From the Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
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ABSTRACT |
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Membrane anchorage of Ras proteins in the inner
leaflet of the plasma membrane is an important factor in their
signaling and oncogenic potential. Despite these important roles, the
precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface
of intact cells. To investigate Ras-membrane interactions in live
cells, we employed studies on the lateral mobility of a constitutively
active Ras isoform to characterize its membrane dynamics, and examined
the effects of the Ras-displacing antagonist S-trans,trans-farnesylthiosalicylic acid (FTS) (Haklai, R.,
Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch,
G., and Kloog, Y. (1998) Biochemistry 37, 1306-1314) on
these parameters. A green fluorescent protein (GFP) was fused to the N
terminus of constitutively active Ki-Ras 4B(12V) to generate
GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was
preferentially localized to the plasma membrane and displayed
transforming activity. The lateral mobility studies demonstrated that
GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma
membrane, rather than exchange between membrane-bound and unbound
states. Treatment of the cells with FTS had a biphasic effect on
GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral
diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is
released from some constraints on its lateral mobility. This was
followed by dislodgment of the protein into the cytoplasm, and a
reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with
other S-prenyl analogs showed that these effects are
specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.
The small G-proteins of the Ras family are essential components of
signaling cascades that regulate important cell functions such as
growth and differentiation (1-4). Wild-type Ras isoforms alternate
between inactive (Ras-GDP) and active (Ras-GTP) states (5, 6).
Mutations at positions 12, 13, or 61 result in constitutively active
Ras isoforms; these mutants bind GTP, have transforming activity, and
contribute to uncontrolled cell growth (7, 8). The function of Ras
proteins as signal transduction regulators and their oncogenic
potential require association with the inner leaflet of the plasma
membrane (2, 3, 9). Membrane anchorage of Ras proteins is promoted by
their C-terminal S-farnesylcysteine and by either a stretch
of lysines (Ki-Ras 4B) or by S-palmitoyl moieties (Ha- and
N-Ras, or Ki-Ras 4A) (10-13). The anchoring moieties of Ras proteins
also appear to target them to the plasma membrane (2), possibly to
specific membrane domains (14, 15).
Although the essential role of membrane tethering in Ras signaling and
transforming activity is well established, the precise mode of
Ras-membrane interactions is not yet understood. It is not clear
whether Ras proteins are stably associated with the plasma membrane or
undergo rapid exchange between membrane-bound and unbound states (16),
whether they form tight complexes with putative membrane receptors
(17), and whether the Ras anchoring moieties (e.g.
farnesylcysteine) interact randomly with the membrane lipid milieu or
associate preferentially with distinctive domains or sites. A possible
role for specific membrane domains is implied by the evidence that
Ha-Ras is enriched in low buoyant density fractions typical of caveolae
or analogous glycosphingolipid/cholesterol-enriched domains (14,
15).
We have recently developed compounds resembling the farnesylcysteine of
Ras proteins (18-20). One of these compounds,
S-trans,trans-farnesylthiosalicylic acid
(FTS),1 inhibited the growth
of cells transformed by Ha-Ras; the inhibition is not further
downstream, since growth of cells transformed by constitutively active
v-Raf was not affected (18, 19). FTS specifically dislodged
farnesylated Ha-Ras from membranes of Rat-1 cells, but not
non-farnesylated N-myristoylated Ras or prenylated G Earlier studies on Ras-membrane interactions either employed cell-free
systems or involved cell or tissue fixation. Obviously, it is important
to characterize these interactions at the surface of intact cells. To
investigate Ras-membrane interactions in live cells, we applied in the
current work fluorescence photobleaching recovery (FPR) studies on the
lateral mobility of green fluorescent protein (GFP)-tagged
constitutively active Ki-Ras 4B(12V) (GFP-Ki-Ras(12V)) protein
expressed in Rat-1 cells. In combination with experiments on the
effects of the Ras antagonist FTS on Ras membrane anchorage, our
results indicate that GFP-Ki-Ras(12V) undergoes fast lateral diffusion
at the plasma membrane, rather than exchange between membrane-bound and
unbound states. FTS was capable of releasing GFP-Ki-Ras(12V) from some
constraints on its mobility during the early phase of FTS treatment,
prior to the dislodgment of Ras from the plasma membrane. These results
have implications for the nature of the interactions of Ki-Ras with
specific membrane anchorage domains or sites.
Materials--
N-Acetyl-S-trans,trans-farnesyl-L-cysteine
(AFC), S-geranylthiosalicylic acid (GTS) and FTS were
prepared and purified as detailed elsewhere (18, 22).
1,1'-Dihexadecyl-3,3,3',3'-tetramethylindocarbocynanine perchlorate
(DiIC16) and octadecyl rhodamine B chloride (R18) were
obtained from Molecular Probes (Eugene, OR). Bovine serum albumin
(BSA), fatty acid-free BSA, Hanks' balanced salt solution (HBSS),
cytochalasin D, peroxidase-conjugated goat anti-mouse or anti-rabbit
IgG, and protein A-Sepharose 4B were from Sigma. Mouse monoclonal
pan-Ras antibody-3 (anti-Ras) was purchased from Calbiochem, and
anti-GFP rabbit polyclonal IgG was from
CLONTECH.
Construction of GFP-Ki-Ras(12V) Chimera--
The entire coding
region of human Ki-Ras 4B(12V) cDNA cloned in pBluescript II SK via
the PstI and BamHI sites of the multiple cloning
region was a gift from P. Gierschik (University of Ulm, Ulm, Germany).
The insertion was performed by the use of polymerase chain reaction to
generate flanking sequences on the 5' (CTGGAGCAT, containing a
PstI site) and on the 3' (GGATCC, a BamHI site)
ends of the Ki-Ras(12V) coding region. It was then excised by the same restriction enzymes, and inserted into
PstI/BamHI-digested pEGFP-C3 (CLONTECH), resulting in a chimeric construct of
enhanced GFP (a red-shifted enhanced GFP variant) fused in frame to the
5' end of Ki-Ras(12V). The coding regions in the final construct were
verified by DNA sequencing.
