MOLECULAR AND CELLULAR BIOLOGY, Oct. 2005, p. 8401–8414
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 19
Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein
Kinase A Signaling Is Attributed to CBP-Mediated
Acetylation of GCMa†
Ching-Wen Chang,1Hsiao-Ching Chuang,2Chenchou Yu,1Tso-Pang Yao,3and Hungwen Chen1,2*
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan1; Graduate Institute of Biochemical Sciences,
National Taiwan University, Taipei 106, Taiwan2; and Department of Pharmacology and Cancer Biology, Duke University,
Durham, North Carolina 277103
Received 15 February 2005/Returned for modification 15 March 2005/Accepted 29 June 2005
Human GCMa is a zinc-containing transcription factor primarily expressed in placenta. GCMa regulates
expression of syncytin gene, which encodes for a placenta-specific membrane protein that mediates tropho-
blastic fusion and the formation of syncytiotrophoblast layer required for efficient fetal-maternal exchange of
nutrients and oxygen. The adenylate cyclase activator, forskolin, stimulates syncytin gene expression and cell
fusion in cultured placental cells. Here we present evidence that cyclic AMP (cAMP) signaling pathway
activates the syncytin gene expression by regulating GCMa activity. We found that forskolin and protein kinase
A (PKA) enhances GCMa-mediated transcriptional activation. Furthermore, PKA treatment stimulates the
association of GCMa with CBP and increases GCMa acetylation. CBP primarily acetylates GCMa at lysine367,
lysine406, and lysine409in the transactivation domain (TAD). We found that acetylation of these residues is
required to protect GCMa from ubiquitination and increases the TAD stability with a concomitant increase in
transcriptional activity, supporting the importance of acetylation in PKA-dependent GCMa activation. Our
results reveal a novel regulation of GCMa activity by cAMP-dependent protein acetylation and provide a
molecular mechanism by which cAMP signaling regulates trophoblastic fusion.
GCM1 (glial cell missing), also named Glide (Glial cell defi-
cient), was first isolated from a Drosophila melanogaster mutant
line that produces additional neurons at the expense of glial
cells. Conversely, ectopic expression of GCM1 in flies gener-
ated excessive numbers of glial cells at the expense of neurons
(24, 26). It is thought that GCM functions as a genetic binary
switch between neuronal and glial determination in Drosoph-
ila. Drosophila GCM1 is transiently expressed in glial precur-
sors and immature glial cells except for mesectodermal midline
glia in the central nervous system and many of the specialized
support cells of PNS sensory neurons (24, 26). Recently, a
GCM1 homologue called GCM2 or Glide2, located 27 kb apart
from the GCM1 locus in the Drosophila genome, was isolated
(2, 27). GCM2 has redundant functions of GCM1 and plays a
minor role during gliogenesis. However, both GCM1 and -2
are required for the proper differentiation of the plasmatocyte/
macrophage lineage of blood cells (2).
Two GCM homologues called GCMa and b have been iden-
tified in mice, rats, and humans (28, 30). In contrast to the
neural expression pattern of Drosophila GCM1 and -2, mouse
GCMa mRNA is highly expressed in the labyrinthine tropho-
blast cells of placenta and at low levels in restricted sites of the
postnatal kidney and thymus (4, 21). GCMa is required for
placental development because genetic ablation of mouse
GCMa leads to failure of labyrinth layer formation and no
fusion of trophoblasts to syncytiotrophoblasts (3, 37). GCMb is
required for the proper development of parathyroid glands
(19). Recently, chicken GCM has been isolated and shown to
be exclusively expressed in extra-embryonic tissues (22). Since
mammalian GCMa is also expressed in extra-embryonic tis-
sues, it has been speculated that GCM evolutionary function is
conserved between mammals and birds. In addition, zebra fish
GCMb has been characterized and shown to be required for
normal development of pharyngeal cartilages (20, 23).
GCM proteins form a novel family of transcription factor
with a conserved DNA-binding domain, termed the GCM mo-
tif, at the N terminus (1, 36). Recent crystallographic analysis
of the GCM motif has revealed that it is a zinc-containing
domain of ? sheets interacting with the major groove of its
cognate DNA element, 5?-ATGCGGGT-3? (14). Transactiva-
tion domain has been identified in the carboxyl terminus of
GCM proteins (1, 38). In terms of physiological function, Dro-
sophila GCM1 regulates expression of repo (reverse polarity)
and pnt (pointed) genes, the principal mediators of glial differ-
entiation, whereas human GCMa regulates expression of the
syncytin gene, which encodes a placental fusogenic membrane
protein mediating trophoblastic fusion (39, 45). Syncytin is an
envelope (Env) protein of the newly identified human endog-
enous retrovirus family W (HERV-W), which is a class I
HERV with sequences homologous to the mammalian type C
retroviruses and a tRNA primer-binding site for tRNATrp(7,
33). Two functional GCMa-binding sites in the 5?-flanking
region of the 5? long terminal repeat (LTR) of the HERV-W
have been identified (45). This suggests that GCMa regulates
syncytin-mediated trophoblastic fusion at the transcriptional
level. Like other retroviral Env proteins, syncytin is posttrans-
lationally cleaved into a surface (SU) subunit and a transmem-
* Corresponding author. Mailing address: Institute of Biological
Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan. Phone:
886-2-27855696, ext. 6090. Fax: 886-2-27889759. E-mail: hwchen@gate
† Supplemental material for this article may be found at http://mcb
brane (TM) subunit, which contains a fusion peptide. Two
sodium-dependent amino acid transporters, ASCT1 and -2,
have been reported as the syncytin receptors (32). It is gener-
ally believed that syncytin binds to its cognate receptor via its
SU subunit and results in a conformational rearrangement in
its TM subunit in the fusion process. Indeed, our recent study
has demonstrated that interaction between two heptad repeat
regions in the TM subunit is required for syncytin-mediated
cell fusion (10). It is feasible to speculate that this interaction
facilitates exposure and insertion of the fusion peptide into the
target cell membrane.
It has been shown that treatment of human placental cells
with the adenylate cyclase activator, forskolin, dramatically
increases cell-cell fusion (29, 40). In addition, the mRNA level
of syncytin in placental BeWo cells is increased after forskolin
stimulation (33). These observations suggest that the cAMP/
PKA signaling pathway is involved in the syncytin-mediated
cell fusion and prompted us to investigate whether GCMa
activity is regulated by the cAMP/PKA signaling pathway. In
the present study, we demonstrate that forskolin and protein
kinase A (PKA) stimulate GCMa-mediated transcriptional ac-
tivation and CBP is involved in this pathway by directly inter-
acting with and acetylating GCMa. Moreover, PKA facilitates
the interaction between CBP and GCMa to promote GCMa
acetylation, which increases the protein stability of GCMa and
enhances GCMa-mediated transcriptional activation. Our
studies help to show how the forskolin-activated cyclic AMP
(cAMP)/PKA signaling pathway regulates trophoblastic fusion
at the molecular level.
MATERIALS AND METHODS
Plasmid constructs. The pHA-GCMa expression plasmid was constructed by
cloning into the pEF1-MycHis expression plasmid (Invitrogen, Carlsbad, CA), a
DNA fragment encoding human GCMa with a triple hemagglutinin (HA) tag at
its N terminus. The pGCMa-Myc expression plasmid was similar to pHA-GCMa
except that it contained a quadruple Myc tag attached to the C terminus of
GCMa. pPKAcata was constructed by cloning into pRcCMV (Invitrogen), a
DNA fragment encoding the catalytic subunit of the cAMP-dependent PKA.
pPKI and pRevAB constructs encoding a peptide inhibitor specific to PKAcata
and a dominant-negative regulatory subunit of PKA, respectively, were kindly
provided by Stanley McKnight (University of Washington, Seattle). Four tandem
copies of the proximal GCMa-binding site (pGBS, 5?-TTCTGGGATGAGGGC
AAAACG-3?) in the 5?-LTR of the syncytin-containing HERV-W was cloned
into pE1bCAT, which harbors a minimal promoter element from the adenovirus
E1B gene and the bacterial CAT coding sequence, to generate the reporter
construct, p(pGSB)4E1bCAT. p(pGBS)4E1bLUC was similar to p(pGBS)4
E1bCAT except that its CAT reporter gene was replaced with a firefly luciferase
gene. p(Mut)5E1bLUC contained five tandem copies of a mutant pGBS (Mut,
5?-TTCTGGGATGATAGCAAAACG-3?, which is not recognized by GCMa) in
pE1bLUC. pLUC(25468-30953) reporter construct containing the syncytin pro-
moter element was similar to pCAT(25468-30953) described previously (45),
except that its CAT reporter gene was replaced with a firefly luciferase gene.
