Activation of Ras leads to the constitutive
activation of a downstream phosphorylation cascade comprised of Raf-1,
mitogen-activated protein kinase (MAPK) kinase, and MAPK. We have
developed a yeast-based assay in which the Saccharomyces
cerevisiae mating pheromone-induced MAPK pathway relied on
co-expression of K-Ras and Raf-1. Radicicol, an antifungal antibiotic,
was found to inhibit the K-ras signaling pathway
reconstituted in yeast. In K-ras-transformed, rat
epithelial, and K-ras-activated, human pancreatic carcinoma
cell lines, radicicol inhibited K-Ras-induced hyperphosphorylation of
Erk2. In addition, the level of Raf kinase was significantly
decreased in radicicol-treated cells, whereas the levels of K-Ras
and MAPK remained unchanged. These results suggest that radicicol
disrupts the K-Ras-activated signaling pathway by selectively depleting
Raf kinase and raises the possibility that pharmacological
destabilization of Raf kinase could be a new and powerful approach for
the treatment of K-ras-activated human cancers.
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INTRODUCTION |
The Ras and Raf protooncogene products are key proteins involved
in the transmission of many proliferative signals. They serve as
intermediates in this signaling pathway by connecting upstream tyrosine
kinases with downstream serine/threonine kinases such as
mitogen-activated protein kinase
(MAPK)1 or MAPK kinase
(MAPKK). A similar MAPK signaling pathway also exists in the mating
pheromone-responding, signaling cascade of Saccharomyces
cerevisiae. This signaling pathway consists of the Ste11, Ste7,
and Fus3/Kss1 kinase, which are homologs of MAPKK kinase, MAPKK, and
MAPK, respectively (1). We have previously reported that expression of
mammalian H-Ras and Raf-1, together with Ste7P368, rescued
the defect in mating pheromone signal transduction because of STE11
deficiency (2). In the present study, we modified this in
vivo system by substituting the K-Ras for H-Ras, because K-ras is the most frequently activated ras gene
in human cancer (3) and, therefore, more important as a target for
cancer therapy than H-ras or N-ras. Using this
modified yeast assay, we screened for inhibitors that block the
Ras-Raf pathway reconstituted in S. cerevisiae and have
identified radicicol (Fig. 1) as a candidate inhibitor of the Ras-Raf
signaling pathway.
Radicicol (Fig. 1), a macrocyclic antifungal antibiotic originally
isolated from the fungus Monosporium bonorden (4), is a
potent tranquilizer of low toxicity (5, 6) and an inhibitor of in
vivo angiogenesis (7). Radicicol induces reversal of the
transformed phenotype of src-transformed cells (8) and has
also been reported to inhibit the phosphorylation and protein kinase
activity of pp60v-src. It does not inhibit the
serine/threonine kinases, such as protein kinase C and protein kinase A
(9-11). In addition to the inhibitory activity of radicicol against
Src kinase, there is evidence that radicicol suppresses transformation
by the ras oncogene. Decreased MAPK activity accompanies
morphological reversion of H-ras-transformed cells by
radicicol (12). However, radicicol does not inhibit the kinase activity
of MAPKK or MAPK in vitro (13). These results suggest that
radicicol does not directly inhibit the MAPK or MAPKK activity in
ras-transformed cells but inhibits MAPK activation by
unknown mechanisms.
Our findings pertaining to radicicol as an inhibitor of the K-Ras
signaling pathway in yeast mutants prompted us to examine its effect on
K-ras-transformed mammalian cell lines. Our present data
reveal that radicicol inhibits ras-dependent phosphorylation of MAPK in K-ras-transformed rat epithelial cell lines and,
further, that disruption of the K-Ras signaling pathway is due to the
destabilization of Raf protein by radicicol.
