Báo cáo khoa học: Akt-dependent phosphorylation negatively regulates the transcriptional activity of dHAND by inhibiting the DNA binding activity pdf

Akt-dependent phosphorylation negatively regulates the
transcriptional activity of dHAND by inhibiting the DNA binding
activity
Masao Murakami
1,2
, Keiichiro Kataoka
1
, Shigetomo Fukuhara
1
, Osamu Nakagawa
2
and Hiroki Kurihara
1,3
1
Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto
University, Japan;
2
Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, TX, USA;
3
Department of Physiological Chemistry and Metabolism, The University of Tokyo Graduate School of Medicine, Japan
HAND2/dHAND is a basic helix-loop-helix transcription
factor expressed in the heart and neural crest derivatives
during embryogenesis. Although dHAND is essential for
branchial arch, cardiovascular and limb development, its
target genes have not been identified. The regulatory mech-
anisms of dHAND function also remain relatively
unknown. Here we report that Akt/PKB, a serine/threonine
protein kinase involved in cell survival, growth and differ-
entiation, phosphorylates dHAND and inhibits dHAND-
mediated transcription. AU5-dHAND expressed in 293T
cells became phosphorylated, possibly at its Akt phos-

phorylation motif, in the absence of kinase inhibitors,
whereas the phosphatidylinositol 3-kinase inhibitor wort-
mannin and the Akt inhibitor NL-71-101, but not the p 70 S6
kinase inhibitor rapamycin, significantly reduced dHAND
phosphorylation. Coexpression of HA-Akt augmented
dHAND phosphorylation at multiple serine and threonine
residues mainly located in the bHLH domain and, as a
result, decreased the transcriptional activity of dHAND.
Consistently, alanine mutation mimicking t he nonphos-
phorylation state abolished the inhibitory effect of A kt on
dHAND, whereas aspartate mutation mimicking the phos-
phorylation state resulted in a loss of dHAND transcrip-
tional activity. These changes in dHAND transcriptional
activity were in parallel with changes in the DNA binding
activity rather than in dimerization activity. These results
suggest that Akt-mediated signaling may regulate dHAND
transcriptional activity through the modulation of its DNA
binding activity during embryogenesis.
Keywords: Akt; bHLH; dHAND; phosphorylation; tran-
scription.
Basic helix-loop-helix (bHLH) proteins are a highly con-
served superfamily of transcriptional factors that commit to
various developmental processes, such as cell f ate determin-
ation, differentiation and tissue-specific gene expression
[1,2]. bHLH proteins form homo- or heterodimers to allow
the basic region to bind to the palindromic DNA sequence,
CANNTG, termed the E-box [3–5]. In general, tissue-
specific bHLH proteins heterodimerize with broadly
expressed bHLH proteins, named E-proteins [2].
The HAND proteins, HAND1/eHAND and HAND2/

dHAND are expressed in the heart, neural crest-derivatives
and extraembryonic tissues during embryogenesis [6–8].
Gene targeting experiments have revealed that the develop-
mental roles of the two HAND proteins are essential and
distinct. dHAND-null mice die at embryonic day 9.5 due
to defects in cardiovascular development [9,10], whereas
eHAND-null mice have lethal defects in early extraembry-
onic t issues and cardiovascular development [11,12].
Despite evidence for the importance of HAND proteins
during embryogenesis, the regulatory mechanisms of tran-
scriptional activity of HANDs have not been analyzed in
detail [13], especially in terms of post-translational modifi-
cations.
Akt is a serine/threonine protein kinase activated down-
stream of phosphatidylinositol 3-kinase (PI3K) [14,15], and
plays central roles in cell survival, growth and differenti-
ation. From detailed biochemical analyses, the consensus
sequence of t he phosphorylation t arget for Akt w as
identified as RXRXXS/T [16]. Glycogen synthase kinase-
3, BAD, and caspase-9 are known as Akt s ubstrates [1 7–21].
Insulin signaling upstream of Akt was previously shown to
stimulate global protein synthesis i n the heart [22,23]. It has
been reported that heart size is increased in transgenic mice
that express a constitutively active form of Akt from a
cardiac specific promoter [23]. In some cases Akt translo-
cated into nucleus after stimulation [24,25]. Although it has
been reported that Akt can phosphorylate transcription
factors such as FKHR [26– 28], CREB [29], and Nur77
[30,31], the physiological importance of Akt phosphoryla-
tion in the transcriptional control has not been fully

established.
In this study, we investigated regulatory mechanisms of
dHAND t ranscriptional activity by Akt-mediated s ignaling.
dHAND is phosphorylated by Akt at serine/threonine
Correspondence to H. Kurihara, D epartment of Physiological Chem-
istry and Metabolism, The University of Tokyo Graduate School of
Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Fax: + 81 3 5684 4958, Tel.: + 81 3 5841 3495,
E-mail:
Abbreviations: bHLH, basic helix-loop-helix; PI 3K, phosphatidyl-
inositol 3-kinase; GST, glutathione S-transferase; MBP, maltose
binding protein.
(Received 29 January 2004, revised 14 June 2004,
accepted 23 June 2004)
Eur. J. Biochem. 271, 3330–3339 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04267.x
residues mainly w ithin t he bHLH domain. Although
phosphorylation of dHAND did not affect its dimerization
with E-proteins, the phosphorylation decreased the tran-
scriptional activity due to inhibition of its DNA binding.
This fact may present a novel model for the regulatory
mechanism of bHLH transcription factors.
Materials and methods
Plasmid construction
pCEFL-HA-Akt, that encodes HA-tagged Akt [32], was a
gift from J. S. Gutkind ( National I nstitutes o f H ealth,
USA). Mouse dHAND, eHAND, E47, and ME2/ITF2a
cDNAs were cloned by PCR using the mouse E 10.5 cDNA
library (Gibco BRL) as a template. Deletion and point
mutants o f dHAND were made by PCR using Quick-
Change

