(Received for publication, January 22, 1996; and in revised form, March 4, 1996)
From the
Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway.
The small guanosine nucleotide binding proteins of the ras gene superfamily act as molecular switches in the transduction of many extracellular signals from cell surface receptors to the nucleus and are involved in a variety of cellular processes from mitosis and differentiation to apoptosis(1, 2) . Moreover, activating mutations in ras genes have been implicated in malignant transformation in mammals(3) . Ras proteins are synthesized as cytosolic precursors and must undergo posttranslational modifications at their C termini before they become biologically functional. These modifications include farnesylation at a cysteine residue located four residues from the C terminus, followed by removal of the C-terminal tripeptide and methylation of the newly exposed C terminus(4) . There is general agreement that farnesylation of Ras is indispensable for its biological functions in vivo. Yeast cells in which farnesylation of Ras is suppressed by pharmacological agents or by mutation are not viable(5, 6, 7) . Likewise, oncogenic mammalian Ras proteins that are not farnesylated are completely nontransforming(8, 9) . Since prenylated Ras proteins are predominantly localized to the cytoplasmic face of the plasma membrane, whereas the nonprenylated forms are cytosolic, prenylation of Ras has been implicated in membrane targeting where it interacts with other components of the signal transduction pathway(4, 10, 11, 12, 13, 14, 15) .
Some Ras proteins (Ha-Ras, N-Ras, Ras2) are further lipidated by
palmitoylation at one or two cysteines near the farnesylated C terminus (7, 10) . Although this modification appears to be
required for high affinity binding of prenylated Ras to the plasma
membrane, its biological significance remains unclear. Interestingly
palmitoylation is dynamic(16) , suggesting the possibility of
regulation by reversible membrane-to-cytosol translocations as has been
documented in the case of the -subunit of heterotrimeric
G-proteins(17) . In yeast, the biological significance of Ras2
palmitoylation has been explored in detail(7) . Yeast Ras2
mutants, which are farnesylated but not palmitoylated, are not
localized to the plasma membrane, and a single copy of a
palmitoylation-defective RAS2 gene confers cell
viability(7) . Although such mutants showed no marked growth
phenotype, they fail to induce a transient increase in intracellular
cAMP in response to glucose addition, suggesting that nonpalmitoylated
Ras2 is unable to activate adenylate cyclase, its main downstream
effector in the glucose signaling pathway in yeast. In mammalian
systems, earlier studies using cell lines overexpressing oncogenic Ras
revealed that mutation of the palmitoylation sites of Ha-Ras caused a
loss of membrane localization(8, 10) . However, the
mislocalized protein retains the ability to cause cell transformation
as long as it is farnesylated(8) . Analogous observations have
been made for the polybasic domain in the hypervariable region of the
K-Ras protein, which led to the interpretation that plasma membrane
localization is not essential for the transforming activity of
oncogenic Ras(10) . This view has been challenged recently by
similar transfection studies where oncogenic K-Ras was expressed at
near-physiological levels in NIH-3T3 cells. The study revealed that the
polybasic domain of K-Ras confers targeting to the plasma membrane and
is absolutely required for cell transformation(18) . The
discrepancies were believed to result from different susceptibilities
of particular cell clones to transformation (18) .
In order
to clarify the functional significance of Ha-Ras palmitoylation and
membrane targeting for the activation of signal transduction cascades
and biological responses in vivo, we have used Xenopus
laevis oocytes as an experimental system. Oocytes are naturally
arrested at the G-M boundary of the first meiotic cell
division. When microinjected with oncogenic Ha-Ras protein, oocytes
resume meiotic maturation (19) in a process that is accompanied
by activation of the mitogen-activated protein kinase (MAPK) (
)cascade(20, 21, 22, 23, 24, 25) and an
increase in the p34
kinase activity of
maturation promoting factor (MPF)(23) . Here we describe a
detailed dose-response study comparing biological properties of
palmitoylated and nonpalmitoylated forms of Ha-Ras in Xenopus oocytes. Our results suggest an important role of Ha-Ras
palmitoylation for signaling functions in vivo.