Generation of Cell Lines and Cell Culture Procedures--
Cells
were routinely grown at 37 °C, 5% CO2, and 100%
humidity, in Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf serum (FCS; Biological Industries, Beth Haemek, Israel) and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). To
generate stably expressing cell lines, Rat-1 cells (1 × 106 in a 35-mm dish) were transfected with 1 µg of DNA
from the pEGFP-C3 vector (for expression of free, unfused GFP) or the
same vector containing the GFP-Ki-Ras(12V) cDNA. The transfection
mixture contained 10 µl of LipofectAMINE (Life Technologies, Inc.) in 1 ml of Opti-MEM (Life Technologies, Inc.). After 5 h of
incubation at 37 °C, 1 ml of DMEM/FCS was added. After 48 h,
the cells were transferred to a 10-cm dish, and incubated with
selection medium (DMEM/FCS supplemented with 800 µg/ml G418; Life
Technologies, Inc.). Single G418-resistant clones were isolated. The
clones transfected with the vector containing the GFP-Ki-Ras(12V)
cDNA were tested for expression of GFP-Ki-Ras(12V) (see
"Results"), and two representative clones were selected for further analysis.
For experiments on guanine nucleotide binding (see below), COS-7 cells
were transiently transfected by the DEAE-dextran method (23) using 4 µg of the GFP-Ki-Ras(12V) construct per 1 × 106
cells in a 10-cm dish.
Detection of Guanine Nucleotide Binding to
GFP-Ki-Ras(12V)--
We have basically employed a protocol described
previously (24, 25). COS-7 cells were transfected as described above. Eighteen hours after transfection, the medium was replaced by serum-free DMEM, and incubation was continued for 18 h. After washing with DMEM devoid of serum and phosphate, the cells were incubated 4 h at 37 °C with 4 ml of the same medium
supplemented with 0.5 mCi of carrier-free
[32P]orthophosphate (Amersham). The cells were washed
three times with ice-cold phosphate-buffered saline (PBS), and lysed
(15 min, 4 °C) with 0.5 ml of lysis buffer (50 mM
Tris-HCl, pH 7.6, 20 mM MgCl2, 150 mM NaCl, 0.5% Nonidet P-40, 5 units/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol). After removal of insoluble material (14,000 rpm,
10-min spin in Eppendorf centrifuge), free nucleotides were removed by
1.4% charcoal (24). From each sample, volumes containing equal amounts
of radioactivity (5 × 107 cpm) were taken for
immunoprecipitation, performed after completing the volume to 500 µl
of lysis buffer, 1% BSA, 2 µg/ml anti-GFP. Following incubation for
2 h at 4 °C, 40 µl of protein A-Sepharose (120 µg of
protein A) were added, and incubated for 1 h at 4 °C. The beads
were washed twice in lysis buffer and once in PBS. The pellets were
suspended in 20 µl of 20 mM Tris-HCl, pH 7.6, containing 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP. The suspension was heated (65 °C, 5 min) and
centrifuged, and samples (11 µl) were spotted onto a
polyethyleneimine cellulose TLC plate (Sigma), which was developed in
0.75 M KH2PO4, pH 3.4. The TLC
plates were analyzed by a phosphorimager (FujiX Bas1000), and the spots
corresponding to guanine nucleotides were quantified using TINA 2.0 software by Ray Test (Staubenhardt, Germany).
Drug Treatment--
Stock solutions of FTS and its analogs were
freshly prepared in dimethyl sulfoxide (Me2SO) and diluted
to working concentrations (10-50 µM) in DMEM
supplemented with 10% FCS; the final Me2SO concentration
was always 0.1%. Rat-1 cells expressing GFP-Ki-Ras(12V) or GFP were
plated at densities specified for each experiment; 24 h later, the
FTS-containing medium (or similar medium containing only 0.1%
Me2SO in control experiments) was added for various time
periods as indicated in the text. To estimate the effect of FTS on cell
growth, the cells were plated in 24-well plates at 5000 cells/well,
treated with the drug at various concentrations, and counted on day 4 of the treatment.
Treatment with cytochalasin D (10 µg/ml, 15 min, 37 °C) was
performed by adding it to the FTS-containing medium (or to the control
medium) for the last 15 min of the incubation. The drug was kept in for
the remainder of the experiment.
Western Immunoblotting--
Western blotting and ECL were
performed as described by us previously (21). Rat-1 cells expressing
GFP-Ki-Ras(12V) or GFP were plated at a density of 1 × 106/10-cm dish. After FTS treatment as described above,
they were homogenized, and the cytosolic (S100) and total
membrane (P100) fractions were obtained by centrifugation
(100,000 × g, 30 min, 4 °C) (21). Samples of the
total homogenate and of the S100 and P100
fractions were calibrated for similar protein content, and subjected to
SDS-polyacrylamide (12.5%) gel electrophoresis followed by
electrophoretic blotting onto nitrocellulose filters. The filters were
blocked and incubated with antibodies as detailed previously (21),
using either mouse anti-Ras (1:2000) or rabbit anti-GFP (1:1000)
followed by peroxidase-goat anti-mouse (1:7500) or anti-rabbit (1:5000)
IgG, respectively. Bands were visualized by ECL and quantified by
densitometry on a BioImaging System 202D (Dynco-Renium, Jerusalem,
Israel), using Tina 2.0 software (Ray Test).
