 signaling induced by the
corresponding membrane-localized forms when co-transfected into NIH 3T3
cells in a transient transcriptional transactivation assay. As shown in
Fig. 1, each cytosolic Ras protein was
able to inhibit signaling induced by the corresponding
membrane-localized Ras protein (45-90% inhibition). Generally, N-Ras
mutants inhibited better (85 and 90%) than H-Ras mutants (55 and
70%). Despite equivalent stability, cytosolic K-Ras(G12V) showed the
lowest and most variable ability to inhibit its membrane-localized
counterpart, suggesting that this Ras isoform may have more limited DN
activity than H- or N-Ras DNs. As expected, the DN Ras proteins alone
showed no activity in this assay.
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Fig. 1.
Cytosolic GTP-bound H-, N-, and K-Ras4B
inhibit Elk-1 activation by their membrane-localized counterparts and
cross-inhibit Elk-1 activation by other Ras isoforms.
Co-transfection of fibroblasts with 100 ng of plasmid coding for each
GTP-bound Ras proteins (H-Ras(G12V), H-Ras(Q61L), N-Ras(G12D),
N-Ras(G61K) or K-Ras(G12V)) along with 1 µg of plasmid coding
for each cytosolic GTP-bound Ras protein (H-Ras(G12V/C186S),
H-Ras(Q61L/C186S), N-Ras(G12D/C186S), N-Ras(G61K/C186S), or
K-Ras(G12V/C185S)) in every combination shows that each cytosolic
GTP-bound Ras can act as a DN inhibitor of Ras signaling regardless of
which Ras protein is inducing Elk-1 activation. Overall,
K-Ras(G12V/C185S) produced the most limited inhibitory effect. Data are
shown as average luciferase activity ± S.D. from at least two
independent experiments done in duplicate. All data using cytosolic Ras
proteins are normalized to luciferase activity generated by GTP-bound
Ras in the presence of vector alone, which is defined as 100%.
Co-transfection is as follows: vector, black bars;
H-Ras(G12V/C186S), dark stippled bars; H-Ras(Q61L/C186S),
medium stippled bars; N-Ras(G12D/C186S), light
stippled bars; N-Ras(G61K/C186S), dotted bars; and
K-Ras(G12V/C185S), white bars.
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The data presented in Fig. 1 also show that each cytosolic Ras protein
could cross-inhibit Elk-1 signaling induced by other membrane-localized
Ras isoforms. In general, each cytosolic GTP-bound N-Ras mutant (G12D
or G61K) most strongly inhibited Elk-1 signaling induced by
membrane-localized N-Ras, whereas this isoform specificity was not seen
with cytosolic GTP-bound H-Ras or K-Ras4B mutants. Moreover, cytosolic
N-Ras(G61K) generally had the highest level of DN activity
regardless of the identity of the membrane-localized Ras protein being
inhibited. Here again, cytosolic K-Ras4B(G12V) displayed the least
ability to inhibit Elk-1 activation and produced the most variable
results between assays. Overall, these data suggest that DN activity is
a general feature of Ras proteins and is not limited to one isoform or
to a particular activating mutation. Nevertheless, K-Ras4B(G12V/C185S)
showed decreased DN ability compared with H-Ras or N-Ras in this assay.
Cytosolic GTP-bound H-Ras Effector Domain Mutants (EDMs) Inhibit
Elk-1 Activation Induced by Activated H-Ras or K-Ras4B--
Although
it is well established that Elk-1 activation results from Ras signaling
through Raf, it is also clear that Elk-1 activation is modulated by
Raf-independent inputs (24-26). Furthermore, since all Ras isoforms
bind to and activate the three major Ras effectors (Raf, RalGDS, and
PI3-K), the DN inhibition of Elk-1 activation by cytosolic Ras proteins
shown in Fig. 1 could be mediated by the sequestration of any (or any
combination of) Ras effector proteins within the cytosol.
Effector domain mutations (EDMs) in Ras proteins that preferentially
permit the association of membrane-localized, GTP-bound Ras with
specific effectors were originally described in yeast two-hybrid
systems using cytosolic Ras (8, 10, 11, 23) and have been widely used
to show that distinct effects are mediated by Ras-dependent
Raf, RalGDS, and PI3-K signaling pathways. Here we have used a
corresponding set of cytosolic GTP-bound H-Ras mutants also containing
EDMs (T35S, E37G, and Y40C) to inhibit signaling by endogenous
wild-type Ras through Raf, RalGDS, and PI3-K, respectively.
The data presented in Fig. 2A
show partial inhibition of Elk-1 signaling by each of the effector
domain mutants (55-60%), compared with 85-90% inhibition by
parental H-Ras(Q61L/C186S). This suggests that all three
signaling pathways are necessary for full activation of Elk-1
by H-Ras and that partial DN activity can occur via inhibition of
different signaling pathways. Similar results were obtained if
farnesylated K-Ras(G12V) (Fig. 2B) or geranylgeranylated K-Ras(G12V/188L) (Fig. 2C) was used to
induce Elk-1 activity, which suggests that both H-Ras and K-Ras
activate Elk-1 by the same multipathway mechanism (compare Fig. 2,
A and B). Because it is well established that
activation of Raf induces Elk-1 activation, it is not surprising that
H-Ras(Q61L/T35S/C186S), which should preferentially block Raf, inhibits
Elk-1 activation. It has also been shown that PI3-K may lead to the
activation of Elk-1 (25). Our observation that H-Ras(Q61L/Y40C/C186S)
inhibits Elk-1 activation supports this conclusion. Our data also
indicate that Ras effectors capable of binding E37G, such as RalGDS or phospholipase C
(27), are involved in Elk-1 activation because the
E37G EDM, which should preferentially inhibit these effectors, reduces
Elk-1 activation to a similar degree as the T35S and Y40C EDMs.