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EXPERIMENTAL PROCEDURES |
Plasmid and Yeast Strains--
The plasmid pVT-VKR expresses
Kirstein sarcoma virus ras gene from the ADH
promoter. It was constructed by ligating the
StuI-BamHI fragment of pHN12 (14) into the
PvuI-BamHI site of pVT101-L in which the
PvuI site was blunt-ended. pVT101-L is a YCp-based plasmid
containing the LEU2 selectable marker (15). pADU-Raf contains c-Raf-1
controlled from the ADH1 promoter. pNC318-P368 carries the
STE7P368 allele controlled from the CYC1
promoter.
Strain SY1984-RP is SY1984 (MAT
leu2 ura3 trp1
ste11
his3
FUS1::HIS3)
transformed with pADU-Raf and pNC318-P368 (2). Yeast strains expressing
K-Ras and Raf-1 were obtained by transducing pVT-VKR into strain
SY1984-RP.
Yeast cells were grown in the synthetic medium SC, which is SD 2%
glucose, 0.7% yeast nitrogen base without amino acid containing appropriate auxotrophic supplements (16). SC lacking amino acids or
other nutrients (e.g. SC-ura, which lacks uracil) was used to select transformants. Yeast transformation was performed by the
method of Itoh et al. (17).
Inhibition of Growth of SY1984-RP Expressing K-Ras--
The
yeast strain SY-1984-RP carrying pVT-VKR was grown at 30 °C to
stationary phase in SC-Ura-Trp-Leu. Agar plates were prepared by adding
50 µl of the above culture to 50 ml of SC agar lacking Trp, Leu, His,
and Ura. Paper discs soaked in drugs were placed on agar plates, the
plates were inoculated at 30 °C for 3 days, and the diameters of the
zones of growth inhibition were measured.
Antibodies and Cell Culture--
The antibodies used were
phospho-specific MAPK antibody, phospho-specific MEK1/2 (Ser217/221)
antibody, pan-MEK1/2 antibody (New England Biolabs), Erk2 antibody
(Upstate Biotechnology Inc.), MEK-1 and MEK-2 antibodies
(Transduction Laboratories), c-K-ras(Ab-1) antibody (Oncogene Science),
and Raf-1(C-12), A-Raf(C-20), and B-Raf(C-19) antibodies (Santa
Cruz Biotechnology).
NRK and KNRK5.2 cells were obtained from Dr. David A. Johnson. PSN-1
cells were obtained from Dr. Ken Yamaguchi. These cells were maintained
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum.
Radicicol Treatment of Cells--
KNRK5.2 cells (1 × 105) or PSN-1 cells (1 × 105) were plated
in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a 24-well tissue culture plate. Approximately 8 h later, various amounts of radicicol were added to each well without adding any
fresh medium, and the cells were cultured for a further period of
40 h. To examine the growth inhibitory effect of radicicol, cells
were dispersed by trypsin-EDTA, and cells in the suspension were
counted by an F-520 cell counter.
Cell Lysis and Western Blotting--
Generally, 24-well tissue
culture plate cells were used for each sample. Cells were washed once
with phosphate-buffered saline. The cells were lysed by the addition of
20 µl/well of ice-cold lysis buffer (50 mM Hepes-NaOH, pH
7.4, 250 mM NaCl, 1 mM EDTA, 1% Nonidet P-40,
1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 mM
Na3VO4, 1 mM NaF, 10 mM
-glycerophosphate).
Cells were lysed for 10 min on ice and clarified by centrifugation. 10 µg of total cell lysate from each sample was electrophoresed on a
10% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride
membranes, and immunoblotted with appropriate antibodies. For
detection, the blots were incubated with the appropriate ECL secondary
antibody (horseradish peroxidase-conjugated anti-Ig antibody) and
developed using the ECL detection system (Amersham Corp.), according to
the instructions of the manufacturer. Films were scanned into a
Macintosh computer and analyzed by using NIH Image software.
 |
RESULTS |
Identification of a K-Ras Signaling Inhibitor Using Yeast Mutants
Expressing K-Ras and Raf-1--
Kirstein sarcoma virus ras
gene was expressed together with c-Raf-1 and Ste7P368 in a
ste11-disrupted mutant strain having a mating
pathway-responsive reporter gene (FUS1::HIS3). Consistent
with our previous results obtained for H-Ras (2), expression of K-Ras
in this strain conferred a His+ phenotype, indicating that
Raf was activated by K-Ras in S. cerevisiae and transmitted
the signal to the yeast pheromone-responding MAPK pathway. Using this
K-Ras expressing mutant strain, we screened our chemical library for
compounds that could inhibit the K-Ras signaling pathway reconstituted
in yeast. One compound, radicicol (Fig.