Labeling of Cells with DiIC16 and R18--
The
lipophilic indocarbocyanine lipid analog DiIC16, which
distributes equally into both the external and internal leaflets of the
plasma membrane in cells (26), was incorporated into the plasma
membrane of Rat-1 cells expressing GFP-Ki-Ras(12V) following the
procedure of Edidin and Stroynowski (27) with minor modifications. The
cells were plated on glass coverslips placed in 35-mm dishes at 10,000 cells/dish. A stock solution of DiIC16 (100 µg/ml) was
prepared in ethanol, and diluted prior to the experiment to 0.3 µg/ml
in HBSS containing 20 mM HEPES, pH 7.4 (HBSS/HEPES). The
cells were incubated with this labeling solution 10 min at 22 °C,
washed three times with HBSS/HEPES supplemented with 0.1% fatty
acid-free BSA to remove free dye, and immersed in HBSS/HEPES containing
BSA (not fatty acid-free; HBSS/HEPES/BSA) and 0.1% Me2SO
(same conditions as for FTS treatment) for FPR studies. In experiments
where the cells were treated with FTS for 30 min or 24 h, they
were washed with HBSS/HEPES prior to labeling, and FTS was added back
at the end of the labeling procedure; other than that, the
DiIC16 labeling was identical to that employed for
untreated cells.
Incorporation of the membrane lipid marker R18 into the cellular plasma
membrane was performed following procedures described previously (28,
29). The procedure was similar to that described above for
DiIC16, except that the labeling solution contained 1.8 µM R18, and the labeling was for 5 min at 4 °C. After
the labeling, the cells were fixed with 4% paraformaldehyde in PBS, mounted in 0.1% gelatin/PBS, and visualized by confocal fluorescence microscopy.
Fluorescence Photobleaching Recovery--
Lateral diffusion
coefficients (D) and mobile fractions
(RF) were measured by FPR (30, 31) using
previously described instrumentation (32). Cells were plated on glass
coverslips at 10,000 cells/coverslip; they were treated where indicated
with FTS or other drugs and/or labeled with DiIC16 as
described above. The coverslip was placed (cells facing downward) over
a serological slide with a depression filled with HBSS/HEPES/BSA and
containing 0.1% Me2SO with or without FTS. The monitoring
laser beam (Coherent Innova 70 argon ion laser; 488 nm and 1 microwatt
for GFP fluorescence or 529.5 nm and 1 microwatt for
DiIC16) was focused through the microscope (Zeiss
Universal) to a Gaussian radius of 0.61 ± 0.02 µm using 100×
oil immersion objective. In some cases, the beam size was changed to
1.23 ± 0.04 µm by using 40× water immersion objective. The
beam radius was determined as described previously (33, 34). The use of
a pinhole in the image plane in the photometer head in front of the
photomultiplier makes the light collection confocal, enabling the
collection of fluorescence from a narrow depth in the focal plane (31),
focusing either on the plasma membrane or in the cytoplasm. A brief
pulse (5 milliwatts, 30-40 ms for the 100× objective, and 40-80 ms
for the 40× objective) bleached 50-70% of the fluorescence in the
illuminated region. The time course of fluorescence recovery was
followed by the attenuated monitoring beam. D and
RF were extracted from the fluorescence recovery
curves by nonlinear regression analysis (35). Incomplete fluorescence
recovery is interpreted to represent fluorescence-labeled molecules
immobile on the FPR experimental time scale (D Expression and Transforming Activity of GFP-Ki-Ras(12V)--
In
order to investigate the interactions of Ki-Ras with the plasma
membrane of intact cells, we have generated a GFP-tagged constitutively
active Ki-Ras 4B (GFP-Ki-Ras(12V)) and stably expressed it in Rat-1
cells (see "Experimental Procedures"). The orientation of the
construct was chosen to retain an intact Ki-Ras C-terminal domain,
which is required for post-translational modification and membrane
anchorage. The GFP-Ki-Ras(12V) was properly synthesized in the cells,
as evidenced by Western immunoblotting using either anti-Ras or
anti-GFP antibodies (Fig. 1A).
Using anti-Ras, both GFP-Ki-Ras(12V) (54 kDa) and endogenous Ras (21 kDa) were detected, and only the latter was observed in homogenates
prepared from cells expressing GFP alone. Upon labeling with anti-GFP,
only the 54-kDa band was detected in cells expressing GFP-Ki-Ras(12V), and a 34-kDa band was observed in GFP-expressing Rat-1 cells. No
labeling was detected in untransfected Rat-1 cells. The apparent molecular weight of GFP-Ki-Ras(12V) fitted that expected for a GFP-Ras
fusion protein, and no smaller fragments were detected. To validate the
constitutively active nature of GFP-Ki-Ras(12V), we employed a guanine
nucleotide binding assay in intact cells. In this assay, we employed
anti-GFP to immunoprecipitate GFP-Ki-Ras(12V), in order to ensure that
the binding measured is to the fusion protein and not to endogenous
Ras. This experiment (Fig. 1B), performed in transiently
transfected COS-7 cells, demonstrated preferential binding of GTP over
GDP to GFP-Ki-Ras(12V) (80% versus 20%, respectively), as
expected for a constitutively active Ras isoform (7, 8).
The expression of GFP-Ki-Ras(12V) in the Rat-1 clones was further
validated by confocal immunofluorescence microscopy. The fluorescent
Ras isoform localized preferentially to the plasma membrane, as opposed
to GFP expressed in Rat-1 cells, which distributed mainly to the
cytoplasm and the nucleus (Fig. 2,
A and B; see also Fig.
3). This indicates that the association
of GFP-Ki-Ras(12V) with the plasma membrane is mediated by the Ras
protein in this construct and not by the GFP. Importantly,
GFP-Ki-Ras(12V) is biologically active, as evidenced by its
transforming activity. Thus, the GFP-Ki-Ras(12V)-expressing Rat-1 cell
lines exhibited anchorage-independent growth in soft agar (Fig. 2,
C and D). Furthermore, these cells were able to
develop tumors in nude mice at a rate similar to that of cells
expressing a constitutively active Ras (5 out of 5 mice in both
cases).