TM
XL Site-Directed Mutagenesis Kit (Stratagene).
They were inserted into pGEX (Amersham Pharmacia
Biotech) or pMAL (NEB) for g lutathione S-transferase
(GST)- or maltose binding protein (MBP)-fusion proteins,
respectively. These cDNAs were also cloned into a mam-
malian e xpression vector, pCEFL-AU5 [32], w hich contains
the AU5-tag at the N-terminus, or pCMV-DBD (Strata-
gene) f or fusion proteins with a Gal4 DNA binding domain.
Cell culture and preparation of cell lysates
293T cells were cultured in DMEM containing 10% (v/v) fetal
bovine serum, 100 UÆmL
)1
penicillin G and 100 lgÆmL
)1
streptomycin at 37 °Cin5%(v/v)CO
2
. T he C2C12 myoblast
cell line w as cultured in DMEM containing 20% fetal
bovine serum, 100 UÆmL
)1
penicillin G and 100 lgÆmL
)1
streptomycin at 37 °Cin5%(v/v)CO
2
. Wortmannin ( a PI3K
inhibitor), NL-71-101 (an Akt inhibitor), and rapamycin (a
p70 S6 kinase inhibitor) were purchased from Calbiochem.
Cells we re lysed in NaCl/P
i

containing 1% (v/v) Triton X -100
and protease i nhibitors, and whole cell lysates were prepared
for immunoprecipitation or pull-down assay. For the
immunocomplex kinase assay, cells were lysed in kinase-lysis
buffer [ 50 m
M
Tris/HCl (pH 7.5), 137 m
M
NaCl, 1 m
M
EDTA, 1 m
M
Na
3
VO
4
,20m
M
b-glycerophosphate, 5 0 m
M
NaF, 1% (v/v) Triton X-100, and 10% (v/v) glycerol]
containing protease inhibitors.
Preparation of GST- or MBP-fusion proteins
GST- or MBP-fusion proteins were expressed in Escherichia
coli DH5a and were induced by adding 0.4 m
M
isopropyl
thio-b-
D
-galactoside. The cells were suspended in lysis buffer

[NaCl/P
i
containing 1% (v/v) Triton X-100 and 1 m
M
phenylmethanesulfonyl fluoride]. After sonication, crude
cell l ysates were centrifuged at 4 °C and the supernatant was
used for the experiments. GST-fusion and MBP-fusion
protein was purified using GSH-Sepharose beads and
Amirose resin, and eluted by 20 m
M
glutathione or
30 m
M
maltose, respectively.
Immunocomplex kinase assay
Cell lysates were prepared from 293T cells transfected
with pCEFL-HA-Akt. Twenty-four hours after transfec-
tion, the c ells were starved for 24 h a nd stimulated by
adding fetal bovine serum at a final concentration of 20%
(v/v) for 40 min. HA-Akt was immunoprecipitated using
a monoclonal antibody raised against HA, and HA-Akt
bound protein-G conjugated sepharose beads were
washed by kinase reaction buffer [ 10 m
M
Tris/HCl
(pH 7.5 ), 25 m
M
MgCl
2
,10m

M
b-glycerophosphate,
0.33 m
M
Na
3
VO
4
, and 0.33 m
M
dithiothreitol not con-
taining ATP]. The substrate, GST- or MBP-dHAND and
its mutants, and 5 lCi [
32
P]ATP[cP] were added t o the
beads. The kinase r eaction was performed a t 37 °Cfor
30 min and the reaction was stopped by adding 2.5· sam-
ple buffer followed by boiling for 5 min. These samples
were applied on 10% (w/v) SDS/PAGE followed by
autoradiography.
Pull-down assay
GST- or MBP-fusion proteins bound to the beads were
incubated at 4 °C for 6 h with cell lysates prepared from
293T cells expressing HA-Akt. The beads were w ashed three
times with NaCl/P
i
containing 1% (v/v) Triton X-100 and
protease inhibitors, and bound proteins were eluted by
adding 2.5· sample buffer followed by boiling for 5 min.
These samples were applied on 10% (v/v) SDS/PAGE a nd

proteins were detected by Western blotting.
Immunoprecipitation, Western blotting, and antibodies
The procedure of immunoprecipitation and Western blot-
ting was described previously [33]. Cell lysates were
incubated with protein G sepharose beads and the mono-
clonal antibody (anti-HA or anti-AU5) at 4 °C for 6 h. The
beads were washed with NaCl/P
i
containing 1% (v/v)
Triton X-100 and protease inhibitors three times an d bound
proteins were eluted by adding 2.5· sample buffer followed
by boiling for 5 min. Antibodies were purchased from
Covance (anti-HA and anti-AU5), Zymed (anti-phospho-
serine and anti-phosphothreonine), and Cell Signaling
[anti-(phospho-Akt substrate) (RXRXXS/T)].
Gel shift analysis
Double strand o ligonucleotide probes w ere end-labeled with
[
32
P]ATP[cP] by T4 polynucleotide kinase. The binding
sequence o f eHAND/E47 heterodimer, reported previously
[34], was used as the probe. The sequence of each probe
was as shown below. Underlining indicates the E-box
sequence: CANNTG. Probe E¢ (E¢); 5¢-TTTGCAAGGGG
CATCTGGCATTCGCCC-3¢, and mutant probe E¢ (m E¢);
5¢-TTTGCAAGGGG
CGAACAGCATTCGCCC-3¢.Two
microliters of cell l ysate prepared from 293T c ells was
incubated with 1 lg poly ( dI-dC) and labeled probe at
room temperature for 30 min. These samples were applied