pG5LUC, a luciferase reporter plasmid containing five tandem copies of a GAL4
binding site, was obtained from Promega (Madison, WI). The pCBP-HA and
pCBP-Flag expression plasmids encoded mouse CBP with a C-terminal HA and
FLAG tag, respectively, under the control of a cytomegalovirus (CMV) enhancer
and promoter. pCBPHAT?-HA is a mutant pCBP-HA harboring leucine1690-to-
lysine and cysteine1691-to-leucine mutations, which cause loss of the intrinsic
histone acetyltransferase (HAT) activity of CBP (41). pCBPHAT?-Flag was sim-
ilar to pCBPHAT?-HA except that its HA tag was replaced with a FLAG tag. The
pHA-EGFP expression plasmid was constructed by cloning into pEF1-MycHis a
DNA fragment encoding EGFP with a triple HA tag at its N terminus. A
pGal4-Flag expression plasmid was constructed by cloning the GAL4 DNA-
binding domain into p3XFLAG-CMV14 (Sigma, St. Louis, MO). Full-length
GCMa and truncated GCMa cDNAs were subcloned into pGal4-Flag to gener-
ate wild-type and mutant pGal4-GCMa-Flag expression plasmids, as indicated in
the legend to Fig. 7A. Site-directed mutagenesis of the lysine residues in pGal4-
GCMa-Flag(300-436) was performed by two-step PCRs with designated primer
pairs to generate K349R, K367R, K406R, K409R, K2R, and K3R constructs. The
K2R construct is a mutant pGal4-GCMa-Flag(300-436) harboring lysine to ar-
ginine mutations in lysine406and lysine409. The K3R construct harbors lysine to
arginine mutations in lysine367, lysine406, and lysine409. Similar strategies were
used to generate full-length mutant GCMa expression plasmids pGCMa-Myc-
K349R, -K2R, and -K3R. All constructs were verified by DNA sequencing by
using the dideoxy chain termination method.
Cell culture, transfection, reporter gene assay, and RNA interference. The
human trophoblast cell lines, BeWo and JAR, were obtained from the American
Type Culture Collection (ATCC; Manassas, VA) and maintained at 37°C in
F-12K medium supplemented with 15% fetal bovine serum (FBS), streptomycin
(100 ?g/ml), and penicillin (100 U/ml). Stable BeWo cells expressing HA-GCMa
described previously (45) were maintained in the same culture conditions for
BeWo cells. 293T and CV1 (ATCC) were maintained at 37°C in HEPES-buff-
ered Dulbecco modified minimal essential medium (DMEM) supplemented with
10% FBS and the same antibiotics mentioned above. For transient expression,
293T and JAR cells were incubated with calcium phosphate-DNA coprecipitates
containing the indicated amounts of reporter plasmid and expression plasmid as
described in the figure legends. Adjusted amounts of the empty expression vector
were also included to maintain a constant amount of total DNA in each trans-
fection assay. Transfection of BeWo and CV1 cells was performed by using the
TransIT LT1 reagent (Mirus, Madison, WI) according to the instructions of the
manufacturer. Cells were harvested in the reporter lysis buffer (Promega) 48 h
posttransfection. CAT and luciferase assays were performed as previously de-
scribed (11). Specific CAT and luciferase activities were normalized by protein
concentration. Protein concentrations were measured by using the BCA protein
assay kit (Pierce, Rockford, IL). For RNA interference, 293T cells were trans-
fected with CBP small interfering RNA (siRNA; Santa Cruz Biotechnology,
Santa Cruz, CA) or GL2 siRNA (Dharmacon Research, Lafayette, CO) by using
the TransIT TKO reagent (Mirus), followed by transfection with the indicated
reporter and expression plasmids by using the TransIT LT1 reagent. The efficacy
of CBP siRNA was verified in 293T cells by reverse transcriptase-PCR (RT-
PCR) with RNeasy reagents (QIAGEN, Hilden, Germany) for RNA purification
and the SuperScript III first-strand synthesis system (Invitrogen) for first-strand
cDNA synthesis. The sequences of the primers were 5?-ACCTTAGACCCCGAA
C-3? and 5?-CCGTGACTTCATCCCG-3? for CBP and 5?-CTCAAGGGCATCCT
GGGCTA-3? and 5?-CTGTTGCTGTAGCCAAATTCGTT-3? for GAPDH.
Coimmunoprecipitation and pull-down assay. To study the interaction be-
tween GCMa and CBP, 293T cells were cotransfected with the indicated com-
binations of pCBP-HA, pHA-GCMa, and pPKAcata as described in the figure
legend. At 48 h posttransfection, cells were harvested in lysis buffer containing 20
mM HEPES (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.05%
Tween 20, 5% glycerol, 1 mM Na3VO4, 5 mM NaF, and 1 mM phenylmethyl-
sulfonyl fluoride. Approximately 180 ?g of cell lysate was immunoprecipitated
with GCMa antibody and protein A-conjugated agarose beads (Roche, Mann-
heim, Germany). After extensive washing, the immune complexes were analyzed
by immunoblotting with a rat monoclonal anti-HA antibody (HA monoclonal
antibody [MAb]; Roche). The interaction between GCMa and CBP in placental
cells were analyzed in stable HA-GCMa-expressing BeWo cells by the above-
mentioned coimmunoprecipitation assay using HA MAb and CBP antibodies
from different sources, including A-22 (Santa Cruz Biotechnology), a rabbit
polyclonal anti-CBP antibody (Upstate, Lake Placid, NY), and AC26 (CBP
MAb) (15). To specify the functional role of PKA in the interaction between
GCMa and CBP, HA-GCMa or CBP-HA was first immunoprecipitated from
293T cells cotransfected with pHA-GCMa and pPKAcata or pCBP-HA and
pPKAcata. Subsequently, the immune complexes were treated with 200 U of
lambda protein phosphatase (?-PPase; NEB, Beverly, MA) with or without 20
mM Na3VO4at 30°C for 1 h, followed by incubation with 1 ?g of recombinant
Flag-CBP or GCMa-Flag proteins in the lysis buffer at 4°C overnight. After
extensive washing, the pull-down complexes were analyzed by immunoblotting
with a mouse monoclonal anti-FLAG antibody (FLAG MAb; Sigma). Recom-
binant Flag-CBP and GCMa-Flag proteins were purified from Sf9 cells infected
with recombinant baculovirus strains using the anti-FLAG M2-conjugated aga-
rose beads (Sigma).
The glutathione S-transferase (GST) fusion protein expression vector
pGEX4T-1 (Amersham Biosciences, Piscataway, NJ) was used to express GST
fusion proteins of full-length GCMa, GST-GCMa(1-436), and truncated GCMa,
GST-GCMa(1-220), -GCMa(1-300), -GCMa(1-349), -GCMa(167-349), -GCMa
(220-330), -GCMa(300-436), and -GCMa(349-436) in the Escherichia coli strain
BL21(DE3). Purification of GST fusion proteins was performed as described by
Frangioni and Neel (16). For GST-CBP fusion proteins, the corresponding
8402CHANG ET AL.MOL. CELL. BIOL.
mouse CBP cDNA fragments were cloned into the pGEX6P-1 vector (Amer-
sham Biosciences) to express GST-CBP(1-451), -CBP(451-721), -CBP(721-
1100), -CBP(1099-1460), -CBP(1460-1891), -CBP(1892-2163), and -CBP(2114-
2441) fusion proteins. To map the interacting domains of GCMa and CBP, GST
pull-down assays were performed. In brief, cell lysates were prepared from 293T
cells transfected with pCBP-HA and pHA-GCMa, respectively. Per reaction, 180
?g of the indicated cell lysate was incubated with 2.5 ?g of the indicated GST
fusion protein prebound in the glutathione beads (Amersham Biosciences) at
4°C overnight. After extensive washing, the pull-down complexes were analyzed
by immunoblotting with HA MAb.
