 |
INTRODUCTION |
Ras proteins are small molecular weight GTPases that link cell
surface receptors to intracellular effector pathways that regulate cell
growth, differentiation, and survival. The mammalian genome contains
three ras genes that encode highly related proteins of either 188 or 189 amino acids: Ha-Ras, N-Ras, K4A-Ras, and K4B-Ras (1).
The first 86 amino acids of Ras proteins are 100% identical, and the
next 80 amino acids exhibit 85% homology between any pair of Ras
proteins. The remaining C-terminal sequence consisting of residues
166-185, known as the hypervariable domain, is highly divergent with
the exception of the last four amino acids (the CAAX motif), which are
required for post-translational processing and plasma membrane
association (2). Although ubiquitously expressed, the three Ras
isoforms have been postulated to perform distinct biological functions.
This notion is supported by the finding that K-Ras but not Ha-Ras or
N-Ras is essential for normal mouse development (3, 4). Additionally,
mutationally activated forms of the three ras genes
are selectively expressed in different human tumors. For example, K-Ras
mutations occur in 90% of pancreatic adenocarcinomas, whereas in acute
myeloid leukemia over 25% of mutations detected involve N-Ras (5).
The biological effects of Ras proteins are exerted through the
activation of several downstream effectors including the Ser/Thr kinase
Raf, the p110 catalytic subunit of phosphoinositide 3-kinase (PI3K),1 and Ral GDS, an
exchange factor for Ral GTPases (6). The interaction of Ras with its
effectors is mediated by the effector binding loop, which spans
residues 32-40. Despite the fact that the amino acid sequence
corresponding to the effector binding loop is identical among Ras
proteins, recent studies have demonstrated that the three Ras isoforms
can differentially activate Raf-1 and PI3K. K-Ras activates Raf-1 more
effectively than Ha-Ras and N-Ras (7, 8). On the other hand, Ha-Ras is
a more potent activator of PI3K than K-Ras (8). These hierarchies
appear to result, at least in part, from differences in the mechanisms
of membrane attachment of the three Ras isoforms (8). However, their
physiological significance remains to be determined.
Through genetic and biochemical studies it has been established that
the growth promoting activity of Ras proteins is dependent on the
synergistic activation of multiple effector pathways (9, 10). Of
particular relevance to the present work are the observations that the
activation of a pathway that engages the small GTPase Rac is critical
for both the mitogenic and oncogenic effects of Ras (11, 12). Rac
itself controls at least three effector pathways leading to
transcriptional activation, rearrangement of the actin cytoskeleton,
and the generation of superoxide (13). Of these, the effector functions
involved in regulating the actin cytoskeleton and production of
superoxide are necessary components of the mitogenic response (14-16).
Additionally, Rac plays an essential role in providing protection
against apoptosis induced by high intensity Ras signaling (17).
In the present study we sought to determine whether the Ha-Ras and
K-Ras isoforms differ with respect to their ability to activate the Rac
pathway. We demonstrate that K-Ras is a more potent activator of Rac
relative to Ha-Ras. Our findings indicate that the efficiency of Rac
activation is dictated by the mode of membrane anchoring of Ras and
impacts on the ability of Ras to regulate cell survival.
 |
EXPERIMENTAL PROCEDURES |
Expression Plasmids--
All Ras constructs were generated by
subcloning the human cDNAs into the mammalian expression vector
pCGT, which is derived from pCGN with a replacement of the HA epitope
with the T7 epitope (18). The Ha-RasV121-164/K-RasV12
cDNA fragments were amplified by polymerase chain reaction using
Pfu DNA polymerase. The primers used are as follows:
Ha-RasV12, 5'-CAATCTAGAATGACGGAATATAAG-3' and
5'-GTCCTGAGCCTGCCGAGATTC-3'; K-Ras V12, 5'-CAGGCTCAGGACTTAGC-3' and
5'-GATGGATCCTTACATAATTACACA-3'. The final polymerase chain reaction
product was digested by the restriction enzymes BamHI and
XbaI and ligated into pCGT. The expression plasmids encoding
T7 epitope-tagged RacV12 and RacWT were kindly provided by Linda
VanAelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
The expression plasmid encoding HA epitope-tagged ERK2 has been
previously described (11). Myr-HRasV12S186 expression plasmid was
kindly provided by J. Gibbs (Merck).
