ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4.

Abstract

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins βTrCP1 or βTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

Full text

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Understanding and controlling the mechanism by which stem cells
balance the critical characteristics of self-renewal versus differentiation
would substantially advance our knowledge of embryogenesis and cancer
biology as well as help to provide a source of cells for transplantation
to replace cells lost through injury or disease. The controlled expres-
sion of the Krüppel-like factor Klf4, together with other transcrip-
tion factors, is capable of reprogramming adult human fibroblasts to
become induced pluripotent stem (iPS) cells, which behave similarly to
embryonic stem cells1,2. These proteins (that is, Oct3, Oct4, Sox2, Klf4,
c-Myc and Nanog) are often referred to as iPS factors. Using these iPS
factors, terminally differentiated mouse and human dermal fibroblasts
can produce iPS cells, which can generate chimeras1 and teratomas in
athymic nude mice3. Recent studies indicate that the activity of iPS
factors is regulated through post-translational modifications. For exam-
ple, phosphorylation of human SOX2 at Ser249, Ser250 and Ser251 by
as-yet-unidentified kinases4 results in the inhibition of SOX2 DNA
binding activity5, whereas acetylation of mouse Sox2 at Lys75 by p300-
CBP family proteins enhances nuclear export and degradation of Sox2
through a ubiquitin-mediated degradation pathway6. Additionally,
phosphorylation of human OCT4 at Ser229 by protein kinase A
might partially regulate OCT4 transactivation activity7. Oct4 is also a
target for small ubiquitin-related modifier (SUMO)-1 modification that
occurs at a single lysine, Lys118, located at the end of the N-terminal
transactivation domain and next to the DNA binding domain8. These
findings indicate that post-translational modifications of iPS factors are
probably involved in the regulation of their activity, which could result
in modulation of embryonic stem cell self-renewal activity.
Klfs are DNA-binding transcription factors that form a subset of
the broad class of Cys2-His2 (C2H2) zinc-finger proteins, a motif that
comprises the second most abundant motif in the human genome and
the most abundant among all transcription factors9. Three members
of this family, Klf2, Klf4 and Klf5, are highly expressed in undiffer-
entiated mouse embryonic stem cells and are rapidly downregulated
during the early stages of differentiation10. Klf4 is widely expressed
in a variety of tissues and has a role in many diverse physiological
processes11, including the regulation of self-renewal of embryonic
stem cells12. However, the precise mechanism regulating the role of
Klf4 in self-renewal is not yet clearly understood. Thus, we set out to
identify potential protein kinases that interact with and regulate Klf4
in embryonic stem cell self-renewal. We found that the C-terminal
region of ERK1 or ERK2 binds the activation domain of Klf4 and
directly phosphorylates Klf4 at Ser123 in mouse embryonic stem
cells. This phosphorylation of Klf4 Ser123 causes the inhibition of
Klf4 transcriptional activity, resulting in the induction of mouse
embryonic stem cell differentiation. By contrast, inhibition of phos-
phorylation of Klf4 by ERKs enhances Klf4 activity, resulting in the
suppression of embryonic stem cell differentiation. Notably, phospho-
rylation of Klf4 Ser123 by ERK1 or ERK2 induces recruitment and
binding of βTrCP2 (a component of ubiquitin E3 ligase) to the Klf4
N-terminal domain (residues 1–140). Furthermore, binding of βTrCP
results in Klf4 ubiquitination and degradation through the protea-
somal degradation pathway. Overall, our data provide a molecular
basis for the role of Klf4 in the direct mediation of embryonic stem
cell self-renewal.
1The Hormel Institute, University of Minnesota, Austin, Minnesota, USA. 2Center for Laboratory Animal Resources, KyungPook National University, Daegu, Republic of
Korea. 3Current Address: College of Pharmacy, The Catholic University of Korea, Bucheon-si, Korea. 4These authors contributed equally to this work. Correspondence
should be addressed to Z.D. (zgdong@hi.umn.edu).
Received 26 July 2010; accepted 2 December 2011; published online 5 February 2012; doi:10.1038/nsmb.2217
ERK1 and ERK2 regulate embryonic stem cell self-renewal
through phosphorylation of Klf4
Myoung Ok Kim1,4, Sung-Hyun Kim1,2,4, Yong-Yeon Cho1,3, Janos Nadas1, Chul-Ho Jeong1, Ke Yao1, Dong Joon Kim1,
Dong-Hoon Yu1, Young-Sam Keum1, Kun-Yeong Lee1, Zunnan Huang1, Ann M Bode1 & Zigang Dong1
Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great 
importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism 
regulating this role of Klf4 is unclear. We found that ERK? or ERK2 binds the activation domain of Klf4 and directly 
phosphorylates Klf4 at Ser?23. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. 
Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. 
Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins bTrCP? or bTrCP2 
(components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. 
Overall, our data provide a molecular basis for the role of ERK? and ERK2 in regulating Klf4-mediated mouse embryonic  
stem cell self-renewal.

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RESULTS
Klf4 is a newly identified substrate of ERK? and ERK2
Based on recent studies describing post-translational modifica-
tions of Sox2 and Oct4, we hypothesized that Klf4 activity might
be regulated by post-translational modification, specifically phos-
phorylation. In order to identify potential upstream kinases of Klf4,
we screened approximately 60 kinases in a mammalian two-hybrid
assay by co-transfecting each pBIND-kinase and the pG5-luciferase
reporter plasmid (pG5-Luc) with pACT-Klf4 as bait (Supplementary
Table 1). We found that Klf4 showed the highest binding affinity
with ERK1 and ERK2 compared with other pBIND kinases or the
pG5-Luc–pBIND mock (Fig. 1a), and confirmed this by coimmuno-
precipitating hemagglutinin (HA)-tagged Klf4 and HisG-tagged
ERK1 from HEK293 cells (Fig. 1b). To examine whether ERK1 or
ERK2 phosphorylates Klf4, we constructed a glutathione S-trans-
ferase (GST)-fusion bacterial expression vector and purified the GST-
Klf4 protein from Escherichia coli BL21. The protein was treated by
in vitro kinase assay with [γ-32P]ATP and commercially available
active ERK1 or ERK2. The results demonstrated that either ERK1
or ERK2 could phosphorylate Klf4 (Fig. 1c). To identify the phos-
phorylation domain, we tested truncated forms of Klf4 in the same
kinase assay. We found strong 32P-labeled bands that were detectable
in full-length Klf4 (GST-Klf41–474) and GST-Klf41–140, which includes
the activation domain (Fig. 1d). These results demonstrate that Klf4
is a newly identified ERK1 or ERK2 substrate.
Identification of Klf4 sites phosphorylated by ERK? or ERK2
ERK1 and ERK2 are members of a well-known MAP kinase family
that phosphorylate the XX(pS)P or XX(pT)P motif of its substrates13.