ChIP assay. To study association of GCMa and CBP with the promoter region
of syncytin gene, 3 ? 106BeWo cells were treated with or without 50 ?M
forskolin for 12 h and analyzed by chromatin immunoprecipitation (ChIP) assays
as described by Boyd and Farnham (8). Associated protein-DNA complexes were
incubated with GCMa antibody or the CBP antibody from Upstate and then
precipitated with protein A-conjugated agarose beads. A specific region contain-
ing the pGBS sequence in the syncytin promoter in the immune complexes was
detected by PCR with specific primers. PCR conditions included denaturation at
94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 50 s for 40
cycles. PCR products were analyzed on 5% polyacrylamide gels. Sequences of
primers were 5?-CTCTCTGGAGAGTGAATTACTGAGTC-3? and 5?-CCTGT
CTCTCAGTTGCAAGATAATTGC-3? for syncytin and 5?-AAAAGCGGGGA
GAAAGTAGG-3? and 5?-CTAGCCTCCCGGGTTTCTCT-3? for GAPDH.
RNA was isolated from BeWo cells treated with or without forskolin using
RNeasy reagents, and the syncytin and ?-actin transcripts were analyzed by
Northern blotting with full-length syncytin and ?-actin cDNAs as probes.
In vitro acetylation study and EMSA. For recombinant MBP-GCMa fusion
proteins, GCMa cDNA was cloned into the pMAL-c2 vector (NEB) and ex-
pressed in BL21(DE3). Affinity purification of MBP-GCMa proteins was per-
formed with maltose agarose beads (NEB) according to the manufacturer’s
instructions. For in vitro acetylation of GCMa, 1.5 ?g of MBP or MBP-GCMa
protein was incubated with 150 ng of the purified recombinant Flag-CBP pro-
teins in a 30 ?l of reaction buffer (50 mM HEPES [pH 8.0], 10 mM sodium
butyrate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) plus or
minus 0.8 mM acetyl coenzyme A (Ac-CoA) at 30°C for 1 h. For acetylation
analysis, the reaction mixture was analyzed by immunoblotting with a mouse
monoclonal anti-acetylated-lysine antibody (Ac-K MAb; Cell Signaling, Beverly,
MA). Immunoblotting of MBP and MBP-GCMa proteins was performed with a
mouse monoclonal anti-MBP antibody (MBP MAb; Clontech, Palo Alto, CA).
For electrophoretic mobility shift assay (EMSA), unacetylated or acetylated
MBP-GCMa was incubated with a32P-labeled pGBS oligonucleotide probe as
previously described (45).
In vivo acetylation study and pulse-chase analysis of protein turnover. To
study CBP-mediated acetylation of GCMa in vivo, 293T cells were transfected
with pGal4-GCMa-Flag alone or with the indicated combinations of pGal4-
GCMa-Flag, pCBP-HA, pCBPHAT?-HA, and pPKAcata. The pHA-EGFP ex-
pression plasmid was included in each transfection group as an internal control
of transfection efficiency. At 48 h posttransfection, cells were harvested for
immunoprecipitation with GCMa Ab. The immune complexes were further
analyzed by immunoblotting with Ac-K MAb. To study the acetylation of GCMa
in placental cells, stable HA-GCMa-expressing BeWo cells were mock treated,
treated with forskolin alone, or treated with forskolin together with trichostatin
A (TSA) for 24 h, followed by acetylation analysis with HA MAb for immuno-
precipitation and Ac-K MAb for immunoblotting. To study the effect of acet-
ylation on protein stability of GCMa, 293T cells in 10-cm culture dishes were
transfected with the indicated combinations of pHA-GCMa, pCBP-Flag, and
pPKAcata. At 18 h posttransfection, cells were subcultured into 3.5-cm culture
dishes for pulse-chase experiments. At 36 h posttransfection, cells were pulse-
labeled with 50 ?Ci of [35S]methionine/ml for 2 h. After labeling, cells were
washed twice with phosphate-buffered saline and incubated in chase medium
(DMEM with 10% FBS plus 50 ?g of methionine/ml) for various time periods.
Radiolabeled HA-GCMa proteins were immunoprecipitated with the HA MAb
and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and fluorography. Quantification of pulse-chase experiments was
performed by using the bioimaging analyzer BAS-1500 (Fujifilm, Kanagawa,
Japan). To study the effect of acetylation on GCMa ubiquitination, 293T cells
were transfected with different combinations of pHA-Ub, pGCMa-Myc, lysine-
to-arginine mutant pGCMa-Myc, pCBP-Flag, pCBPHAT?-Flag, and pPKAcata,
followed by immunoprecipitation with a mouse monoclonal anti-Myc antibody
(Myc MAb; Roche) and immunoblotting with HA MAb.
Mapping of CBP acetylation sites in GCMa. To map the acetylation domains
in GCMa in vitro, 0.2 ?g of GST or the indicated GST-GCMa fusion protein was
incubated with 0.4 ?g of Flag-CBP under the same reaction conditions as de-
scribed for in vitro acetylation analysis. To identify the acetylation domain in
GCMa in vivo, 293T cells were cotransfected with pCBP-HA and pPKAcata and
a series of pGal4-GCMa-Flag expression plasmids containing truncated regions
of GCMa. Gal4-GCMa-Flag proteins were immunoprecipitated with GCMa Ab
and analyzed for acetylation as described above. To identify the CBP acetylation
sites in the C-terminal TAD of GCMa, 293T cells were cotransfected with
pCBP-HA, pPKAcata, and the wild-type or mutant pGal4-GCMa-Flag(300-436)
expression plasmids, followed by acetylation analysis. Similar acetlyation site
analyses of full-length GCMa were also performed using pGCMa-Myc and
lysine-to-arginine mutant pGCMa-Myc.
Forskolin regulates the transcriptional activity of GCMa.
Since syncytin is a target gene of GCMa and its expression is
stimulated by the adenylate cyclase activator, forskolin, we
tested whether forskolin could also stimulate the transcrip-
tional activity of GCMa. The p(pGBS)4E1bCAT reporter plas-
mid containing four copies of the proximal GCMa-binding site
(pGBS) in the promoter region of syncytin was constructed. In
transient-expression experiments, 293T cells, which lack en-
dogenous GCMa, were transfected with p(pGBS)4E1bCAT
alone or plus the GCMa expression plasmid pHA-GCMa and
then further treated with or without forskolin. As shown in Fig.
1A, chloramphenicol acetyltransferase (CAT) activity directed
by p(pGBS)4E1bCAT was positively regulated by GCMa.
Moreover, this GCMa-mediated transcriptional activation was
further stimulated about four fold in the presence of forskolin
(Fig. 1A). To investigate the signaling pathway activated by
forskolin, several kinase inhibitors, including H89, PD 98059,
and SB 203580, were tested in transient-expression experi-
ments for blockage of the stimulatory effect of forskolin. As
shown in Fig. 1A, the stimulatory effect of forskolin was sig-
nificantly inhibited by H89, a specific inhibitor of the cAMP-
dependent PKA, but not by the MEK inhibitor, PD 98059, nor
by the p38 mitogen-activated protein kinase inhibitor, SB
Since forskolin stimulates syncytin gene expression and cell-
cell fusion in placental BeWo cells, we tested whether forskolin
also stimulates endogenous GCMa activity in BeWo cells. To
this end, BeWo cells were transfected with p(pGBS)4E1bLUC
or p(Mut)5E1bLUC, which contains a mutant pGBS not rec-
ognized by GCMa, and treated with or without forskolin. The
luciferase activity directed by p(pGBS)4E1bLUC was signifi-
cantly higher than that shown by p(Mut)5E1bLUC in the ab-
sence of forskolin, a finding suggestive of a specific response of
p(pGBS)4E1bLUC to the endogenous GCMa proteins (Fig.
1B). Moreover, the luciferase activity directed by p(pGBS)4
E1bLUC, but not p(Mut)5E1bLUC, was significantly stimu-
lated by forskolin (Fig. 1B). This forskolin-upregulated GCMa
activity was counteracted by the addition of H89 or upon pPKI
or pRevAB cotransfection (Fig. 1B). pPKI and pRevAB con-
structs encode a peptide inhibitor specific to PKAcata and a
dominant-negative regulatory subunit of PKA, respectively.
Therefore, these results suggested that PKA regulates GCMa
activity in the forskolin-activated cAMP signaling pathway.
This notion was also supported by the observation that the
luciferase activity directed by p(pGBS)4E1bLUC was signifi-
cantly increased upon pPKAcata cotransfection in the absence
of forskolin (Fig. 1B).
CBP is involved in the PKA-upregulated GCMa activity.
Examination of the protein sequence of GCMa by PROSITE
VOL. 25, 2005ACETYLATION OF GCMa INCREASES ITS PROTEIN STABILITY8403
(25) and NetPhos (6) suggested several PKA consensus phos-
phorylation sites in GCMa. Indeed, GCMa was phosphory-
lated by the catalytic subunit of PKA in vitro (see Fig. S1A to
D in the supplemental material). However, transient-expres-
sion experiments using mutant GCMa expression plasmids
harboring mutations in these PKA phosphorylation sites did
not demonstrate any adverse effect on the PKA-upregulated
GCMa activity (see Fig. S1E in the supplemental material).