FTS Mediates Dislodgment of GFP-Ki-Ras(12V) to the Cytoplasm and
Inhibits Cell Growth--
We have recently demonstrated that FTS, a
compound resembling farnesylcysteine, specifically dislodges
farnesylated Ha-Ras(12V) from membranes of Rat-1 cells (21). To examine
the effect of FTS on GFP-Ki-Ras(12V), Rat-1 cells stably expressing the
fusion protein were subjected to FTS treatment and to analysis by
confocal fluorescence microscopy. The results of a typical experiment
are depicted in Fig. 3. Prior to FTS treatment, GFP-Ki-Ras(12V) was localized mainly at the rim of the cells, exhibiting typical plasma membrane labeling and co-localization with R18, a fluorescent membrane
marker (Fig. 3, panels A-C; see also the insets
for optical scanning along the z axis). Following FTS
treatment (50 µM, 24-48 h), a significant amount of
GFP-Ki-Ras(12V) was dislodged from the membrane into the cytoplasm; the
dislodgment was not complete, leaving some GFP-Ki-Ras(12V) at the
plasma membrane (Fig. 3, panels D-F; see insets
for z-scan). As expected, R18 remained at the plasma membrane.
The dislodgment of GFP-Ki-Ras(12V) from the membrane was also followed
biochemically, quantifying GFP-Ki-Ras(12V) in the membrane pellet and
in the cytosolic fractions by immunoblotting with anti-GFP antibodies.
Incubation of Rat-1 cells expressing GFP-Ki-Ras(12V) with 50 µM FTS for 24 h mediated a reduction in the relative
amount of the protein associated with the membranes and a parallel
increase in its cytosolic fraction, although a significant fraction
remained in the membrane pellet (Fig. 4).
This suggests that GFP-Ki-Ras(12V) is dislodged by FTS from the
membrane and accumulates in the cytoplasm, in accord with the confocal
microscopy results (Fig. 3). This differs from our former observations
on the fate of Ha-Ras(12V), which was degraded after the dislodgment
(21), suggesting that the GFP-Ki-Ras(12V) is more stable.
Interestingly, incubation with FTS for periods up to 4 h did not
reduce the level of GFP-Ki-Ras(12V) associated with the membrane (Fig.
4), suggesting that relatively long incubation times (>4 h) are
required to dislodge GFP-Ki-Ras(12V) from the membrane.
We have formerly demonstrated that FTS mediates dislodgment followed by
degradation of Ha-Ras(12V) (21) and Ki-Ras
4B(12V)2 in fibroblasts
transformed by these constitutively active Ras proteins. The above
phenomena have biological consequences, as evidenced by the ability of
FTS to inhibit the growth of these cells2 (18, 19). Fig.
5 demonstrates that FTS concentrations
(10-50 µM), similar to those that dislodge
GFP-Ki-Ras(12V) from the membrane, inhibit the growth of Rat-1 cells
transformed by the constitutively active fusion protein. This
inhibition depends on the structure of FTS, specifically on the length
of its farnesyl moiety and its rigid backbone (18, 20), as indicated by
the failure of GTS (10-carbon farnesyl chain versus
15-carbon in FTS) and AFC (15-carbon farnesyl chain but no rigid
backbone) to inhibit the growth of the transformed cells (Fig. 5).
Interestingly, the growth inhibitory response of FTS occurs despite the
absence of accelerated degradation of GFP-Ki-Ras(12V), which
accumulates in the cytoplasm (Figs. 3 and 4). This suggests that
dislodgment, rather than accelerated degradation, of GFP-Ki-Ras(12V)
from the membrane is responsible for the FTS growth-inhibitory
effect.
GFP-Ki-Ras(12V) Diffuses Laterally in the Inner Leaflet of the
Plasma Membrane--
Ras proteins do not span the membrane, and are
anchored to the cytoplasmic face of the plasma membrane via their
farnesyl group and additional moieties. Therefore, they can either show continuous association with the inner lipid leaflet or rapid dynamic exchange between membrane-bound and unbound states. To differentiate between these possibilities, we conducted FPR experiments on
GFP-Ki-Ras(12V) expressed in Rat-1 cells, employing laser beams of
different sizes. In these experiments, fluorescence recovery after
photobleaching may occur either as a result of lateral diffusion of
GFP-Ki-Ras(12V) in the plasma membrane (in the case of continuous
association), or due to exchange between membrane-bound and cytoplasmic
GFP-Ki-Ras(12V) (dynamic exchange). It has been shown (34, 36, 37) that these two mechanisms lead to highly different dependence on the laser
beam size in the FPR experiment. The characteristic fluorescence recovery time
Typical FPR curves obtained with two different laser beam sizes
are shown in Fig. 6. The
The lateral diffusion of GFP-Ki-Ras(12V) (Figs. 6 and
7) is characterized by D = (1.9 ± 0.14) × 10 FTS Induces a Transient Increase in the Lateral Diffusion Rate of
GFP-Ki-Ras(12V)--
To examine the effects of FTS on the interactions
of GFP-Ki-Ras(12V) with the plasma membrane, we studied the lateral
diffusion of GFP-Ki-Ras(12V) in the plasma membrane of Rat-1 cells as a function of the time of incubation with 50 µM FTS (Fig.
7). FTS induced a rapid (within 30 min) and transient (lasting 2-4 h) increase in the lateral diffusion rate (D) of
GFP-Ki-Ras(12V) without affecting its high mobile fraction. This
increase occurs prior to the dislodgment of GFP-Ki-Ras(12V) to the
cytoplasm (see Figs. 4 and 7). At longer incubations (6-48 h),
D of GFP-Ki-Ras(12V) was reduced to about 30% below the
value measured prior to FTS treatment, with no effect on
RF (Fig. 7A). The effects of FTS on
the lateral mobility of GFP-Ki-Ras(12V) are not due to changes in the
dynamic properties of the bulk membrane lipids, since neither the
D nor the RF values of the
DiIC16 lipid probe were altered by FTS (Fig.