Purified Ha-Ras proteins were loaded with
GTPS by incubation in buffer containing 20 mM Tris, pH
7.4, 10 mM EDTA, 5 mM MgCl
, 1 mM dithiothreitol, 10-30 µM Ras, and 250
µM GTP
S (Sigma) for 20 min at 30 °C. Nucleotide
exchange was stopped on ice by the addition of MgCl
to a
final concentration of 25 mM, and free nucleotides were
removed by repeated cycles of ultrafiltration in a Centricon-10 device
(Amicon) as described previously(31) . Ras proteins were
finally concentrated to 1-2 mg/ml and stored at -80 °C
in small aliquots in Ras buffer (20 mM Tris-HCl, pH 7.4, 100
mM NaCl, 4 mM MgCl
, 0.1 mM dithiothreitol).
Ras proteins were diluted in Ras buffer, and 50 nl
was microinjected into the cytoplasm. Oocytes were kept in Barth medium
and scored for germinal vesicle breakdown (GVBD) by the appearance of a
white spot on the animal hemisphere. For biochemical analysis,
5-10 oocytes/group were randomly harvested and homogenized in 20
µl/oocyte of ice-cold lysis buffer (60 mM -glycerophosphate, pH 7.4, 20 mM EGTA, 15 mM MgCl
, 1 mM Na
VO
, 1
mM NaF, 1 mM benzamidine, 5 µg/ml leupeptin, 1
mM phenylmethylsulfonyl fluoride). The crude lysates were
either processed immediately or flash-frozen in liquid nitrogen and
stored at -80 °C.
Like progesterone, the physiological stimulus for oocyte
maturation, oncogenic Ha-Ras (Val-12), has been shown to activate the
MAPK cascade (21, 22) and to induce maturation in Xenopus oocytes(19) . To analyze the significance of
Ha-Ras palmitoylation for biological activity and activation of
downstream effectors in vivo, GTPS-bound forms of WT
Ha-Ras and the mutant 181/4S in which the palmitoylated cysteines have
been substituted with serine were microinjected into oocytes.
Epitope-tagged, nononcogenic (Gly-12) WT Ha-Ras and its 181/4S mutant
were purified in the nonprocessed form from Sf9 cells and were
activated by loading with GTP
S, a nonhydrolyzable GTP analogue. In vitro farnesylation followed by SDS-PAGE analysis resulted
in complete conversion of WT and mutant Ras proteins into the
processed, faster migrating form(10, 27) , and
approximately 1 mol of [
H]farnesyl/mol of Ras
(estimated by [
H]GTP binding) was incorporated
into both proteins (data not shown). Thus, the C-terminal prenylation
sequence, which is indispensable for farnesylation and subsequent
processing steps, is functional in these Ras preparations.
Fig. 1shows that mutation of cysteines 181 and 184 prevents
palmitoylation and membrane localization but does not affect
farnesylation, C-terminal proteolysis, and methylation of Ha-Ras in Xenopus oocytes. [H]FPP, the farnesyl
donor for Ha-Ras prenylation in Xenopus oocytes(35) ,
is readily incorporated into microinjected Ha-Ras proteins (Fig. 1A), indicating that WT and mutant Ha-Ras are
comparably good substrates for the protein farnesyltransferase present
in Xenopus oocytes. Similarly, WT Ha-Ras and 181/4S
incorporate alkali-labile
[methyl-
H]methionine with comparable
kinetics (Fig. 1B), indicating that both proteins are
equally proteolyzed and carboxymethylated in oocytes. Farnesylation is
also accompanied by an increase in electrophoretic mobility and
hydrophobicity, which can be monitored by Triton X-114
phase-partitioning(10, 33) . Immunoblot analysis of
phase-partitioned oocytes microinjected with WT Ha-Ras or 181/4S shows
that both proteins undergo farnesylation with comparable kinetics (Fig. 1C). Both proteins show a time-dependent shift
from the hydrophilic, slower migrating, microinjected form to the more
hydrophobic, faster migrating form with a half-life of about
2.5-5 h. After 7.5 h, farnesylation is essentially complete, and
WT Ha-Ras and 181/4S are stable under these conditions for at least 24
h. Thus, as in the case of ras-transfected COS
cells(10) , mutation of the Ha-Ras palmitoylation sites does
not noticeably affect the other processing steps in Xenopus oocytes. When oocytes microinjected with WT Ha-Ras or 181/4S were
labeled with [
H]palmitate, WT but not mutant
Ha-Ras incorporated palmitate (Fig. 1D), suggesting
that the same cysteine residues of Ha-Ras are palmitoylated in Xenopus oocytes as those identified in COS cells(10) .