on 4% (w/v) native PAGE followed by autoradiography.
Transfection and reporter assay
Transfection into 293T or C 2C12 cells was carried out using
Lipofectamine Plus reagent (Life Technologies). G al4-DBD
or Gal4-DBD constructs, pFR-Luc (Stratagene), pRL-
SV40 (Promega), and pCEFL-HA-Akt were transfected
Ó FEBS 2004 dHAND phosphorylation by Akt (Eur. J. Biochem. 271) 3331
and firefly luciferase units were determined 48 h after
transfection. Transfection efficiency was normalized on the
basis of r enila luciferase activity. All these a ssays were
performed in triplicate.
Results and Discussion
Phosphorylation of dHAND by Akt
Although g ene targeting analyses in mice demonstrated the
importance of HAND proteins during development, the
regulatory mechanisms of HAND transcriptional activity
are not well understood. In this study, we examined the
possibility that Akt might regulate dHAND activity,
because d HAND has a consensus motif for A kt phos-
phorylation (RXRXXS) at amino acids 108–114. First, we
analyzed the phosphorylation status of dHAND in 293T
cells in the absence or presence of several kinase inhibi-
tors. AU5-dHAND was immunoprecipitated by anti-
AU5 Ig and the phosphorylation status of dHAND was
analyzed b y Western b lotting using antibody raised against
the phospho-Akt substrate. As shown in Fig. 1A, AU5-
dHAND expressed in 293T cells became phosphorylated
possibly at i ts Akt phosphorylation motif in the a bsence of
kinase inhibitors. This signal was decreased by treatment
with 200 n

M
wortmannin, a PI3K inhibitor (Fig. 1A). Fifty
micromoles of NL-71-101, an Akt inhibitor, also signifi-
cantly reduced dHAND phosphorylation (Fig. 1A). In
contrast, 20 n
M
rapamycin, a p70 S6 kinase inhibitor, did
not largely affect the phosphorylation status of dHAND
(Fig. 1A). These results suggest that dHAND is phosphory-
lated in a PI3K/Akt-dependent, but p70 S6 kinase-inde-
pendent manner.
To determine whether dHAND was phosphorylated by
Akt in vivo, we transiently expressed AU5-dHAND and
HA-tagged Akt in 293T cells. Western blotting with anti-
(phospho-Akt substrate) Ig revealed that overexpression of
HA-Akt upregulated dHAND phosphorylation (Fig. 1B).
Fig. 1. Phosphorylation status of dHAND in 293T cells. (A) Effec t of kinase inhibitors on the b asal phosphorylation of d HAND. 293T cells expressing
AU5-dHAND w ere t reated with 200 n
M
wortmannin, 50 l
M
NL-71-101 or 20 n
M
rapamycin fo r 1 20 min. Dimethylsulfoxide w as used as a vehic le.
AU5-dHAND was imm unopre cipitated us ing a nti-AU5 Ig and applied on 10% SDS/PAGE. The phos phorylation status of dHAND was a nalyzed
by Western blotting using anti-(phospho-Akt substrate) Ig (top). The same memb rane was reblotted by an ti-AU 5 Ig (bottom). (B) Effects of Akt
overexpression on dHAND phosphorylation. Cell lysates were prepared from 293T cells expressing AU5-dHAND and HA-Akt, and the phos-
phorylation status of d HAND was analyzed by immunoblotting with anti -(p hospho-Ak t substrate) Ig (top). The same membrane was reblotted by
anti-AU5 Ig (bottom). ( C) Effects of Akt overexpression on dHAND phosphorylation on serine/threon ine residues. Cell lysates were prepared from
293T cell expressing AU5-dHAND and/or HA-Akt. Phosphorylation status of dHAND was an alyzed b y Western blotting using anti-phosphoserine

Ig (top ) or anti-p hosphot hreonine Ig (m iddle). The prot ein amount o f AU5-dHAND was estimated by Western blotting using anti-AU5 Ig (b ottom).
Fig. 2. Akt phosphorylated dHAND in vitro. HA-Akt was immuno-
precipitated from the cell lysate prepared from 293T cells expressing
HA-Akt, and immunocomplex kinase assay was performed in the
presence of [
32
P]ATP[cP] usin g GST or G ST-dHAND as a su bstrate.
Protein sample w as applied on 1 0% SDS/PAGE and incorporation of
32
P was detected by autoradiography.
3332 M. Murakami et al.(Eur. J. Biochem. 271) Ó FEBS 2004
We also analyzed the phosphorylation status of AU5-
dHAND by immunoprecipitation and Western blotting
methods using antibodies specific to phosphoserine or
phosphothreonine. In the cells expressing AU5-dHAND
alone, the anti-AU5 immunoprecipitants gave signals for
both anti-phosphoserine and anti-p hosphothreonine immu-
noreactivity, suggesting that dHAND may be basally
phosphorylated at serine and threonine residues in the cells
(Fig. 1C). These results indicate that the Akt signal m ay pos-
itively modulate the phosphorylation status of dHAND.
We further examined whether Akt directly phosphory-
lates dHAND using immunocomplex kinase assay. HA-
Akt was transiently expressed in 293T cells and cells were
starved a nd stimulated by 20% (v/v) fetal bovine serum for
40 min to activate HA-Akt. HA-Akt was immunoprecip-
itated using anti-HA Ig and the kinase reaction was started
by adding GST-dHAND to the immunoprecipitants in the
reaction buffer containing [
32

P]ATP[cP]. As shown in
Fig. 2, strong incorporation of
32
P into GST-dHAND
was detected when incubated with HA-Akt immunoprecip-
itates. Autophosphorylation of Akt was also detected as
described previously [35]. In contrast, GST was not
phosphorylated at all, and immunoprecipitates from the
mock transfected cell lysates did not phosphorylate GST-
dHAND, indicating that Akt specifically phosphorylates
dHAND.
To locate the dHAND phosphorylation sites by Akt, we
constructed MBP-dHAND mutants in w hich the C-terminal
region was serially deleted (dHAND-(1–102),-(1–132),
and -(1–196) (Fig. 3A). Comparable phosphorylation was
detected in dHAND-(1–196) and dHAND-(1–132), in which
the bHLH domain was preserved and partially deleted,
respectively (Fig. 4A). In contrast, no significant phosphory-
lation was d etected in dHAND(1–102) (Fig. 4A). This result
indicates that the major phosphorylation sites reside in the
region of amino acids 103–132 of dHAND.
The region of amino acids 103–132 contains several
serine and threonine residues and Ser114 matches the
consensus motif for Akt phosphorylation ( RXRXXS/T)
(Fig. 3B). T hus, t o i dentify the phosphorylation site of
dHAND, we first substituted Ser114 with alanine and
examined whether this mutant (named dHAND-3SA;
Fig. 3A) c an be phosphorylated by Akt. Unexpectedly,
the phosphorylation signal was comparable with that of
Fig. 3. Structures and putative phosphorylation