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Fig. 2.
Cytosolic GTP-bound H-Ras EDMs partially
inhibit Elk-1 signaling by H-Ras, farnesylated K-Ras4B, and
geranylgeranylated K-Ras4B. NIH 3T3 fibroblasts were transiently
cotransfected as described in Fig. 1 with H-Ras(Q61L), farnesylated
K-Ras(G12V), or geranylgeranylated K-Ras(G12V/C188L) plus
H-Ras(Q61L/C186S), H-Ras(Q61L/T35S/C186S), H-Ras(Q61L/E37G/C186S), or
H-Ras(Q61L/Y40C/C186S). In comparison to maximal inhibition of Elk-1
activation by H-Ras(Q61L/C186S), each H-Ras EDM had limited inhibitory
ability regardless of which Ras protein was used to activate Elk-1.
Data are shown as average luciferase activity ± S.D. from at
least two independent experiments done in duplicate. All data using
cytosolic Ras proteins are normalized to luciferase activity generated
in the presence of vector alone, which is defined as 100%. Similar
results were obtained using H-Ras(G12V/C186S) and corresponding
EDMs.
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Although this shows that cytosolic H-Ras can inhibit signaling by
membrane-associated, GTP-bound K-Ras, treatment of cells with
farnesyltransferase inhibitors not only generates cytosolic H-Ras but
also generates alternatively prenylated (geranylgeranylated) K-Ras (28,
29), whose signaling properties may differ from those of the
farnesylated form (30). Thus we evaluated the DN activity of
H-Ras(Q61L/C186S) on signaling by K-Ras mutated at position 188 to be
exclusively geranylgeranylated (K-Ras(G12V/188L)) and whether such
inhibition was affected by EDMs within cytosolic H-Ras (Fig.
2C). Similar to results with farnesylated K-Ras(G12V), cytosolic GTP-bound H-Ras strongly inhibited Elk-1 activation (90%) by
K-Ras(G12V/188L), supporting the possibility that DN Ras activity may
indirectly contribute to farnesyltransferase inhibitor action. In
contrast, each H-Ras(Q61L/C186S) EDM produced only partial inhibition
(60%), suggesting that Raf, RalGDS, and PI3-K all participate in Elk-1
activation by K-Ras(G12V/188L), and further suggesting that activated
geranylgeranylated K-Ras functions similarly to activated
farnesylated K-Ras in this process.
Dominant Negative Ras Mutants Remain Cytosolic--
We confirmed
that Ras proteins containing mutations of the prenylated cysteine at
position 185/186 were cytosolic and did not significantly associate
with the membrane by performing immunofluorescence analysis on cells
stably expressing each unprenylated protein. Fig.
3 shows that all 185/186 Ras mutants are
exclusively cytosolic within the limits of detection in this assay.
None shows significant membrane localization, which contrasts sharply
with the characteristic membrane fluorescence seen in
H-Ras(Q61L)-expressing cells. That the cytosolic fluorescence observed
in these cells is not due simply to nonspecific staining is confirmed
by the lack of cytosolic fluorescence in vector cells. Qualitatively
similar levels of fluorescence observed in each cell line are
consistent with similar overall protein expression levels.
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Fig. 3.
Dominant negative Ras mutants are
cytosolic. Sparsely plated NIH 3T3 fibroblasts stably expressing
the indicated DN Ras mutants were fixed in formaldehyde and stained
with either anti-Ras or anti-HA antibodies followed by FITC-conjugated
secondary antibodies. Cells expressing empty vector or prenylated
H-Ras(Q61L) served as negative and positive controls, respectively.
Representative examples of stained cells are shown. Significant
cytosolic fluorescence seen in DN cell lines compared with vector
indicates that DN mutant proteins are expressed at significant
levels. The lack of membrane-specific fluorescence compared with
prenylated H-Ras(Q61L) demonstrates that DN proteins do not localize to
membranes to any significant degree.