To determine the specific site or sites of Klf41–140 that could be phos-
phorylated by ERK1 and ERK2, we used the Human Protein Reference
Database (http://www.hprd.org/; see Supplementary Fig. 1a) for
sequence comparison between human and mouse Klf4. The results
showed that Klf41–140 contains three putative SP motifs (Supplementary
Fig. 1b,c) and that Ser123 is a predicted phosphorylation site of Klf4
(Supplementary Fig. 1d). (Note that this is the shorter amino acid
sequence for Klf4 downloaded in 2007 that has since been updated to
include nine more amino acids in front of the former number 1 amino
acid, methionine). To verify that Ser123 of Klf4 is a target amino acid
for ERK1 or ERK2, we constructed a point mutant of GST-Klf41–140,
in which Ser123 was replaced by alanine (S123A). Results from an
in vitro kinase assay with ERK1 indicated that the Klf4 S123A mutation
abolished most of the phosphorylation signal compared to wild-type
Klf41–140 (Fig. 2a). Furthermore, an ex vivo immunoprecipitation assay
using deletion mutants of Klf4 and full-length ERK1 indicated that the
N-terminal domain of Klf4 interacts with ERK1 (Fig. 2b). Overall,
these results suggested that Klf4 Ser123 is a target phosphorylation site
of ERK1 or ERK2. Based on this result, a visual inspection of the full-
length protein sequence was conducted to determine whether the pro-
tein possessed the common docking (CD) and DEF motifs. A potential
common docking motif was identified at 79-LARRETEEFNDLLDL-94
and then aligned to other known ERK2 substrate protein sequences14,15
using EMBOSS16 for validation. We found that the common docking
motif that binds to the D domain of ERK is (Arg or)Lys2-Xaa2-6-Faa-
Xaa-Faa. A surface representation of ERK2 and the binding sites of
its substrate is shown (Fig. 2c). The crystal structures of substrates
mitogen-activated protein kinase 3 (MPK3) and hematopoietic tyro-
sine phosphatase non-receptor type 7 (HePTP) bound to ERK2 (PDB
2FYS45 and PDB 2GPH46, respectively) possess similar sequences.
A manual alignment was carried out in order to align the appropriate
residues with each other (Supplementary Fig. 1e). The ERK2–Klf4
computational model shows the D domain of ERK2 interacting with
abc
GST-KIf41–474
Active ERK1
Active ERK2
+
–
–
–
+
+
–
–
+
+
+
–
–
–
+
–
+
–
+
–
+
–
–
–
GST-mock
32P-GST-KIf4
32P-auto-ERK1
Coomassie
blue
staining
7
6
5
4
3
2
1
*
*
0
pG5-Luc
pACT-KIf4
pBIND
Relative luciferase
activity (fold)
+
–
+
+
–
+
+
+
+
+
+
+
+
+
+
+
+
+
ERK1
ERK2ERK8
LCK
IKKg
ALK3
d
GST
Klf41–140
Klf4141–250
Klf4251–370
Klf4371–474
GST-KIf4371–474
GST-KIf4251–370
GST-KIf4141–250
GST-KIf41–140
Active ERK1
200 aa
ID
SRR
NLS
Zinc fnger
AD
HA-Klf41–474
His-ERK1
HA-mock
His-mock
IP: HisG
–
–
+
+
–
–
+
–
+
+
–
–
–
+
++
WB: HA
WB: His
WB: β-actin
Cell lysate
WB: HA
32P-GST-KIf4
WB: ERK1
WB: ERK2
32P-GST-KIf4
Coomassie
blue staining
+
+
–
+
–
+
–
+
–
–
+
–+
–
+
–
–
+
–
+
+
–
+
–
–
–
–
––
––
––
–
––
–
––
–
–
–
GST-Klf41–474
GST-mock
Figure 1 Klf4 is a substrate of ERK1 or ERK2. (a) Assessment of interactions of Klf4 with protein kinases by
mammalian two-hybrid assay. Activity is indicated as relative luminescence normalized to a negative
control (value for cells transfected with only pG5-luciferase (pG5-Luc)/pACT-Klf4 = 1.0). Data are shown
as mean ± s.d. of values from triplicate experiments. (*P < 0.05). (b) Confirmation of Klf4 and
ERK1 binding by immunoprecipitation with a HisG antibody. Coimmunoprecipitated Klf4 was detected by
western blot (WB) using an HRP-conjugated HA antibody. IP, immunoprecipitate. (c) Klf4 is phosphorylated
by ERK1 or ERK2. The GST-Klf41–474 fusion protein was analyzed by in vitro kinase assay with [γ-32P]ATP
and active ERK1 or ERK2. Each arrow indicates a GST fusion protein. (d) Identification of the
Klf4 domain phosphorylated by ERK1 or ERK2. GST fusions to truncated forms of Klf4 are shown
schematically (top) and were used for an in vitro kinase assay with [γ-32P]ATP and active ERK1 or ERK2.
The arrows indicate the GST fusion proteins. SRR, serine-rich region; NLS, nuclear localization signal;
AD, activation domain; ID, inhibitory domain.

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© 2012 Nature America, Inc. All rights reserved.
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the arginine residues on Klf4 and highlights the importance of hydro-
gen bonding for the interaction (Fig. 2d). Mutating both arginine resi-
dues in Klf4 to methionine negates hydrogen bonding to the D domain
of ERK2 and prevents Klf4 from binding to ERK2. Mutation of both
leucine residues to alanine prevents hydrogen bonding of Asp93 with
Thr108 and Lys112 (Fig. 2e). To confirm the common docking motif of
Klf4 binding, we constructed 80-AAMMETEEFNDLADA-94 mutants
for 80-LARRETEEFNDLLDL-94. We transfected wild-type pCMV-
HA-Klf4 or mutants together with pcDNA4-HisG-ERK1 into HEK293
cells and found that Klf4 and ERK1 coimmunoprecipitated. ERK1
binding was abrogated with the 80-AAMM-83 mutant. This result
indicated that 80-LARR-83 is important for ERK1 binding (Fig. 2f).
The phosphorylation status of these mutants was determined in an
in vitro kinase assay using ERK1. Results showed that phosphorylation
of ERK1 was reduced by the LRR mutant but was not affected by the
LL mutant (Fig. 2g).
Inhibiting ERK signaling induces stem cell self-renewal
Recent studies indicated that the MAP kinases have an important
role in the homeostasis of stem cell self-renewal17. To examine Klf4
phosphorylation during embryonic stem cell differentiation, we
examined the phosphorylation level of ERK1 and ERK2 with a phos-
pho-MAP kinase array using protein samples from undifferentiated
or four-day differentiated embryonic stem cells. Results showed that
phosphorylation of ERK1 and ERK2 was increased from 2.5- to 3.3-fold
at the four-day differentiation stage induced by withdrawal of leukemia
inhibitory factor (LIF) (Fig. 3a). To examine this closely, we ana-
lyzed total and phosphorylated protein levels of ERK1 or ERK2 and
Klf4 at 24-h intervals during the differentiation process induced
by withdrawal of LIF. We found that phosphorylation of ERK1 or
ERK2 was increased gradually during the differentiation process
without altering the total ERK1 or ERK2 protein levels (Fig. 3b). We
simultaneously found that phosphorylation of Klf4 Ser123 was also
increased during embryonic stem cell differentiation (Fig. 3b). The
antibody to detect phosphorylation of Klf4 Ser123 was produced
and tested in an in vitro kinase assay and cell transfection system
(Supplementary Fig. 2a,b). Notably, the total protein level of Klf4
was markedly suppressed at 3 d after induced differentiation (Fig. 3b).