Cell Culture and Transient Transfections--
REF-52 cells were
cultured in DMEM supplemented with 10% fetal calf serum in a
humidified incubator with 6.3% CO2 at 37 °C. COS-1
cells were grown in DMEM supplemented with 5% fetal calf serum in a
humidified incubator with 5% CO2 at 37 °C. Transient transfections using a calcium phosphate method were performed as
described previously (19). Briefly, DNA was diluted in Tris/EDTA buffer
(pH 7) containing 2 M CaCl2 and added to
HEPES/Na2HPO4 buffer. The solution was
incubated at room temperature for 15 min prior to the addition to the
cells. After 12-16 h the cells were washed with DMEM and fed with DMEM
supplemented with 5% fetal calf serum. The cells were serum-starved
for 24 h prior to harvesting.
Mitogen-activated Protein Kinase Assay--
REF-52 cells were
washed twice with cold PBS and lysed in (10 mM Tris, pH
7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1%
Triton X-100, 50 mM NaF, 1 mM sodium vanadate,
10 µg/ml leupeptin, 1% aprotinin, 10 µg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml soybean
trypsin inhibitor, 1 mM okadaic acid, 10 mM
benzamidine). The lysates were clarified by centrifugation for 20 min
at 14,000 × g and then incubated with anti-HA antibody
(12CA5) for 2 h. The immune complexes were captured by incubation
with protein A beads for 45 min. The immune complexes were washed twice
in cold lysis buffer and subsequently washed twice in cold kinase reaction buffer (25 mM Tris, pH 7.4, 0.5 mM
sodium vanadate, 20 mM MgCl2, 2 mM
MnCl2, 20 µM ATP). The ERK2 kinase reaction
was performed by incubating the immune complexes with 0.2 mg/ml myelin basic protein (Sigma) and 10 µCi of [
-32P] ATP
(PerkinElmer Life Sciences; 6000 Ci/mmol) in kinase reaction buffer.
The reactions were incubated for 30 min at 30 °C and terminated by
the addition of SDS sample buffer. The products were resolved by
SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.
Pak Binding Assay--
Serum-starved COS-1 cells were washed
twice with cold PBS supplemented with 1 mM
MgCl2 and 0.5 mM CaCl2 and lysed in
(50 mM Tris, pH 7.4, 10 mM MgCl2,
1% Nonidet P-40, 10% glycerol, 100 mM NaCl, 0.5% sodium
deoxycholate, 10 mg/ml leupeptin, 1% aprotinin, 10 mg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 10 mg/ml soybean trypsin
inhibitor, 10 mM benzamidine). The lysates were clarified
by centrifugation for 5 min at 14,000 × g. Aliquots from the supernatant were taken to compare protein amounts. The lysates
were incubated with either GST alone or GST-PBD (p21-binding domain)
fusion protein bound to glutathione-coupled to Sepharose beads at
4 °C for 30 min. The beads and the proteins bound to the fusion
protein were washed three times with cold lysis buffer, and the eluted
bound Rac proteins were analyzed by Western blotting using a monoclonal
mouse antibody against Rac1.
Immunoblot Analysis--
Samples were resolved on
SDS-polyacrylamide gels and transferred to nitrocellulose membranes
(Schleicher & Schuell). The membranes were incubated with either
anti-T7 monoclonal (Novagen, 1:10,000), anti-Rac1 monoclonal
(Transduction Laboratories, 1:1000), or anti-HA 12CA5 (1:5,000) primary
antibodies and goat anti-mouse secondary antibody coupled to
horseradish peroxidase (Cappel, 1:20,000). The immunoreactive bands
were visualized using enhanced chemiluminescence reagents (PerkinElmer
Life Sciences), and densitometry scanning was performed using Image Pro
Plus computer software.