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Cytosolic GTP-bound H-Ras Inhibits Phosphorylation of Akt and Erk
Stimulated by PDGF or Oncogenic Ras--
Our data suggest that
cytosolic GTP-bound Ras proteins inhibit Ras-induced Elk-1 activation
through Raf, RalGDS, and PI3-K (Fig. 2). Because PI3-K is an upstream
activator of Akt (31, 32) and Raf is an upstream activator of Erk, we
asked whether Akt or Erk activation were also affected by cytosolic
GTP-bound Ras proteins. To stimulate phosphorylation of Akt or Erk, NIH 3T3 cells stably expressing cytosolic H-Ras(Q61L), corresponding EDMs
(T35S, E37G, or Y40C), or vector were either stimulated with PDGF to
trigger endogenous Ras signaling or co-transfected with membrane-localized, oncogenic H-Ras(Q61L). Phospho-Akt (P-Akt) and
phospho-Erk (P-Erk) levels were assessed by Western analysis. As shown
in Fig. 4, cytosolic H-Ras(Q61L)
significantly reduced PDGF-stimulated phospho-Akt or phospho-Erk levels
compared with vector cells (Fig. 4A). These results
demonstrate that cytosolic, active Ras can act as a DN inhibitor of
growth factor-stimulated Akt or Erk activation, presumably by
inhibiting Ras signaling through PI3-K or Raf, respectively. However,
partial inhibition of P-Akt resulted from both the T35S and Y40C DN
EDMs, whereas only T35S significantly inhibited P-Erk. This suggests
that both Raf and PI3-K are involved in the activation of Akt, whereas
only Raf is involved in Erk activation. That inhibition of
PI3-K-reduced Akt phosphorylation is not surprising given the well
established functional connection between these proteins. However, the
fact that inhibition was only partial and that inhibition of Raf also resulted in reduced Akt phosphorylation is surprising because it
implies an as yet unidentified role for Raf in Akt activation. The
inability of the cytosolic E37G EDM to inhibit Akt or Erk activation
suggests that the Ras effector RalGDS is not involved in these
processes. Similar results were obtained when P-Akt and P-Erk levels
were stimulated by co-transfection with active Ras (Fig.
4B), demonstrating that active, cytosolic Ras proteins can act as DN inhibitors of both normal and oncogenic Ras signaling.
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Fig. 4.
Cytosolic GTP-bound H-Ras inhibits
phosphorylation of Akt and Erk in a Raf- and
PI3-K-dependent manner. NIH 3T3 fibroblasts stably
expressing cytosolic H-Ras(Q61L), corresponding EDMs, or vector were
serum-starved overnight, stimulated with PDGF for 5 min, and lysed in
the presence of protease and phosphatase inhibitors (A).
Alternatively, the same series of stable cell lines was transfected
with fully prenylated, oncogenic Ras, serum-starved overnight, and
lysed (B). Lysates containing 10 µg of total protein were
subjected to SDS-PAGE and Western analysis using antibodies against
phospho-(Ser-473)-Akt, total Akt, phospho-Erk, total Erk, or H-Ras.
Proteins were visualized by ECL. The lower panel in each
pair demonstrates that differences in phospho-Akt and -Erk levels were
not the result of differences in total Akt or Erk levels.
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Cytosolic GTP-bound Ras Induces Cell Flattening in Stably
transfected Cells--
Ras is known to participate in numerous aspects
of cellular metabolism including changes in the cytoskeleton and
adhesion resulting from integrin activation. The most striking feature of cells stably expressing cytosolic GTP-bound Ras proteins was their
altered morphology and reduced growth rate. In the data presented in
Fig. 5 each cell line shown is expressing
only the Ras protein indicated. Whereas vector cells have a morphology characteristic of normal NIH 3T3 fibroblasts, those expressing only
cytosolic GTP-bound H-, N-, or K-Ras had a distinctly flattened appearance characterized by low refractility and more extensive spreading, suggesting that these dominant negative proteins are affecting a process in normal cells that modulates cell adhesion through endogenous signaling proteins. This effect contrasts sharply with the highly refractile, spindle-like transformed morphology of
cells expressing dominant active H-Ras(Q61L), suggesting, as expected,
that dominant active Ras and dominant negative Ras have opposing
effects. Interestingly, cytosolic H-Ras(G12V) did not induce
flattening. It is not clear why this is the case because this protein
was expressed at similar levels to the other proteins (not shown), was
cytosolic (Fig. 3), and inhibited Elk-1 activation as effectively as
other cytosolic Ras proteins (Fig. 1). However, its inability to induce
cell flattening is consistent with its relative inability to inhibit
focus formation (Fig. 7). This inability is unlikely to be specific to
H-Ras (versus N-Ras or K-Ras4B) because cytosolic
H-Ras(Q61L) is able to induce this flattened morphology very
efficiently. Also, the possibility that the Q61L mutation confers
greater DN potency than the G12V mutation is not supported by the
ability of cytosolic N-Ras(G12D) and K-Ras(G12V) to induce a flattened
morphology. Finally, cytosolic K-Ras(G12V) can induce morphological
changes even though it poorly inhibits Elk-1 activation (Fig. 1)
whereas the reverse is true of cytosolic H-Ras(G12V), suggesting that
Elk does not play a major role in cell morphology. Moreover, because of
the central role played by PI3-K in modulating cell morphology (11), we
predicted that inhibition of this pathway by the cytosolic Ras
Y40C EDM would revert cells to a normal phenotype, whereas the T35S and
E37G EDMs would have a lesser effect. However, none of the cytosolic Ras EDMs produced a flattened morphology, suggesting either that signaling through multiple Ras effectors is necessary for this phenotypic change or that these effects are too weak to be detected in
this system. A role for multiple effector pathways in cell flattening
is consistent with the relative inability of any of the H-Ras EDMs to
fully inhibit Elk-1 activation (Fig. 2). Because simple visual
inspection is not quantitative enough to conclude that each pathway has
equivalent influence, further analysis is warranted.
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Fig. 5.