Thus, the processing enzymes present in Xenopus oocytes are
functionally comparable with their mammalian counterparts. Analysis of
the cellular localization of WT Ha-Ras or 181/4S by oocyte
fractionation shows that 7.5 h after microinjection, about 80% of WT
Ha-Ras is associated with the membrane compartment, whereas 181/4S
remains entirely cytosolic (Fig. 1E). This pattern did
not change significantly for the subsequent 24-h incubation period (not
shown), suggesting that the cellular distribution has reached steady
state.
Figure 1:
Posttranslational processing and
cellular localization of WT Ha-Ras and 181/4S in Xenopus oocytes. A, oocytes were coinjected with 1 pmol of
[H]FPP and WT or mutant Ha-Ras. After a 2.5-h
incubation in Barth medium, oocytes were lysed, and Ras proteins were
immunoprecipitated, resolved on 15% polyacrylamide gels, and analyzed
by fluorography. The exposure time was 14 days. B, oocytes
injected with 1 pmol of WT Ha-Ras or 181/4S were labeled in Barth
medium containing [methyl-
H]methionine.
After overnight culture, oocytes were lysed and Ras proteins were
immunoprecipitated and analyzed by SDS-PAGE followed by fluorography.
The exposure time was 4 days. 5800 (WT) and 8400 (181/4S) cpm were released as
[
H]methanol if the excised Ras gel slices were
treated with 2 M NaOH(51) . If oocytes were analyzed
after a 5-h labeling period, 1900 and 2150 cpm were released from WT
and mutant Ha-Ras, respectively. C, oocytes microinjected with
0.5 pmol of WT Ha-Ras or 181/4S were lysed 2.5, 5, 7.5, or 24 h after
injection and subjected to Triton X-114 partitioning. Equal portions of
detergent (d) or aqueous (a) phase were resolved by
SDS-PAGE (15%), and Ras proteins were detected by immunoblotting with
anti-Ras mAb as described under ``Experimental Procedures.'' i, Ha-Ras before injection (purified from the aqueous phase of
Sf9 cells). Similar results were obtained with anti-Glu-Glu antibody
(not shown). D, mutant- or WT-injected oocytes were cultured
in cold medium for 4 h. After a 30-min pulse in medium containing 2
mCi/ml [
H]palmitic acid, oocytes were lysed, Ras
was immunoprecipitated, loaded onto polyacrylamide gels (15%), and
visualized by fluorography. The exposure time was 14 days. Immunoblot
analysis showed that comparable amounts were immunoprecipitated (not
shown) E, 7.5 h after microinjection with WT Ha-Ras or 181/4S,
oocytes were fractionated into cytosolic (s) and particulate (p) fractions. Total cell lysate (t) and equivalent
portions of s and p fractions were resolved by 15%
SDS-PAGE and analyzed by immunoblotting with anti-Ras
mAb.
A typical time-course of oocyte maturation following microinjection of 1 pmol of WT Ha-Ras or palmitoylation-site mutant 181/4S is shown in Fig. 2. While WT Ha-Ras induces near 100% maturation after a 24-h incubation period, no significant GVBD was detected in oocytes injected with 181/4S after 48 h. This lack of biologic activity of 181/4S could not be overcome by larger doses (up to 2.5 pmol) and extended culture periods (up to 72 h) in several experiments using oocytes from different frogs.
Figure 2: Kinetics of oocyte maturation induced by WT Ha-Ras and the 181/4S mutant. Stage VI oocytes were either treated with progesterone (10 µM) as a positive control or microinjected with 1 pmol of WT Ha-Ras, nonpalmitoylated mutant 181/4S, or buffer alone and incubated in Barth medium at 18-20 °C. After the indicated time periods, oocytes were monitored for GVBD. Results are expressed as a percentage of the number of injected oocytes (30-40/group).
