Selective Inhibition of MAPKK Wis1 in the
Stress-activated MAPK Cascade of Schizosaccharomyces
pombe by Novel Berberine Derivatives*
Myoung Jin
Jang
,
Miri
Jwa,
Jung-Ho
Kim§, and
Kiwon
Song¶
From the Department of Biochemistry, and Institute of
Life science and Biotechnology, College of Science, Yonsei University,
Seoul 120-749, Korea and § Hanwha Chemical Research and
Development Center, Taejeon 305-345, Korea
Intracellular molecular targets of novel
berberine derivatives, HWY 289 and HWY 336, were identified by a screen
of a variety of mutants in fission yeast Schizosaccharomyces
pombe. HWY 289 and HWY 336 completely inhibited the proliferation
of wild type as well as various mutant fission yeast cells (minimal
inhibitory concentrations were 29.52 µM for HWY 289 and
11.83 µM for HWY 336), but did not affect the
proliferation of Wis1 mitogen-activated protein kinase kinase (MAPKK)
deletion mutants. In addition, HWY 289 with an IC50 value
of 7.3 µM or HWY 336 with IC50 of 5.7 µM specifically inhibited in vitro kinase
activities of purified Wis1, whereas either compound did not affect the
activities of other kinases in the mitogen-activated protein kinase
(MAPK) cascades of fission yeast. These genetic and biochemical results
demonstrate the high degree of specificity of HWY 289 and HWY 336 to
MAPKK Wis1 and suggest that the cytotoxicity of these compounds is not simply due to the inhibition of Wis1 kinase activity. High salt wash
experiments have shown that strong noncovalent binding occurs between
Wis1 and either HWY 289 or HWY 336. The preincubation of Wis1 kinase
with ATP did not affect the inhibition of Wis1 by HWY 289 and HWY 336, but when Wis1 was preincubated with MBP, a protein substrate, Wis1
kinase activity was no longer inhibited. These observations demonstrate
that HWY 289/HWY 336 do inhibit Wis1 kinase, not by binding to the
ATP-binding site but by disturbing the binding of substrate to the
kinase. Target validation of the complex of HWY 289/HWY 336 and Wis1
kinase will provide important clues for the mechanism of specific
cytotoxicity of these compounds in S. pombe. On a broader
aspect, it would create an initiative to further modify and develop
compounds that selectively inhibit kinases and cause cytotoxicity in
various MAPK cascades including those of mammals.
*
This work was supported by Critical Technology Program Grant
00-J-BP-01-B-64 from the Korea Institute of Science and Technology Evaluation and Planning.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.
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