Microinjection and Microscopy--
For microinjection
experiments, REF-52 cells were plated on gridded glass coverslips,
grown to confluency, and serum-starved for 24 h. 50 ng/µl of
Ha-RasV12, K-RasV12, or Ha-RasV121-164/K-Ras expression
plasmids, each resuspended in microinjection buffer (50 mM
HEPES, pH 7.2, 100 mM KCl and 5 mM
NaH2PO4), were injected into the nucleus of
cells located on selected grids of the coverslip. The membrane ruffles
were scored 4 h after injection by the appearance of transient
phase dense bands traversing across the upper surface of the cell. To
quantify membrane ruffling, the surface area of the membrane ruffles
was measured by autotracing the membrane ruffles based on pixel density
using Metamorph computer software. The average surface area per
injected cell was then normalized to the total area of the injected
cells. Fluid phase pinocytic vesicles were scored as phase bright
vesicles and counted 6 h after injection. The membrane ruffles and
pinocytic vesicles were photographed using a Zeiss Axiovert
Fluorescence microscope. The diameter of the pinocytic vesicles was
measured using Image Pro Plus computer software.
For immunofluorescence, the injected cells were fixed in 3.7%
formaldehyde in PBS for 1 h. The cells were permeabilized with 0.1% Triton X-100 for 3 min at room temperature. Anti-T7 (1:400) or
Y13-259 (1:200) primary antibodies were diluted in 1% bovine serum
albumin/PBS and incubated with the cells for 1 h at 37 °C in a
humidified incubator. The cells were washed three times with PBS and
subsequently incubated with rhodamine-conjugated goat anti-mouse or
rhodamine-conjugated goat anti-rat in 1% bovine serum albumin/PBS for
1 h at 37 °C in a humidified incubator (Cappel, 1:100). The
coverslips were mounted with 5 µl of 2%
p-phenylenediamine (Sigma) dissolved in 200 µl of
Immu-mount (Shandon). The cells were photographed using a Zeiss
Axiovert Fluorescence microscope.
Wound Healing--
REF-52 cells were plated on gridded glass
coverslips, grown to confluency, and serum-starved for 24 h. A
wound diameter between 140 and 180 µm was introduced into a confluent
monolayer of quiescent REF-52 cells with a pair of extra fine
microforceps. Immediately following the introduction of the wound, the
cells aligning the edges of the wound, and the adjacent row of cells
were injected with 50 ng/µl of Ha-RasV12 or K-RasV12 expression
plasmids. The wound width was measured at the time of wounding and
6 h after injection. A mean wound width was calculated for each
time point by measuring the diameter of the wound every 60 µm over
the length of the wound for ~400-500 µm. The expression of the
protein in the injected cells was verified by indirect immunofluorescence.
Apoptosis Analysis--
The detection of apoptotic cells using
fluorescein isothiocyanate (FITC)-annexin V was performed as described
previously (17). Briefly, 16 h after the microinjection of
Ha-RasV12, K-RasV12, and Ha-Ras1-164/K-Ras expression
plasmids (50 ng/µl), the cells were washed once with PBS and
incubated in a buffer containing FITC-annexin V
(CLONTECH Laboratories Inc.) for 15 min at
37 °C. The cells were then washed with PBS and fixed with 3.7%
formaldehyde in PBS. The FITC-annexin V positive cells were visualized
using fluorescence microscopy.
 |
RESULTS |
Ha-RasV12 and K-RasV12 Induce Distinct Membrane Ruffling
Patterns--
Among the earliest cellular responses to Ras activation
is the induction of membrane ruffling (20). To compare the effects of
Ha-Ras and K-Ras on membrane ruffling, quiescent REF-52 cells were
microinjected with T7-tagged Ha-RasV12 and K-RasV12 4B (hereafter referred to as K-RasV12) expression plasmids. 4 h after injection, the cells were analyzed by time lapse video microscopy. Membrane ruffles were readily detected by the appearance of phase dense bands at
the cell periphery or the dorsal surface of the cell (Fig.
1A, arrowheads).
