Akt-dependent phosphorylation negatively regulates thetranscriptional activity of dHAND by inhibiting the DNA binding activity Masao Murakami1,2, Keiichiro Kataoka1, Shigetomo Fukuhara1,
Trang 1Akt-dependent phosphorylation negatively regulates the
transcriptional activity of dHAND by inhibiting the DNA binding
activity
Masao Murakami1,2, Keiichiro Kataoka1, Shigetomo Fukuhara1, Osamu Nakagawa2and Hiroki Kurihara1,3 1
Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan;2Department 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 p70 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 the nonphos-phorylation state abolished the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phos-phorylation state resulted in a loss of dHAND transcrip-tional activity These changes in dHAND transcriptranscrip-tional 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 fate
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 tissues 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 the phosphorylation target for Akt was identified as RXRXXS/T [16] Glycogen synthase
kinase-3, BAD, and caspase-9 are known as Akt substrates [17–21] Insulin signaling upstream of Akt was previously shown to stimulate global protein synthesis in 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 transcripphosphoryla-tional control has not been fully established
In this study, we investigated regulatory mechanisms of dHAND transcriptional activity by Akt-mediated signaling dHAND is phosphorylated by Akt at serine/threonine
Correspondence to H Kurihara, Department 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: kuri-tky@umin.ac.jp
Abbreviations: bHLH, basic helix-loop-helix; PI3K,
phosphatidyl-inositol 3-kinase; GST, glutathione S-transferase; MBP, maltose
binding protein.
(Received 29 January 2004, revised 14 June 2004,
accepted 23 June 2004)
Trang 2residues mainly within the 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 Institutes of Health,
USA) Mouse dHAND, eHAND, E47, and ME2/ITF2a
cDNAs were cloned by PCR using the mouse E10.5 cDNA
library (Gibco BRL) as a template Deletion and point
mutants of dHAND were made by PCR using
Quick-ChangeTMXL Site-Directed Mutagenesis Kit (Stratagene)
They were inserted into pGEX (Amersham Pharmacia
Biotech) or pMAL (NEB) for glutathione S-transferase
(GST)- or maltose binding protein (MBP)-fusion proteins,
respectively These cDNAs were also cloned into a
mam-malian expression vector, pCEFL-AU5 [32], which contains
the AU5-tag at the N-terminus, or pCMV-DBD
(Strata-gene) for 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)1penicillin G and 100 lgÆmL)1
streptomycin at 37C in 5% (v/v) CO2 The C2C12 myoblast
cell line was cultured in DMEM containing 20% fetal
bovine serum, 100 UÆmL)1penicillin G and 100 lgÆmL)1
streptomycin at 37C in 5% (v/v) CO2 Wortmannin (a PI3K
inhibitor), NL-71-101 (an Akt inhibitor), and rapamycin (a
p70 S6 kinase inhibitor) were purchased from Calbiochem
Cells were lysed in NaCl/Picontaining 1% (v/v) Triton X-100
and protease inhibitors, 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 mM Tris/HCl (pH 7.5), 137 mM NaCl, 1 mM
EDTA, 1 mMNa3VO4, 20 mMb-glycerophosphate, 50 mM
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
coliDH5a and were induced by adding 0.4 mMisopropyl
thio-b-D-galactoside The cells were suspended in lysis buffer
[NaCl/Pi containing 1% (v/v) Triton X-100 and 1 mM
phenylmethanesulfonyl fluoride] After sonication, crude
cell lysates were centrifuged at 4C 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 mM glutathione or
30 mMmaltose, respectively
Immunocomplex kinase assay
Cell lysates were prepared from 293T cells transfected
with pCEFL-HA-Akt Twenty-four hours after
transfec-tion, the cells were starved for 24 h and 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 mM Tris/HCl (pH 7.5), 25 mM MgCl2, 10 mM b-glycerophosphate, 0.33 mM Na3VO4, and 0.33 mM dithiothreitol not con-taining ATP] The substrate, GST- or MBP-dHAND and its mutants, and 5 lCi [32P]ATP[cP] were added to the beads The kinase reaction was performed at 37C for