Since CBP is an important downstream effector in the cAMP/
PKA signaling pathway and functions as a coactivator for many
transcription factors, we therefore tested whether CBP was
involved in the regulation of GCMa activity by PKA. 293T cells
were transfected with p(pGBS)4E1bLUC, pHA-GCMa, and
plus increasing amounts of pCBP-HA. As shown in Fig. 2A,
CBP enhanced the transcriptional activity of GCMa in a dose-
dependent manner. Interestingly, this positive effect of CBP
was further increased when pPKAcata was cotransfected (Fig.
2A). We also tested the effect of a CBP siRNA on the forsko-
lin-upregulated GCMa activity by cotransfecting 293T cells
with p(pGBS)4E1bCAT, pHA-GCMa, and CBP siRNA. As
shown in Fig. 2B (left panel), the CBP siRNA, but not the
unrelated GL2 siRNA, efficiently knocked down the endoge-
nous CBP transcript in 293T cells based on RT-PCR (Fig. 2B,
left panel). Correspondingly, CBP siRNA, but not GL2 siRNA
reduced GCMa-mediated transcriptional activation in the
presence or absence of forskolin (Fig. 2B, right panel), sug-
gesting that CBP is an important regulator for GCMa-medi-
ated transcriptional activation.
We further tested whether GCMa physically interacts with
CBP by transfecting 293T cells with pHA-GCMa and pCBP-
HA, followed by immunoprecipitation with GCMa antibody
and immunoblotting with HA MAb. As shown in Fig. 2C (left
panel), GCMa specifically interacted with CBP. Moreover, this
interaction was stimulated when pPKAcata was cotransfected
(Fig. 2C, left panel). To investigate the role of PKA in regu-
lating the interaction between GCMa and CBP, we tested
whether dephosphorylation of GCMa and CBP by ?-PPase
affects the interaction between the two proteins. 293T cells
were cotransfected with pHA-GCMa and pPKAcata or with
pCBP-HA and pPKAcata, and the HA-GCMa and CBP-HA
proteins were immunoprecipitated, respectively. The immune
complexes were treated with ?-PPase or ?-PPase plus Na3VO4,
a phosphatase inhibitor. The treated HA-GCMa and CBP-HA
immune complexes were incubated with recombinant Flag-
CBP and GCMa-Flag proteins, respectively, in pull-down as-
says. As shown in Fig. 2C (right panel), the interaction between
GCMa and CBP was decreased when the precipitated HA-
GCMa, but not CBP-HA, was pretreated with ?-PPase. How-
ever, this interaction between HA-GCMa and Flag-CBP was
not affected when ?-PPase was inhibited by Na3VO4. These
results suggested that PKA may modify the phosphorylation
status of GCMa thereby increasing its CBP-binding activity.
To study the interaction between GCMa and CBP in pla-
cental cells, previously established stable BeWo cells express-
ing HA-GCMa (43) were used for coimmunoprecipitation
analyses. Interaction between HA-GCMa and endogenous
CBP was barely detectable in the stable BeWo cells (data not
shown). However, when the stable BeWo cells were treated
with forskolin, specific interaction between HA-GCMa and
endogenous CBP was detected by immunoprecipitation and
immunoblotting with HA MAb and CBP antibodies from dif-
ferent sources (Fig. 3A). As a control, this interaction was not
FIG. 1. Forskolin and PKA stimulate the transcriptional activity of GCMa. (A) 293T cells were transfected with 0.5 ?g of p(pGBS)4E1bCAT
alone or together with 0.5 ?g of pHA-GCMa. At 24 h posttransfection, cells were mock-treated or treated with 50 ?M forskolin or 50 ?M forskolin
plus 3 ?M H89, 10 ?M PD 98059, or 3 ?M SB 203580 for another 24 h. Mean values and the standard errors of the mean (SEM) obtained from
four independent transfection experiments are provided. The protein levels of HA-GCMa and ?-actin (as a loading control) in each transfection
group were detected by immunoblotting with HA-MAb and ?-actin MAb, respectively. PD, PD 98059; SB, SB 203580. (B) BeWo cells were
transfected with 0.5 ?g of p(pGBS)4E1bLUC or p(Mut)5E1bLUC alone or together with 0.2 ?g of pPKAcata, pPKI, or pRevAB. At 24 h
posttransfection, cells were mock treated or treated with 50 ?M forskolin or 50 ?M forskolin plus 3 ?M H89 for another 24 h. Mean values and
the SEM obtained from three independent transfection experiments are provided. FSK, forskolin.
8404CHANG ET AL.MOL. CELL. BIOL.
detected using an unrelated antibody against the GAL4 DNA-
binding domain, Gal4 antibody, for immunoprecipitation (Fig.
We further investigated whether forskolin could stimulate
the occupancy of syncytin promoter by GCMa and CBP in
BeWo cells by ChIP assays. As expected, the level of syncytin
transcript was significantly increased in BeWo cells treated
with forskolin in Northern analysis (Fig. 3B, lower panel).
Under ChIP analysis, occupancy of GCMa and CBP on pGBS
in the syncytin promoter was significantly increased in forsko-
lin-treated BeWo cells compared to the untreated BeWo cells
(Fig. 3B, upper panel). To test the effect of GCMa and CBP on
syncytin promoter activity, we performed transient-expression
experiments in JAR cells, in which syncytin gene expression has
been shown to be stimulated by forskolin (35). JAR cells were
transfected with pHA-GCMa, pCBP-HA, and pLUC(25468-
30953), a reporter construct of syncytin promoter with two
functional GBSs. As shown in Fig. 3C, the luciferase activity
directed by pLUC(25468-30953) was positively stimulated by
GCMa or CBP. Moreover, the luciferase activity was further
stimulated when pHA-GCMa and pCBP-HA were cotrans-
fected (Fig. 3C). Taken together, these results suggested that
the cAMP/PKA signaling pathway activated by forskolin leads
to an increased association of GCMa and CBP with the syn-
cytin promoter and a concomitant increase in syncytin gene
Mapping of the interacting domains of GCMa and CBP. We
next characterized the interaction between GCMa and CBP by
mapping their interacting domains. To map the CBP-interact-
ing domain(s) of GCMa, GST pull-down assays were per-
formed by incubating a series of GST-GCMa fusion proteins
with cell lysate of 293T cells transfected with pCBP-HA, fol-
FIG. 2. Functional and physical interaction of GCMa and CBP. (A) CBP and PKA synergistically enhance GCMa-mediated transcriptional
activation. 293T cells were transfected with 0.3 ?g of p(pGBS)4E1bLUC alone or together with 0.3 ?g of pHA-GCMa or 0.3 ?g of pHA-GCMa
plus the indicated amount of pCBP-HA (■). In a separate set of experiments, 0.1 ?g of pPKAcata was included in each transfection group (u).
Mean values and SEM obtained from four independent transfection experiments are provided. (B) Inhibition of GCMa-mediated transcriptional
activation by CBP siRNA. 293T cells were transfected with the indicated combinations of 0.3 ?g of p(pGBS)4E1bCAT, 0.3 ?g of pHA-GCMa, and
10 nM GL2 or CBP siRNA. At 24 h posttransfection, cells were mock-treated or treated with 30 ?M forskolin for another 24 h. Mean values and
the SEM obtained from three independent transfection experiments are provided. The efficacy of CBP siRNA was analyzed by RT-PCR of 293T
cells transfected with 10 nM GL2 or CBP siRNA. (C) GCMa interacts with CBP. 293T cells were transfecetd with 1 ?g of pCBP-HA alone or
together with 1 ?g of pHA-GCMa or 1 ?g of pHA-GCMa plus 0.1 ?g of pPKAcata. At 48 h posttransfection, cells were harvested for
coimmunoprecipitation assays as described in Materials and Methods. The protein level of CBP-HA in the whole-cell lysate (WCL) is presented.
Of note, the interaction of GCMa and CBP was enhanced in the presence of PKAcata. IP, immunoprecipitation; IB, immunoblot. (D) PKA
regulates the CBP-binding activity of GCMa. 293T cells were transfected with 5 ?g of pHA-GCMa or pCBP-HA. At 48 h posttransfection, cells
were harvested and immunoprecipitated with HA-MAb. The immune complexes were mock treated or treated with 200 U of ?-PPase plus or minus
20 mM Na3VO4. The treated HA-GCMa and CBP-HA complexes were then incubated with 1 ?g of recombinant Flag-CBP and GCMa-Flag
proteins, respectively, for pull-down analysis as described in Materials and Methods.