Cytosolic GTP-bound Ras induces a
flattened morphological phenotype in stably transfected
cells. Untransformed NIH 3T3 cells stably expressing each
cytosolic GTP-bound Ras protein displayed a morphology distinct from
vector-expressing cells, characterized by extensive cell spreading and
low refractility indicative of cell flattening. This effect
was observed in cells expressing cytosolic H-Ras(Q61L),
N-Ras(G12D), N-Ras(G61K), and K-Ras(G12V) but not in cell
expressing cytosolic H-Ras(G12V) or its EDMs. In contrast, cells
expressing only membrane-localized, GTP-bound H-Ras(Q61L) were
highly refractile and spindle-shaped, characteristic of Ras
transformation. This suggests that DN Ras (cytosolic) and dominant
active Ras (membrane-localized) have opposing effects on cell
morphology.
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Cytosolic GTP-bound Ras Proteins Enhance Cell Adhesion to
Fibronectin--
Overexpression of GTP-bound H-Ras has been reported
to inhibit cell adhesion in Chinese hamster ovary cells by
down-regulating integrins (33). Hence, cytosolic GTP-bound H-Ras might
be expected to increase cell adhesion by blocking H-Ras signaling to
integrins. To determine whether this was the case, we first assessed
the adhesion to fibronectin of NIH 3T3 fibroblasts stably expressing each cytosolic GTP-bound Ras protein used in Fig. 1. Consistent with previous studies (33), prenylated GTP-bound H-Ras decreased cell
adhesion to fibronectin by over 50% compared with vector alone, as
shown in Fig. 6A. GTP-bound
R-Ras(38V) served as a positive control; cells expressing it adhered to
fibronectin 2-fold higher than control cells transfected with vector
alone (34) (Fig. 6, A and B). The cytosolic form
of GTP-bound H-Ras increased adhesion to levels comparable with that of
active R-Ras, suggesting that DN Ras can counteract a Ras-induced
decrease in adhesion in a manner similar to R-Ras. We also show that
cytosolic GTP-bound N-Ras and K-Ras were also able to increase
adhesion, presumably by blocking endogenous Ras signaling to integrins
(Fig. 6A). These data are consistent with a dominant
negative mode of action for cytosolic GTP-bound Ras mutants and with
the idea that reversal of basal Ras signaling can result in
enhanced integrin-dependent adhesion to extracellular
matrices.
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Fig. 6.
Cytosolic GTP-bound Ras proteins enhance cell
adhesion to fibronectin. NIH 3T3 fibroblasts stably
expressing membrane-localized H-Ras(Q61L) or
R-Ras(G38V), cytosolic H-Ras(Q61L/C186S), and corresponding
EDMs, N-Ras(G12D/C186S), N-Ras(G61K/C186S),
K-Ras(G12V/C185S) or vector were non-enzymatically removed from tissue
culture plates, washed, counted, and loaded onto a fibronectin-coated
96-well plate for 40 min at 37 °C. After washing, total adherent
cells were quantitated using a standard curve based on intracellular
ATP content. Specific adhesion was determined by subtracting adhesion
to BSA (see "Materials and Methods"). A,
Vector represents the negative control of cells transfected
with empty plasmid. The positive adhesion control is R-Ras
("38V" is an activating mutation analogous to G12V in
H-Ras). B, H-Ras with no EDM is the cytosolic GTP-bound form
H-Ras(Q61L/C186S). T35S, E37G, and Y40C represent individual point
mutations in the H-Ras effector loop in the context of cytosolic
GTP-bound H-Ras (Q61L/C186S). C, cytosolic Ras-induced
increase in cell adhesion to fibronectin is inhibited by preincubation
with anti-1 antibody (white bars) compared
with preincubation with control antibody (black bars)
showing that 1 is involved in the adhesive process.
D, surface expression levels of the 1
integrin subunit were evaluated by fluorescence labeling with
anti-1 antibody and quantitated by flow cytometry.
Gray peak, control antibody; white peak,
1-integrin antibody. Mean fluorescence intensity
(MFI) correlates with 1 expression. In all
panels error bars depict S.E. of duplicate means from
representative experiments. Each experiment was performed at least
three times.
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Because H-Ras stimulates at least three downstream effector pathways,
we assessed which effector(s) is required for Ras-dependent inhibition of adhesion using cells stably expressing cytosolic H-Ras
and corresponding EDMs (T35S, E37G, or Y40C). As shown in Fig.
6B, the T35S and Y40C EDMs showed significant 2-fold
increases in cell adhesion to fibronectin. To a lesser extent the E37G
EDM also increased adhesion relative to vector. These data suggest that
Raf and PI3-K, and to a lesser extent RalGDS, act downstream of H-Ras
to down-regulate the fibronectin adhesion response in NIH 3T3 cells.
The cytosolic E37G Ras EDM is able partially to block Elk activation
(Fig. 1) demonstrating that this mutant is functional and that its
relative inability to block adhesion to fibronectin is significant.
That the T35S and Y40C EDMs can increase adhesion as effectively as
parental H-Ras(Q61L/C186S) suggests that both pathways are
necessary for decreased adhesion and that inhibiting either one is
sufficient to block this effect. Alternatively, T35S or Y40C may be
more complete blockers of a particular pathway than the parental
mutants because their selectivity allows the whole population of that
protein to associate with and inhibit a single effector rather than
having to simultaneously titrate three (or more) effectors.