It has been shown
that oncogenic Ha-Ras activates MAPK in Xenopus oocytes (20, 21) and that MAPK activation is necessary to
mediate Ras-induced GVBD(25, 36) . Thus, we
investigated whether the inability of Ha-Ras 181/4S to induce oocyte
maturation was associated with a lack of activation of components of
the MAPK cascade. Fig. 3shows that MAPK activity in extracts
from oocytes injected with WT Ha-Ras is increased by 4-8-fold and
reaches levels comparable with those measured in progesterone-treated
oocytes. In marked contrast, MAPK activity in oocytes injected with
181/4S remained at control levels. MAPK activation was also evident in
anti-phosphotyrosine immunoblots revealing a prominent band of 42
kDa in WT-injected or progesterone-treated oocyte extracts, which is
absent in 181/4S- or buffer-injected cells (Fig. 3B).
This tyrosine phosphorylation was paralleled by the characteristic
electrophoretic mobility shift of MAPK in anti-ERK2 immunoblots (Fig. 3C). Oncogenic Ha-Ras has also been shown to
induce activation of MPF(23, 24) , a cell cycle
regulatory element that controls the G
-M transition in
eukaryotic cells and in Xenopus oocytes(37, 38) . When the histone kinase
activity associated with MPF was measured in these extracts, oocytes
microinjected with WT Ha-Ras or treated with progesterone exhibited
increased MPF activity, whereas 181/4S-injected cells remained at
control levels (Fig. 3D). Taken together, these results
suggest that activation of the MAPK cascade, the histone kinase
activity of MPF, and the initiation of meiosis in Xenopus oocytes by Ha-Ras require palmitoylation.
Figure 3:
Ha-Ras 181/4S fails to activate MAPK and
MPF in Xenopus oocytes. Oocytes were microinjected or treated
with progesterone (prog) as described in the legend to Fig. 2. After 4-24 h of culture in Barth medium, groups of
5-10 oocytes were randomly harvested, and cleared extracts were
prepared. A, MAPK activity was measured by incorporation of P into myelin basic protein(34) . The -fold MAPK
activation represents the cpm of Ras-injected/cpm of buffer-injected
oocytes, after subtracting the minus substrate control from each data
point. The mean ± S.D. from six independent experiments is
shown. B, oocyte extracts (0.1 oocyte equivalent/lane) were
separated by SDS-PAGE (10%), transferred onto nitrocellulose, and
probed with antibody to phosphotyrosine (
-PY). Immunoreactive
bands were visualized by ECL. C, the same blot was stripped
and reprobed with antibody to MAPK (
-ERK2). D,
MPF activity was measured by incorporation of
P into
histone H1 substrate as described under ``Experimental
Procedures.'' After terminating reactions by boiling for 5 min in
SDS sample buffer, reaction mixtures were separated by SDS-PAGE, and H1
phosphorylation was analyzed by autoradiography. The arrow indicates the position of the H1 band.
Since the apparent lack of biological activity of 181/4S might be due to a low sensitivity of oocytes to Ras-mediated signals, Ras-injected oocytes were costimulated with insulin in a second series of experiments. Insulin has been shown to induce oocyte maturation (39) possibly by a Ras-mediated pathway (40) and was found to synergize with oncogenic Ha-Ras in the induction of oocyte maturation(41) . Oocytes were microinjected with WT or mutant Ha-Ras, cultured for 8 h to allow completion of posttranslational processing, and then transferred into fresh medium supplemented with suboptimal doses of insulin. Costimulation of Ha-Ras-injected oocytes by insulin accelerated GVBD and greatly increased their sensitivity to Ras. The time course of maturation (Fig. 4A) shows that Ha-Ras 181/4S can also promote oocyte maturation under these conditions, whereas insulin treatment on its own exhibits only marginal effects. Maturation induced by Ha-Ras 181/4S, however, proceeds with slower kinetics, and the time required to achieve 50% maturation was typically 2-3 times longer than for WT-injected cells. Insulin also substantially reduced the minimal dose of Ras needed to accelerate oocyte maturation. Fig. 4B shows the maturation of oocytes microinjected with various amounts of WT Ha-Ras or 181/4S and costimulated with insulin. Both WT and mutant Ha-Ras accelerated GVBD in a dose-dependent fashion, and significant maturation could be observed with as little as 10 fmol of WT Ha-Ras. However, the amount of 181/4S required to induce the same extent of maturation as WT was 25-50-fold higher. Although the sensitivity of oocytes to Ras- and insulin-induced GVBD varied considerably between different batches of oocytes, comparable differences in biological activity between WT and mutant Ha-Ras were obtained with oocytes obtained from different females.