VOL. 25, 2005 ACETYLATION OF GCMa INCREASES ITS PROTEIN STABILITY8405
lowed by immunoblotting with HA MAb. As shown in Fig. 4A,
CBP interacted with two domains in GCMa, i.e., amino acids 1
to 220 and amino acids 349 to 436. The former essentially
contains the GCM motif, whereas the later contains a C-ter-
minal TAD. Likewise, to map the GCMa-interacting do-
main(s) of CBP, a series of GST-CBP fusion proteins were
incubated with HA-GCMa-containing cell lysate, followed by
immunoblotting with HA MAb. As shown in Fig. 4B, two
domains in CBP were identified for interaction with GCMa,
i.e., amino acids 1 to 451 and amino acids 1460 to 1891. The
former contains a region from the N terminus to the C/H1
domain, whereas the later contains a partial HAT domain and
the C/H3 domain.
Acetylation of GCMa by CBP does not affect its DNA-bind-
ing activity. Since CBP interacts with GCMa and has an in-
trinsic HAT activity that acetylates nonhistone proteins, we
now tested whether CBP could acetylate GCMa. In vitro acet-
ylation reactions were performed by incubating purified re-
combinant Flag-CBP and MBP or MBP-GCMa proteins in the
presence or absence of Ac-CoA, followed by immunoblotting
with Ac-K MAb. As shown in Fig. 5A, CBP specifically acet-
ylated MBP-GCMa only in the presence of Ac-CoA. Similar
reactions with MBP as the substrate did not reveal any acet-
ylation signals. We further tested whether GCMa acetylation
in the stable HA-GCMa-expressing BeWo cells could be reg-
ulated by forskolin. As shown in Fig. 5B, acetylation of HA-
FIG. 3. Regulation of syncytin promoter by GCMa and CBP. (A) Physical interaction between GCMa and CBP in placental cells. Stable BeWo
cells expressing HA-GCMa were treated with 30 ?M forskolin for 24 h and then analyzed for the interaction between HA-GCMa and endogenous
CBP by coimmunoprecipitation assays with the indicated combinations of antibodies. Asterisk and arrow indicate the CBP and the HA-GCMa
protein, respectively. SC, Santa Cruz; US, Upstate. (B) Association of GCMa and CBP with the syncytin promoter is stimulated by forskolin. BeWo
cells were treated with or without 50 ?M forskolin for 12 h and analyzed by ChIP assays for a promoter region covering pGBS in the syncytin gene
or for a promoter region in the GAPDH gene. A reaction was performed in the absence of antibody as a control (no Ab). Serial dilutions of input
chromatin DNA were analyzed in PCRs with primers for the syncytin promoter. Mock- or forskolin-treated BeWo cells were also analyzed for the
levels of syncytin and ?-actin transcripts by Northern blotting (NB) using radioactive syncytin and ?-actin cDNAs as probes. (C) Stimulation of
syncytin promoter activity by GCMa and CBP. JAR cells were transfected with the indicated combinations of 0.1 ?g of pLUC(25468-30953), 0.1
?g of pHA-GCMa, 0.1 ?g of pCBP-HA, and 0.1 ?g of pCBPHAT?-HA. Mean values and the SEM obtained from five independent transfection
experiments are provided.
8406CHANG ET AL.MOL. CELL. BIOL.
GCMa was detected in the stable BeWo cells treated with
forskolin, which was further enhanced in cells treated with
forskolin and TSA, a histone deacetylase inhibitor. To test
whether acetylation of GCMa affects its DNA-binding activity,
unacetylated or acetylated MBP-GCMa was incubated with a
radiolabeled pGBS probe in an EMSA. We first performed in
vitro acetylation reactions with 1.5 ?g of MBP-GCMa and
increasing amounts (100, 200, 400, and 800 ng) of CBP in the
presence or absence of Ac-CoA. The level of acetylated MBP-
GCMa increased with increasing amounts of CBP and reached
a plateau at 400 ng of CBP in the presence of Ac-CoA (Fig.
5C). A portion of each reaction was used for band-shift reac-
tions to compare the DNA-binding activity of acetylated and
unacetylated MBP-GCMa. As shown in Fig. 5C, although the
fraction of acetylated MBP-GCMa was gradually increased in
the total amount of MBP-GCMa used in the band-shift reac-
tions, we did not observe a significant difference in the forma-
tion of DNA-protein complex for acetylated MBP-GCMa
compared to unacetylated MBP-GCMa. Taken together, these
results suggested that GCMa is an acetylation substrate of
CBP. GCMa acetylation by CBP does not significantly affect
the DNA-binding activity of GCMa.
CBP-mediated acetylation of GCMa increases the protein
stability of GCMa. We further examined whether CBP could
acetylate GCMa in vivo. Since GCMa has a similar mobility to
the heavy chain of immunoglobulin G, which may impede
acetylation analysis with Ac-K MAb, we therefore constructed
the Gal4-GCMa fusion expression construct, pGal4-GCMa-
Flag. 293T cells were cotransfected with different combinations
pPKAcata. As shown in Fig. 6A, acetylation of Gal4-GCMa-
Flag was detected when pCBP-HA was cotransfected. More-
over, this acetylation was further enhanced in the presence of
PKAcata (Fig. 6A, compare lanes 3 and 4). The observed
acetylation depended on the HAT activity of CBP because the
HAT-null mutant CBPHAT?-HA failed to acetylate GCMa
(Fig. 6A, lane 5). Interestingly, we also observed an increased
protein level of Gal4-GCMa-Flag in 293T cells cotransfected
with pGal4-GCMa-Flag, pCBP-HA, and pPKAcata (Fig. 6A,
lane 4). This was unlikely to be due to differential transfection
efficiencies because the level of the internal control protein,
HA-EGFP, was similar in each transfection group. Therefore,
we speculated that acetylation of GCMa by CBP increases the
protein stability of GCMa. To test this hypothesis, pulse-chase
experiments were performed in 293T cells transfected with
pHA-GCMa alone, pHA-GCMa and pCBP-Flag, or pHA-
GCMa and pCBP-Flag plus pPKAcata. As shown in Fig. 6B,
HA-GCMa maintained a half-life of ca. 90 min, which was
FIG. 4. Mapping of the interacting domains of GCMa and CBP. (A) CBP interacts with the GCM motif and the C-terminal TAD of GCMa.
(B) GCMa interacts with a region from the N terminus to the C/H1 domain of CBP, as well as a partial HAT domain and the C/H3 domain of
CBP. 293T cells were transfected with 5 ?g of pCBP-HA and 2 ?g of pHA-GCMa, respectively. At 48 h posttransfection, cells were harvested,
and 200 ?g of WCL was incubated with 2.5 ?g of the indicated GST fusion protein for GST pull-down assays as described in Materials and
Methods. Western analyses of 1/20 of whole-cell lysate (WCL) in panel A and 1/50 of WCL in panel B were performed. The lower panels are
Coomassie brilliant blue stainings of GST fusion proteins used in pull-down assays.
VOL. 25, 2005ACETYLATION OF GCMa INCREASES ITS PROTEIN STABILITY8407
further prolonged in the presence of Flag-CBP or Flag-CBP
Recently, we demonstrated that GCMa can be ubiquitinated
and degraded by the 26S proteasome (43). Since both ubiquiti-
nation and acetylation occur in the ε-amino group of lysine resi-
dues in substrate proteins, we now tested whether ubiquitination
of GCMa could be counteracted upon GCMa acetylation by
pGCMa-Myc, pHA-Ub, pCBP-Flag, and pPKAcata. The level of
ubiquitinated GCMa-Myc was analyzed by immunoprecipitation
with Myc MAb and immunoblotting with HA MAb. As shown in
Fig. 6C, the level of ubiquitinated GCMa-Myc was decreased in
the presence of pPKAcata and was further decreased in the pres-
ence of CBP or CBP plus PKAcata. Taken together, these results
suggest that CBP-mediated acetylation of GCMa prevents
ubiquitination of GCMa and thereby increases the protein
stability of GCMa.