Membrane ruffles induced by Ha-RasV12 occurred predominantly along the
cell periphery and had a long wavelike appearance. K-RasV12 induced
elaborate and highly convoluted membrane ruffles at multiple sites both
on the dorsal surface and the periphery of the cell. In addition to the
distinct morphological differences, the membrane ruffles induced by the
two Ras isoforms displayed different kinetic behavior. The
K-RasV12-induced membrane ruffles were highly dynamic as indicated by
the pronounced changes in ruffle appearance over a 120-s interval (Fig.
1A). In comparison, the Ha-RasV12-induced membrane ruffles
remained mostly unchanged over a 120-s interval. Furthermore, based on
the sampling of 10 cells, the average surface area of the
K-RasV12-induced membrane ruffles was ~2-fold greater than the
Ha-RasV12 membrane ruffle surface area (Fig. 1B). The Ras
isoforms were expressed to similar levels and displayed an identical
cellular distribution as determined by indirect immunofluorescence
(Fig. 1C). Moreover, the distinct ruffling patterns
displayed by Ha-RasV12 and K-RasV12 were retained at higher
concentrations of expression plasmid and occurred irrespective of cell
density (data not shown). Because Ras-induced membrane ruffling is
mediated by Rac (21), the differences in the membrane ruffling activity
of the Ras isoforms suggest that Ha-Ras and K-Ras are differentially
coupled to the Rac pathway.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1.
Ha-RasV12 and K-RasV12 induce distinct
membrane ruffling patterns and exhibit similar subcellular
localizations. A, REF-52 cells were serum-starved for
24 h prior to the microinjection of T7-tagged Ha-RasV12 and
K-RasV12 expression vectors (50 ng/µl). 4 h after injection, the
cells were imaged at 60-s intervals by time lapse video microscopy.
Bar, 20 µm. B, examples of membrane ruffle
traces of three cells injected with Ha-RasV12 (top panels)
and three cells injected with K-RasV12 (bottom panels). The
phase images corresponding to the first cell from the left
are shown. The traces were generated and analyzed as described under
"Experimental Procedures." The average surface area per injected
cell is denoted in pixels ± standard deviations. The
results represent the average of at least five experiments with two
injected cells analyzed per experiment (p = <0.001).
C, REF-52 cells were immunostained for the expression and
subcellular localization of Ha-RasV12 and K-RasV12 with anti-T7
monoclonal antibody followed by rhodamine-conjugated goat anti-mouse
secondary antibody. Bar, 20 µm.
|
|
Ha-RasV12 and K-RasV12 Differentially Stimulate Fluid Phase
Pinocytosis and Cell Motility--
Ras-induced membrane ruffling is
accompanied by the stimulation of fluid phase pinocytosis (20).
Pinosomes are derived from membrane ruffles that close to form
intracellular vesicles. They form at the cell margins and subsequently
move inwardly toward the perinuclear region of the cell. Because the
membrane ruffles induced by each of the Ras isoforms had a unique
pattern, we hypothesized that there would be
isoform-dependent differences in the stimulation of fluid
phase pinocytosis. To test this hypothesis, quiescent REF-52 cells were
microinjected with T7-tagged expression plasmids and analyzed by phase
contrast microscopy at 6 h after injection. The pinocytic vesicles
can be readily identified under these conditions by their refractile
appearance (Fig. 2B), and we
have established previously that they correspond to pinosomes (20). The
number of K-RasV12-induced pinocytic vesicles per cell was ~2-fold
greater than that induced by Ha-RasV12 (Fig. 2, A and
B). Moreover, the average size of pinocytic vesicles formed
in the K-RasV12-injected cells was twice as large when compared with
Ha-RasV12-injected cells (Fig. 2C). As has been shown for
Ha-RasV12 (21), the stimulation of membrane ruffling and pinocytosis by
K-RasV12 was blocked by the co-injection of dominant negative Rac,
RacN17 (data not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of Ha-RasV12 and K-RasV12 on fluid
phase pinocytosis. REF-52 cells were serum-starved for 24 h
prior to the microinjection of T7-tagged Ha-RasV12 and K-RasV12
expression vectors (50 ng/µl). A, 6 h after
injection, the number of pinocytic vesicles per cell were counted in
each injected field. Results represent the average of at least three
experiments ± standard deviations (p = <0.001).
