Eriodictyol Inhibits RSK2-ATF1 Signaling and Suppresses
EGF-induced Neoplastic Cell Transformation*□
Kangdong Liu‡§1, Yong-Yeon Cho‡1, Ke Yao‡§1, Janos Nadas‡, Dong Joon Kim‡, Eun-Jin Cho‡, Mee-Hyun Lee‡,
Angelo Pugliese‡, Jishuai Zhang‡, Ann M. Bode‡, Ziming Dong§, and Zigang Dong‡2
University,ZhengZhou 450001, China
RSK2 is a widely expressed serine/threonine kinase, and its
activation enhances cell proliferation. Here, we report that
ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signal-
ing plays an important role in EGF-induced neoplastic cell
transformation. RSK2 phosphorylated ATF1 at Ser-63 and en-
hanced ATF1 transcriptional activity. Docking experiments
using the crystal structure of the RSK2 N-terminal kinase do-
main combined with in vitro pulldown assays demonstrated
that eriodictyol, a flavanone found in fruits, bound with the
N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal
kinase activity. In cells, eriodictyol inhibited phosphorylation
of ATF1 but had no effect on the phosphorylation of RSK,
MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol spe-
cifically suppresses RSK2 signaling. Furthermore, eriodictyol
inhibited RSK2-mediated ATF1 transactivation and tumor
promoter-induced transformation of JB6 Cl41 cells. Eriodic-
tyol or knockdown of RSK2 or ATF1 also suppressed Ras-me-
diated focus formation. Overall, these results indicate that
RSK2-ATF1 signaling plays an important role in neoplastic
cell transformation and that eriodictyol is a novel natural com-
pound for suppressing RSK2 kinase activity.
RSK2 (ribosomal S6 kinase 2) is a member of the p90RSK
protein family that is activated by ERK1/2 and PDK1 (phos-
phoinositide-dependent kinase 1) (1, 2). RSK2 translocates to
the nucleus when activated by growth factors, peptide hor-
mones, or neurotransmitters (3, 4). Numerous proteins, such
as the cAMP response element (CRE)3-binding protein
(CREB), Elk-1, histones (5–9), ATF4 (activating transcription
factor 4) (10), p53 (11), and NFAT3 (12), are phosphorylated
by active RSK2. Based on its broad substrate specificity, the
RSK2 protein mediates many cellular processes, including
proliferation and transformation, as well as the cell cycle. Our
recent study provided evidence indicating that RSK2 plays an
important role in cell transformation induced by tumor pro-
moters such as EGF and 12-O-tetradecanoylphorbol-13-ace-
tate (13). Furthermore, RSK2 knock-out mice display reduced
c-Fos-dependent osteosarcoma formation through the regula-
tion of c-Fos protein stability (14). Thus, RSK2 likely plays a
key role in cell proliferation and transformation.
ATF1 is a member of the CREB family, which includes
ATF1, CREB1, and the CRE modulator (15). In response to
growth factors, stress signals, neurotransmitters, or other
agents that elevate intracellular cAMP or Ca2?levels, CREB
family members are activated and promote the expression of
numerous cellular target genes that contain CREs in their
promoters (16), including proto-oncogenes such as c-fos and
c-jun (17, 18) and cell cycle genes such as cyclins D and A and
other genes related to cell growth, proliferation, and neuronal
activities (19, 20). Phosphorylation of ATF1 at Ser-63 in its
kinase-inducible domain by serine/threonine kinases en-
hances its transactivation activity by promoting recruitment
of the coactivator CREB-binding protein/p300 (21). ATF1 is
overexpressed in lymphomas and transformed lymphocytes
(22), suggesting that ATF1 may contribute to the growth of
these tumor cells. ATF1 is up-regulated in metastatic mela-
noma cells, and inhibition of ATF1 suppresses their tumorige-
nicity and metastatic potential in nude mice (23). Constitutive
activation of ATF1 mediates EWS-ATF1 (Ewing sarcoma pro-
tein), transforming phenotypes and unique features of clear
cell sarcoma (24). However, the upstream kinases and the role
of ATF1 in proliferation and cell transformation have not
been completely elucidated.