Identification of CBP acetylation sites in GCMa. Inspection
of GCMa protein sequence revealed 28 lysine residues as po-
tential acetylation sites for CBP. Therefore, we now attempted
to identify the CBP acetylation sites by first characterizing the
domains in GCMa acetylated by CBP. We performed in vitro
acetylation assays with recombinant Flag-CBP and a series of
GST-GCMa fusion proteins. As shown in Fig. 7 (left panel),
CBP-mediated acetylation was detected in most domains
(amino acids 1 to 220, 1 to 300, 300 to 436, and 349 to 436)
covering the whole GCMa polypeptide except the domain of
amino acids 167 to 349. Therefore, the CBP acetylation sites in
GCMa were localized to its N-terminal domain of amino acids
1 to 167 and C-terminal domain of amino acids 349 to 436,
which also well correlated with the CBP-interacting domains in
In addition, we also performed acetylation analysis of GCMa
in vivo by cotransfecting 293T cells with pCBP-HA, pPKAcata,
and truncated pGal4-GCMa-Flag expression plasmids contain-
ing different regions of GCMa. As shown in Fig. 7A (right
panel), among the three domains in the C terminus of GCMa
(amino acids 300 to 349, 349 to 436, and 300 to 436) tested, two
(amino acids 300 to 436 and amino acids 349 to 436) were
found to be acetylated by CBP. Surprisingly, unlike the in vitro
acetylation results, pGal4-GCMa-Flag expression plasmids en-
coding the N-terminal regions of GCMa were not acetylated by
CBP. Based on these in vivo results, the CBP acetylation sites
in GCMa were localized to its C-terminal domain of amino
acids 349 to 436.
Since the GCMa N-terminal domain of amino acids 1 to 167
harbors a DNA-binding domain motif, but acetylation of re-
combinant GCMa proteins by CBP did not change the DNA-
binding activity of GCMa (Fig. 5C), we concentrated on iden-
tifying CBP acetylation sites in the C terminus of GCMa, which
FIG. 5. In vitro and in vivo acetylation of GCMa. (A) In vitro acetylation of GCMa by CBP. 1.5 ?g of MBP or MBP-GCMa fusion protein was
incubated with 150 ng of recombinant Flag-CBP protein in a reaction mixture with or without Ac-CoA. The reaction mixtures were analyzed by
immunoblotting with Ac-K MAb and MBP MAb, respectively. (B) In vivo acetylation of GCMa in placental cells. Stable BeWo cells expressing
HA-GCMa were mock treated or treated with 10 ?M forskolin alone or together with 50 ng of TSA/ml for 24 h. Cells were then harvested for
the acetylation analysis of HA-GCMa as described in Materials and Methods. (C) Acetylation of GCMa does not significantly affect the
DNA-binding activity of GCMa. A total of 1.5 ?g of MBP or MBP-GCMa fusion protein was incubated with the indicated amount of recombinant
Flag-CBP protein in a reaction mixture with or without Ac-CoA. One-thirtieth of each reaction mixture was analyzed by EMSA with a32P-labeled
pGBS oligonucleotide. The complex of MBP-GCMa and pGBS was further verified by supershift reactions with GCMa antibody. For comparison,
the levels of unacetylated and acetylated MBP-GCMa used for EMSA were analyzed by immunoblotting with MBP MAb and Ac-K MAb.
8408CHANG ET AL.MOL. CELL. BIOL.
also harbors a C-terminal TAD. Inspection of the protein
sequence in amino acids 300 to 436 of GCMa revealed four
potential lysine residues that could be acetylated, lysine349,
lysine367, lysine406, and lysine409. To identify which of these
lysine residues was actually acetylated by CBP, we constructed
mutant pGal4-GCMa-Flag(300-436) expression plasmids har-
boring single or combined lysine to arginine mutations in the
four lysine residues and tested their susceptibilities to CBP-
mediated acetylation in 293T cells. As shown in Fig. 7B, com-
pared to wild-type Gal4-GCMa-Flag(300-436), mutations at
lysine367, lysine406, or lysine409residues, but not lysine349, resulted
in reduced acetylation of mutant Gal4-GCMa-Flag(300-436)
by CBP. Moreover, combined mutation of both lysine406and
lysine409residues in Gal4-GCMa-Flag(300-436) (K2R) com-
pletely eliminated its acetylation by CBP (Fig. 7B). Similarly,
combined mutation of lysine367, lysine406, and lysine409resi-
dues (K3R) also eliminated its acetylation by CBP (data not
shown). The observed differential acetylation of wild-type and
mutant Gal4-GCMa-Flag(300-436) was not due to differential
interactions of CBP with wild-type and mutant Gal4-GCMa-
Flag(300-436) because a similar level of interaction was de-
tected in coimmunoprecipitation experiments (data not
shown). Taken together, these results suggested that of the
three CBP acetylation sites (lysine367, lysine406, and lysine409)
in the C-terminal TAD of GCMa, lysine406and lysine409are
the primary ones.
CBP enhances the transcriptional activity of GCMa via its
HAT and coactivator activities. We now tested whether CBP-
mediated acetylation affects the activity of GCMa C-terminal
TAD. CV1 cells, which have a very low level of endogenous
CBP (47), were cotransfected with pG5LUC, pGal4-GCMa-
Flag(300-436), and pCBP-HA or mutant pCBPHAT?-HA.
pG5LUC is a luciferase reporter plasmid containing five tan-
dem copies of a GAL4 binding site. As shown in Fig. 8A (left
panel), CBP significantly, whereas CBPHAT?only marginally,
increased the transcriptional activity of Gal4-GCMa-Flag(300-
436). When the level of Gal4-GCMa-Flag(300-436) in each
cotransfection group was analyzed, coexpression with CBP-
HA, but not CBPHAT?-HA, also increased the protein level of
Gal4-GCMa-Flag(300-436) (Fig. 8A, right panel). Therefore,
CBP-driven acetylation may stabilize Gal4-GCMa-Flag(300-
436) and enhances the transcriptional activity of Gal4-GCMa-
Flag(300-436). Correlatively, the enhancement effect of CBP
FIG. 6. CBP acetylates GCMa in vivo and increases GCMa protein stability. (A) 293T cells were transfected with 3 ?g of pGal4-GCMa-Flag
alone or together with the indicated combinations of 0.1 ?g of pPKAcata, 3 ?g of pCBP-HA, and 3 ?g of pCBPHAT?-HA. As an internal control
of transfection efficiency, 0.3 ?g of pHA-EGFP was also included in all transfection groups. At 48 h posttransfection, cells were harvested for
acetylation analysis as described in Materials and Methods. The asterisk, arrow, and arrowhead indicate the heavy chain of IgG, the HA-EGFP
protein, and the wild-type or mutant CBP-HA protein, respectively. Of note, CBP-HA further increased the level of acetylated Gal4-GCMa-Flag
protein in the presence of PKAcata. (B) CBP-mediated acetylation increases the protein stability of GCMa. 293T cells were transfected with 3 ?g
of pHA-GCMa alone or together with 3 ?g of pCBP-Flag or 3 ?g of pCBP-Flag plus 0.1 ?g of pPKAcata. At 36 h posttransfection, cells were
analyzed by pulse-chase experiments as described in Materials and Methods. The lower panel shows the quantification of pulse-chase experiments
by the bioimaging analyzer BAS-1500. Symbols: ■, pHA-GCMa plus empty vector; }, pHA-GCMa plus pCBP-Flag; Œ, pHA-GCMa plus
pCBP-Flag plus pPKAcata. (C) Acetylation of GCMa by CBP prevents GCMa from ubiquitination. 293T cells were transfected with the indicated
combinations of 7 ?g of pGCMa-Myc, 7 ?g of pHA-Ub, 3.5 ?g of pCBP-Flag, and 1 ?g of pPKAcata. At 24 h posttransfection, cells were treated
with 40 ?M MG132 for another 16 h, followed by immunoprecipitation with Myc MAb and immunoblotting with HA MAb or Myc MAb. The
upper and lower panels indicate the levels of HA-ubiquitinated GCMa-Myc and total GCMa-Myc in the immune complexes, respectively.
VOL. 25, 2005 ACETYLATION OF GCMa INCREASES ITS PROTEIN STABILITY8409
on GCMa-upregulated syncytin promoter activity was also
stronger than that of CBPHAT?in JAR cells (Fig. 3C).