B, phase contrast micrographs of Ha-RasV12 and K-RasV12
injected cells. Pinocytic vesicles appear as highly refractile circular
structures (arrowheads). Bar, 20 µm.
C, the diameters of the Ha-RasV12 and K-RasV12-induced
pinocytic vesicles were measured using Image Pro Plus software. Results
are represented as histograms, and the median diameters are represented
as dotted lines. The histograms represent three experiments
with the number of pinocytic vesicles tabulated from two or three
injected cells/experiment.
|
|
Membrane ruffling is a critical event both in cell spreading and
migration (22). Therefore, we compared the effects of Ha-RasV12 and
K-RasV12 on cell motility using a wound healing assay. A 140-180-µm scratch was introduced into a monolayer of quiescent REF-52 cells, and
the cells aligning the edges of the wound were injected with T7-tagged
Ha-RasV12 and K-RasV12. The K-RasV12 injected cells migrated at a rate
of 18 µm/h (± 1.5), whereas the Ha-RasV12 injected cells migrated at
a rate of 12 µm/h (± 1.2) (Fig.
3).

View larger version (97K):
[in this window]
[in a new window]
|
Fig. 3.
Ha-RasV12 and K-RasV12 exhibit differential
effects on cell motility. REF-52 cells were serum-starved for
24 h prior to the introduction of a wound across a confluent
monolayer. The cells surrounding the wound were microinjected with
empty vector (top panels) or Ha-RasV12 or K-RasV12
expression plasmids (50 ng/µl), and the distance was measured between
the edges of the wound at the time points indicated. The experiment
shown is representative of three experiments that gave similar results.
Bar, 60 µm.
|
|
K-RasV12 Is a More Potent Activator of Rac than Ha-RasV12--
The
enhanced motility induced by K-RasV12 together with the more potent
effects of this isoform on membrane ruffling and pinocytosis suggests
that K-Ras might be a more effective activator of the Rac pathway
compared with Ha-Ras. To directly compare the abilities of Ha-RasV12
and K-RasV12 to activate Rac, we have used a Pak binding assay (23).
COS-1 cells were transiently transfected with T7 epitope-tagged RacWT
and Ha-RasV12 or K-RasV12 expression plasmids. Following 24 h of
serum starvation, cells were lysed, and the lysates were incubated with
the GTPase binding domain (CRIB domain) of the kinase Pak, a downstream
effector of Rac, fused to GST. This domain has been found to
specifically bind to the activated GTP-bound form of Rac (24). The
eluted proteins bound to the fusion protein GST-PBD were analyzed by
Western blotting. As shown in Fig. 4
(A and B), the binding of Rac-GTP to GST-PBD was
~2-fold higher in the presence of K-RasV12 than Ha-RasV12, which is
consistent with K-Ras being a stronger activator of Rac. It is
important to note that the differential effects of Ha-RasV12 and
K-RasV12 on membrane ruffling displayed by REF-52 cells were also
observed in COS-1 cells (Fig. 4C).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4.
K-RasV12 is a more potent activator of Rac
than Ha-RasV12. A, COS-1 cells were transfected with
T7-tagged RacWT and Ha-RasV12 or K-RasV12 expression plasmids or with
RacV12 expression plasmid as a positive control. Transfected cells were
serum-starved for 24 h prior to lysis. Equal amounts of cell
lysates were incubated with GST-PBD fusion protein, and the bound
active Rac-GTP molecules were analyzed by Western blotting using
anti-Rac monoclonal antibody. Densitometric quantitation of immunoblots
is presented as the fold increase over Rac-GTP binding induced by
Ha-RasV12 (bottom panel). Expression of transfected proteins
was determined by immunoblotting the cell lysates with anti-T7
monoclonal antibody (top panel). B, quantitation
of the effects of Ha-RasV12 and K-RasV12 on Rac activation.