Ras is known to affect cell adhesion in part through the modulation of
integrins (33) and that adhesion to fibronectin is mediated largely
through the 
51 integrin (35). Therefore, DN Ras may be increasing adhesion by inhibiting the deactivation of a
1-containing integrin by endogenous Ras. To support this possibility we assessed the expression level of the 1
integrin subunit in DN Ras cells to confirm that changes in adhesion
did not result from changes in expression of this integrin subunit. First, we demonstrated the ability of anti-integrin antibody to prevent
adhesion to fibronectin in cell expressing cytosolic Ras proteins. As
shown in Fig. 6C pretreatment of cells with
anti-1 integrin antibody (white bars) reduced
adhesion of each cell line compared with corresponding cells pretreated
with a control antibody (black bars). This demonstrates that
adhesion is mediated in part by a 1-containing integrin.
Residual adhesion after pretreatment with 1 antibody
implies that 1-independent mechanisms also participate in this adhesive process. We also analyzed cell surface expression using 1-specific antibody quantitated by flow cytometry.
The left peak in each panel of Fig. 6D
corresponds to basal fluorescence using a control antibody, whereas the
right peak shows 1-specific fluorescence. The
mean fluorescence intensity of the right peak correlates with
1 expression. All DN cell lines express the
1 integrin subunit suggesting that modulation of
integrin activation state may be affected by DN Ras.
Cytosolic GTP-bound Ras Proteins Inhibit Focus Formation by
Membrane-localized H-Ras--
Because cytosolic GTP-bound Ras proteins
were able to inhibit Elk-1 and Akt activation, alter the normal
morphology of NIH 3T3 fibroblasts, and increase adhesion to
fibronectin, it was of interest to next determine their effectiveness
in preventing the more complex process of Ras-induced transformation,
which involves each of these elements. NIH 3T3 fibroblasts stably
expressing cytosolic GTP-bound Ras proteins were transfected with
constitutively active, GTP-bound H-Ras to induce focus formation. Only
H-Ras(Q61L)-transfected cells are shown in
Fig. 7; identical results were also
obtained when foci were induced with H-Ras(G12V). All cells
co-expressing active H-Ras and the active cytosolic Ras proteins
produced fewer foci than did cells co-transfected with H-Ras and empty
vector. Interestingly, cells expressing cytosolic H-Ras(G12V) showed
the least inhibition of focus formation, whereas cytosolic H-Ras(Q61L), N-Ras(G12D), N-Ras(G61K), and K-Ras(G12V) all completely blocked focus formation. The reduced ability of cytosolic H-Ras(G12V) and its
corresponding EDMs to inhibit focus formation is consistent with its
reduced ability to induce morphological changes in NIH 3T3s (Fig. 4),
although the reason for this is not clear because all cytosolic
H-Ras(G12V) proteins are expressed at comparable levels to the other
Ras proteins. Cytosolic EDMs T35S and Y40C but not E37G showed reduced
inhibition, suggesting that the Raf and PI3-K but not the RalGDS
effector pathways are involved in the induction of foci by H-Ras. The
general ability of all Ras isoforms to inhibit focus formation is
consistent with our observation that all isoforms also inhibit Elk-1
activation and alter the morphology of NIH 3T3 cells. It is also
consistent with our observation that NIH 3T3 cells stably expressing
cytosolic GTP-bound Ras proteins generally proliferated more slowly
than vector-transfected cells (not shown). To demonstrate that cells
stably expressing cytosolic GTP-bound Ras could be successfully
transfected, we transfected each line with Raf(22W), a
Ras-independent form of the c-Raf kinase that is able, by itself, to
induce foci in NIH 3T3 fibroblasts (36). Raf(22W) induced foci
in all cell lines (not shown) demonstrating that these cells could be
transfected and that the lack of H-Ras(Q61L)- and H-Ras(G12V)-induced
foci shown in Fig. 7 is the result of inhibition of Ras-induced focus
formation by cytosolic GTP-bound Ras proteins.
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Fig. 7.
Cytosolic GTP-bound Ras proteins inhibit
focus formation by membrane-localized H-Ras. NIH 3T3 fibroblasts
stably expressing each cytosolic GTP-bound Ras protein or vector were
transfected with 200 ng of pZIP H-Ras(Q61L) to induce foci. Vector
cells were also transfected with empty pZIP vector as a negative
control. All plates are labeled with the identity of the stably
expressed protein. All plates, except where indicated, were transfected
with H-Ras(Q61L). Stained cells are shown after 2 weeks of growth
under standard conditions. All cytosolic GTP-bound Ras proteins
inhibited focus formation in comparison to vector cells transfected
with H-Ras(Q61L), although cytosolic H-Ras(G12V) showed the most
limited inhibition. Raf and PI3-K but not RalGDS are implicated in
focus formation by the observed reduction in inhibition in cells
expressing the T35S or Y40C EDMs of cytosolic H-Ras(Q61L),
respectively. Similar results were obtained using H-Ras(G12V) to
induce foci. All focus assays were performed in duplicate with
identical results. XF, transfected.