Flavonoids are ubiquitously found in fruits and vegetables
as well as popular beverages, including wine, tea, and coffee
(25). Flavonoids also exhibit antioxidant, antitumor, and anti-
inflammatory effects (25). In particular, their antitumor activ-
ity has attracted much attention as a possible dietary preven-
tion strategy against carcinogenesis (26, 27). Our recent study
demonstrated that kaempferol is a natural compound that
specifically inhibits RSK2 N-terminal kinase activity (28). Eri-
odictyol is a major flavonoid extracted from Yerba Santa (Eri-
odictyon californicum) and has a similar structure to
kaempferol. Eriodictyol is found in lemon, lime, sour orange,
and peppermint. Research data have shown that eriodictyol
also exerts antioxidant, antitumor, and anti-inflammatory
effects (29), suggesting that eriodictyol might also inhibit cell
proliferation and transformation. However, the underlying
* This work was supported, in whole or in part, National Institutes of Health
Grants CA111536, CA120388, CA077646, R37CA081064, and ES016548.
This work was also supported by The Hormel Foundation.
SThe on-line version of this article (available at http://www.jbc.org) con-
tains supplemental Figs. 1–4.
1These authors contributed equally to this work.
2To whom correspondence should be addressed: The Hormel Inst., Univer-
sity of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-
9600; Fax: 507-437-9606; E-mail: firstname.lastname@example.org.
3The abbreviations used are: CRE, cAMP response element; CREB, CRE-bind-
ing protein; MEF, mouse embryonic fibroblast; DMSO, dimethyl sulfoxide;
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 3, pp. 2057–2066, January 21, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
JANUARY 21, 2011•VOLUME 286•NUMBER 3JOURNAL OF BIOLOGICAL CHEMISTRY 2057
mechanism and target protein(s) of eriodictyol have not yet
In this study, we show that ATF1 is a novel substrate of
RSK2. The phosphorylation of ATF1 at Ser-63 by RSK2 in-
duces ATF1 transactivation and transcriptional activity. Inhi-
bition of RSK2 activity by eriodictyol suppressed ATF1 activi-
ties and RSK2-ATF1-mediated cell transformation. These
results demonstrate that the inhibition of RSK2 activity by
eriodictyol modulates RSK2-ATF1 signaling in cell prolifera-
tion and transformation. Therefore, we suggest that eriodic-
tyol is a potential natural compound for chemoprevention.
Materials—Eriodictyol (?95% purity), Tris, NaCl, and SDS
were from Sigma. CNBr-Sepharose 4B, glutathione-Sepharose
4B, and the chemiluminescence detection kit were from Am-
ersham Biosciences. The protein assay kit was from Bio-Rad.
[?-32P]ATP was purchased from New England Biolabs. G418
and the luciferase assay substrate were from Promega. Eagle’s
minimal essential medium was from Invitrogen. Antibodies
for Western blot analysis were from Cell Signaling Technol-
ogy, Santa Cruz Biotechnology, Abcam, and Millipore.
Culture of JB6 Cl41 cells and RSK2?/?and RSK2?/?mouse
embryonic fibroblasts (MEFs) was described previously (13).
Transfection of the various expression vectors and luciferase
reporter plasmids was conducted using jetPEI (Polyplus
Transfection, New York, NY) according to the manufacturer’s
instructions. Eriodictyol was dissolved in dimethyl sulfoxide
(DMSO; 50 mM stock solution), aliquoted, and stored at
?20 °C. Eriodictyol was mixed with complete cell culture me-
dium at various doses, and the final DMSO concentration was
not ?0.1% of the total media volume.
