Radicicol Leads to Selective Depletion of Raf Kinase and Disrupts
K-Ras-activated Aberrant Signaling Pathway*
(Received for publication, June 9, 1997, and in revised form, September 15, 1997)
Shiro Soga‡, Takako Kozawa‡, Hiroaki Narumi§, Shiro Akinaga§, Kenji Irie¶,
Kunihiro Matsumoto¶, Sreenath V. Sharma?, Hirofumi Nakano‡, Tamio Mizukami‡,
and Mitsunobu Hara‡**
From the ‡Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Asahi-machi 3-6-6, Machida-shi, Tokyo 194,
Japan, §Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Shimotogari 1188, Nagaizumi-cho,
Shuntou-gun, Shizuoka 411, Japan, ¶Department of Molecular Biology, Faculty of Science, Nagoya University,
Chikusa-ku, Nagoya 464-01, Japan, and ?Department of Microbiology and Immunology, University of Tennessee,
Memphis, Tennessee 38163
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 reconsti-
tuted in yeast. In K-ras-transformed, rat epithelial, and
K-ras-activated, human pancreatic carcinoma cell lines,
radicicol inhibited K-Ras-induced hyperphosphoryla-
tion 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 pharmacolog-
ical destabilization of Raf kinase could be a new and
powerful approach for the treatment of K-ras-activated
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 con-
necting upstream tyrosine kinases with downstream serine/
threonine kinases such as mitogen-activated protein kinase
(MAPK)1or MAPK kinase (MAPKK). A similar MAPK signal-
ing 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 recon-
stituted 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 origi-
nally 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 trans-
formed 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 transforma-
tion by the ras oncogene. Decreased MAPK activity accompa-
nies 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 sug-
gest 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.
Plasmid and Yeast Strains—The plasmid pVT-VKR expresses
Kirstein sarcoma virus ras gene from the ADH promoter. It was con-
structed 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 STE7P368allele controlled from the
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 appro-
priate auxotrophic supplements (16). SC lacking amino acids or other
nutrients (e.g. SC-ura, which lacks uracil) was used to select transfor-
mants. Yeast transformation was performed by the method of Itoh et al.
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
* The costs of publication of this article were defrayed in part by the
payment of page charges. This 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.: 81-427-25-
2555; Fax: 81-427-26-8330; E-mail: email@example.com.
1The abbreviations used are: MAPK, mitogen-activated protein ki-
nase; MAPKK, MAPK kinase; MEK, same protein as MAPK kinase;
Erk, extracellular signal-regulated protein kinase (same protein as
MAP kinase); hsp90, heat shock protein 90; GAP, GTPase-activating
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 2, Issue of January 9, pp. 822–828, 1998
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on December 28, 2015
J. Biol. Chem.
Nakano, Tamio Mizukami and Mitsunobu
Matsumoto, Sreenath V. Sharma, Hirofumi
Shiro Akinaga, Kenji Irie, Kunihiro
Shiro Soga, Takako Kozawa, Hiroaki Narumi,
Aberrant Signaling Pathway
Raf Kinase and Disrupts K-Ras-activated
Radicicol Leads to Selective Depletion of
CELL BIOLOGY AND METABOLISM:
Find articles, minireviews, Reflections and Classics on similar topics on the
http://www.jbc.org/content/273/2/822 Access the most updated version of this article at
. JBC Affinity Sites
When a correction for this article is posted•
When this article is cited •
to choose from all of JBC's e-mail alertsClick here
This article cites 36 references, 14 of which can be accessed free at
by guest on December 28, 2015