1), was found to have such an activity.
As shown in Fig. 2A, the
yeast strain expressing K-Ras and Raf-1 could grow in agar medium
lacking exogenous histidine, but its growth was inhibited by radicicol
as evidenced by a halo of growth inhibition around the paper disc
soaked with radicicol. When histidine or alanine was added to the
drug-containing paper disc and incubated for 2 days, the halo of growth
inhibition by radicicol disappeared upon exogenous addition of
histidine but not alanine. One simple explanation of these results was
that growth inhibition by radicicol was due to the inhibition of the
K-Ras-MAPK pathway in the mutant yeast strain. We could exclude the
possibility that radicicol inhibited some enzyme associated with
histidine biosynthesis because radicicol was not active against a yeast
two-hybrid strain that also had a HIS3 reporter gene and has been used
previously to detect the interaction between SNF1 and SNF4 (18).

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Fig. 2.
Effect of radicicol on the growth of
ste11 disruption mutant strain expressing K-Ras and Raf-1
(A) or an activated Raf-1 (Raf N) (B).
Agar plates were prepared as described under "Experimental
Procedures." Paper discs were soaked with radicicol (20 nmol),
dissolved in ethanol, dried, and soaked with an aqueous solution
containing 50 µg of alanine (left) or histidine
(right). These paper discs were placed on agar plates. After
3 days at 30 °C, the plates were photographed.
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Previous studies have shown that Raf-1 required the co-expression of
activated Ras for rescuing Ste11 deficiency but that an activated form
of Raf-1 (Raf
N) alone could compensate for Ste11 deficiency (2). We
therefore examined the effect of radicicol on the growth of the mutant
expressing Raf
N (Fig. 2B). Radicicol was effective in
growth inhibition of this mutant strain. Similar results were obtained
for two different mutant strains and suggested that the molecular
target of radicicol in yeast could be a molecule that is uniquely
common between the two strains. Such a molecule might include, for
example, Raf-1 itself, factors downstream of Raf-1 in the pathway, or a
protein that could interact with and regulate Raf-1 kinase.
Radicicol Inhibits K-ras-dependent MAPK Phosphorylation
in a K-ras-transformed Cell Line--
Based upon the effects of
radicicol on yeast signal transduction, we decided to examine its
effect on the K-ras-MAPK signal transduction pathway in
cultured mammalian cells. To this end, the cell line KNRK5.2 was
selected for further analysis. KNRK5.2 was derived from the rat kidney
epithelial cell line, NRK, by transfection of an activated
K-ras gene cloned from the human colon carcinoma cell line,
SW480. Immunoblot analysis with anti-phosphotyrosine-specific Erk2
antibody revealed that the K-ras-transformed cell line,
KNRK5.2, had a marked elevation in the level of phosphorylation of
Erk2, whereas NRK cells had no phosphorylation signal (compare Erk2 and
phospho-Erk2 signal in lanes 1 and 2, Fig.