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DISCUSSION |
Here we present the first extensive characterization of cytosolic
GTP-bound Ras proteins and of their function as dominant negative (DN)
inhibitors of Ras signaling. In contrast to normal, fully processed
Ras, the cytosolic forms of GTP-bound Ras associate with effectors but
fail to translocate them to the plasma membrane, thus
sequestering them in non-productive complexes and blocking signaling.
We have also used these proteins, and corresponding versions containing
Ras effector binding domain mutations, to study the role of Ras
effector pathways in various aspects of Ras signaling and
transformation. The Ras EDMs T35S, E37G, and Y40C preferentially retain
the ability to associate with Raf, RalGDS, and PI3-K, respectively.
We and others (4, 7) have shown previously that cytosolic GTP-bound
H-Ras inhibits activation of the Ras effector Raf. Extending that
observation, we show here for the first time that cytosolic GTP-bound
N-Ras and K-Ras can also act as DNs, that the potency of inhibition is
independent of the activating mutation (position 12 versus
position 61) in the cytosolic protein, and that signaling through
another canonical Ras effector, PI3-K, can also be inhibited.
Specifically, cytosolic GTP-bound H-, N-, and K-Ras4B can inhibit the
activation of Elk-1 by any membrane-localized GTP-bound Ras in
transient transcriptional transactivation reporter assays, regardless
of the activating mutation. Maximal inhibition of Elk-1 by cytosolic
GTP-bound H-Ras results from simultaneous inhibition of the Raf,
RalGDS, and PI3-K pathways, because single effector domain mutations
(T35S, E37G, and Y40C) in the cytosolic protein each yielded only
partial inhibition. Cytosolic H-Ras inhibited phosphorylation of Akt
and Erk, whereas introduction of additional mutations at T35S or Y40C
limited this inhibition and E37G prevented it completely. In stably
expressing NIH 3T3 fibroblasts, cytosolic Ras proteins were expressed
at similar levels, remained cytosolic, and in the absence of EDMs,
induced cellular flattening compared with vector-expressing cells.
Consistent with this, we show that cytosolic GTP-bound Ras proteins
increase NIH 3T3 cell adhesion to fibronectin and that a
1-subunit-containing integrin may be involved in this
process. Finally, we show that transformation as measured by focus
formation is inhibited by cytosolic GTP-bound H-, N-, and K-Ras and
that this effect requires inhibition of Raf and PI3-K but not
RalGDS.
Defining the roles of Ras and its effector pathways in either wild-type
or oncogenic Ras signaling has been accomplished primarily through two
approaches. The first involves expressing in cells membrane-localized,
constitutively GTP-bound Ras proteins or their EDMs, which selectively
permit Ras to signal through particular effector proteins, and showing
that this is sufficient for the induction of a corresponding phenotype,
such as altered cell morphology, adhesion, or motility, increased
proliferation, or reduced apoptosis (8-15, 23). A second,
complementary approach involves small chemical inhibitors of several
kinases downstream of Ras to block Ras-induced aspects of
transformation, showing that either Ras or particular Ras effectors are
necessary to induce a given a phenotype. Cytosolic GTP-bound Ras
proteins are similar in function to chemical inhibitors but should also
inhibit effectors even if those effectors are not well characterized.
For example, in addition to Raf, RalGDS, and PI3-K, many other
proteins, such as phospholipase C (27), AF-6 (37), Rin1 (38),
p120Ras-GAP (39), and NF-1 (40), for which chemical inhibitors are not available, also interact with Ras and may also be effectors of Ras
function. Moreover, collectively Raf, RalGDS, and PI3-K are insufficient to reconstitute all aspects of Ras transformation, implicating other additional effectors in Ras signaling. Because they
contain an intact Ras effector binding domain, cytosolic GTP-bound Ras
DN mutants should effectively inhibit all Ras effectors, even those
that are as yet unknown. In this way cytosolic Ras DN mutants would be
more effective inhibitors of Ras-mediated signaling than chemical
inhibitors directed against specific proteins downstream of Ras.
Our observation that cytosolic H-, N-, and K-Ras could each
significantly inhibit Elk-1 activation demonstrates that each Ras
isoform could act as a dominant negative inhibitor of Ras signaling and
that the identity of the activating mutation (12 versus 61)
had no effect on this ability. Furthermore, our data show that DN
activity is independent of the identity of the membrane-localized Ras
that is used to induce Elk-1 activation, consistent with the fact that
these proteins act through direct association with effector proteins.
However, cytosolic GTP-bound K-Ras showed the least inhibition. This
finding is inconsistent with a previous report (41) showing that K-Ras
is a more potent activator of the canonical Ras/Raf/MEK/Erk/Elk-1
cascade than is H-Ras, suggesting that cytosolic K-Ras should sequester
Raf and inhibit Elk-1 more effectively than cytosolic H-Ras. Neither
the expression level nor the stability of cytosolic K-Ras was below
that of H- or N-Ras constructs. It is possible that the relative
inability of cytosolic K-Ras to inhibit Ras-induced Elk-1 activation is
due to differences in affinity of K-Ras versus H-Ras for
effectors. Such differences might be unmasked only in the cytosolic
proteins, by loss of proximity to different effector pools due to
membrane localization of the normal fully processed Ras proteins in
(H-Ras) or out (K-Ras) of lipid rafts (42).