30 min and the reaction was stopped by adding 2.5· sam-ple buffer followed by boiling for 5 min These samsam-ples 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 4C for 6 h with cell lysates prepared from 293T cells expressing HA-Akt The beads were washed three times with NaCl/Picontaining 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 and 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 4C for 6 h The beads were washed with NaCl/Pi containing 1% (v/v) Triton X-100 and protease inhibitors three times and 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 oligonucleotide probes were end-labeled with [32P]ATP[cP] by T4 polynucleotide kinase The binding sequence of 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¢ (mE¢); 5¢-TTTGCAAGGGGCGAACAGCATTCGCCC-3¢ Two microliters of cell lysate prepared from 293T cells 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 C2C12 cells was carried out using Lipofectamine Plus reagent (Life Technologies) Gal4-DBD
or Gal4-DBD constructs, pFR-Luc (Stratagene), pRL-SV40 (Promega), and pCEFL-HA-Akt were transfected
FEBS 2004 dHAND phosphorylation by Akt (Eur J Biochem 271) 3331
Trang 3and firefly luciferase units were determined 48 h after
transfection Transfection efficiency was normalized on the
basis of renila luciferase activity All these assays were
performed in triplicate
Results and Discussion
Phosphorylation of dHAND by Akt
Although gene 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 dHAND has a consensus motif for Akt
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 by Western blotting using antibody raised against
the phospho-Akt substrate As shown in Fig 1A,
AU5-dHAND expressed in 293T cells became phosphorylated
possibly at its Akt phosphorylation motif in the absence of
kinase inhibitors This signal was decreased by treatment
with 200 nMwortmannin, 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 nMrapamycin, 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) Effect of kinase inhibitors on the basal phosphorylation of dHAND 293T cells expressing AU5-dHAND were treated with 200 n M wortmannin, 50 l M NL-71-101 or 20 n M rapamycin for 120 min Dimethylsulfoxide was used as a vehicle AU5-dHAND was immunoprecipitated using anti-AU5 Ig and applied on 10% SDS/PAGE The phosphorylation status of dHAND was analyzed
by Western blotting using anti-(phospho-Akt substrate) Ig (top) The same membrane was reblotted by anti-AU5 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 dHAND was analyzed by immunoblotting with anti-(phospho-Akt substrate) Ig (top) The same membrane was reblotted by anti-AU5 Ig (bottom) (C) Effects of Akt overexpression on dHAND phosphorylation on serine/threonine residues Cell lysates were prepared from 293T cell expressing AU5-dHAND and/or HA-Akt Phosphorylation status of dHAND was analyzed by Western blotting using anti-phosphoserine
Ig (top) or anti-phosphothreonine Ig (middle) The protein amount of AU5-dHAND was estimated by Western blotting using anti-AU5 Ig (bottom).
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] using GST or GST-dHAND as a substrate Protein sample was applied on 10% SDS/PAGE and incorporation of
32
P was detected by autoradiography.
Trang 4We 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-phosphothreonine
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 may
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 and 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 [32P]ATP[cP] As shown in
Fig 2, strong incorporation of 32P 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 which 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 detected 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) Thus, to identify the phosphorylation site of dHAND, we first substituted Ser114 with alanine and examined whether this mutant (named dHAND-3SA; Fig 3A) can 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 aspartic 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).
FEBS 2004 dHAND phosphorylation by Akt (Eur J Biochem 271) 3333
Trang 5wild type dHAND (dHAND-WT) (Fig 4B, lanes 1 and
4), leading us to speculate that additional serine/threonine
residues are phosphorylated by Akt There are no 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 decreases in phosphorylation were
detected in mutants 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 dHANDin vitro and in vivo
To examine whether Akt can directly interact with dHAND, we performed pull-down assay using
GST-Fig 4 In vitro kinase assay using MBP-dHAND mutants
Immuno-complex kinase assay 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 bands were
detected by staining with Coomassie Brilliant Blue (bottom)
Arrow-heads indicate the positions of dHAND mutants.