3A). Thus, the K-Ras signaling
pathway was constitutively activated in this cell line. We examined the
effect of radicicol on K-ras-induced phosphorylation of
Erk2. Cells were treated with varying amounts of radicicol for 40 h, and cell lysates were subjected to immunoblot analysis with
anti-phosphotyrosine-specific Erk2 antibody. Radicicol inhibited phosphorylation of Erk2, as revealed by the decrease in the
phospho-Erk2 band. This was also evident from the decrease in the
phospho-Erk2 band, which migrated slightly more slowly than
unphosphorylated Erk2 in gels immunoblotted with anti-Erk2 antibody
(Fig. 3). Intracellular amounts of Erk2 protein remained relatively
unchanged with increasing concentrations of radicicol. Inhibition of
Erk2 phosphorylation by radicicol was quantitated by calculating the
relative amount of phospho-Erk2 to total Erk2 protein as a function of
drug concentration. The IC50 for Erk2 phosphorylation was
1.1 µM, which was comparable with the IC50
for cell growth inhibition, 0.89 µM (Fig. 3B).
Radicicol also inhibited K-Ras-dependent phosphorylation of
Erk2 in K-ras-transformed rat 3Y1 fibroblasts with an
IC50 of 0.78 µM (data not shown). It has
previously been reported that immunoprecipitated MAPK from radicicol-treated H-ras-transformed fibroblasts showed
decreased activity in MAPK assays employing myelin basic protein as a
substrate (12). The present results were consistent with this and
indicated that the decreased MAPK activity was due to the inhibition of phosphorylation of MAPK by radicicol.

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Fig. 3.
Radicicol inhibits K-Ras-induced Erk2
phosphorylation by depleting Raf-1 kinase in KNRK5.2 cells.
A, KNRK5.2 cells were grown for 40 h in the presence of
various concentrations of radicicol. 10 µg of each cell lysate was
analyzed by immunoblotting with an antibody against Raf-1, the
phosphorylated form of MEK1/2, MEK1/2, the phosphorylated form of
Erk1/2, or Erk2, respectively (from top to
bottom). B, immunoblotted gel was analyzed as
described under "Experimental Procedures." The levels of Raf-1,
phosphorylated Erk2, and phosphorylated MEK1/2 were plotted as a
percentage of the control.
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As previously reported for H-ras-transformed fibroblastic
cell lines (12, 13), radicicol caused a morphological reversion of
K-ras-transformed epithelial cells to a more normal
phenotype (Fig. 4). Morphological
reversion was apparent at drug concentrations as low as 0.625 µM. At this drug concentration, radicicol did not inhibit
phosphorylation of Erk2. Therefore, morphological changes appeared to
be more sensitive to radicicol than the inhibition of phosphorylation
of Erk2.

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Fig. 4.
Morphological change of KNRK5.2 cells caused
by radicicol. KNRK5.2 cells were treated with various
concentrations of radicicol for 40 h, and then photographs were
taken with a phase-contrast microscope.
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Radicicol Decreases the Steady-state Level of Raf-1--
To gain
further insight into the inhibition of
K-ras-dependent Erk2 phosphorylation in
radicicol-treated KNRK5.2 cells, we examined the level of Raf-1 and
MEK1/2 by immunoblot analysis (Fig. 3). Raf-1 protein levels were
significantly decreased with increasing concentrations of radicicol.
The relative amount of Raf-1 protein, compared with Erk2 protein, at
each drug concentration was calculated, and the IC50 value
of Raf-1 depletion was determined to be 3.1 µM. Besides
Raf-1, Raf is known to have two other isoforms, A-Raf and B-Raf. B-Raf
was not expressed in KNRK5.2 cells, as determined by immunoblot
analysis (data not shown). A-Raf was expressed and its levels were
significantly decreased with increasing concentrations of
radicicol (Fig. 5).
IC50 values for A-Raf and Raf-1 depletion were 5.7 and 5.0 µM, respectively. Thus, radicicol induced the
destabilization of all isoforms of Raf protein expressed in KNRK5.2
cells.

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Fig. 5.
Radicicol destabilizes the A-Raf protein in
KNRK5.2 cells. A, KNRK5.2 cells were grown for 40 h in
the presence of various concentrations of radicicol. 10 µg of each
cell lysate was analyzed by immunoblotting with antibody against Raf-1,
A-Raf, or Erk2, respectively (from top to
bottom). B, the immunoblotted gel was analyzed as
described under "Experimental Procedures." The levels of Raf-1 and
A-Raf were plotted as a percentage of the control.