sites of dHAND mutants. (A) Schematic
representation of the structure of MBP- or
GST-fused dHAND mutants. Serine (S) and
threonine (T) residues that were substituted
with alanine (A) or aspart ic acid (D) are
indicated by circles. Hatched box, black boxes,
and gray box indicate basic region, helix motif,
and loop structure, respectively. (B) Amino
acid sequence of mouse dHAND. The posi-
tion of the bHLH domain is indicated. The
mutated sites in this study are shown by
asterisks (Ser114, Thr103, Thr112, Thr140,
Thr145, and Thr204).
Ó FEBS 2004 dHAND phosphorylation by Akt (Eur. J. Biochem. 271) 3333
wild type dHAND (dHAND-WT) (Fig. 4B, lanes 1 and
4), leading us to speculate that additional serine/threonine
residues are pho sphorylated by A kt. There are n o sites
other than Ser114 that match completely to the canonical
consensus motif (Fig. 3B). However, five additional
threonine residues in the C-terminal half of dHAND fit
the R/KXXS/T motif, which is similar to the consensus
of Akt phosphorylation (Fig. 3). Therefore we substituted
Ser114 and all of the five threonines with alanine or
aspartic acid (dHAND-Ala and dHAND-Asp, respect-
ively; Fig. 3A) and examined whether these mutants can
be phosphorylated by Akt. No phosphorylation was
detected in these mutants, suggesting that these six
residues contain the targets for Akt phosphorylation
(Fig. 4B). Then we substituted each of the six serine/
threonine residues one by one with alanine (dHAND-

1TA, 2TA, 3SA, 4TA, 5TA and 6TA; Fig. 3A). Among
these mutants, dHAND-1TA demonstrated partial but
substantial decrease in phosphorylation (Fig. 4B). In
addition, further d ecreases in phosphorylation were
detected in mutants dHAND-Ala-X and dHAND-Ala-
XZ (Fig. 4B). These results indicated that the major
phosphorylation sites of dHAND were located within
three sites (Thr102, Thr112, and Ser114) mutated in
dHAND-Ala-X.
Akt binds to dHAND
in vitro
and
in vivo
To examine whether Ak t c an dire ctly inte ract with
dHAND, we performed pull-down assay using GST-
Fig. 4. In vitro kinase assay using MBP-dHAND mutants. Imm uno-
complex kinase a ssay was performed using dHAND deletion mutants
(A) or dHAND point mutants (B) as described in Fig. 2. Each protein
used as a substrate was subjected to 10% SDS/PAGE and b ands were
detected by stain ing with Coomassie Brilliant B lue (bottom). Arro w-
heads indicate the positions of dHAND mutants.
Fig. 5. Analysis o f the interaction between d HAND and Akt. (A) GST-
pull down assay was pe rformed using ce ll extract of 293T cells
expressing HA-Akt (top). GST or GST-dHANDs used in this assay
wereappliedon10%SDS/PAGEandproteinwasdetectedbystaining
with Coomassie Brilliant Blue (bottom). Arrowheads indicate the
positions of dHAND mutants. (B) Coimmunoprecipitation assay of
dHAND and Akt. AU5-dHAND was immunoprecipitated from the
cell lysate of 293T c ells e xpressing AU5-dHAND and HA-Akt. These
samples were applied on 10% SDS/PAGE and bound proteins were

detected by Western blotting using anti-HA Ig.
3334 M. Murakami et al.(Eur. J. Biochem. 271) Ó FEBS 2004
dHAND and cell extracts of 293T cells expressing HA-Akt.
As shown in Fig. 5A, HA-Akt interacted with GST-
dHAND but not with GST. To identify the domain of
dHAND interacting with Akt, we performed pull-down
assay using dHAND deletion mutants. As shown in
Fig. 5A, HA-Akt was also found to interact with GST-
dHAND-(1–132) and -(1–196). In contrast, Akt did not
bind to GST-dHAND-(1–102) efficiently, indicating that
dHAND may bind to Akt via its bHLH domain.
To detect the interaction between dHAND and Akt
in viv o, we coexpressed AU5-dHAND and HA-Akt in 293T
cells and performed a coimmunoprecipitation experiment.
As shown i n Fig. 5B, interaction b etween HA-Akt and
AU5-dHAND was detected. This result indicated that A kt
could bind to dHAND in the cells.
Transcriptional activity of dHAND was decreased
by Akt phosphorylation
Interestingly, major phosphorylation sites of dHAND are
located within the bHLH domain. Therefore, we speculated
that phosphorylation of these sites might affect the DNA
binding and/or dimerization activities of dHAND. We first
analyzed the effect of phosphorylation on the transcriptional
activity of dHAND using reporter assay (Fig. 6A). AU5-
dHAND, -dHAND-Ala-X, -dHAND-Ala, and -E47 were
cotransfected into 293T cells together with reporter plasmid
driven by three E-box sequences. Although AU5-dHAND,
-dHAND-Ala-X, -dHAND-Ala, and E47 alone showed
relatively low transcriptional activity, coexpression of E47