To investigate the possible effect of ERK signaling on mediating
Klf4 activity in embryonic stem cells, we determined the optimum
90°
bc
Glu79
Arg82
Asn80
Thr108
Asp93
Lys112
Thr116
Ser318
Arg81
Arg133
Tyr129
Asp316
Asp319
gf
a
d
–
–
+
–
–
+
–
–
–
–
–
–
–
–
++++
+
+HA-Klf4
His-ERK1
HA-mock
His-mock
IP: His
WT
LL mtLRR mtLRR + LL mt
WB: HA
WB: His
WB: HA
Cell lysate
–
–
–
–
+
–
–
+
–
+
–
––
–
–
–
–
++
–
–
–
+
+
+Active ERK1
GST-Klf4 (LL mt)
GST-Klf4 (LRR mt)
GST-Klf4
GST-mock
32P-GST-Klf4
Coomassie
Klf4
–
–
–
–
+
–
+
–
–
+
–
–
–
+
–
–
–
–
–
–
+
–
+
++–
–
–
+
+
IP: HA
Cell lysate
WB: His
WB: His
His-ERK1
HA-Klf41–140
HA-Klf41–250
HA-Klf41–370
HA-Klf41–474
WB: HA
WB: HA
WB: β-actin
Active ERK1–
–
+
–
+–
++
+
Coomassie
blue staining
WB: ERK1
32P-GST-Klf4
GST-Klf41–140-S123A
GST-Klf41–140
Klf4
Arg133
Asp93
Lys112
Asp319
D domain ERK2
e
Figure 2 ERK1 phosphorylates Klf4 Ser123. (a) In vitro kinase
assay of GST-Klf41–140-S123A with [γ-32P]ATP and active ERK1.
The arrows indicate the respective GST-Klf4 fusion protein.
(b) Confirmation of ERK1 and Klf4 binding. HEK293 cell lysates
expressing various constructs and ERK1 were immunoprecipitated
with anti-HA, and ERK1 was visualized with anti-histidine. IP, immunoprecipitate. (c) Surface representation
of ERK2 showing binding regions of substrates. Green, D domain where the substrate common docking motif
binds. Purple, ERK2 hinge; pink, activation loops. Red, ATP-binding cavity with ATP mimic 5-iodotubercidin
(PDB 2Z0Q). Blue, substrate traverses to place phosphorylated residue near activation loop. Yellow, recognition domain where DEF motif inserts.
(d) ERK2–Klf4 model showing substrate and protein interactions. Green box and left panel, D domain of ERK2 interacting with Klf4 (Arg81 hydrogen
bonding to Ser318 and Asp316, and Arg82 hydrogen bonding to Asp319 and Glu79). Yellow box and right panel, anchor point of Klf4 sequence to
ERK2 (Asp93 hydrogen bonding to Thr108, Thr116 and Lys112). Orange box, ERK2 ATP-binding cavity. (e) Orientations of Klf4 and mutated Klf4
with ERK2 after molecular dynamics simulation. (f) Confirmation of Klf4 binding to ERK1. Full-length of wild type or mutant (mt) constructs were
co-transfected with pcDNA4-HisG-ERK1. Klf4 was detected with anti-HA. (g) Verification of Klf4 phosphorylation by ERK1. Results from in vitro kinase
assay of GST proteins with [γ-32P]ATP and active ERK1 are shown. The arrows indicate the respective GST fusion protein.

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concentration of LIF needed to obtain the maximum difference for
studying the inhibitory effect of ERK signaling induced by PD98059,
an MEK inhibitor. Results indicated that ten units of LIF showed the
most gradual differentiation during the four-day differentiation proc-
ess (Supplementary Fig. 2c). Therefore, we treated embryonic stem
cells with LIF (10 U) and PD98059 (0, 5, 10 and 20 µM) and found that
ERK phosphorylation could be inhibited by increasing PD98059 in
complete embryonic stem culture medium without altering total ERK
protein level (Fig. 3c). Notably, inhibition of Klf4 phosphorylation at
Ser123 corresponded with the downregulation of ERK1 or ERK2 phos-
phorylation (Fig. 3c). However, total Klf4 protein was increased in a
dose-dependent manner by PD98059, suggesting that ERKs might have
a role in stabilizing Klf4 (Fig. 3c). To examine the effect of inhibiting
ERK signaling on the activity of Klf4, we chose the Lefty1 promoter,
because Klf4 is a major transcription factor for Lefty1 gene expres-
sion18. The results indicated that inhibiting ERK signaling by PD98059
enhanced Lefty1 promoter activity in a dose-dependent manner (Fig. 3d).
Furthermore, we found that inhibiting ERK signaling by PD98059 treat-
ment enhanced the number of embryonic stem colonies positive for
alkaline phosphatase (alkaline phosphatase is an embryonic stem cell
marker for undifferentiated embryonic stem cells) in a dose-dependent
manner (Fig. 3e). Overall, these results indicate that inhibition of ERK
signaling enhances self-renewal activity in embryonic stem cells.
Phosphorylation is crucial in stem cell self-renewal
To examine the functional significance of phosphorylation of Klf4
Ser123 by ERKs, we assessed Klf4 activity using the Lefty1 promoter
luciferase reporter plasmid. We found that Lefty1 promoter activ-
ity was suppressed in a dose-dependent manner by overexpression
of constitutively active MEK1 (CA-MEK1) in embryonic stem cells
cultured with complete embryonic stem culture medium (Fig. 4a).
Furthermore, we found that Klf4 transactivation activity was inhibited
by overexpression of ERK1 (Fig. 4b). Notably, we found that activa-
tion or inhibition of ERK signaling by overexpression of CA-MEK or
dominant negative ERK1 (DN-ERK1), respectively, regulated Lefty1
promoter activity in embryonic stem cells in an opposite manner in
the presence or absence of LIF (Fig. 4c). These results indicated that
Klf4 activity might be modulated by ERK signaling. Furthermore,
we found that activation of ERK signaling by overexpression of
CA-MEK1 decreased the number of alkaline phosphatase–positive
embryonic stem colonies (Fig. 4d, left), and suppression of ERK sig-
naling by overexpression of DN-ERK1 increased the number (Fig. 4d,
right). By transactivation activity analysis, we found that CA-MEK1
attenuated the transactivation activity of wild-type Klf4, but not of
mutant Klf4 (S123A) (Fig. 4e). We also found that ERK1 or ERK2
induces Klf4 phosphorylation with overexpression of CA-MEK in
HeLa cells but not in Klf4 S123A mutant cells (Supplementary Fig. 3).