Fig 5 Analysis of the interaction between dHAND and Akt (A) GST-pull down assay was performed using cell extract of 293T cells expressing HA-Akt (top) GST or GST-dHANDs used in this assay were applied on 10% SDS/PAGE and protein was detected by staining 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 cells expressing 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.
Trang 6dHAND 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 vivo, we coexpressed AU5-dHAND and HA-Akt in 293T
cells and performed a coimmunoprecipitation experiment
As shown in Fig 5B, interaction between HA-Akt and
AU5-dHAND was detected This result indicated that Akt
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 and data not shown) It was of interest that coexpression of Akt reduced the transcriptional activity of dHAND-WT/E47 The activity of dHAND-Ala-X/E47 was partially 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, WT, 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 transcripphosphoryla-tional 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 activity of dHAND/E47 heterodimer
Fig 6 The effects of Akt expression on the transcriptional activity of dHAND (A) AU5-dHAND, -dHAND-Ala-X, -dHAND-Ala, and -E47 were cotransfected into 293T cells with reporter plasmid encoding luciferase gene, whose expression is driven 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 analyzed by Western blotting using anti-AU5 Ig (bottom) Note that 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 dHAND, its mutants, and E47 were analyzed by Western blotting using anti-AU5 Ig (bottom).
FEBS 2004 dHAND phosphorylation by Akt (Eur J Biochem 271) 3335
Trang 7Phosphorylation 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 heterodimer independently of their DNA binding activity (Fig 7, upper panel) 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 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 detect the effects of Akt phosphoryla-tion on dimerizaphosphoryla-tion 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 binding domain (DBD)-fused
dHAND and reporter plasmid containing the luciferase gene, whose
expression was driven by five Gal4 binding sites, were transfected to
C2C12 cells, and luciferase assay was performed Fold activation is the
ratio of the luciferase activity in cells transfected with E47 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 was compared after in vitro phosphorylation procedure in the presence (+) or absence (–) of Akt Protein samples of AU5-dHAND or AU5-ME2 were mixed with 32 P-labeled oligonucleotide probe containing E-box sequence, 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).
Trang 8ME2/ITF2a tagged with AU5 as a heterodimeric partner in
this experiment
We phosphorylated dHAND by Akt in vitro, mixed with
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 of
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 to dHAND
and phosphorylate it at serine 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 on these three aspects: (a) dimerization activity with
E-protein, (b) DNA binding activity of dHAND/E-protein,
and (c) transcriptional activity of dHAND/E-protein
heterodimer Our results indicated that the effect of
Akt-dependent phosphorylation acted on (b), but not (a) and (c)
After phosphorylation by Akt, dHAND/E-protein
hetero-dimer can be present, but this heterohetero-dimer 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 consensus phosphorylation 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 be indirect Our present study is unique in that
the phosphorylation sites exist within the bHLH domain and
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 the 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 been reported that heart size is increased in
transgenic mice that express a constitutively active form of
Akt [23], and that down regulation of 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 because 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 regulated
by Akt signaling in vivo
In conclusion, dHAND was phosphorylated by Akt and the phosphorylation inhibited 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
We thank Dr J S Gutkind for plasmids, Ms S Okamura, Ms K Shin-Fukuhara, Ms M Yonemitsu, and Dr M Nakagawa for technical assistance, Dr H Saya and Dr T Hirota for advice and discussion on the phosphorylation analysis, and Dr E N Olson and
Dr H Iba for critical comments on this paper This work was supported
by JSPS (Japan Society for the Promotion of Science) Research for the Future Program; Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan; and the Research Grant for Cardiovascular Diseases (14C-1) from the Ministry of Health, Japan.
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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, the heterodimer of phosphorylated dHAND can not bind to the E-box sequence (2) As a result, the transcriptional activity of dHAND is inhibited by Akt signaling.
FEBS 2004 dHAND phosphorylation by Akt (Eur J Biochem 271) 3337
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FEBS 2004 dHAND phosphorylation by Akt (Eur J Biochem 271) 3339