MTS Assay—Proliferation of JB6 Cl41 cells and viability of
RSK2?/?and RSK2?/?MEFs were measured using the MTS
assay kit (Promega) according to the manufacturer’s
In Vitro Kinase Assay—The plasmids for GST-tagged or
His-tagged fusion proteins including ATF1 and ATF1-S63A
were purified from BL21 bacteria using glutathione-Sepha-
rose 4B or nickel-nitrilotriacetic acid-agarose beads. The puri-
fied fusion proteins (1 ?g; ?90% purity) were used for in vitro
kinase assays with 20 ng of active RSK2 (Millipore) and visual-
ized by autoradiography or Western blotting as described
Cell Cycle Analysis—The cell cycle was analyzed by pro-
pidium iodide staining using the FACSCalibur flow cytometer
(BD Biosciences) as described by Ahmad et al. (30).
Mammalian Two-hybrid Assay—To screen for protein-
binding partners, we used the mammalian two-hybrid assay
following the Promega CheckMateTMmammalian two-hybrid
system protocol as described previously (11). The relative lu-
ciferase activity was calculated using the pG5-luciferase basal
control by transfection of pG5-luciferase/pACT-mock/
pBIND-RSK2 and normalized against Renilla luciferase activ-
ity, which included the pBIND vector.
ATF1 Transactivation Assay—JB6 C141 cells (6 ? 104)
were cultured in 12-well plates for 24 h before transfection.
The p5?Gal4-luciferase reporter plasmid was transfected
with pcDNA4-RSK2 and the expression vector for Gal4-ATF1
or Gal4-ATF1-S63A. Cells were cultured for 36 h and then
disrupted for firefly luciferase activity analysis. The reporter
gene vector phRL-SV40 (Promega) was cotransfected into
each cell line, and the transfection efficiencies were normal-
ized to the Renilla luciferase activity generated by this vector.
Anchorage-independent Cell Transformation Assay—The
effects of eriodictyol on EGF-induced transformation were
investigated in JB6 C141 cells as described by Colburn et al.
(31). Colonies were counted under a microscope with the Im-
age-Pro Plus software program (Version 6, Media Cybernet-
ics, Silver Spring, MD).
Preparation of Sepharose 4B Beads—Sepharose 4B beads
(0.3 g) were washed with 30 ml of 1 mM HCl three times for 5
min each by gentle inversion and then incubated with 3 mg of
eriodictyol or DMSO in coupling buffer (0.1 M NaHCO3and
0.5 M NaCl (pH 8.3)) at 4 °C overnight. The samples were
washed five times with coupling buffer and incubated with
blocking buffer (0.1 M Tris-HCl (pH 8.0)) at 4 °C overnight.
The samples were alternatively washed with 0.1 M acetic acid
buffer (pH 4.0) and with 0.1 M Tris-HCl and 0.5 M NaCl (pH
8.0) three times and then resuspended in 1 ml of PBS for use.
Pulldown Assays—For pulldown assays, eriodictyol-Sepha-
rose 4B beads (100 ?l, 50% slurry) were combined with puri-
fied RSK2 or a cellular supernatant fraction of JB6 C141 cells
(500 ?g) overnight. Bound RSK2 proteins were visualized by
Western blotting as described (28).
Focus Formation Assay—A focus formation assay using
NIH3T3 cells was conducted according to standard protocols
(24). Foci were fixed and stained with 0.5% crystal violet
and counted using the Image-Pro Plus software program
To build a model of the active form of the RSK2 N-terminal
kinase domain, we conducted homology modeling based on
the active conformation of the RSK1 N-terminal kinase do-
was used to align the RSK2 and RSK1 sequences, wherein they
were shown to possess an identity of 85% and a similarity of
90% (32). Using Modeller 9v4 (33), we created a template
structure of RSK2 based on the active structure of RSK1. To
ensure an adequate bound protein form, waters and stauros-
porine were added from the crystal structure of RSK1 using
MAESTRO 9.0 and were allowed to relax with OPLS_2005
force field. This was performed using MacroModel 9.7 follow-
ing standard minimization procedures (34).