Rac-GTP binding activities were determined by densitometry scanning and
normalized to the levels of expression of transfected Rac. Shown are
the averages of three independent experiments ± standard
deviations. C, COS-1 cells were serum-starved for 24 h
prior to the microinjection of Ha-RasV12 and K-RasV12 expression
plasmids (50 ng/µl). 4 h after injection the cells were imaged
using phase contrast microscopy. Bar, 20 µm.
|
|
Ras Membrane Attachment Influences the Activation of the Rac
Pathway--
The attachment of Ras to the membrane is dependent on
specific modifications of the C terminus. The CAAX motifs of
Ha-Ras and K-Ras undergo the same post-translational modifications
involving farnesylation, proteolytic cleavage, and carboxyl methylation (25). In contrast, the hypervariable region preceding the CAAX motif is
processed differently between Ha-Ras and K-Ras. Ha-Ras becomes
palmitoylated on cysteine residues within the hypervariable region,
whereas K-Ras lacks these cysteine residues but contains a polybasic
domain that is required for effective membrane association (2). To test
whether these differences could account for the differential activation
of the Rac pathway, a chimeric Ras protein was constructed containing
the first 164 amino acids of Ha-RasV12 and the last 25 amino acids of
K-RasV12 (Ha-Ras1-164/K-Ras). The chimeric protein showed
the same subcellular distribution as Ha-RasV12 following injection into
quiescent REF-52 cells (Fig. 5A). Significantly, the
membrane ruffling phenotype of the Ha-Ras1-164/K-Ras
protein resembled closely the phenotype induced by K-RasV12 including
an enhanced accumulation of pinocytic vesicles (Fig. 5A).
These results suggest that the membrane attachment mechanisms can
impact the efficiency of Ras-dependent activation of the
Rac pathway.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
The interaction of Ras with the plasma
membrane influences the activation of the Rac pathway.
A, REF-52 cells were serum-starved for 24 h prior to
the microinjection of Ha-RasV12 and Ha-RasV121-164/K-Ras
expression vectors (50 ng/µl). Left panels, the cells were
immunostained for the expression and localization of Ha-RasV12 and
Ha-RasV121-164/K-Ras with anti-T7 monoclonal antibody
followed by rhodamine-conjugated goat anti-mouse secondary antibody.
Bar, 20 µm. Right panels, phase contrast
micrographs of cells expressing Ha-RasV12 and
Ha-RasV121-164/K-Ras, 4 h after microinjection.
Bar, 20 µm. B, REF-52 cells were serum-starved
24 h prior to the microinjection of Ha-RasV12 and Myr-HRasV12S186
expression plasmids (50 ng/µl). Top panels, the cells were
immunostained for the expression and localization of Ha-RasV12 and
Myr-HRasV12S186 with Y13-259 antibody followed by rhodamine-conjugated
goat anti-rat secondary antibody. Bar, 20 µm. Middle
panels, phase contrast micrographs of cells expressing Ha-RasV12
and Myr-HRasV12S186 4 h after microinjection. Bar, 20 µm. Bottom panel, REF-52 cells were transiently
transfected with HA-tagged ERK2 and Ha-RasV12, Ha-RasV12S186, or
Myr-HRasV12S186. ERK2 was immunoprecipitated with anti-HA monoclonal
antibody (12CA5) and incubated with myelin basic protein
(MBP) in the presence of [ -P32]ATP for 30 min at 30 °C. The myelin basic protein phosphorylation was
visualized by autoradiography (bottom panel).
|
|
To further test this idea, we compared the signaling activities of
Ha-RasV12 and Myr-HRasV12S186, which contains a mutation in the
farnesylation site and a myristoylation signal on the N terminus,
enabling the protein to be anchored to the membrane via the N terminus.
Myr-HRasV12S186 and Ha-RasV12 had a similar subcellular distribution
and activated the ERK kinase cascade to a similar level (Fig.
5B). However, Myr-HRasV12S186 failed to induce membrane
ruffling when microinjected into quiescent REF-52 cells, indicating
that activation of the Rac pathway is uniquely sensitive to the
positioning of Ras in the membrane.