We further tested whether CBP-mediated acetylation of ly-
sine367, lysine406, and lysine409in GCMa affects its TAD activ-
ity. CV1 cells were transfected with different combinations of
pG5LUC, pGal4-GCMa-Flag(300-436), K2R, K3R, and pCBP-
HA. Both K2R and K3R had a lower transcriptional activity than
Gal4-GCMa-Flag(300-436) in the absence CBP (Fig. 8B, left
panel). When cotransfected with pCBP-HA, CBP enhanced the
transcriptional activity of Gal4-GCMa-Flag(300-436), K2R, and
K3R, with the highest response being that of Gal4-GCMa-
Flag(300-436) (Fig. 8B, left panel). Since CBP was unlikely to
have acetylated K2R and K3R and CBP did not increase the
protein levels of K2R and K3R as it did for the wild type (Fig. 8B,
right panel), the mutated lysine residues may play important roles
in maintaining the transcriptional activity and the protein stability
of GCMa C-terminal TAD. Moreover, CBP’s enhancement of
due to the coactivator activity of CBP. Taken together, these
results suggested that lysine367, lysine406, and lysine409in the
GCMa C-terminal TAD modulate its transcriptional activity and
that CBP-mediated acetylation of these residues increases its pro-
tein stability with a concomitant increase in transcriptional acti-
GCMa ubiquitination is regulated by CBP-mediated acet-
ylation of the lysine367, lysine406, and lysine409residues in
GCMa. We next investigated whether acetylation of lysine367,
lysine406, and lysine409by CBP has any effect on GCMa ubiq-
uitination. We first assayed CBP-mediated acetylation of full-
length wild-type and lysine-to-arginine mutant GCMa by trans-
FIG. 7. Characterization of CBP-mediated GCMa acetylation sites. (A) Mapping of GCMa domains acetylated by CBP. To map the acetylation
domains in GCMa in vitro (left panel), 0.2 ?g of GST or the indicated GST-GCMa fusion protein was incubated with 0.4 ?g of Flag-CBP and
analyzed by in vitro acetylation reactions. To map the acetylation domains in GCMa in vivo (right panel), 293T cells were transfected with 0.5 ?g
of pCBP-HA, 0.1 ?g of pPKAcata, and 2 ?g of the indicated pGal4-GCMa-Flag expression plasmids for truncated GCMa proteins. At 48 h
posttransfection, cells were harvested for acetylation analysis as described in Materials and Methods. The levels of immunoprecipitated Gal4-
GCMa-Flag truncated proteins are given (lower right panel). (B) Mapping of CBP acetylation sites in the GCMa C-terminal TAD. 293T cells were
transfected with 0.5 ?g of pCBP-HA, 0.1 ?g of pPKAcata, and 2 ?g of the indicated wild-type or mutant pGal4-GCMa-Flag(300-436) expression
plasmid. At 48 h posttransfection, cells were harvested for acetylation analysis as described in Materials and Methods. The levels of immuno-
precipitated wild-type and mutant Gal4-GCMa-Flag(300-436) proteins are shown in the lower panel.
8410CHANG ET AL.MOL. CELL. BIOL.
fecting 293T cells with pCBP-Flag and wild-type pGCMa-Myc
or mutant pGCMa-Myc-K349R, -K2R, and -K3R. As shown in
Fig. 9A, acetylation of wild-type GCMa-Myc was specifically
detected when CBP-Flag, but not CBPHAT?-Flag, was coex-
pressed, suggesting that this acetylation was dependent on the
HAT activity of CBP. Acetylation of wild-type GCMa-Myc and
mutant GCMa-Myc-K349R by CBP was detected to a similar
level, whereas mutants GCMa-Myc-K2R and -K3R were only
weakly acetylated by CBP (Fig. 9A). The lysine-to-arginine
mutation did not have an adverse effect on the interaction
between CBP and the individual mutant GCMa-Myc com-
pared to wild-type GCMa-Myc (Fig. 9A). These results sug-
gested that lysine367, lysine406, and lysine409are the major CBP
acetylation sites in the full-length GCMa. Moreover, there are
other lysine residues functioning as minor CBP acetylation
sites because weak acetylation signals of GCMa-Myc-K2R and
-K3R were still detected in the presence of CBP.
Subsequently, we tested the effect of acetylation of lysine367,
lysine406, and lysine409on GCMa ubiquitination. We compared
ubiquitination of wild-type and lysine-to-arginine mutant
GCMa by transfecting 293T cells with different combinations
of pCBP-Flag, pHA-Ub, pGCMa-Myc, and pGCMa-Myc-
K349R, -K2R, and -K3R. As shown in Fig. 9B, ubiquitination
of wild-type and all mutant GCMa was detected to a similar
level in the absence of CBP. Therefore, the three major acet-
ylation sites and other lysine residues (possibly including the
minor acetylation sites) are susceptible to ubiquitination when
they are not acetylated by CBP. Interestingly, ubiquitination of
wild-type GCMa-Myc and mutant GCMa-Myc-K349R, but not
GCMa-Myc-K2R and -K3R, was counteracted in the presence
of CBP (Fig. 9B). Therefore, when the major acetylation sites
are not acetylated or are mutated into arginines (as in GCMa-
Myc-K3R), other lysine residues (possibly including the minor
acetylation sites) are more susceptible to ubiquitination than to
CBP-mediated acetylation. These results suggested that acet-
ylation of lysine367, lysine406, and lysine409significantly protects
GCMa from ubiquitination.
Trophoblastic fusion is essential for formation of the syncy-
tiotrophoblast layer during placental development (5). It has
long been known that fusion of cultured placental cells can be
stimulated by forskolin (29, 40). However, the underlying
mechanism of this phenomenon remained elusive until two
recent observations shed new light on it. First, a placenta-
specific fusogenic protein termed syncytin was identified and
shown to mediate fusion of cultured placental cells (33). Sec-
ond, forskolin was shown to stimulate expression of the syncy-
tin gene in placental cells (33). These observations suggest that
forskolin stimulates placental cell fusion via transcriptional
regulation of syncytin gene expression.
FIG. 8. CBP-mediated acetylation enhances GCMa-mediated transcriptional activation by increasing its protein stability. (A) Acetylation of
GCMa TAD by CBP increases its transcriptional activity and protein stability. CV1 cells were cotransfected with 0.5 ?g of pG5LUC, 0.5 ?g of
pGal4-GCMa-Flag(300-436), and 0.5 ?g of pCBP-HA or mutant pCBPHAT?-HA. Mean values and the SEM obtained from three independent
transfection experiments are provided. The protein level of Gal4-GCMa-Flag(300-436) was analyzed by immunoblotting with FLAG MAb (right
panel). (B) Roles of lysine367, lysine406, and lysine409in the CBP-enhanced transcriptional activity of GCMa C-terminal TAD. CV1 cells were
transfected with different combinations of 0.5 ?g of pGal4-GCMa-Flag(300-436), pGal4-GCMa-Flag(300-436)K2R, pGal4-GCMa-Flag(300-
436)K3R, and pCBP-HA. Mean values and the SEM obtained from three independent transfection experiments are listed. The protein level of
Gal4-GCMa-Flag(300-436) and its mutants was analyzed by immunoblotting with FLAG MAb (right panel).
VOL. 25, 2005 ACETYLATION OF GCMa INCREASES ITS PROTEIN STABILITY8411
Earlier we had shown that the placental transcription factor,
GCMa, regulates expression of syncytin gene (45). In the
present study, we further confirmed that forskolin is able to
stimulate GCMa-mediated transcriptional activation via PKA
and CBP, two key components of the cAMP signaling pathway.
Several lines of evidence support that PKA modulates the
transcriptional activity of GCMa. First, forskolin was able to
stimulate the transcriptional activity of GCMa in transient-
expression experiments, while this effect was negated by the
addition of H89, an inhibitor of cAMP-dependent PKA. Sec-
ond, this forskolin-induced upregulation of GCMa transcrip-
tional activity was also inhibited by the peptide inhibitor, PKI,
and the dominant-negative regulatory subunit of PKA,
RevAB, both of which specifically blocked the catalytic activity
of PKA. Third, the transcriptional activity of endogenous and
ectopic GCMa was enhanced in transient-expression experi-
ments in the presence of the PKA catalytic subunit. Although
we identified several major PKA phosphorylation sites in
GCMa by in vitro kinase assays, serine-to-alanine mutagenesis
of these sites did not have any adverse effect on PKA-upregu-
lated GCMa transcriptional activity (see Fig. S1 in the supple-
mental material). Two possibilities may explain this observa-
tion. First, cryptic PKA phosphorylation sites may exist in
GCMa that were not identified by our in vitro assay, and these
may play important roles in regulation of GCMa activity. A
more sensitive analytical tool such as mass spectrometry could
characterize these cryptic PKA phosphorylation sites in future
investigations. Second, PKA may indirectly modulate GCMa
activity by cross-talking with other signaling pathways. Since
the MEK inhibitor PD 98059 and the p38 mitogen-activated
protein kinase inhibitor SB 203580 did not alter the effect of
forskolin on GCMa activity (Fig. 1), the role of other signaling
pathways on direct regulation of GCMa activity needs to be
In order to ascertain whether PKA phosphorylation of
GCMa directly enhances GCMa transcriptional activity, we
investigated the possibility that PKA may regulate the interac-
tion between GCMa and other effectors in the cAMP/PKA
signaling pathway. We demonstrated that CBP interacts with
and acetylates GCMa and that PKA can positively regulate
both events. In addition, PKA very likely modifies the phos-
phorylation status of GCMa to facilitate an interaction be-
tween GCMa and CBP because treatment of GCMa with
?-PPase decreases the interaction between GCMa and CBP.