7B). It should be noted that the 30-min FTS treatment raised
the D value of GFP-Ki-Ras(12V) to the same value measured for DiIC16, in accord with the notion that GFP-Ki-Ras(12V)
is released from some mobility constraints during early phases of FTS
treatment. The increase in the fluorescence recovery rate is not due to
elevated exchange rates of GFP-Ki-Ras(12V) in the FTS-treated cells,
since FPR experiments analogous to those described in Fig. 6 (using
different laser beam sizes) on cells incubated for 30 min with 50 µM FTS yielded the same dependence on laser beam size
(
Ras proteins are involved in the regulation of
Rac/Rho-dependent cytoskeletal rearrangements (42-44), and
Ras-transformed cells were shown to lack stress fibers (43).
Furthermore, Ras inhibitors (45) and
FTS3 are capable of restoring
actin stress fibers in Ras-transformed Rat-1 cells. To examine the
possible dependence of the FTS-mediated modulation of GFP-Ki-Ras(12V)
mobility on actin stress fibers, we employed cytochalasin D. The
results depicted in Fig. 8 show that the
transient increase in D of GFP-Ki-Ras(12V) after a short incubation with FTS persisted in cytochalasin D-treated cells. Cytochalasin treatment by itself mediated a small but significant reduction in D of GFP-Ki-Ras(12V), most likely due to the
morphological changes in the cell membrane and/or the formation of
actin aggregates. However, FTS treatment for 30 min increased
D of GFP-Ki-Ras(12V) by the same factor as in the absence of
cytochalasin (Fig. 8), suggesting that at the early stages the FTS
effect is independent of stress fibers. On the other hand, the
reduction in the lateral mobility of GFP-Ki-Ras(12V) after prolonged
incubation (24 h) with FTS was not detected after cytochalasin D
treatment (Fig. 8). Thus, the long term reduction in GFP-Ki-Ras(12V)
mobility by FTS may depend to some extent on cytoskeletal
interactions.
Different S-prenyl analogs vary in their ability to inhibit
the growth of Ras- or GFP-Ki-Ras(12V)-transformed cells (Refs. 18 and
19; see Fig. 5). It was therefore important to examine whether the
effects of S-prenyl analogs on the lateral diffusion of
GFP-Ki-Ras(12V) correlate with their ability to inhibit cell growth.
Fig. 9 demonstrates that AFC and GTS,
which fail to inhibit the growth of Rat-1 cells expressing
GFP-Ki-Ras(12V), have no effect on the lateral diffusion of
GFP-Ki-Ras(12V) at either short or long incubation periods. This
contrasts with the modulation of GFP-Ki-Ras(12V) mobility by FTS, which
effectively inhibits cell growth (Figs. 5 and 7).
In the current studies, we employed FPR and the Ras-displacing
antagonist FTS to characterize directly and quantitatively the
interactions of GFP-Ki-Ras(12V), a constitutively active GFP-Ki-Ras 4B
mutant, with the plasma membrane in live cells. The GFP-Ras fusion
protein retained Ras biological activity, as evidenced by several
parameters. (a) It was constitutively active, demonstrating preferential binding of GTP (Fig. 1B); (b) it had
transforming activity, mediating anchorage-independent growth of the
expressing cell lines in soft agar (Fig. 2) and rendering them
tumorigenic in nude mice; (c) it was preferentially
localized to the plasma membrane (Figs. 2 and 3), as reported for
Ki-Ras.
FPR studies were conducted on GFP-Ki-Ras(12V) stably expressed in Rat-1
cells to explore its mode of interactions with the plasma membrane.
Changing the laser beam size in the FPR experiments provides a
sensitive way to differentiate between lateral diffusion within the
membrane or dynamic exchange between membrane bound and unbound forms
(34, 36, 37). Our results (Fig. 6) show an increase in the fluorescence
recovery rate that is proportional to the illuminated area,
demonstrating that the lateral motion of GFP-Ki-Ras(12V) occurs by
lateral diffusion rather than by exchange. While this suggests that
exchange does not contribute significantly to the mobility measured on
the FPR time scale, it may still occur at a slower rate, being
undetectable after diffusion of membrane-anchored molecules had already
taken place. Our results contrast with the interpretation in a former
report (16), which concluded that a cycle 3 GFP-Ki-Ras 4B chimera
diffuses mainly by exchange between membrane and cytosolic forms. Some of the differences may be attributed to cell type differences or to the
use of transient expression. However, this former study relied on the
detection of time-dependent edge-widening of a Gaussian fluorescent spot on the cell surface, an insensitive parameter due to
the very weak intensity at the periphery of the Gaussian spot. This
method is therefore much less sensitive than the FPR beam-size test.
Our conclusion that GFP-Ki-Ras(12V) is not subject to rapid exchange is
in accord with previous findings, which demonstrated that Ras proteins
remain associated with the plasma membrane even after cell
homogenization and isolation of membranes (46).
The lateral diffusion of GFP-Ki-Ras(12V) is less restricted than that
of transmembrane or GPI-anchored proteins (Fig. 6; see also Refs. 27
and 38-41). However, its D value is 1.6-fold lower than
that of the lipid probe DiIC16, suggesting that it
experiences some mobility restricting interactions. GFP-Ki-Ras(12V) is
released from these constraints during the early phase of FTS treatment (within 30 min), as indicated by the elevation of its diffusion rate to
that of DiIC16 (Fig. 7B). This effect is clearly
due to modulation of GFP-Ki-Ras(12V) lateral diffusion, as demonstrated by the lack of effect on its exchange rate using the FPR beam-size test, and by the lack of FTS effect on the dynamic properties of the
bulk membrane lipids (see "Results"). A plausible mechanism that
could give rise to the FTS early-phase effect is competition by the
farnesyl-like FTS for sites that interact with GFP-Ki-Ras(12V) and
retard its lateral mobility. This notion is supported by the lack of
effect of structurally different S-prenyl analogs (Fig. 9).