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To clarify the relationship between the inhibition of phosphorylation
of Erk2 and Raf depletion, we examined the time dependence of both
events in the presence of 10 µM radicicol. Raf-1
depletion and Erk2 phosphorylation inhibition occurred with similar
kinetics (Fig. 6). Taken together, these
results strongly suggested that radicicol inhibited Erk2
phosphorylation by depleting Raf kinase in KNRK5.2 cells. As evidenced
by the decrease in the MEK signal detected by the anti-MEK antibody
(which can recognize MEK doubly phosphorylated at Ser-217 and Ser-221),
radicicol also inhibited the phosphorylation of MEK without affecting
the level of MEK protein significantly (Fig. 3). The simplest
interpretation of these results was that the inhibition of MEK
phosphorylation was due to the depletion of Raf kinases.

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Fig. 6.
Time course of Raf-1 depletion and inhibition
of Erk2 phosphorylation. A, KNRK5.2 cells were grown with 10 µM radicicol (lanes 1-7) or without radicicol
(lane 8). At the indicated times, cells were lysed and
analyzed by immunoblotting with antibody against Raf-1, the
phosphorylated form of Erk1/2, or Erk2, respectively (from
top to bottom). B, band intensities
were determined as described under "Experimental Procedures" and
plotted as a percentage of the control.
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The effect of radicicol on Raf depletion in KNRK5.2 cells was
reversible (Fig. 7). Cells were treated
initially for a period of 24 h with radicicol, which significantly
depleted Raf-1 protein and inhibited Erk2 phosphorylation in a
dose-dependent manner. Radicicol was then removed, and the
cells were washed and incubated in drug-free media for a further 24-h
period. Removal of radicicol restored both Erk2 phosphorylation and
levels of protein Raf-1. Therefore, continuous exposure to radicicol
was required to attenuate Raf-1 protein expression.

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Fig. 7.
Effect of drug removal on radicicol-induced
Raf depletion in KNRK5.2 cells. A, cells were treated with
various concentrations of radicicol in duplicate. At 24 h, one set
of cells was lysed and analyzed by immunoblotting (lanes
1-4). Another set of cells received radicicol for 24 h and
was then transferred to a drug-free medium. After another 24-h period,
cells were lysed and analyzed by immunoblotting (lanes
5-8). B, immunoblotted gel was analyzed as described
under "Experimental Procedures." The levels of Raf-1 and
phosphorylated Erk2 were plotted as a percentage of the control.
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Effect of Radicicol on the Ras Signaling Pathway in Activated K-ras
Human Pancreatic Carcinoma Cell Line, PSN-1--
Previous studies have
demonstrated the ability of radicicol to inhibit the
anchorage-independent growth in soft agar of the human bladder
carcinoma cell line, EJ, that harbors a point mutationally activated
allele of endogenous H-ras oncogene and the human
fibrosarcoma cell line, HT1080, with a point mutationally activated
allele of N-ras oncogene (12). Given our present findings in
K-ras-transformed NRK cells, we wanted to extend our
observation to human tumor cell lines in which the endogenous
K-ras gene was activated by a point mutation. The human
pancreatic carcinoma cell line, PSN-1, has a Gly to Val mutation at
amino acid 12 in its K-ras gene (19). Immunoblot analysis of
PSN-1 cell lysate revealed the presence of the phosphorylated form of
Erk2, indicating the activation of the K-Ras signaling pathway in cells
(Fig. 8). Radicicol inhibited the
tyrosine phosphorylation of Erk2 with an IC50 of 1.2 µM. Interestingly, Erk1 was minimally phosphorylated in
the PSN-1 cell line. Depletion of Raf-1 was also observed in
radicicol-treated PSN-1 cells. Although the level of MEK1/2 remained
unchanged in radicicol-treated KNRK5.2 cells (Fig. 3), a slight
decrease in the level of MEK1/2 was observed in PSN-1 cells treated
with radicicol. In PSN-1 cells, endogenous K-Ras protein was fully
farnesylated, and it migrated to the same position as a standard sample
of farnesylated K-Ras protein in SDS-polyacrylamide gel electrophoresis
(data not shown). As shown in Fig. 8, the level of K-Ras protein in
PSN-1 cells remained unchanged in the presence of different
concentrations of radicicol. These observations further supported our
conclusion obtained from the KNRK5.2 cell study that the primary target
of radicicol in the K-Ras signaling pathway was, in fact, the Raf-1
kinase.