Partial inhibition of H-Ras-induced Elk-1 activity by each cytosolic
GTP-bound Ras EDM suggests that each of these Ras effector pathways is
necessary for full Elk-1 activity. We obtained similar results
regardless of whether Elk-1 activation was stimulated by H-Ras,
farnesylated K-Ras(G12V), or K-Ras(G12V) mutated in the
carboxyl-terminal CAAX motif to be exclusively
geranylgeranylated. Although H- and K-Ras have been reported to
preferentially utilize either PI3-K or Raf, respectively (41, 43),
these results suggest that K-Ras, like H-Ras, also requires multiple
effector pathways for full activation of Elk-1. That inhibition of Raf with the cytosolic GTP-bound Ras T35S EDM reduces Elk-1 activity was
not surprising because the functional connection between Raf and Elk-1
via the Raf/MEK/Erk/Elk-1 pathway is well characterized. Similarly,
inhibition of PI3-K with the cytosolic GTP-bound Ras Y40C EDM resulted
in lower Elk-1 activity, consistent with the work of others (25, 44)
who showed that inhibition of PI3-K with LY294002 or wortmannin could
block both Raf and Erk activation, possibly by blocking PI3-K-mediated
effects on p21-activated kinase (26). Also, both wortmannin and a
dominant negative p85 regulatory subunit of PI3-K (
p85) have been
shown to block A-Raf and Erk activation (24). However, activation of
Elk-1 by PI3-K is not observed in all contexts. For example, in tumors
in nude mice caused by NIH 3T3 cells expressing the membrane-localized,
GTP-bound H-Ras Y40C EDM (12), no increased Erk activation was
observed, suggesting that PI3-K is not upstream of Erk. Although our
data support a role for PI3-K in Elk-1 activation, we cannot rule out the possibility that inhibition of Akt by the T35S EDM results from
residual association with PI3-K. Perhaps more importantly, the presumed
ability of parental unprenylated Ras proteins to bind and inhibit all
cytosolic Ras effectors suggests that at least some of the various
effects we have observed may result from inhibition of effectors other
than Raf, RalGDS, or PI3-K and may even involve as yet unidentified proteins.
Our observation that the cytosolic GTP-bound H-Ras E37G EDM partially
blocks Elk-1 activation suggests that RalGDS is also upstream of Elk.
However, the preponderance of literature suggests that Ras-induced
RalGDS signaling does not result in Erk activation. For example, it has
been shown (9) that neither RalGDS nor the membrane-localized,
GTP-bound H-Ras E37G EDM, which preferentially signals through RalGDS,
activated Erk in NIH 3T3 fibroblasts, the same cell type used in our
studies. Other evidence exists as well (12, 14, 15, 45). However, the
effector binding specificity of the Ras E37G EDM may not be complete.
For example, although Ras(E37G) associates primarily with RalGDS (8) or phospholipase C (27), it has been shown to retain some Raf-binding ability (12). Furthermore, RalGDS and oncogenic Ras(E37G) were not
functionally interchangeable in a muscle differentiation assay (46),
suggesting other downstream targets of Ras(E37G). Thus, more than one
Ras effector pathway may be partially inhibited by cytosolic GTP-bound Ras(E37G).
Although cytosolic GTP-bound Ras strongly inhibits PDGF- and
Ras-induced phosphorylation of Akt and Erk presumably by sequestering PI3-K and Raf, respectively, the corresponding Y40C EDM, which selectively impairs the PI3-K pathway, had a limited effect on phospho-Akt levels. These results suggest that another signal(s) is
required for full Akt activation. Our data show that such a pathway may
involve Raf, because blocking Raf with the cytosolic GTP-bound Ras T35S
EDM inhibits Akt to approximately the same degree as does blocking
PI3-K with the corresponding Y40C EDM. Several studies
(24-26)2 have demonstrated modulation of Raf
activity by PI3-K, primarily by showing that blocking PI3-K activity
with LY294002 or wortmannin inhibits Raf-dependent
phenotypes. It has also been shown that Akt directly phosphorylates Raf
resulting in inhibition of Raf kinase activity (48-50). However, our
observation that the cytosolic T35S EDM also partially inhibits P-Akt
suggests that the reverse is also true, that Raf contributes to PI3-K
activity. It has been shown (51) that Raf is not a direct activator of
PI3-K. However, Raf activation of PI3-K may be indirect. A recent
report (52) demonstrates that Raf-induced transformation of NIH 3T3
cells requires an interleukin 1 autocrine loop, suggesting that
blocking Raf may cause a decrease in interleukin 1-induced stimulation of PI3-K activity by the interleukin-1 receptor, thereby
resulting in the reduced Akt activation that we observed. The
dependence of Elk-1 activity on PI3-K may also be explained in this
way, and it will be interesting to distinguish between direct and
autocrine effects of Raf inhibition on Akt activation.