significantly enhanced the activity of dHAND-WT, -Ala-X,
or -Ala (Fig. 6A a nd data not shown). I t w as of interest that
coexpression of Akt reduced the transcriptional activity of
dHAND-WT/E47. The activity of dHAND-Ala-X/E47
was p artially reduced by coexpression of Akt. But this
inhibitory effect of Akt was not detected in the case of
dHAND-Ala/E47 (Fig. 6A).
Luciferase assay was performed using dHAND mutants
to estimate the effect of phosphorylation. As shown
in Fig. 6B, dHAND-WT, dHAND-Ala-X, and dHAND-
Ala showed strong transcriptional activity in the presence
of E47. But dHAND-Asp-X, that mimics a phosphoryla-
tion form, showed weak transcriptional activity (Fig. 6B).
The expression level of dHAND-Asp-X was lower than
those of others in the absence of E47. But in the presence of
E47, the expression level of this mutant was equivalent to
wild type dHAND or other mutants (Fig. 6B, lower panel).
This indicated the transcriptional activity of dHAND-
Asp-X/E47 was much lesser than those of wild type. These
results indicated that phosphorylation of dHAND
decreased the transcriptional a ctivity of dHAND/E47
heterodimer.
Fig. 6. The effects of A kt expression on the transcriptional activity of dHAND. (A) AU5-d HAND, -dHAND-Ala-X, -dHAND-Ala, and -E 47 were
cotransfected into 293T cells with reporter plasmid encoding luciferase g ene, whose expression is d riven by three E-box sequences ( CATCTG). The
effect of Akt expression was monitored by luciferase assay. The expression levels of dHAND and its mutants were an alyzed b y West ern blotting
using anti-AU5 Ig (bott om). Note t hat dHAND transcriptional activity requires the presence of E47. (B) Transcriptional activity of dHAND
mutants was analyzed by luciferase assay using 293T cells. The expression levels of dH AND, its mutants, and E47 were analyzed by Western
blotting using anti-AU5 Ig (bottom).
Ó FEBS 2004 dHAND phosphorylation by Akt (Eur. J. Biochem. 271) 3335
Phosphorylation of dHAND did not affect the

dimerization activity with E47
To clarify the mechanisms of the inhibition of dHAND
activity by Akt phosphorylation, we examined whether
dimerization activity of dHAND with E47 could be
affected by Akt phosphorylation or not. We selected the
Gal4-fusion system because we can estimate the dimeri-
zation activity with E47 and the transcriptional activity of
the dHAND/E47 h eterodimer independently of their
DNA binding activity (Fig. 7, upper p anel). As shown
in Fig. 7, coexpression of E47 enhanced the transcrip-
tional activity of Gal4-dHAND-WT, indicating that E47
bound to Gal4-dHAND and functioned as a strong
transcriptional activator. Gal4-dHAND-Ala-X and Gal4-
dHAND-Ala also showed similar results. The transcrip-
tional activity of dHAND-Asp-X was also enhanced
by coexpression of E47. This result suggested that
phosphorylated dHAND could dimerize with E47 and
function as a transcriptional activator complex in a Gal4-
fusion system.
DNA binding activity of dHAND was decreased
by Akt phosphorylation
Because we did not d etect the effects of Akt phosphoryla-
tion on dimerization activity of dHAND, we next examine
whether the DNA binding activity of dHAND could be
affected by Akt phosphorylation. The influence of phos-
phorylation by Akt on the DNA binding activity was
examined by gel shift analysis. We included the E-protein
Fig. 7. Transcriptional activity of dHAND/E47 was analyzed using
Gal4-fusion system. Gal4 DNA bindin g domain (DBD)-fused
dHAND and reporter plasmid co ntaining t he luc iferase ge ne, whose

expression was driven by five G al4 binding sites, were transfecte d t o
C2C12 cells, a nd luciferase assay w as performed. Fold activation is the
ratio of the luciferase activity in cells transf ected with E 47 construct to
cells transfected with empty vector.
Fig. 8. Gel shift analysis of dHAND. (A)
DNA binding activity of dHAND/ME2 or
dHAND-Ala/ME2 to probe E w as compared
after in vitro phosphorylation procedure in t he
presence (+) or absence (–) o f Akt. Protein
samples of A U5-dH AND or AU5-ME2 were
mixed with
32
P-labeled oligonucleotide probe
containing E-box seq uence, probe E. After
incubation at room temperature for 30 min,
samples were applied on 4% native PAGE
followed by autoradiography. (B) Protein
amounts of dHAND and dHAND-Ala used
in this experiment were estimated by Western
blotting using anti-AU5 Ig. (C) DNA binding
activities of the heterodimers between
dHAND mutants and ME2 were compared to
that of wild type dHAND using probe
E. Protein amounts of dHAND, dHAND-
Asp-X, and dHAND-Asp used in this
experiment were estimated by Western
blotting using anti-AU5 Ig (bottom).
3336 M. Murakami et al.(Eur. J. Biochem. 271) Ó FEBS 2004
ME2/ITF2a tagged with AU5 as a heterodimeric partner in
this experiment.

We phosphorylated dHAND b y Akt in vitro,mixedwith
ME2, and performed gel shift analysis. As shown in
Fig. 8A, DNA binding activity of wild type dHAND/
ME2 was reduced after phosphorylation by Akt. In
contrast, dHAND-Ala showed a shifted band with higher
intensity, and this was not affected by the phosphorylation
procedure (Fig. 8A,B). The higher DNA binding activity o f
dHAND-Ala may be due to escaping endogenous phos-
phorylation. To investigate the effect of phosphorylation on
DNA binding activity, we performed the same assay using
phosphorylation mimicking mutants. As shown in Fig. 8C,
dHAND-Asp-X and -Asp almost lost their DNA binding
activity. These results indicated that the phosphorylation of
Thr102, Thr112, and Ser114 inhibited DNA binding activity
of dHAND.
In this study, we showed that Akt could bind t o d HAND
and phosphorylate i t at s erine and threonine residues within
the bHLH domain. Phosphorylation by Akt inhibited the
transcriptional activity of dHAND. We analyzed the
mechanisms of this inhibitory effect of Akt-dependent
phosphorylation on the transcriptional activity of dHAND
focused o n t hese th re e a spects: ( a) dimeriza t ion a ctivity w ith
E-protein, (b) DNA binding activity of dHAND/E-protein,
and (c) transcriptional a ctivity of dHAND/E-protein
heterodimer. Our results indicated that the effect of Akt-
dependent p hosphorylation acted on (b), but not (a) and (c).
After phosphorylation by Akt, dHAND/E-protein hetero-
dimer can be present, but this heterodimer can not bind to
DNA, leading to a decrease in the transcriptional activity
(Fig. 9) .