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Fig. 8.
Radicicol inhibits K-Ras-induced Erk2
phosphorylation by depleting Raf-1 kinase in PSN-1 cells. A,
PSN-1 cells were grown for 40 h in the presence of various
concentrations of radicicol. 33 µg of each cell lysate was analyzed
by immunoblotting with antibody against K-Ras, Raf-1, the
phosphorylated form of MEK1/2, MEK1/2, the phosphorylated form of
Erk1/2, or Erk2, respectively (from top to
bottom). B, the immunoblotted gel was analyzed as described under "Experimental Procedures." The levels of Raf-1, phosphorylated Erk2, and phosphorylated MEK1/2 were plotted as a
percentage of the control.
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DISCUSSION |
Genetic and biochemical studies demonstrate that Raf
functions downstream of Ras in many signaling pathways, and activation of Ras leads to constitutive activation of a downstream phosphorylation cascade comprising Raf proteins, MAPKK and MAPK. A small molecule inhibitor of Ras-Raf signal transduction could not only be a valuable tool in elucidating that signal transduction pathway but could also
have a therapeutic potential. We have developed a yeast assay system in
which the yeast pheromone-induced MAPK pathway is dependent upon
co-expression of K-Ras and Raf-1. Using this system, we have screened
for inhibitors of the Ras signaling pathway reconstituted in yeast and
rediscovered radicicol as an inhibitor of the Ras pathway. Radicicol
inhibited the K-Ras signaling pathway in mammalian epithelial cells as
well.
Although radicicol has been shown to inhibit activation of MAPK in
H-ras-transformed fibroblastic cell lines (12), the precise mechanism by which radicicol disrupts Ras signal transduction has not
been unambiguously determined. Radicicol did not affect the biochemical
function of Ras protein, such as GTP binding or membrane localization
(13). Furthermore, it inhibited neither MAPKK nor MAPK in enzyme assays
using purified enzyme from Xenopus oocytes (13). Our present
results clearly demonstrate a molecular mechanism by which radicicol
inhibits the Ras signaling pathway. Radicicol inhibits
K-Ras-dependent phosphorylation of MAPK through the
destabilization of Raf protein in a K-ras-transformed rat kidney epithelial cell line and in K-ras-activated human
pancreatic carcinoma cells. The level of Ras and MAPK did not change in
the cells treated with radicicol. Depletion of Raf-1 by radicicol can
also explain why radicicol is active against the yeast mutant strains
expressing Raf-1
N.
Selective destabilization of Raf was also reported for the benzoquinone
ansamycin family antibiotic, geldanamycin (20, 21). Although both
geldanamycin and radicicol have a macrocyclic moiety in their
structure, radicicol differs structurally from ansamycin family
antibiotics. Raf is known to exist as part of a multimolecular complex
that contains hsp90, Cdc37/p50, and other proteins (22, 23). This
complex may function as a transportosome directing Raf to its proper
subcellular localization. Geldanamycin has been shown to bind directly
to hsp90 and disrupts the complex of which it is a part (20).
Disruption of the Raf-hsp90 association by geldanamycin leads to
destabilization of Raf protein. In KNRK5.2 cells, we also found that
geldanamycin caused a depletion of Raf protein and inhibited the
phosphorylation of Erk2 (data not shown). These results are similar to
those observed for radicicol. Therefore, it is possible that radicicol
may affect hsp90, which in turn regulates the stability of Raf kinases.