These interactions appear to be independent of the actin cytoskeleton, since they persisted in cytochalasin D-treated cells (Fig. 8). Elements
that can be involved in such interactions are putative Ras-binding
proteins and/or specific membrane domains (e.g.
glycosphingolipid/cholesterol-enriched domains). Caveolae are not
likely candidates for mediating these mobility-restricting
interactions, since Ras-transformed cells display a large reduction in
caveolin (47), as was validated for the GFP-Ki-Ras(12V)-expressing
Rat-1 cells.4 This is
consistent with reports that Ras is not restricted to caveolae or to
glycosphingolipid/cholesterol-enriched domains (14, 15). However,
transient association of GFP-Ki-Ras(12V) with the latter domains cannot
be dismissed altogether. The size of caveolae (~0.1-0.3 µm
diameter) and the estimated size of sphingolipid rafts (47, 48) is such
that their lateral motion is likely too slow to be detected on the time
scale of the lateral diffusion of free lipids or lipid-anchored
proteins; indeed, GPI-anchored proteins preferentially localized to
glycosphingolipid/cholesterol enriched domains exhibit large immobile
fractions in FPR studies (27, 41). Together with the finding that
GFP-Ki-Ras(12V) displays high mobile fractions
(RF > 0.93) and that the early FTS effects are
on its D values, this indicates that the
mobility-restricting interactions of GFP-Ki-Ras(12V) with such
microdomains (or with membrane proteins that diffuse at a rate much
slower than lipids) must be transient (dynamic) rather than stable.
This conclusion is based on the demonstration (37, 49, 50) that
transient interactions of a labeled membrane component with an immobile entity reduce its apparent D value (since each labeled
molecule undergoes several association/dissociation events during the
measurement). In contrast, stable complex formation reduces
RF, since a bound labeled molecule remains
associated with the immobile component throughout the measurement.
The FTS effect on GFP-Ki-Ras(12V) mobility is biphasic, shifting from
an initial elevation in the diffusion rate to a reduction in
D (Fig. 7A). A plausible mechanism is that once
GFP-Ki-Ras(12V) is released from its original sites of interaction due
to FTS competition (resulting in the early-phase elevation in its
D value), its association with the membrane is altered,
enabling enhanced interactions with novel domains or elements. The
long-term FTS treatment does not affect the bulk of the membrane lipids
(no effect on DiIC16 mobility; Fig. 7B);
however, FTS may induce the formation of new membrane domains or
stabilize pre-existing ones that interact with Ras. Interactions with
actin stress fibers may also contribute to the reduction in
GFP-Ki-Ras(12V) mobility, since cytochalasin D eliminated the long-term
FTS effect (Fig. 8). In accord with this notion, cytoskeletal
interactions were shown to impede the lateral motion of membrane
proteins (38, 39, 51, 52). Indeed, Ras-transformed cells lack stress
fibers (43), which reappear following treatment with FTS3
or with farnesyl transferase inhibitors (45). Finally, since long
incubation with FTS blocks the constitutive activity of
GFP-Ki-Ras(12V), this blockade may be involved in altering the state of
the membrane (domain organization) and/or of the cytoskeleton.
The current experiments showing FTS-mediated growth inhibition of cells
expressing GFP-Ki-Ras(12V), which accumulates in the cytoplasm after
dislodgment (Fig. 4), indicate that the association of GFP-Ki-Ras(12V)
with the plasma membrane is important for its activity. Furthermore,
FTS inhibited the growth of these cells under conditions where a
significant amount of GFP-Ki-Ras(12V) remained in the plasma membrane,
although with altered lateral mobility (Figs. 3, 4, and 7). Since the
release of GFP-Ki-Ras(12V) from the initial constraints on its mobility
at the early phase of FTS treatment occurs prior to its dislodgment, we
propose that GFP-Ki-Ras(12V) interacts preferentially with specific
membrane domains and/or associated proteins, and that these
interactions (which are disrupted by FTS) play a role in Ras activity.
Along with the previous demonstration of FTS-mediated inhibition of Ras-dependent signaling to mitogen-activated protein kinase
(20), the present experiments suggest that altering the mode of
Ras-membrane interactions is sufficient for disruption of Ras signaling.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
of heterotrimeric G-proteins (21), suggesting that it disrupts the
interactions of Ras with specific anchorage domains. Since FTS appears
to affect directly membrane-bound Ras, it can serve as a tool to
investigate Ras-membrane interactions, and has a potential therapeutic value.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
5 × 10
12 cm2/s). All the FPR
measurements were conducted at 22 °C.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Western immunoblotting of and guanine
nucleotide binding to GFP-Ki-Ras(12V). A,
immunoblotting. Total homogenates of Rat-1 cell lines stably expressing
either GFP-Ki-Ras(12V) or GFP were prepared and subjected to gel
electrophoresis followed by immunoblotting with anti-Ras (15 µg of
homogenate protein/lane; left panel) or anti-GFP
(25 µg/lane; right panel) (see "Experimental
Procedures"). Visualization was by peroxidase-conjugated secondary
antibodies and ECL. The lanes are labeled as GFP-Rat-1
(cells expressing GFP) and GFP-Ras-Rat-1(1) and
GFP-Ras-Rat-1(2) (two independent clones of Rat-1 cells
stably expressing GFP-Ki-Ras(12V)). Untransfected Rat-1 cells serve as
a control in the anti-GFP panel. B, guanine nucleotide
binding: COS-7 cells transiently expressing GFP-Ki-Ras(12V) were loaded
with [32P]orthophosphate and lysed. Samples containing
equal amounts of radioactivity (5 × 107 cpm) were
subjected to immunoprecipitation using anti-GFP (+) or normal rabbit
IgG (control; ) as described under "Experimental Procedures." The
nucleotides were extracted, spiked with GDP and GTP, and separated by
TLC. The radiolabeled GTP and GDP spots were analyzed by a
phosphorimager (see "Experimental Procedures"), identifying the
spots by comparison to the spots of unlabeled GTP and GDP, which were
visualized under UV light. The experiment shown is representative of
three carried out. The lanes shown are phosphorimages of duplicate
samples immunoprecipitated by anti-GFP (+), and of a control sample
immunoprecipitated by an equal amount of normal rabbit IgG (
).