Functional modulation by phosphorylation has been
reported in some bHLH proteins. For example, the amino
acid sequences of the various myogenic regulatory factors
contain co nsensus pho sphorylation motifs for cAMP-
dependent protein kinase, protein kinase C and casein
kinase II [36–42]. The activation of cAMP-dependent
protein kinase or protein kinase C can inhibit muscle
differentiation, whereas casein kinase II stimulates myogen-
esis by increasing the transcriptional activities of MRF4 and
MyoD [43]. In these cases, however, phosphorylation or
mutagenesis of each site does not affect the transcriptional
activity with an exception that phosphorylation of the
protein kinase C site of myogenin blocks its DNA binding
activity [40,43], suggesting that the effects of protein kinases
are likely to b e indirect. Our present study is unique in that
the phosphorylation sites exist w ithin the bHLH domain a nd
their phosphorylation can directly affect the function as a
transcriptional factor. Although p70 S6 kinase can phos-
phorylate the R/KXXS/T motif, rapamycin did not inhibit
dHAND phosphorylation. This result and t he evidence that
Akt can bind to dHAND and phosphorylate it strongly
suggest that Akt directly regulates dHAND activity.
Akt signaling is known to be involved in not only many
developmental processes but also homeostasis in adults [44–
47]. It has b een reported that h eart size is increased i n
transgenic mice that express a constitutively active form of
Akt [23], and that down regulation o f HAND gene
expression is observed in rodent hypertrophy and human
cardiomyopathy [48]. The modulation of dHAND tran-
scriptional activity by Akt phosphorylation may explain a

part of this involvement b ecause there is the report that Akt
is expressed in developing heart [49]. Interestingly, the
sequence of phosphorylation sites in the bHLH domain is
well conserved within the Twist-related bHLH subfamily, to
which dHAND and eHAND belong [6]. The activity of
some of these bHLH family proteins might also be r egulated
by Akt signaling in vivo.
In conclusion, dHAND was phosphorylated by Akt and
the phosphorylation i nhibited the transcriptional activity of
dHAND. Although the phosphorylated form of dHAND
could dimerize with E-protein, this complex could not
activate the transcription due to a loss of the DNA binding
activity. The present finding may be an important clue of the
regulation mechanisms of HAND proteins.
Acknowledgements
WethankDrJ.S.Gutkindforplasmids,Ms.S.Okamura,Ms.K.
Shin-Fukuhara, Ms. M. Yonemitsu, and Dr M . N akagawa f or
technical assistance, Dr H. Saya and Dr T. Hirota for advice and
discussion on the phosp horylation analysis, and D r E. N. Olson and
Dr H. Iba f or critical comments on this paper. Th is work was supported
by JSPS (Japan Society for t he Promotion of Science) Research for the
Future Pro gram; Grants-in-Aid for Scientific R esearch from th e
Ministry of Education, Science and Culture, Japan; and the Research
Grant for Cardiovascular Diseases (14C-1) from t he Ministry of
Health, Japan.
References
1. Atchley, W. & Fitch, W . (1997) A natural classification of the b asic
helix-loop-helix class of transcription factors. Proc. Natl Acad. Sci.
USA 94, 5172–5176.
2. Massari, M.E. & Murre, C. (2000) Helix-Loop-Helix Proteins:

Regulators of TranscriptioninEucaryoticOrganisms.Mol. Cell.
Biol. 20, 429–440.
Fig. 9. A model of the regulation mechanisms of dHAND activity.
Phosphorylation of dHAND by Akt does not affect on the dimeriza-
tion with E-proteins. Phosphorylated dHAND can dimerize with
E-protein (1) and this complex can function as a transcriptional acti-
vator if recruited to DNA (3). But, th e heterodimer of phospho rylated
dHAND can not bind to the E-box sequence (2). As a result, the
transcriptional activity of dHAND is inhibited by Akt signaling.
Ó FEBS 2004 dHAND phosphorylation by Akt (Eur. J. Biochem. 271) 3337
3. Ephrussi, A., Church, G.M., Tonegawa, S. & Gilbert, W. (1985)
B-lineage-specific interactions of an immunoglobulin enhancer
with cellular factors in vivo. Science 227, 134–140.
4. Murre, C., McCa w, P.S. , V aessin, H., Caudy, M., Jan, L.Y., Jan,
Y.N., Cabrera, C .V., Buskin, J.N., Hauschka, S .D., Lassar, A.B.,
Weintraub, H. & Baltimore, D. (1989) Interactions between het-
erologous h elix-loop-helix proteins generate co mplexes that bind
specifically to a common DNA sequen ce. Cell 58, 537–544.
5. Ellenberger, T., Fass, D., Arnaud, M. & Harrison, S. (1994)
Crystal structure of transcription fac tor E47: E-box recognitio n by
a basic region helix-loop-helix dimer. Genes Dev. 8, 970–980.
6. Srivastava, D., Cserjesi, P. & Olson, E.N. (1995) A subclass of
bHLH proteins required for cardiac morphogenesis. Science 270,
1995–1999.
7. McFadden, D.G., Charite, J., R ichardson, J.A., S rivastava, D.,
Firulli, A.B. & Olson, E.N. (2000) A GATA-dependent right
ventricular enhancer controls dHAND transcription in the
developing heart. Developmen t 127, 5331–5341.
8. Yamagishi, H., Olson, E.N. & Srivastava, D. (2000) The basic
helix-loop-helix transcription factor, dHAND, is required for