This prompted us to study whether radicicol could bind hsp90. Our
preliminary data show that radicicol binds specifically to the
chaperone protein hsp90. Furthermore, radicicol is able to compete for
binding with geldanamycin to hsp90. Taken together, destabilization of
Raf kinases by radicicol may result from the ability of the drug to
inhibit the binding of hsp90 to Raf kinases. Thus, radicicol is the
first non-benzoquinone ansamycin capable of binding to hsp90 and
interfering with its function.
Radicicol has been reported to inhibit Mos-induced MAPK activation
(12). Since Raf is probably not involved in this activation, molecular
target(s) for the inhibition of Mos-stimulated MAPK activation could be
some other factor(s), which could potentially interact with hsp90. The
present observation that radicicol induced morphological reversion of
KNRK5.2 cells at concentrations that were lower than those that
affected the Raf-MAPK pathway also implies the presence of molecular
target(s) that are not involved in the linear pathway from Ras to MAPK
but are associated with cell morphological changes.
There is increasing evidence that Ras may mediate its action by
stimulating multiple downstream targets, of which Raf is only one.
Oncogenic Ras activation of Rac1 and RhoA, coupled with activation of
the Raf/MAPK pathway, is required to trigger the full morphologic and
mitogenic consequence of oncogenic Ras transformation (24-26). Dominant inhibitory mutants of Rac1 and RhoA block oncogenic
ras-transforming activity and partially reverse the
morphology of ras-transformed NIH3T3 cells. On the other
hand, activated Rac1 and RhoA further enhance oncogenic
ras-mediated morphologic transformation and cell motility
(27). Thus, Rac- and Rho-induced changes in actin cytoskeleton could
contribute significantly to oncogenic transformation of fibroblastic
cells. If the same were true in epithelial cells, morphological
reversion of KNRK5.2 cells at lower concentrations of radicicol may be
due to the inhibition of some molecule(s) in the signaling pathway
involving Rac and/or Rho. One of the components that links Ras and Rho
is p120 Ras-GTPase-activating protein (GAP). The N-terminal region of
GAP has been shown to regulate cytoskeletal structure and cell adhesion
(28), and it is the site of interaction with two cytoplasmic
phosphoproteins, p190 and p62 (29, 30). GAP-associated p190 protein
itself functions as a GTPase activation protein for Rho (31, 32). GAP-associated protein p62, which has recently been cloned and renamed
as p62dok, is a novel protein with features of a signaling
molecule in a pathway downstream of receptor tyrosine kinases (33, 34). It should be noted that radicicol has previously been shown to inhibit
the tyrosine phosphorylation of p62dok in
ras-transformed NIH3T3 cells (12). Taken together with
previous studies, radicicol might cause morphological reversion of
KNRK5.2 cells by interfering with the interaction of hsp90 with some
tyrosine kinase that is involved in the phosphorylation of
p62dok.
Several lines of evidence show that inhibition of the function of Raf
could lead to the disruption of an aberrant mitogenic signal
originating from an activated ras gene. A loss-of-function mutation of the raf gene in Caenorhabditis
elegans suppresses the phenotype resulting from an activated
ras gene (35). Antisense oligodeoxynucleotides targeted
against raf have been shown to suppress the growth of
ras-activated human tumors inoculated into nude mice (36,
37). Our present results support the notion that Raf is a crucial
pharmacological target for human malignancy that is closely associated
with ras gene activation and highlight the possibility that
pharmacological destabilization of Raf protein could be one potential
approach for inhibiting the function of Raf in
K-ras-activated human cancers.
We are grateful to D. A. Johnson for
KNRK5.2 cells, K. Yamaguchi for PSN-1 cells, and F. Tamanoi for
pVT101-L plasmid. We thank L. M. Neckers for communicating
unpublished results. We acknowledge F. Tamanoi for critical reading of
the manuscript.