Furthermore, we confirmed that PD98059 treatment increased the
number of alkaline phosphatase–positive embryonic stem colonies
(Fig. 4f, bar 2 versus bar 3). We also found that the mutant sub-
stantially increased the number of embryonic stem colonies positive
for alkaline phosphatase, compared to wild-type Klf4 (Fig. 4f, bar 4
versus bar 6). These results demonstrate that regulation of Klf4 by
phosphorylation at Ser123 has an important role in embryonic stem
cell self-renewal activity.
Klf4 Ser?23 phosphorylation induces bTrCP binding
Although expression of stem cell factors is downregulated by methylation
in their gene promoters after differentiation19, our results demonstrate
that ERK-mediated phosphorylation of Klf4 could also downregu-
late Klf4 activity as well as the total Klf4 protein level (Fig. 4b–d),
suggesting that phosphorylation of Klf4 may affect protein stability.
To examine this hypothesis, we co-treated embryonic stem cells with
cyclohexamide (CHX) and/or MG132 (a proteasome inhibitor) and
found that the Klf4 protein level was gradually decreased by CHX treat-
ment and then restored by co-treatment with MG132 and CHX (Fig. 5a),
indicating that Klf4 might be regulated by its protein stability medi-
ated through the ubiquitin-proteasomal protein degradation pathway.
5
a
4
LIF (+)
LIF (–)
3
2
0
ERK1
Phosphorylation (fold)
ERK2
1
e
–
1,000
101010
5–
10
90
1020
–
1,000
101010
5
*
**
**
–
10
Number of colonies (AP positive)
1020
LIF(+) (U)
AP staining
PD98059 (µM)
LIF(+) (U)
PD98059 (µM)
80
70
60
50
40
30
20
10
0
d
c
p-ERKs
Total-ERKs
p-KIf4 Ser123
Total-KIf4
β-actin
––
1,000
10
1010
5–
– 1010
20
LIF (U)
PD98059 (µM)
b
Time (d)
LIF
p-ERKs
p-KIf4 Ser123
Total-KIf4
βTrCP1
βTrCP2
β-actin
Total-ERKs
+
–
1
–––
23
After differentiation
4
2,000
1,600
1,200
800
400
0
Luciferase activity (×104)
LIF (1,000 U)
pLefty1-luc (300 ng)
PD98059 (µM)
+
–
+
10
–
+
20
––
**
**
Figure 3 Inhibiting ERK signaling induces self-renewal of
embryonic stem cells. (a) ERK1 and ERK2 phosphorylation
in differentiated and undifferentiated embryonic stem
cells was compared by phosphokinase protein array and
analyzed with ImageJ (version 1.41). (b) ERK-mediated
Klf4 Ser123 phosphorylation assessed during E14Tg2a differentiation induced to differentiate by LIF withdrawal and harvested at various times.
(c) Effect of inhibiting ERK signaling on Klf4 Ser123 phosphorylation. E14Tg2a cells were cultured with LIF-reduced embryonic stem cell medium
and the MEK inhibitor PD98059. Media were changed every 24 h over 4 d. For b and c, proteins were visualized by western blotting, and protein
loading was verfied by β-actin expression. (d) Effect of inhibiting ERK signaling on Lefty1 promoter activity. E14Tg2a cells were co-transfected with
pLefty1-luciferase (pLefty1-luc) and phRL-SV40 Renilla luciferase reporter plasmids (50 pg) and incubated with PD98059. At 36 h after transfection,
luciferase activity was measured and normalized against Renilla luciferase activity. (e) Effect of inhibiting ERK signaling on embryonic stem cell
differentiation. E14Tg2a cells were cultured for 4 d with LIF-reduced embryonic stem cell medium and PD98059. Cells were fixed and stained, and
colonies positive for alkaline phosphatase (AP) were counted under a light microscope. For d and e, data are shown as mean ± s.d. of values obtained
from triplicate experiments (*P < 0.05, **P < 0.005).

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βTrCP is an F-box protein with E3 ligase activity that has an important
role in the regulation of protein levels through the proteasomal protein
degradation pathway20. We found that Klf4 contains a βTrCP consensus
motif that is adjacent to Ser123 (Supplementary Fig. 4a), suggesting that
Klf4 protein stability might be regulated through the βTrCP-mediated
proteasomal protein degradation pathway. To examine this idea, we
transfected cells with pcDNA3-Flag-βTrCP1 or pcDNA3-Flag-βTrCP2
expression vectors and HA-Klf4. Immunoprecipitation and western blot-
ting results showed that both βTrCP1 and βTrCP2 strongly bind with Klf4
(Fig. 5b). Klf4 specifically bound with βTrCP1 or βTrCP2, but did not
a
*
*
Relative luciferase activity (×104)3,000
2,500
2,000
1,500
1,000
500
0
LIF (1,000 U)
pLefty1-luc (300 ng)
CA-MEK1 (ng)
+
+
–
+
+
20
+
+
40
+
+
80
**
40
*
AP staining positive (%)
+
1010
––
–
DN-ERK1
LIF (U)
30
20
10
0
d
70
*
AP staining positive (%)
+
++
––
–
60
50
40
30
20
10
CA-MEK1
LIF (1,000 U)
0
b
600
***
*
Gal4-luciferase activity
(×103)
p5xGal4-Luc
pGal4-Klf41–474
pcDNA4-ERK1
+
–
–
+
+
–1020
+
+
+
+
500
400
300
200
100
0
f
*
**
***
AP staining positive (%)
100
80
60
40
20
LIF (U)
pCMV-HA-Klf41–474-S123A
pCMV-mock
PD98059 (10 µM)
pCMV-HA-Klf41–474
0
10
–
–
–
+
–
–
–
–
+
–
+
–
–
–
–
+
–
–
–
+
–
+
––
+
–
–
+
+
+
–
–
+
10101010 10 1,000
c
6,000
4,000
*
*
5,000
Relative luciferase activity
(×104)
3,000
2,000
1,000
pLefty1-Luc (20 ng)
CA-MEK1 (40 ng)
DN-ERK1 (40 ng)
e
8,000
LIF
0
+
–
––
+
+
+
–
+
+
–+ –+ –
*
*
Gal4-luciferase activity
(×104)
6,000
4,000
2,000
+
+
–
–
–
–+
+
+
–
–
–+
+
+
–
+
–+
+
–
+
+
–
0
LIF
p5xGal4-Luc (20 ng)
pGal4-Klf41–474 (40 ng)
pGal4-Klf41–474-S123A (40 ng)
CA-MEK1 (40 ng)
Figure 4 Klf4 Ser123 phosphorylation has a role in
embryonic stem cell self-renewal. (a) Effect of ERKs
on Lefty1 promoter activity. E14Tg2a cells were
co-transfected with pLefty1-luciferase and phRL-SV40
Renilla luciferase reporter plasmids and CA-MEK1.
(b) Effect of ERK1 on Klf4 transactivation. p5xGal4-
luciferase reporter and pGal4-Klf41–474 plasmids were
co-transfected with ERK1 into HEK293 cells.