vascular development. J. Clin. Invest. 105, 261–270.
9. Srivastava, D., Thomas, T., Lin, Q., Kirby, M.L., Brown, D. &
Olson, E.N. (1997) Regulation of cardiac mesodermal and neural
crest development by the bHLH transcription facto r, dHAND.
Nat. Genet. 16, 154–160.
10. Thomas, T., Kurihara, H., Yamagishi, H., Kurihara, Y ., Yazaki,
Y., Olsen, E.N. & Srivastava, D. (1998) A signaling cascade in-
volving endothelin-1, dHAND and Msx1 regulates development
of neural-crest-derived branchial arch mesenchyme. Developm ent
125, 3005–3014.
11. Firulli, A.B., McFadden, D.G., Lin, Q., Srivastava, D. & Olson,
E.N. (1998) Heart and extra-embryonic mesodermal defects in
mouse embryos lackin g the bHLH transcription factor Hand1.
Nat. Genet. 18, 266–270.
12. Riley, P., Anson-Cartwright, L. & Cross, J.C. (1998) The Hand1
bHLH transcription factor is essential for placentation and car-
diac morphogenesis. Nat. Genet. 18, 271–275.
13. Dai, Y.S. & Cserjesi, P. (2002) The basic helix-loop-helix factor,
HAND2, functions as a transcriptional activator by binding to
E-boxes as a heterodimer. J. Biol. Chem. 277, 12604–12612.
14. Burgering, B.M. & Coffer, P .J. (1995) Protein k inase B (c-Akt) in
phosphatidylinositol-3-OH kinase s ignal t ransduction. Nature
376, 599–602.
15. Franke, T.F., Yang, S.I., Chan, T.O., Datta, K., Kazlauskas, A.,
Morrison, D.K., Kaplan, D.R. & T sichlis, P.N. (1995) The
protein kinase encoded by th e Akt proto- oncogene is a target o f
the PD GF-activated phosphatidylinositol 3-kinase. Cell 81, 727–
736.
16. Obata, T., Yaffe, M.B., Leparc, G.G., Piro, E.T., Maegawa, H.,
Kashiwagi, A., Kikkawa, R. & Cantley, L.C. (2000) Peptide and

protein library screening defines optimal substrate motifs for
AKT/PKB. J. Biol. Chem. 275, 36108–36115.
17. Cross, D.A., Alessi, D.R., Cohen, P., Andjelkovich, M. & Hem-
mings, B.A. (1995) Inhibition of glycogen synthase kinase-3 by
insulin mediated by protein kinase B. Nature 378, 785–789.
18. Alessi, D.R., Caudwell, F.B., Andjelkovic, M., Hemmings, B.A. &
Cohen, P. (1996) Molecular basis for the substrate specificity of
protein k inase B; comparison with MAPKAP kinase-1 a nd p70,
S6 kinase. FEBS Lett. 399, 333–338.
19. Datta,S.R.,Dudek,H.,Tao,X.,Masters,S.,Fu,H.A.,Gotoh,Y.
& Greenberg, M.E. (1997) Akt phosphorylation of BAD couples
survival signals to the cell-intrinsic death machinery. Cell 91, 231–
241.
20. delPeso,L.,Gonzalez-Garcia,M.,Page,C.,Herrera,R.&Nunez,
G. (1997) Interleukin-3-induced phosphorylation of BA D through
the protein kinase Akt. Science 27 8, 687–689.
21. Cardone, M.H., R oy, N ., S ten nicke, H.R ., S alv esen, G .S., F ranke,
T.F., Stan bridge, E., Frisch, S. & Reed, J .C. (1998) Regulation of
cell death protease caspase-9 by phosphorylation. Science 282,
1318–1321.
22. Shiojima, I., Yefremashvili, M., Luo, Z., Kureishi, Y., Takahashi,
A., T ao, J., Rosenzweig, A ., Kahn, C.R., Abel, E.D. & Walsh, K.
(2002) Akt signaling mediates postnatal heart growth in response
to insulin and nutritional status. J. Biol. Chem. 277, 37670–37677.
23. Shioi, T., McMullen, J.R., Kang, P.M., D ouglas, P.S., Obata, T.,
Franke, T.F., Cantley, L.C. & Izumo, S. (2002) Akt/protein kinase
B promotes organ growth in transgenic mice. Mol. Cell. Biol. 22,
2799–2809.
24. Andjelkovic, M., Alessi, D.R., Meier, R., Ferna ndez, A., Lamb,
N.J., F rech, M., Cron, P., Coh en, P., Lucocq, J.M. & Hemmings,

B.A. (1997) R ole of translocation in the activation and function of
protein kinase B. J. Biol. Che m. 272, 31515–31524.
25. Meier, R., Alessi, D.R., Cron, P., Andjelkovic, M. & Hemmings,
B.A. (1997) Mitogenic activation, phosphorylation, and nuclear
translocation of protein kinase Bbeta. J. Biol. Chem. 272, 30491–
30497.
26. Brunet, A., Bonni, A., Zigmond,M.J.,Lin,M.Z.,Juo,P.,Hu,
L.S., Anderson, M.J., Arden, K.C., Blenis, J. & Greenberg, M.E.
(1999) Akt promotes cell survival by phosphorylating and-
inhibiting a Forkhead transcription factor. Cell 96, 857–868.
27. Brownawell, A.M., Kops, G.J., Macara, I.G. & B urgering,
B.M. (2001) Inhibition of nuclear import by protein kinase B
(Akt) regulates the subcellular distribution and activity of the
forkhead transcription factor AFX. Mol. Cell. Biol. 21, 3534–
3546.
28. Tang. E.D., Nune z, G., Barr, F.G. & Guan, K .L. (1999) Negative
regulation of the forkhead transcription f actor FKHR by Akt.
J. Biol. Chem. 274, 16741–16746.
29. Du, K. & Montminy, M. (1998) CREB is a regulatory target for
the protein kinase Akt/PKB. J. Biol. Chem. 273, 32377–32379.
30. Masuyama, N., Oishi, K., Mori, Y., Ueno, T., Takahama, Y. &
Gotoh, Y. (2001) Akt inhibits the orphan nuclear rec ept or Nu r77
and T-cell apoptosis. J. Biol. Chem. 276, 32799–32805.
31. Pekarsky. Y., Hallas, C., Palamarchuk, A., Koval, A., B ullrich, F.,
Hirata, Y., Bichi, R., Letofsky, J. & Croce, C.M. (2001) Akt
phosphorylates and regulates the o rphan n uclear receptor Nur77.
Proc.NatlAcad.Sci.USA98, 3690–3694.
32. Murga, C., Laguinge, L ., Wetzker, R., Cuadrado, A. & Gutkind,
J.S. (1998) A ctivation of Akt/protein kinase B by G protein-cou-
pled receptors. A role for alpha and beta gamma subunits of