Molecular Docking to a Homology Model of the RSK2 N-
terminal Kinase Domain—Kaempferol and eriodictyol were
both built and minimized and underwent extensive confor-
mational searches followed by subsequent minimization using
MacroModel to determine the lowest energy conformations
for docking. Staurosporine as the reference compound was
subjected to similar strategies except starting from its crystal-
lized bound orientation. The Induced Fit module was used for
docking because both ligand and binding site residue flexibil-
ity are provided for, thereby mimicking reality (34). The rec-
2058 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 286•NUMBER 3•JANUARY 21, 2011
22. Hsueh, Y. P., and Lai, M. Z. (1995) J. Immunol. 154, 5675–5683
23. Jean, D., Tellez, C., Huang, S., Davis, D. W., Bruns, C. J., McConkey,
D. J., Hinrichs, S. H., and Bar-Eli, M. (2000) Oncogene 19, 2721–2730
24. Brown, A. D., Lopez-Terrada, D., Denny, C., and Lee, K. A. (1995) Onco-
gene 10, 1749–1756
25. Hollman, P. C., and Katan, M. B. (1999) Food Chem. Toxicol. 37,
26. Zhou, Q., Yan, B., Hu, X., Li, X. B., Zhang, J., and Fang, J. (2009) Mol.
Cancer Ther. 8, 1684–1691
27. Li, Z. D., Hu, X. W., Wang, Y. T., and Fang, J. (2009) FEBS Lett. 583,
28. Cho, Y. Y., Yao, K., Pugliese, A., Malakhova, M. L., Bode, A. M., and
Dong, Z. (2009) Cancer Res. 69, 4398–4406
29. Clavin, M., Gorzalczany, S., Macho, A., Mun ˜oz, E., Ferraro, G., Acevedo,
C., and Martino, V. (2007) J. Ethnopharmacol. 112, 585–589
30. Ahmad, N., Feyes, D. K., Nieminen, A. L., Agarwal, R., and Mukhtar, H.
(1997) J. Natl. Cancer Inst. 89, 1881–1886
31. Colburn, N. H., Wendel, E. J., and Abruzzo, G. (1981) Proc. Natl. Acad.
Sci. U.S.A. 78, 6912–6916
32. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,
P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R.,
Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics
33. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779–815
34. Schroginger Suite (2009) Induced Fit Docking Protocol, Glid Version
5.5, Prime version 2.1., Schrondinger, LLC, New York
35. Zheng, D., Cho, Y. Y., Lau, A. T., Zhang, J., Ma, W. Y., Bode, A. M., and
Dong, Z. (2008) Cancer Res. 68, 7650–7660
36. Kang, S., Elf, S., Dong, S., Hitosugi, T., Lythgoe, K., Guo, A., Ruan, H.,
Lonial, S., Khoury, H. J., Williams, I. R., Lee, B. H., Roesel, J. L., Karsenty,
G., Hanauer, A., Taunton, J., Boggon, T. J., Gu, T. L., and Chen, J. (2009)
Mol. Cell. Biol. 29, 2105–2117
37. Zucman, J., Delattre, O., Desmaze, C., Epstein, A. L., Stenman, G., Spele-
man, F., Fletchers, C. D., Aurias, A., and Thomas, G. (1993) Nat. Genet.
38. Shimomura, A., Ogawa, Y., Kitani, T., Fujisawa, H., and Hagiwara, M.
(1996) J. Biol. Chem. 271, 17957–17960
39. Arthur, J. S., and Cohen, P. (2000) FEBS Lett. 482, 44–48
40. Wiggin, G. R., Soloaga, A., Foster, J. M., Murray-Tait, V., Cohen, P., and
Arthur, J. S. (2002) Mol. Cell. Biol. 22, 2871–2881
2066 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 286•NUMBER 3•JANUARY 21, 2011