(c) Comparison of Lefty1 promoter activity between
DN-ERK1 and CA-MEK1. DN-ERK1 or CA-MEK1
was co-transfected with pLefty1- and phRL-SV40
Renilla luciferase plasmids into E14Tg2a cells.
(d) Confirmation of ERK1 signaling in embryonic
stem cell differentiation. E14Tg2a cells were
transfected with CA-MEK1 or DN-ERK1. At 4 d,
colonies positive for alkaline phosphatase were counted under a light microscope. (e) Effect of inhibiting Klf4 Ser123 phosphorylation on CA-MEK1–
mediated suppression of Klf4 transactivation. The p5xGal-Luc and phRL-SV40 Renilla luciferase reporter plasmids were co-transfected into E14Tg2a cells
with pGal4-Klf41–474, pGal4-Klf41–474-S123A or pCA-MEK1. For a–c and e, at 36 h, luciferase activities were measured and normalized against Renilla
luciferase activity. (f) Effect of inhibiting Klf4 Ser123 phosphorylation on self-renewal. The pCMV-mock, pCMV-HA-Klf41–474 or pCMV-Klf41–474-S123A
plasmid was introduced into E14Tg2a cells and treated with PD98059. At 3 d, cells were analyzed as in d. For a–f, data are mean values ± s.d. from
triplicate experiments (*P < 0.05, **P < 0.005, ***P < 0.001).
Flag-βTrCP2
Flag-βTrCP1
HA-Klf41–474
–
+
–
–
–
+
–
+
+
+
–
–
+
–
++
–
–
Flag
IP: HA
HA
HA
Cell lysate: WB
Flag
β-actin
β-actin
f
LIF(–)
shMock
102401240124
shβTrCP1 shβTrCP2
KIf4
d
CHX
KIf4
shMock
01240124h
shβTrCP1,2
β-actin
–
–
–
2
–
+
4
–
+
6
–
+
2
+
+
4
+
+
6
+
+
WB: KIf4
WB: β-actin
d
MG132
CHX
Time (h)
IP: (Ab)
IgG KIf4
WB
Cell Iysate: WB
βTrCP2
IgG L.C.
IgG L.C.
βTrCP2
KIf4
KIf4
β-actin
LIF(+) shMockLIF(–) shMock
90
80
70
60
50
40
30
20
10
0
LIF
shmock
**
*
No. of AP+ colonies
+
shmock
–––
shβTrCP1
shβTrCP2
bce
a
shβTrCP1shβTrCP2
Figure 5 Klf4 Ser123 phosphorylation induces βTrCP binding. (a) Role of proteasomal
degradation pathway in regulation of Klf4 stability. E14Tg2a cells were treated with
cyclohexamide (CHX) with or without MG132. Klf4 was visualized by western blotting.
(b) The pcDNA3-Flag-βTrCP1 or pcDNA3-Flag-βTrCP2 plasmid was co-transfected
with pCMV-HA-Klf41–474 into HEK293 cells. Klf4 proteins were immunoprecipitated with anti-HA, and βTrCP1 and βTrCP2 proteins were visualized by
western blotting with anti-Flag. (c) Endogenous protein binding of βTrCP2 and Klf4. Klf4 was immunoprecipitated using an embryonic stem cell lysate,
and βTrCP2 and Klf4 were visualized by western blotting using the same cell lysate. IgG L.C., IgG light chain. Ab, Antibody (d) E14 cells were infected
with a lentivirus with pLK0.1-mock or pLK0.1-shβTrCP1 or pLK0.1-shβTrCP2 (shβTrCP1,2) plasmids. The half-life of Klf4 in control or βTrCP1,2
knockdown cells was assessed by cyclohexamide (CHX) chase assay. (e) Klf4 protein level in shMock-, shβTrCP1- or shβTrCP2-transfected E14 cells
during embryonic stem cell differentiation. (f) Effect of inhibiting βTrCP on stem cell self-renewal. The pLK0.1-mock, pLK0.1-βTrCP1 or pLK0.1-
βTrCP2 lentivirus was introduced into E14Tg2a cells. At 3 d, cells were fixed and stained, and colonies positive for alkaline phosphatase were counted
under a light microscope. Data are shown as mean values ± s.d. from triplicate experiments (*P < 0.05, **P < 0.005).

Page 6

© 2012 Nature America, Inc. All rights reserved.
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advance online publication nature structural & molecular biology
articles
bind the F-box proteins FBXW5 or FBXW7 (Supplementary Fig. 4b).
We used the binding of Klf5 with FBXW7 as a positive control21. Notably,
the total protein level of βTrCP1 and βTrCP2 was markedly increased
at 2 d and 3 d after differentiation was induced for 4 d (see Fig. 3b).
Furthermore, we immunoprecipitated Klf4 and detected βTrCP2 by
western blotting to confirm that endogenous βTrCP2 binds with Klf4
in embryonic stem cells (Fig. 5c). To study the degradation of Klf4 by
βTrCP1 or βTrCP2 we introduced shMock, shβTrCP1, or shβTrCP2 into
embryonic stem cells. The protein level of βTrCP1 or βTrCP2 was reduced
in each knockdown cell type (Supplementary Fig. 5a). Transduction
with shβTrCP1 or shβTrCP12 substantially extended the half-life of
endogenous Klf4 (Fig. 5d) and knockdown of βTrCP1 or βTrCP 2
delayed Klf4 protein degradation after CHX treatment (Supplementary
Fig. 5b). Double knockdown of βTrCP1 or βTrCP2 also delayed the
turnover of endogenous Klf4 during embryonic stem cell differentiation
(Fig. 5e) and hindered embryonic stem cell differentiation (Fig. 5f). To
identify the Klf4 domain that is responsible for binding with βTrCP2, we
constructed Klf4 truncated mutants using the pCMV-HA overexpres-
sion vector and co-transfected each with the pcDNA3-Flag-βTrCP2.
Immunoprecipitation and western blotting results indicated that
full-length Klf4 (Klf41–474) and the Klf4 N-terminal domain, including
Ser123 (Klf41–140), coimmunoprecipitated with βTrCP2 (Supplementary
Fig. 5c). Furthermore, by immunoprecipitating Flag-βTrCP2 with the
wild-type or the S123A mutant Klf4 and mutants of the Klf4-related
βTrCP2 consensus motif protein, we found that binding of βTrCP2
and Klf4 was substantially decreased in the mutants S123A, S127A and
S131A, compared with wild-type Klf4 (Fig. 6a). We found that the Klf4
mutants enhanced the number of alkaline phosphatase–positive embry-
onic stem colonies in a dose-dependent manner (Fig. 6b). The half-life
of the Klf4 protein was increased substantially in the Klf4 S127A and
S131A mutants, compared to controls (Fig. 6c, left). These data (Fig. 6c,
left) were normalized to the Klf4 level at 0 h (Fig. 6c, right). Overall,
the results indicate that βTrCP1 and βTrCP2 are previously unknown
binding proteins with Klf4 and regulate Klf4 protein stability through
the proteasomal protein degradation pathway.