Ha-RasV12 but Not K-RasV12 Can Induce Apoptosis--
We have
recently shown that Rac-mediated signals are essential for protection
against Ha-RasV12-induced apoptosis (17). Overexpression of Ha-RasV12
promotes apoptosis, and this response is blocked by the concurrent
expression of dominant active RacV12 and potentiated by the
co-expression of dominant negative RacN17. If Ha-Ras and K-Ras
differentially activate the Rac pathway, then they might be expected to
differ in their ability to induce apoptosis. To test this prediction,
quiescent REF-52 cells were microinjected with 50 ng/µl Ha-RasV12,
K-RasV12, and Ha-Ras1-164/K-Ras expression plasmids, and
apoptosis was visualized by the appearance of rounded cells after
16 h (Fig. 6A). To
quantitate the apoptotic effect, the number of cells stained with
FITC-annexin V, an apoptosis marker, was determined in each injected
area. In agreement with our earlier observations, microinjection of Ha-RasV12 induced apoptosis in 38% (± 4.5) of the cells (Fig. 6B). In contrast, microinjection of K-RasV12 had no apparent
effect on cell viability (Fig. 6). Thus, the differential effects of Ha-RasV12 and K-RasV12 on apoptosis correlate well with the differences in their ability to activate Rac. Furthermore, it appears that the capacity to exert these effects is influenced by the mode of
anchoring of Ras to the plasma membrane because similarly to K-RasV12,
the chimeric Ha-Ras protein Ha-Ras1-164/K-Ras had also no
observable effect on cell viability (Fig. 6B).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of Ha-RasV12, K-RasV12 and
Ha-Ras1-164/K-Ras on apoptosis. REF-52 cells were
serum-starved for 24 h prior to the microinjection of Ha-RasV12,
K-RasV12, and Ha-Ras1-164/K-Ras expression plasmids (50 ng/µl). A, phase contrast micrographs of Ha-RasV12 and
K-RasV12 injected cells. Apoptotic cells appear rounded and refractile
(arrowheads). Images were captured 16 h after
injection. Bar, 80 µm. B, quantitation of
Ras-induced apoptosis. The percentage of apoptotic cells was determined
by counting the number of FITC-annexin V-positive cells as a proportion
of the total number of cells present in the injected field 16 h
after injection. Results represent the averages of at least three
experiments ± standard deviations.
|
|
 |
DISCUSSION |
It is well established that the signaling activities of Ras are
mediated by multiple effector pathways. In the present study we have
demonstrated that the activation of the Rac effector pathway is
differentially regulated by Ha-Ras and K-Ras. A primary determinant in
conferring this difference is the hypervariable region located in the
C-terminal portion of Ras proteins. Because this region specifies the
membrane targeting of Ras, the distinct properties of Ha-Ras and K-Ras
with respect to Rac activation most likely reflect variations in their
membrane anchoring mechanisms.
Several lines of evidence support the notion that Ha-Ras and K-Ras are
localized to distinct membrane domains. First, Ha-Ras traffics to the
plasma membrane via the endomembrane system, whereas K-Ras appears to
reach the plasma membrane through a different transport pathway (26,
27). Second, interfering with the function of caveolin, an integral
membrane protein that binds cholesterol (28) impairs Ha-Ras but not
K-Ras-dependent signaling, indicating that Ha-Ras is
selectively localized to cholesterol-rich membrane domains (29).
Third, K-Ras but not Ha-Ras binds to tubulin, and this
association is critical for the plasma membrane targeting of K-Ras
(30).
It is possible to envisage at least two mechanisms by which the site of
anchoring of Ras to the membrane might influence effector interactions.
Ras effectors or their co-activators could themselves be localized to
distinct subdomains within the plasma membrane. Indeed, a number of
signaling molecules involved in Ras signaling, including the Ras target
Raf, have been shown to be preferentially localized to specialized
cholesterol-rich microdomains also known as caveolea (31). Thus,
differences in the spatial availability of effector molecules could
account for the unique signaling properties of Ha-Ras and K-Ras.
Alternatively, the efficiency of interaction with effector molecules
might be influenced by the mode of anchoring to the plasma membrane.