Densitometric analysis of the spots showed that about 80% of the bound
radioactivity was in the GTP spot and 20% in the GDP spot (calculated
based on 100% as the sum of the GTP and GDP spots). Radioactivity in
these spots was, respectively, 10- and 1.5-fold higher than the
control.
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Fig. 2.
Fluorescence microscopy and transforming
activity of Rat-1 cells expressing GFP-Ki-Ras(12V).
Panels A and B depict confocal
fluorescent images of Rat-1 cells stably expressing GFP-Ki-Ras(12V)
(A) or GFP (B). In A, stronger
fluorescence at the rim is observed, typical of plasma membrane
labeling, while, in B, cytoplasmic and nuclear labeling are
observed. The cells were grown on glass coverslips and fixed with
paraformaldehyde as detailed under "Experimental Procedures."
Images were collected on a laser-scanning confocal microscope (Zeiss
model LSM 410) fitted with fluorescein filters. Bar, 10 µm. Panels C and D show colony formation
of Rat-1 cells stably expressing GFP-Ki-Ras(12V) in soft agar. The
cells (in DMEM containing 10% FCS) were mixed with 0.5 ml of 0.33%
Noble agar, and poured (104 cells/30-mm dish) onto a layer
of 1.5 ml of 0.5% Noble agar in the same medium. The upper layer was
covered with 0.25 ml of medium, and placed for 11 days in a
CO2 incubator. The dishes were photographed using an
inverted microscope (Olympus IX70). C and D are
fluorescent (using fluorescein filters) and phase contrast images of
the same field, respectively. Bar, 50 µm.
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Fig. 3.
Confocal microscopy demonstrates a shift of
GFP-Ki-Ras(12V) from the plasma membrane to the cytoplasm following FTS
treatment. Rat-1 cells stably expressing GFP-Ki-Ras(12V) were
plated on glass coverslips, treated with 50 µM FTS for
48 h, and then labeled with R18 as described under "Experimental
Procedures." After fixation with paraformaldehyde, dual images
(green fluorescence for GFP, red for R18, and
yellow where the two dyes coincide) were collected on the
LSM 410 confocal microscope fitted with fluorescein and rhodamine
filters. The images were exported in TIFF format to Adobe Photoshop and
printed. Bar, 10 µm. The insets in each panel
depict a z-scan analysis of the same cell (bar,
20 µm). A and D, GFP (green)
fluorescence; B and E, R18 (red)
fluorescence; C, superposition of the images in panels
A and B; F, superposition of the images in
panels D and E. A-C, no FTS treatment (48-h
incubation in medium with 0.1% Me2SO). D-F,
FTS-treated cells.
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Fig. 4.
Dislodgment of GFP-Ki-Ras(12V) by FTS
detected by Western immunoblotting. Rat-1 cells expressing
GFP-Ki-Ras(12V) were plated and treated with 50 µM FTS
(or no FTS) for 4 or 24 h (see "Experimental Procedures").
After homogenization, GFP-Ki-Ras(12V) was determined in the particulate
(P100) and the cytosolic (S100) fractions by
immunoblotting and ECL (see "Experimental Procedures"). Equal
amounts of protein (15 µg) were loaded on each lane. The blots shown
are of a typical experiment (one out of three). Densitometric analysis
indicated that a 4-h treatment with FTS did not result in a significant
change (p > 0.25; Student's t test) in the
distribution of GFP-Ki-Ras(12V) between the membrane and cytosolic
fractions; in the P100 fractions, GFP-Ki-Ras(12V) was
(95 ± 18)% of control, and in the S100 fractions it
was (103 ± 15)%. On the other hand, incubation with FTS for
24 h induced a significant (p < 0.05) shift of
GFP-Ki-Ras(12V) from the membrane to the cytosol; GFP-Ki-Ras(12V) was
(72 ± 8)% of control in the P100 fractions, and
(174 ± 24)% of control in the S100 fractions.
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Fig. 5.
Growth inhibition of
GFP-Ki-Ras(12V)-transformed Rat-1 cells by FTS. Rat-1 cells
expressing GFP-Ki-Ras(12V) were plated in 24-well plates and treated
with FTS, GTS, or AFC at various concentrations (see "Experimental
Procedures"). The cells were counted on day 4 of the treatment. Data
(mean ± S.E. of quadruplicate samples) are presented as the ratio
between the number of cells in drug-treated cultures and in similar
cultures prepared in parallel and incubated with 0.1%
Me2SO.
(the time required to attain half of the recoverable fluorescence intensity in the case of a Gaussian bleach profile; see
Ref. 30) reflects different mechanisms in each case. For lateral
diffusion (continuous association),
is essentially the characteristic diffusion time
D, and is
proportional to the area illuminated by the beam
(
D =
2/4D, where
is the Gaussian radius of the laser beam, and D is the
lateral diffusion coefficient). In the case of dynamic exchange between
membrane-bound and cytosolic forms,
reflects the chemical relaxation time due to exchange, which is equal on all surface regions
regardless of whether they are illuminated by the beam, and therefore
does not depend on the beam size (34, 36, 37).
values
clearly depended on the beam size, and the dependence was proportional
to the area illuminated by the beam (increasing
2 by a
factor of 4 resulted in a parallel increase of
by a factor of
3.7 ± 0.5; n = 30 for each beam size). This
clearly demonstrates that GFP-Ki-Ras(12V) diffuses laterally in the
plasma membrane, and that the contribution of dynamic exchange to the
fluorescence recovery is negligible. Thus, GFP-Ki-Ras(12V) is
continuously associated with the plasma membrane, and does not undergo
appreciable exchange on the time scale of the FPR measurement (up to
~15 s).