Klf4 Ser?23 phosphorylation induces protein degradation
To further assess the role of Ser123 phosphorylation in regulating
Klf4 protein stability, we co-transfected pCMV-HA-Klf41–474 and
pCMV-HA-Klf41–474-S123A plasmids into embryonic stem cells,
treated the cells with CHX and assessed the Klf4 protein level by western
blotting. We found that the wild-type Klf4 protein could not be detected
at 2 h after CHX treatment. However, the mutant Klf41–474-S123A
still showed a strong band (Supplementary Fig. 6a), indicating that
Klf4 Ser123 phosphorylation might trigger Klf4 protein degradation.
To compare the susceptibility of wild-type and mutant Klf4 proteins
to ubiquitination, we overexpressed HA-Klf4-1-474 or HA-Klf4-
1-474-S123A together with Flag-Ubiquitin in embryonic stem cells
and cultured cells with or without MG132 for 2 h. Results showed that
ubiquitination of Klf4 was increased by MG132 treatment in wild-
type Klf4 cells (Supplementary Fig. 6b), whereas it was abolished in
the Klf4-1-474-S123A mutant cells (Supplementary Fig. 6b, lane 3
versus lane 4). We confirmed that coexpression of CA-MEK1 with
HA-Klf4-1-474-S123A substantially decreased ubiquitination compared
with cells expressing both CA-MEK1 and HA-Klf4-1-474 (Fig. 6d).
To confirm that knockdown of βTrCP1 or βTrCP2 reduces Klf4
ubiquitination, we co-transfected pCMV-HA-Klf41–474 and pCMV-
HA-Klf41–474-S123A plasmids with pCDNA3.1-Flag-Ubiquitin into
embryonic stem cells and infected them with shMock, shβTrCP1 or
shβTrCP2 separately. Knockdown of βTrCP1 or βTrCP2 substantially
reduced ubiquitination in Klf4 wild-type cells but not in Klf4 S123A
mutant cells (Supplementary Fig. 6c). Overall, our results demon-
strate that Klf4 Ser123 phosphorylation by ERK1 or ERK2 has an
important role in maintaining Klf4 protein stability, which is mediated
through the proteasomal protein degradation pathway, resulting in
downregulation of embryonic stem cell self-renewal.
Figure 6 Effect of the βTrCP2 phosphodegron
in Klf4 on binding with βTrCP2. (a) Klf4
phosphorylation at Ser123, Ser127 and
Ser131 affect binding with βTrCP2. The
pCMV-HA-Klf4-140 wild-type or bearing
substitutions of single Ser residues were
co-transfected with pcDNA3-Flag-βTrCP2 into
HEK293 cells. Protein binding was confirmed
by immunoprecipitation with anti-Flag and
by western blotting with anti-HA in the same
cell lysate. β-Actin was used to verify equal
protein loading. (b) Effect of inhibiting Klf4
phosphorylation (Ser127 or Ser131) on stem
cell self-renewal. A pCMV-mock, pCMV-HA-
Klf41–474, pCMV-Klf41–474-S127A or pCMV-
Klf41–474-S131A plasmid was introduced into
E14Tg2a cells. Data are shown as mean values
± s.d. Significant differences were evaluated
using the Student’s t-test (*P < 0.05,
**P < 0.005). LIF+, LIF present; LIF−, LIF
absent. (c) HA-tagged wild-type Klf4 and
its single-point mutants were individually
transfected into 293 cells, and their expression
levels were detected by immunoblotting.
Quantitative expression of data from a was
analyzed using ImageJ and normalized to the
Klf4 level at 0 h. Relative degradation of Klf4
was calculated on the basis of the protein levels
of cell lysates. Ub, ubiquitin. Untreated density, protein level at 0 h. (d) Effect of the S123A mutation of Klf4 on CA-MEK1–mediated ubiquitination.
Various combinations of expression vectors were co-transfected into HeLa cells. At 36 h, the ubiquitination of Klf4 proteins was visualized by
immunoprecipitation with anti-HA and western blotting with anti-Flag, using the same cell lysate. Arrows indicate position of 55 kDa marker.
a
b
c
100
80
60
40
20
Untreated density (%)
0 0.5124 h
WT
S126A
S123A
S131A
S127A
100
90
80
70
60
50
40
30
20
10
**
*
No. of AP+ colonies
0
LIF
Klf4
+
–
–
–
–
S127A
–
S131A
WT
–
Klf41–140
βTrCP2
+
–
–
+
+
+
S123A
+
S126A
+
S127A
+
S131A
+
Smt3
+
Smt4
+
IP: Flag
Cell lysate
HA
Flag
Flag
HA
d
MG132
CA-MEK
HA-Klf41–474
–
–
–
–
+
–
+
–
+ +
+
+
–
+
–
+
–+
+++ + +
+
+++
+
–
++
–
+
+
+
+
–
–
–
––
––––
Cell lysate
IP:
Flag
HA-Klf41–474-S123A
Flag-Ub
HA
Flag
Flag
β-actin
Klf1–474
Klf41–474 S123A
Klf41–474 S127A
Klf41–474 S131A
β-actin
0
CHX (10 µg ml–1) h
0.5124
Klf41–474 S126A
Klf4 S131A
Mock (LIF–)
Klf4 S127A
Mock (LIF+)
Klf4 WT

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nature structural & molecular biology  advance online publication
?
articles
DISCUSSION
Self-renewal and maintenance of pluripotency of mouse embryonic
stem cells requires LIF, a member of the IL-6 cytokine family used for
maintenance of embryonic stem cells in an undifferentiated state22,23.
The JAK-STAT3 signaling pathway has a crucial role in the maintenance
of pluripotency of embryonic stem cells24,25 by inducing transcrip-
tion of STAT3 target genes, including Klf4 (ref. 26). At the same time,
LIF stimulates and activates MAP kinases, inducing differentiation
of embryonic stem cells. However, suppression of ERK signaling
promotes the self-renewal activity of mouse embryonic stem cells27,
making the role of ERK signaling in self-renewal of embryonic stem
cells controversial.
Many transcription factors are involved in regulating embryonic
stem cell self-renewal and pluripotency. Klf4 regulates the expression
of many target genes involved in cellular functions, including cell pro-
liferation, differentiation and apoptosis28. Although one recent study
demonstrated that Klf4 prevents embryonic stem cell differentiation
by regulating expression of the Nanog gene29, a direct mechanism
for regulating Klf4 activity has not been determined. Our findings
demonstrate that ERK1 or ERK2 directly phosphorylates Klf4 Ser123,
inhibiting Klf4 transcriptional and transactivation activities and pro-
moting differentiation. These results correspond well with previous
studies showing that the MEK-ERK signaling pathway negatively
regulates embryonic stem cell self-renewal activity27.
Information is accumulating regarding the binding of substrates
to ERK1 or ERK2 docking domains, and it indicates that numerous
substrates bind to the ERK1 or ERK2 substrate recognition motifs30,31.