Ras is known to modulate cell adhesion. Oncogenic H-Ras-induced
signaling through Erk correlates with a decrease in integrin activation
(33), suggesting that one potential role for GTP-bound Ras mutants in
human cancer is to decrease cell adhesion, thereby contributing to cell
survival and proliferation in the absence of substratum and to
metastasis. In contrast, the Ras-related protein R-Ras increases cell
adhesion to fibronectin (34) and collagen (53). Our results show that
cytosolic GTP-bound H-, N- and K-Ras can increase the adhesion of NIH
3T3 fibroblasts to fibronectin to levels at least as high as those
obtained using cells stably expressing GTP-bound R-Ras, possibly by
inhibiting Ras-induced inactivation of integrins via the Erk kinase
pathway (33). If integrins are involved in the increase in adhesion we
observed, our results also suggest that inactivating integrins and
reducing adhesion to the extracellular matrix are normal functions of
endogenous wild-type Ras. Previous studies (33, 34, 54-56) utilized
mutationally active Ras to modulate integrin activation making it
difficult to evaluate the physiological relevance of these observations.
Our data also show that the cytosolic T35S and Y40C EDMs increase
adhesion to the same degree as parental, cytosolic H-Ras, whereas the
E37G EDM has a reduced effect. These results suggest that a Ras-induced
decrease in cell adhesion is primarily dependent on Raf and PI3-K and
to a lesser but significant extent on RalGDS. This is consistent with a
study (33) showing that suppression of Ras-induced integrin activation
correlated with activation of the Erk pathway, and with our observation
that Elk-1 activation is dependent on multiple effector pathways
including Raf and PI3-K. Because none of the cytosolic Ras EDMs (T35S,
E37G, and Y40C) were able to induce cell flattening, whereas all were
able to increase adhesion to fibronectin, it seems likely that the
morphological changes we observed are not the result of inhibition of
integrin deactivation alone but also involve other
Ras-dependent events.
Cellular transformation, as demonstrated by the formation of
Ras-induced foci in an NIH 3T3 cell monolayer, is a complex process that involves reduced contact inhibition of growth and alterations in
cell morphology. Induction of foci by oncogenic H-Ras was completely inhibited by cytosolic GTP-bound H-, N-, and K-Ras, further confirming the general ability of Ras proteins to act as DN inhibitors regardless of activating mutation. The inability of cytosolic H-Ras(G12V) to
inhibit focus formation relative to the other cytosolic Ras proteins
was consistent with its inability to induce apparent cell flattening
but was unexpected given its comparable level of expression, cytosolic
localization, and ability to strongly inhibit Elk-1 activation. In
contrast, cytosolic K-Ras gave the opposite results, producing only
limited Elk-1 inhibition while strongly inhibiting focus formation. The
explanation for this differential inhibition is unclear. That
cytosolic K-Ras is less able than other cytosolic Ras proteins to
inhibit Elk-1 activation, but is equally capable of inhibiting focus
formation, supports the conclusion that multiple Ras effectors are
required for maximum Ras-induced transformation. Further, the relative
failure of cytosolic GTP-bound K-Ras to inhibit Elk-1 activation does
not preclude its inhibiting other Ras effectors that are required for
transformation. Alternatively, even weak inhibition of Elk-1 by
cytosolic K-Ras may reduce Elk-1 activity below a threshold level
necessary for transformation. That parental cytosolic H-Ras but none of
the corresponding EDMs could produce complete inhibition of focus formation also supports the requirement for multiple Ras effectors in
Ras-induced transformation.
The relative inability of cytosolic H-Ras(G12V) and its EDMs to fully
inhibit foci or induce cell flattening compared with corresponding
H-Ras(Q61L) proteins may be the result of previously observed
functional differences between these activating mutations. For example,
although both Ras(G12V) and Ras(Q61L) bind with similar affinities to
the Ras effector Raf (57, 58), the G12V mutation reduces Ras affinity
for Ras-GAP compared with wild-type, whereas the Q61L mutation has the
opposite effect, increasing affinity by almost 50-fold (59). Because
Ras-GAP not only modulates Ras activity but may itself be an effector
of Ras function, differential inhibition of Ras-GAP by cytosolic
H-Ras(G12V) or H-Ras(Q61L) may account for the functional differences
we observed. That both cytosolic Ras mutants inhibit Elk activation
equally well suggests that any presumed effector function of Ras-GAP is
not involved in this process.
Partial inhibition of Ras-induced foci by the cytosolic Ras T35S or
Y40C EDMs demonstrates that Raf and PI3-K, but not RalGDS, are
necessary for full Ras-induced focus formation. This is consistent with
previous observations that membrane-localized, GTP-bound Ras T35S (Raf
signaling) and E37G (RalGDS signaling) EDMs were partially impaired or
fully impaired, respectively, in generating foci (8, 10), suggesting
that signaling through Raf or RalGDS alone is insufficient for maximal
Ras-induced morphological transformation. However, signaling through
RalGDS is capable of synergistically enhancing foci produced by Ras
T35S EDM (Raf signaling) (8), suggesting that RalGDS is necessary but
not sufficient for maximal focus formation. Although our observation
that the cytosolic GTP-bound Ras E37G EDM completely fails to inhibit
Ras-induced foci confirms the insufficiency of RalGDS for this
phenotype, it is inconsistent with the assertion that RalGDS is
necessary for maximal Ras-induced focus formation. The unexpected
observation that inhibition of PI3-K by LY294002 blocked Raf-induced
focus formation2 supports our observation that both Raf and
PI3-K are necessary for maximal Ras-induced focus formation. Whether
these Ras effectors exert their transforming effects through parallel
pathways or sequentially in the same pathway as suggested by our data
remains to be resolved.
§,
, and
TOP
ABSTRACT
INTRODUCTION