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Fig. 6.
Fluorescence recovery after photobleaching of
GFP-Ki-Ras(12V) in Rat-1 cells depends on the laser beam size.
Rat-1 cells expressing GFP-Ki-Ras(12V) were grown on glass coverslips
and subjected to FPR measurements at 22 °C as detailed under
"Experimental Procedures." The dots represent the
fluorescence intensity, and the solid lines are
the best fit to the lateral diffusion equation using nonlinear
regression (35). A, measurement performed using 100× oil
immersion objective ( = 0.61 µm). The specific curve shown yielded
D = 1.91 × 10
9 cm2/s
and RF = 0.95. B, measurement
performed using 40× water immersion objective (
= 1.23 µm). This
curve yielded D = 1.98 × 10
9
cm2/s and RF = 0.96.
9 cm2/s and a high
mobile fraction (RF = 0.94 ± 0.01). It is
significantly faster than the lateral mobility of transmembrane
proteins or even of glycosylphosphatidylinositol (GPI)-anchored
membrane proteins (27, 38-41). This mobility is slightly but
significantly slower than that of the lipid probe DiIC16 in
the GFP-Ki-Ras(12V)-expressing Rat-1 cells (D = (3.3 ± 0.37) × 10
9 cm2/s,
RF = 0.84 ± 0.04, n = 29, 22 °C). The lateral diffusion rate of GFP-Ki-Ras(12V) is in the
range expected for a lipid-anchored protein; a soluble cytoplasmic
protein diffuses at a much faster rate, as we observed for GFP
expressed in Rat-1 cells, whose fluorescence recovered on a much faster
time scale. The extremely fast recovery (
around 0.015 s)
prevented an accurate determination of the diffusion rate of free GFP,
but suggests a lower limit for D (D
4 × 10
7 cm2/s).
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Fig. 7.
Time-dependent alterations in the
lateral mobility of GFP-Ki-Ras(12V) by FTS. The experiments were
conducted as described under "Experimental Procedures" and in Fig.
6, using 100× objective. The times indicate incubation periods with 50 µM FTS. All measurements were at 22 °C. Each
bar is the mean ± S.E. of 30-70 measurements.
A, GFP-Ki-Ras(12V) mobility. The mobile fractions were high
throughout and were not affected by the treatment
(RF = 0.94 ± 0.01 in all cases).
Student's t test showed that the differences between the
D values measured on FTS-treated cells as compared with
untreated cells ("0" incubation time) were significant
(p < 0.001 for all incubation times except the 6- and
48-h time points, where p < 0.005 was obtained.
B, DiIC16 mobility. The mobile fractions were
similar in all cases (RF = 0.84 ± 0.04).
The use of Student's t test indicated that the differences
between the D values on FTS-treated as compared with
untreated cells were not significant (p > 0.25 in all
cases).
was increased 3.7-fold following a 4-fold elevation of
2).
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Fig. 8.
The effects of FTS on the lateral mobility of
GFP-Ki-Ras(12V) persist after cytochalasin D treatment. The
experiments were conducted as described under "Experimental
Procedures" and in Fig. 7. Cells were incubated with 50 µM FTS for 30 min or 24 h, and cytochalasin D
(cyto; 10 µg/ml) was added 15 min prior to the FPR
measurements, which were performed at 22 °C. Each bar is
the mean ± S.E. of 30-34 measurements. The mobile fractions were
not significantly affected by the FTS treatments, but were slightly
reduced by cytochalasin D (RF = 0.94 ± 0.01 and RF = 0.90 ± 0.02 in the absence
and presence of cytochalasin D, respectively). For the 30-min FTS
treatment, Student's t test showed that the D
values of cells treated with cytochalasin D and FTS were significantly
different from those of cells treated with cytochalasin alone
(p < 0.001). No significant difference
(p > 0.25) was observed between cytochalasin-treated
cells incubated with or without FTS for 24 h.
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Fig. 9.
The S-prenyl analogs AFC and GTS
do not affect the lateral mobility of GFP-Ki-Ras(12V). The
experiments were conducted as in Fig. 7. The times indicate the
incubation periods with 50 µM AFC or GTS, or with 0.1%
Me2SO in the control ( ). The RF
values were similar in all cases (0.94 ± 0.01). Each
bar is the mean ± S.E. of 50-80 measurements.
Student's t test reveals no significant differences between
the D values measured on control versus
drug-treated cells (p > 0.25 in all cases, except for
control versus AFC-treated cells after 30 min, which yielded
p > 0.1).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENT |
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We are grateful to Dr. Leonid Mittelman (Interdepartmental Core Facility, Sackler School of Medicine, Tel Aviv University) for invaluable assistance with the confocal microscopy.
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FOOTNOTES |
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* This work was supported in part by Grant 97-00141 from the United States-Israel Binational Science Foundation, Jerusalem, Israel (to Y. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 972-3-640-9699;
Fax: 972-3-640-7643; E-mail: kloog{at}post.tau.ac.il.
The abbreviations used are: FTS, S-trans,trans-farnesylthiosalicylic acid; AFC, N-acetyl-S-trans,trans-farnesyl-L-cysteine; BSA, bovine serum albumin; D, lateral diffusion coefficient; DiIC16, 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocynanine perchlorate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FPR, fluorescence photobleaching recovery; GFP, green fluorescent protein; GFP-Ki-Ras(12V), GFP-tagged constitutively active Ki-Ras 4B(V12); GPI, glycosylphosphatidylinositol; GTS, S-geranylthiosalicylic acid; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; R18, octadecyl rhodamine B chloride; RF, mobile fraction.
2 G. Elad, A. Paz, R. Haklai, D. Marciano, and Y. Kloog, unpublished observations.
3 Y. Egozi, M. Gana-Weisz, and Y. Kloog, unpublished results.
4 H. Niv and Y. Kloog, unpublished observations.
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