The design of peptide substrates for ERK2 revealed that the substrate
probably binds across the front of the kinase, anchoring its N-terminal
domain at the D domain, straddling the ATP-binding site, and orient-
ing the phosphorylated sequence of the C-terminal domain near the
activation loop of ERK2 (ref. 32). A similar experiment showed the
plausibility of such a binding orientation, because the DEF docking
motif, typically found after the phosphorylation site, binds below the
activation loop of ERK2 (ref. 33). Our computational model showing
Klf4 or mutant Klf4 binding with ERK2 provides the visual evidence
for our experimental observations. These models show that the two
arginine residues form important hydrogen bonds to two aspartate
residues in the D domain of ERK2 (Fig. 2e). Hydrogen bonds between
Asp93 of Klf4 and Thr108, Thr116 and Lys112 of ERK2 create anchor
points for the Klf4 common docking sequence motif. These studies
provide further evidence that Klf4 is a substrate of ERK2. Protein
sequence alignment confirmed that Klf4 possesses the necessary
common docking motif sequence located roughly 20 amino acids
away from the Ser123 phosphorylation site. Furthermore, the model
shows that mutation of this sequence disrupts the network of hydro-
gen bonds that allows Klf4 to bind with ERK2.
Methylation on the gene promoter of stem cell factors is believed
to be a major regulatory mechanism governing stem cell self-renewal.
Although improvement of the demethylation status of Oct4 and Nanog
was observed in iPS cells compared with murine embryonic fibro-
blasts, substantial differences in the demethylation status in the Oct4
promoter were observed in embryonic stem and iPS cells12. These
results suggest that regulation of gene expression might not be suf-
ficient to regulate the activity of stem cell factors. Recently, several
groups showed that Sox2 and Oct4 could be post-translationally mod-
ified by phosphorylation, SUMOylation or ubiquitination, resulting
in modulation of transactivation and transcriptional activity4–7. Klf4
promotes differentiation by TGF-β receptor-mediated Smad and p38
MAPK signaling in vascular smooth muscle cells, and phosphorylation
mediates activation of Klf4, but the mechanism is not understood34.
Proteomics data identified the ubiquitination sites in Klf4 (ref. 35)
and indicated that one of the sites is in the Klf4 N terminal. However,
this site was not affected by Klf4 ubiquitination induced by ERKs
(data not shown). Although cDNA microarray analyses revealed a
list of genes whose transcript levels are changed markedly during
embryonic stem cell differentiation36,37, the mechanism explaining
the role of post-translational modification in embryonic stem cell
differentiation is not fully understood.
To date, many studies have investigated the role of Klf4 in normal
physiological homeostasis, cell differentiation and cancer formation
and have shown that Klf4 can either activate or repress transcrip-
tion and, in certain cellular contexts, can function as an oncogene
or a tumor suppressor28,38,39. Together with our findings regarding
the regulation of Klf4, we suggest that the self-renewal program of
cancer cells might resemble the self-renewal program of embryonic
stem cells. Although the functions of Klf4 in cancer are controversial,
several reports suggest that Klf4 is involved in human cancer develop-
ment28,40,41. Thus, our results might contribute to understanding how
the cancer cell self-renewal program functions to maintain uncon-
trolled cancer growth.
METhODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/nsmb/.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
ACKNOwLeDgMeNtS
We thank M. Pagano (New York University School of Medicine) for the kind
gift of plasmids, including βTrCP1/2 and several F boxes. We thank H. Niwa
(RIKEN Center for Developmental Biology (CDB)) for the kind gift of plasmids,
including the Lefty1 core promoter. We wish to thank T.M. Poorman for secretarial
assistance. This work was supported by The Hormel Foundation.
AUtHOR CONtRIBUtIONS
M.O.K. and S.-H.K. designed experiments, participated in writing the manuscript,
cultured embryonic stem cells and conducted immunoprecipitation experiments
and screening. Y.-Y.C. designed experiments and participated in writing the
manuscript. J.N. and Z.H. carried out the computational biology and modeling.
C.-H.J. participated in experimental design and cultured embryonic stem cells. K.Y.
cultured embryonic stem cells and did the immunofluorescence and kinase assays.
D.J.K. did the kinase assays. D.-H.Y. cultured embryonic stem cells. Y.-S.K.
participated in designing the experiments and cultured the lentivirus for
knockdown of Klf4. K.-Y.L. cultured embryonic stem cells. A.M.B. and Z.D.
supervised the design of the experiments and the manuscript editing and writing.
COMPetINg FINANCIAL INteReStS
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.

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Page 9

© 2012 Nature America, Inc. All rights reserved.
nature structural & molecular biology
doi:10.1038/nsmb.2217
ONLINE METhODS
Reagents. DMEM and FBS were from Invitrogen, and the restriction enzymes
were from New England BioLabs. The DNA ligation kit (version 2.0) was from
TaKaRa Bio. Lipofectamine 2000 was from Invitrogen and JetPEI reagent was
from Q-Biogene. The pcDNA3.1 plasmid was from Life Technologies and luci-
ferase assay substrate from Promega. The monoclonal-HA, histidine or Flag
antibodies were from Sigma-Aldrich and Invitrogen. Antibodies to Klf4, phos-
phorylated ERKs and total ERKs were from R&D and Cell Signaling Technology.
Anti-phospho-Klf4 Ser123 was produced by Biosynthesis.
Cell culture. HEK293 and HeLa cells were cultured in DMEM-10% FBS at 37 °C,
5% CO2. Mouse embryonic stem cells (E14Tg2a) were maintained on standard
complete embryonic stem culture medium with 15% embryonic stem-qualified
FBS (Millipore, Billerica, MA), 0.055 mM β-mercaptoethanol (Invitrogen),
2 mM L-glutamine, 0.1 mM MEM non-essential amino acid and 1,000 U ml−1
LIF (Millipore).
Expression vector construction. For mammalian two-hybrid system assay and
Klf4 overexpression, the open reading frame of mouse Klf4 was amplified by
PCR and introduced into the pACT mammalian two-hybrid system vector or
pCMV-HA expression vector. For the Klf4-GST fusion protein, full-length Klf4
(Klf41–474) and truncated deletion mutants, including the activation domain
(Klf4-1-140), two inhibitory domains (Klf4-141-250 and Klf4-251-370) and the
zinc-finger domain (Klf4-371-474) were constructed by PCR-based amplification
and introduced into pGEX-5X-C42. The S123A mutation was carried out using
the QuickChange II site-directed mutagenesis kit (Strategene). Constructs were
confirmed by restriction enzyme mapping and DNA sequencing. The amino
acid sequence used for all studies herein was the shorter version from 2007, since
updated to include nine additional amino acids, MRQPPGESD, in front of the
former number 1, methionine.
Western blotting. Cells were harvested and disrupted with NP-40 lysis buffer
(Roche). Proteins were resolved by SDS-PAGE, transferred onto polyvinyli-
dene difluoride membranes and visualized by enhanced chemiluminescence
(ECL) detection.