The association of Ha-Ras with the membrane is mediated by a
farnesyl/palmitoyl anchor, whereas K-Ras uses farnesylation and a
polybasic domain to bind to the plasma membrane (32). Palmitate
contributes to membrane interactions by virtue of its ability to insert
deep into the lipid bilayer, whereas the polybasic domain facilitates
membrane association through ionic interactions with negatively charged
phospholipid head groups (33, 34). These differences could influence
the positioning of each Ras isoform at the inner face of the plasma
membrane, thereby affecting accessibility to effector molecules.
The lack of a detailed understanding of the effector pathways that
couple Ras to Rac makes it difficult to investigate the mechanistic
basis for the differential activation of Rac by Ha-Ras and K-Ras. Like
all GTP-binding proteins, the activation of Rac is mediated by guanine
nucleotide exchange factors that promote the exchange of GTP for GDP.
Guanine nucleotide exchange factors for Rac belong to the Dbl family of
proteins, all members of which contain the tandem arrangement of a PH
domain adjacent to the catalytic Dbl homology domain (35). In certain
cases, the PH domains have been shown to regulate the targeting of Dbl
homology domains to the appropriate subcellular location (36, 37). Although the determinants that specify the targeting properties of PH
domains are not well defined, it has been proposed that, depending on
their lipid binding properties, different PH domains could be recruited
to different membrane domains (38-40). Hence, activators of Rac might
be differentially accessible to Ha-Ras and K-Ras.
PI3K has been implicated in the regulation of Rac activation by Ras
(41, 42). At least one mechanism by which this might occur involves the
generation of lipid mediators that modulate the guanine nucleotide
exchange activity of the Dbl homology domain containing proteins Vav
and Sos (41, 43). It has been shown that Ha-Ras is a more potent
activator of PI3K than K-RasV12 (8). However, we have shown K-Ras to be
a more effective activator of Rac than Ha-RasV12, suggesting the
existence of other coupling mechanisms linking Ras to the Rac cascade.
In addition, it is important to note that mammalian cells express three
isoforms of the catalytic subunit of PI3K, p110
,
, or
(44).
Moreover, it has been recently shown that each isoform has distinct
signaling properties in the context of receptor-mediated regulation of
the actin cytoskeleton (45). Thus, differential interactions of Ha-Ras
and K-Ras with p110 isoforms could contribute to functional specificity. A biochemical assay for isoform-specific activation of
PI3K by Ras will be required to test this hypothesis.
Ras proteins play a role in regulating the turnover of focal adhesions
and are essential for cell movement (22). In agreement with an earlier
report (7), we have found that K-Ras is more effective in stimulating
cell motility compared with Ha-Ras. This difference is most likely a
consequence of the preferential activation of Rac by K-Ras because the
movement of cells requires the coordinated assembly and disassembly of
stress fibers (46). In colon epithelial cells K-Ras but not Ha-Ras
disrupts basolateral polarity by altering the expression of several
intercellular adhesion proteins including
1 integrin and
N-cadherin (47). The preferential ability of K-Ras to induce
loss of cell-to-cell and cell-to-substratum adherence and to stimulate
cell motility could account for the highly invasive and metastatic
phenotype of K-Ras-derived tumor cells (48, 49).
The frequency of K-Ras mutations in human malignancies is considerably
higher compared with Ha-Ras (5). We have found that K-Ras displays a
reduced capacity to induce apoptosis presumably because of its ability
to activate more efficiently the anti-apoptotic cascade. The capacity
of oncogenes to induce apoptosis or cell cycle arrest is thought to
represent safeguard mechanisms to limit the growth of tumor cells.
Thus, the compromised ability of K-Ras to induce apoptosis might
explain, at least in part, the selective growth advantage of tumor
cells harboring K-Ras mutations.
All Ras effectors interact with a common and highly conserved region
within Ras known as the effector binding loop. Our observation that
myristoylated Ha-Ras retains the ability to activate mitogen-activated protein kinase but has lost the ability to activate Rac raises the
possibility that each effector interaction might be uniquely regulated
by the mode of attachment of Ras to the membrane. This is supported by
a recent study suggesting that the C-terminal domain of Ras influences
effector interaction (50). This adds another level of complexity to the
regulation of Ras signaling, which along with the multiplicity of
effector pathways and their cross-interactions enable the finely tuned
control of cellular events that mediate cell growth and differentiation.