Figure 4: Insulin enhances the responsiveness of oocytes to Ha-Ras and partially rescues the biological activity of 181/4S. A, kinetics. Oocytes microinjected with 0.5 pmol of WT Ha-Ras, 0.5 pmol of 181/4S mutant or buffer alone were cultured for 8 h in Barth medium and then transferred to medium supplemented with 5 µM insulin. Incubation was continued at 18-20 °C, and the percentage of GVBD at indicated time points was determined as in Fig. 2. B, dose response. Oocytes (30-40/group) were microinjected with WT Ha-Ras or 181/4S serially diluted in buffer and costimulated with suboptimal insulin concentrations as described above. After an overnight incubation, oocytes were scored for GVBD. After shorter incubation periods or when lower concentrations of insulin were used, only WT Ha-Ras at doses of 0.2-1 pmol induced maturation (not shown).
The effect of synergistic stimulation of oocytes by various
doses of Ha-Ras proteins and insulin on MAPK activity is shown in Fig. 5. Both WT Ha-Ras and 181/4S induced a dose-dependent rise
in MAPK activity at a time when insulin treatment alone showed no
measurable effect. However, about a 25 times higher dose of mutant
Ha-Ras was needed to achieve the same degree of MAPK activation. MAPK
activity in these extracts correlated with tyrosine phosphorylation of
a 42-kDa protein and a reduction of the electrophoretic mobility of
endogenous p42 as revealed by immunoblot analysis (data
not shown). Taken together these results show that the inability of
nonpalmitoylated Ha-Ras to activate MAPK and induce meiosis in oocytes
can be partially rescued by costimulation with insulin. Even under
these conditions, however, about 25 times higher doses of mutant Ha-Ras
are required to activate downstream effectors and induce biological
responses in Xenopus oocytes.
Figure 5: Activation of MAPK in oocytes synergistically stimulated with WT Ha-Ras or 181/4S and insulin. Oocytes were microinjected and cultured as in Fig. 4B. After overnight incubation in Barth medium containing 5 µM insulin, groups of 5-10 oocytes were randomly selected and MAPK activity was measured in cleared extract. MAPK activity is expressed relative to the activity in control extracts (oocytes injected with buffer alone and treated with insulin), which was set as 1.
We have analyzed the functional significance of Ha-Ras palmitoylation for signal transduction in vivo using the Xenopus oocyte system. Our results show that while WT Ha-Ras is predominantly membrane-bound and promotes activation of MAPK and meiotic maturation when microinjected into stage VI oocytes, a nonpalmitoylated but farnesylated and methylated mutant remains cytosolic and is inactive under these conditions (Fig. 2). This result would suggest that, like a C-terminal truncation mutant of oncogenic Ras1 that lacks all posttranslational modifications(42) , the farnesylated but nonpalmitoylated mutant 181/4S is biologically inactive in Xenopus oocytes. These two mutants are both cytosolic, however, their biological properties are distinctly different if oocytes are costimulated with a second agonist. While the C-terminal truncation mutant of oncogenic Ras1 was shown to inhibit maturation induced by insulin-like growth factor(42) , the nonpalmitoylated but farnesylated mutant 181/4S accelerates maturation if oocytes are costimulated with insulin (Fig. 4). Under these conditions, 25-50-fold higher doses of 181/4S are required to elicit responses comparable to its WT counterpart (Fig. 4B). These results suggest that for the induction of meiotic maturation of Xenopus oocytes by Ha-Ras, farnesylation alone converts an antagonist into a partial agonist, whereas farnesylation together with palmitoylation is required to achieve full biological activity. Interestingly, a C-terminally truncated form of oncogenic Ras1 in which a farnesylation site was added also showed partial biological activity in Xenopus oocytes(42) , suggesting that this concept might also apply to other Ras proteins.