heterotrimeric G proteins acting thro ugh ph osphatidylin ositol-3-
OH kinasegamma. J. Biol. Chem. 273, 19080–19085.
33. Murakami, M., Sonobe, M.H., Ui, M., Kabuyama, Y., Watanabe,
H., Wada, T., Handa, H. & Iba, H. (1997) Phosphorylation and
high level expression of Fra-2 in v-src transformed cells: a pathway
of activation of endogenous AP-1. Oncogene 14, 2435–2444.
34. Hollenberg, S.M., Sternglanz, R., Cheng, P.F. & Weintraub, H.
(1995) Identification of a new family of tissue-specific basic helix-
loop-helix proteins with a two-hybrid system. Mol. Cell. Biol. 15,
3813–3382.
35. Ravichandran, L.V., Chen, H., Li, Y. & Quon, M.J. (2001)
Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability t o
dephosphorylate the insulin receptor. Mol. Endocrinol. 15, 1768–
1780.
36. Winter, B., Braun, T. & Arnold, H.H. (1993) cAMP-dependent
protein kinase represses m yogenic differentiation and the ac tivity
of the muscle-specific helix-loop-helix transcription facto rs Myf-5
and MyoD. J. Biol. Chem. 268, 9869–9878.
37. Tapscott, S.J., Davis, R.L., Thayer, M.J., Cheng, P.F., Weintraub,
H. & Lassar, A.B. (1988) MyoD1: a nuclear phosphoprotein
requiring a Myc homology region to convert fibroblasts to myo-
blasts. Science 242, 405–411.
3338 M. Murakami et al.(Eur. J. Biochem. 271) Ó FEBS 2004
38. Lassar, A.B., Davis, R.L., Wright, W.E., Kadesch, T., M urre, C.,
Voronova, A., Baltimore, D. & Weintraub, H. (1991) Functional
activity of myogenic H LH proteins requires hetero-oligomeriza-
tion with E12/E47-like proteins in vivo. Cell 66, 305–315.
39. Li, L., Heller-Harrison, R., Czech, M. & Olson, E .N. (1992) Cyclic
AMP-dependent protein kinase inhibits the activity of m yogenic
helix-loop-helix proteins. Mol. Cell. Biol. 12, 4478–4485.

40. Li,L.,Zhou,J.,James,G.,Heller-Harrison,R.,Czech,M.P.&
Olson, E.N. (1992) FGF inactivates myogenic helix-loop-helix
proteins through phosphorylation of a conserved protein kinase C
site in their DNA-binding domains. Cell 71, 1181–1194.
41. Hardy, S., Kong, Y. & Konieczny, S.F. (1993) Growth factor
inhibits MRF4 activity independently of the phosphorylation
status of a conserved threonine residue within the DNA-binding
domain. Mol. Ce ll . Bi ol . 13, 5943–5956.
42. Mitsui, K., Shirakata, M. & Paterson, B.M. (1993) Ph osphory-
lation inhibits the DNA-binding activity of MyoD homodimers
but not MyoD-E12 heterodimers. J. Biol. Chem. 268, 24415–
24420.
43. Johnson, S.E., Wang, X., Hardy, S., Taparowsky, E.J. &
Konieczny, S.F. (1996) Casein k inase II increases the transcrip-
tional activities of MRF4 and MyoD independently of t heir direct
phosphorylation. Mol. Cell. Biol. 16, 1604–1613.
44. Datta,S.R.,Brunet,A.&Greenberg, M.E. (1999) Cellular sur-
vival: a play in three Akts. Genes Dev. 13, 2905–2927.
45. Galetic, I., Andjelkovic, M., Meier, R., Brodbeck, D., Park, J. &
Hemmings, B.A. (1999) Me chanism of protein kina se B activation
by insulin/insulin-like growth factor-1 revealed by specific inhibi-
tors of phosphoin ositide 3-k inase – significance for diab etes and
cancer. Pharmacol. Ther. 82, 409–425.
46. Kandel, E .S. & Hay, N. (1999) The regulation and activities of the
multifunctional/threonine kinase Akt/PKB . Exp. Cell Res. 253,
210–229.
47. Brazil, D.P. & Hemmings, B.A. (2001) Ten years of protein kinase
B signaling: a hard Akt to follow. Trends Biochem. Sci. 26, 657–
664.
48. Thattaliyath, B.D., Livi, C.B., Steinhelper, M.E., Toney, G.M. &

Firulli, A.B. (2002) HAND1 and HAND2 are expressed in the
adult-rodent heart and a re mo dulated during cardiac hypertrophy.
Biochem. Biophys. Res. Commun. 297, 870–875.
49. Altomare, D.A., Lyons, G.E., Mitsuuc hi, Y., Ch eng, J .Q. & T esta,
J.R. (1998) Akt2 mRNA is highly expressed in embryonic brown
fat and the AKT2 kinase is activated by insulin. Oncogene 16,
2407–2411.
Ó FEBS 2004 dHAND phosphorylation by Akt (Eur. J. Biochem. 271) 3339