Mammalian two-hybrid assay. The mammalian two-hybrid (M2H) assay was
conducted using suggested protocols (Checkmate mammalian two-hybrid system
assay; Promega). Luciferase activity was measured by luminometer (MTX Lab)
and normalized against Renilla activity.
Immunoprecipitation. Wild-type pCMV-HA-Klf4, pcDNA4-His-ERK1,
pcDNA3-Flag-βTrCP1 or pcDNA3-Flag-βTrCP2 was transfected into HEK293
cells and cultured for 36 h, then disrupted with NP-40 lysis buffer. Binding was
confirmed by immunoprecipitation, western blotting and ECL.
In vitro kinase assay. Wild-type GST-Klf4, truncated deletion or point mutant
proteins were used for an in vitro kinase assay with active ERK1 or ERK2
(Millipore). Reaction experiments were conducted at 30 °C for 30 min with
50 µM unlabeled ATP and 10 µCi of [γ-32P]ATP. The reaction was stopped with
6× SDS sample buffer, and samples were separated by 10% SDS-PAGE and visual-
ized by autoradiography.
Prediction of Klf4 phosphorylation site(s). The mouse Klf4 (NP_034767)
sequence was downloaded from http://www.ncbi.nlm.nih.gov/ in 2007. The ERK
phosphomotif was from the Human Protein Reference Database, and the puta-
tive phosphorylation sites were analyzed using the group-based phosphorylation
prediction system (version 2.1)43.
Computational modeling. The complex between the common docking motif
in Klf4 and ERK2 was created using Schrödinger (Schrödinger). The common
docking motif binding to the D domain of ERK is Arg-Lys2-Xaa2–6-Faa-Xaa-Faa
(where Faa stands for the hydrophobic residues leucine, isoleucine and possibly
valine), usually 20 amino acids before the phosphorylation site44. The sequence
of 79-LARRETEEFNDLLDL-94 was identified in mouse Klf4 as the potential
common docking motif. This sequence was aligned to other known common
docking motif peptide sequences crystallized and bound to ERK2. The two crystal
structures used were ERK2 bound to peptide sequences of mitogen-activated
protein kinase 3 (MPK3, PDB 2FYS)45 and hematopoietic tyrosine phosphatase
non-receptor type 7 (HePTP, PDB 2GPH)46.
The complexes Klf4–ERK2 (PDB 2FYS), Klf4–ERK2 (PDB 2GPH),
ERK2–MPK3 and ERK2–HePTP all underwent energy minimizations using
MacroModel (Schrödinger). The environment for carrying out the minimiza-
tion involved the OPLS_20005 (refs. 47,48) force field in a solvent of water with
a constant dielectric of 1.0, and the actual minimization parameters used the low-
memory Broyden-Fletcher-Goldfarb-Shanno method48 with maximum iterations
of 500 converging on the gradient with a threshold of 0.05.
After Klf4 mutant ERK2 complexes were minimized, the Klf4 amino acid
sequences were mutated to 79-AAMMETEEFNDLADA-94, and these new com-
plexes underwent further minimization. The complex of ERK2(2FYS)–Klf4 as
well as the ERK2(2FYS)–Klf4 mutant were analyzed using molecular dynamics
simulations. The same molecular environment was maintained with the following
molecular dynamics protocols: stochastic dynamics with SHAKE (Schrödinger Suite
2010 induced fit docking protocol; Glid version 5.5 (Schrödinger); Prime version 2.1
(Schrödinger); Impact version 5.5 (Schrödinger)), which were applied to the hydro-
gens at a simulation temperature of 300.0 K, a time step of 1.5 fs, an equilibration
time of 25.0 ps, followed by an additional 75.0 ps of simulation. The resulting struc-
tures were analyzed to determine the effects of mutations on Klf4 binding.
Klf4 activity assay. To examine Klf4 activity, E14Tg2a cells (2.0 × 104) were
seeded into 48-well plates and cultured for 18 h with embryonic stem cell medium
containing LIF before transfection. The Lefty1 promoter-luc reporter plasmid49,
comprising the Klf4 binding consensus sequences on the 5′ end upstream of the
Lefty1 promoter, was transfected with expression vectors. To examine transactiva-
tion activity, p5xGal4-luciferase and pGal4-Klf4 wild-type or mutant plasmids
and 50 pg of Renilla luciferase reporter plasmid (phRL-SV40) were transfected
with expression vectors, and cells were cultured for 36 h. Cells were disrupted and
luciferase activity was measured and normalized against Renilla activity.
Alkaline phosphatase staining. Embryonic stem cells were cultured, fixed
with 2% formalin for 2 min, and alkaline phosphatase staining was carried out
with the Alkaline Phosphatase Detection Kit (Millipore), following suggested
protocols. Colonies positive for alkaline phosphatase were observed using a
light microscope.
Protein stability. To examine Klf4 stability, E14Tg2a cells were treated with
10 µM MG132 and 10 µg ml−1 cyclohexamide and analyzed by western blot-
ting. For assessing ubiquitination, the pcDNA3-Flag-Ub expression vector was
co-transfected with combinations of wild-type or mutant Klf4 and CA-MEK1
into E14Tg2a or HeLa cells. Cells were cultured for 36 h, and ubiquitination of
Klf4 was visualized by using immunoprecipitation with anti-Flag or anti-HA and
western blotting with anti-HA or anti-Flag and ECL.
42. Cho, Y.Y. et al. RSK2 mediates muscle cell differentiation through regulation of
NFAT3. J. Biol. Chem. 282, 8380–8392 (2007).
43. Zhou, F.F., Xue, Y., Chen, G.L. & Yao, X. GPS: a novel group-based phosphorylation
predicting and scoring method. Biochem. Biophys. Res. Commun. 325, 1443–1448
(2004).
44. Sharrocks, A.D., Yang, S.H. & Galanis, A. Docking domains and substrate-specificity
determination for MAP kinases. Trends Biochem. Sci. 25, 448–453 (2000).
45. Liu, S., Sun, J.P., Zhou, B. & Zhang, Z.Y. Structural basis of docking interactions
between ERK2 and MAP kinase phosphatase 3. Proc. Natl. Acad. Sci. USA 103,
5326–5331 (2006).
46. Zhou, T., Sun, L., Humphreys, J. & Goldsmith, E.J. Docking interactions induce
exposure of activation loop in the MAP kinase ERK2. Structure 14, 1011–1019
(2006).
47. Jorgensen, W.L. & Tirado-Rives, J. The OPLS [optimized potentials for liquid
simulations] potential functions for proteins, energy minimizations for crystals of
cyclic peptides and crambin. J. Am. Chem. Soc. 110, 1657–1666 (1988).
48. Head, J.D. & Zerner, M.C. A Broyden-Fletcher-Goldfarb-Shanno optimization
procedure for molecular geometries. Chem. Phys. Lett. 122, 264–270 (1985).
49. Nakatake, Y. et al. Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1
core promoter in embryonic stem cells. Mol. Cell. Biol. 26, 7772–7782 (2006).