The induction of oocyte maturation
correlates with phosphorylation and activation of MAPK regardless of
the combination of stimuli (Fig. 2, 3, 4B, and 5),
which is in agreement with the pivotal role of the MAPK cascade in
meiotic cell division in Xenopus oocytes(24, 36, 43, 44, 45) .
Another kinase that becomes activated during oocyte maturation is MPF,
a complex of cyclin B and p34 kinase, which controls
entry of cells into mitosis(37, 38) . Consistent with
this functional role, MPF activity closely correlates with MAPK
activity and the induction of maturation (Fig. 3D and
not shown), further corroborating the differences in biological
activity between WT Ha-Ras and 181/4S.
These results can be interpreted to mean that Ha-Ras has to be palmitoylated and consequently membrane-localized in order to efficiently activate downstream effectors. This would support a proposed biochemical function for Ras in recruiting effector molecules to the plasma membrane where they undergo activation(13, 14, 15) . The Raf-1 kinase is a candidate for such an effector molecule, and translocation of Raf from the cytosol to the plasma membrane either by activated Ras or by the addition of a membrane targeting signal to Raf results in phosphorylation, activation, and stable association with the membrane compartment(13, 14, 46) .
In Xenopus oocytes, Raf appears to be an important downstream effector in the Ras-induced activation of MAPK and meiotic maturation. Raf becomes phosphorylated during meiotic maturation induced by Ras(47) , and kinase-defective Raf blocks Ras-induced maturation and activation of MAPK(22) . Since Raf has to be localized to the plasma membrane for activation, our observation that nonpalmitoylated, cytosolic Ha-Ras is a poor activator of MAPK is consistent with the proposed role of Raf as an intermediate in the Ras-MAPK cascade in Xenopus oocytes.
A novel Ras-dependent activator of the MAPK cascade termed REKS has recently been purified from Xenopus oocytes(48) . In vitro, REKS is activated by posttranslationally processed Ras in the absence of membrane components(49) , and farnesylation but not palmitoylation of Ha-Ras is required for this activity(27) . In light of the present study showing that farnesylated but nonpalmitoylated Ha-Ras is a poor activator of MAPK, REKS appears to play a minor role in the Ras-mediated MAPK activation in vivo. However, the biological activity of 181/4S detected in oocytes costimulated with insulin might be mediated by REKS. Alternatively it is also possible that small amounts of nonpalmitoylated Ha-Ras not detectable by cell fractionation are present in the membrane compartment and are responsible for the residual biological activity of 181/4S.
Our findings also reveal a striking similarity with studies in yeast. Ras is essential for viability in yeast, and farnesylation is required for viability if Ras is expressed at physiological levels(5, 7) . Recent studies have shown that yeast cells containing a single copy of a RAS2 gene defective in palmitoylation are viable but fail to activate adenylate cyclase, its presumed downstream effector in yeast(7) . Thus, Ras proteins that are farnesylated but not localized to membranes appear to lack the ability to couple to some effector molecules, while they can still perform essential functions. Since farnesylation of yeast Ras has been shown to enhance interaction between Ras and adenylate cyclase in vitro(50) , it has been suggested that distinct cytosolic and membrane-bound forms of adenylate cyclase might be involved in cell viability and glucose response (7) .
In mammalian cells, previous studies by
Hancock and co-workers (8) using NIH-3T3 cells transfected with
oncogenic ras genes indicated that membrane localization might
not be important for cell transformation. In the case of K-Ras, which
is not palmitoylated but contains a string of polybasic residues
required for membrane binding, this view has been challenged recently.
Performing similar transfection studies, Jackson and co-workers (18) found that cytosol-localized K-Ras, even when
farnesylated, was completely nontransforming(18) ; these
discrepancies have been ascribed to differences in sensitivity of the
particular cell clones used(18) . Although the induction of
meiosis in naturally G-arrested oocytes is not directly
comparable with transformation of mammalian cell lines by Ras, our
findings would suggest that palmitoylation and membrane binding might
also play an important role in cell transformation by oncogenic Ha-Ras.
In light of the discrepancies found with K-Ras, this issue should be
addressed in future studies.