We are currently investigating how PKA modification of
GCMa phosphorylation increases the CBP-binding activity of
GCMa. Our results of this study are similar to what is known
about the pituitary-specific transcription factor, Pit-1, which is
a POU domain-containing protein. Pit-1 is required to mediate
PKA-regulated expression of growth hormone (GH), prolac-
tin, GH-releasing hormone receptor, and thyrotropin ?-sub-
unit genes (31, 46). Although Pit-1 is a direct target of PKA,
mutation of the PKA phosphorylation sites in Pit-1 does not
affect PKA-regulated Pit-1 activity (13, 41). Instead, PKA-
regulated Pit-1 activity has been attributed to a physical inter-
action between CBP and Pit-1 (41, 46). Xu et al. (41) have
further demonstrated that a C-terminal consensus PKA site in
CBP is required for PKA to regulate Pit-1 activity. However,
Zanger et al. (46) have reported contradictory results showing
that this consensus PKA site in CBP does not play any signif-
icant role in PKA-regulated Pit-1 activity. Nevertheless, it is a
common mechanism by which cAMP/PKA signaling pathway
regulates GCMa and Pit-1 activity via a direct interaction be-
tween CBP and GCMa or Pit-1.
FIG. 9. GCMa ubiquitination is regulated by CBP-mediated acetylation. (A) Lysine367, lysine406, and lysine409are the major CBP acetylation
sites in GCMa. 293T cells were transfected with 4 ?g of wild-type or mutant pGCMa-Myc and 1 ?g of pCBP-Flag or pCBPHAT?-Flag. At 48 h
posttransfection, cells were harvested for acetylation analysis as described in Materials and Methods. In a separate set of experiments, cells were
harvested for coimmunoprecipitation assays to verify similar levels of interaction between CBP-Flag and wild-type or mutant GCMa-Myc.
(B) Acetylation of lysine367, lysine406, and lysine409counteracts GCMa ubiquitination. Ubiquitination of wild-type and mutant GCMa-Myc in
transfected 293T cells was analyzed as in Fig. 6C.
8412 CHANG ET AL.MOL. CELL. BIOL.
Protein acetylation regulates the biological activities of his-
tone and nonhistone proteins. Acetylation occurs on all core
histones (H3, H4, H2A, and H2B) at the evolutionarily con-
served lysine residues located at the N terminus and conse-
quently changes chromatin architecture thereby increasing
transcriptional activity. In general, acetylation of transcription
factors can alter their activities at various levels, including
DNA binding, transcriptional activity, their interactions with
other proteins, nuclear transport, and protein turnover. A va-
riety of nonhistone proteins have been demonstrated to be
acetylated by CBP, including p53, c-Myc, and NF-?B, to name
a few (9, 12). In the present study, we also identified GCMa as
a bona fide CBP acetylation substrate. Acetylation of GCMa
by CBP is stimulated by PKA, which is most likely due to the
increased interaction between GCMa and CBP. Moreover,
acetylation of GCMa by CBP increases the protein stability of
GCMa by blocking GCMa ubiquitination. Although Pit-1 can
interact with CBP, it is a poor CBP acetylation substrate and
requires an additional factor to enhance its acetylation by CBP
(34). Although CBP efficiently acetylates GCMa in vitro, the
possibility of additional factors participating in CBP-mediated
acetylation of GCMa in vivo cannot be ruled out since CBP can
associate with other acetyltransferases, including P/CAF (44)
and GCN5 (42).
In terms of transcriptional activation, we found that CBP
enhances the transcriptional activity of GCMa C-terminal
TAD via its HAT and coactivator activities. Several lines of
evidence support this conclusion. First, the transcriptional ac-
tivation mediated by Gal4-GCMa-Flag(300-436) was signifi-
cantly enhanced by CBP, but not by CBPHAT?, with a corre-
sponding increase in protein level of Gal4-GCMa-Flag(300-
436). Correlatively, the full enhancement effect of CBP on
GCMa-regulated syncytin promoter activation requires the
acetyltransferase activity of CBP. Second, acetylation of
GCMa by CBP prevented GCMa from ubiquitination. Third,
the CBP-enhanced transcriptional activity of K3R, which had
the CBP acetylation sites (lysine367, lysine406, and lysine409)
mutated was still detected, although to a lesser degree. There-
fore, stabilization of GCMa via the HAT activity of CBP, and
connection between GCMa and the transcription machinery
via the coactivator activity of CBP underlies the observed CBP-
enhanced transcriptional activity of GCMa. Unfortunately,
whether CBP-mediated acetylation of the C-terminal TAD of
GCMa upregulates its transcriptional activity is complicated by
the acetylation-dependent protein stabilization of TAD. An in
vitro transcription assay for acetylated and unacetylated
GCMa may help to resolve this issue.
Although GCMa contains 28 lysine residues as potential
acetylation and ubiquitination sites, lysine367, lysine406, and
lysine409were identified as the major CBP acetylation sites but
not the major ubiquitination sites because changing these ly-
sine residues into arginines in GCMa-Myc-K3R significantly
reduced its level of acetylation mediated by CBP but not ubiq-
uitination (Fig. 9A and B). Moreover, when the major acet-
ylation sites are not acetylated or are mutated into arginines
(as in GCMa-Myc-K3R), other lysine residues (possibly includ-
ing the minor acetylation sites) are more susceptible to ubiq-
uitination than to CBP-mediated acetylation. The ETS protein,
ER81, is stabilized by p300-mediated acetylation at lysine33
and lysine116(18). A conformational change in the acetylated
ER81 or a shielding effect from acetylated ER81-associated
factors has been proposed to prevent ER81 from ubiquitina-
tion. Whether similar mechanisms are applicable to the stabi-
lization of acetylated GCMa is still an open question. How-
ever, considering the tremendously counteracting effect of
CBP-mediated acetylation on ubiquitination of wild-type
GCMa-Myc and mutant GCMa-Myc-K349R, it is feasible to
speculate that acetylation of lysine367, lysine406, and lysine409
residues may either further promote acetylation or simply pro-
tect ubiquitination of other lysine residues (perhaps including
the minor acetylation sites) in GCMa.
Previous studies have demonstrated a good correlation be-
tween increased cAMP synthesis and cell fusion in BeWo cells
treated with forskolin (40). Moreover, addition of cAMP ana-
logues to primary-culture trophoblast cells led to stimulation of
syncytium formation and redistribution of PKA type II? in
syncytial cells (29). Recently, Frendo et al. (17) have demon-
strated that suppression of syncytin expression by antisense
oligonucleotides resulted in a decrease in fusion and differen-
tiation of primary-culture trophoblast cells. These observations
indicate that cAMP and PKA are upstream mediators and
syncytin is a downstream effector involved in trophoblastic
fusion. Since the interaction between syncytin and its recep-
tors, ASCT1 and -2, can be a rate-limiting step in the fusion
process, controlling expression of syncytin and its receptors by
mediators provides a hierarchical regulation of trophoblastic
fusion in placental development. Indeed, it is known that the
level of ASCT1 mRNA was low and that forskolin can induce
syncytin, but not ASCT2, gene expression in placental cells
(35). Therefore, regulation of syncytin gene expression is an
important step in forskolin-stimulated placental cell fusion.
Together with the fact that GCMa is a key factor regulating
syncytin gene expression, the importance of the forskolin-
cAMP/PKA-GCMa-syncytin signal transduction pathway in
controlling trophoblastic fusion during placental development
is evident. Although the effect of the cAMP-PKA signaling
pathway is multifaceted, and it is highly probable that addi-
tional mechanisms are involved in trophoblastic fusion induced
by this pathway, based on the results of the present study, we
propose a model for forskolin-stimulated placental cell fusion
as follows. Treatment of placental cells with forskolin activates
PKA, which in turn modifies the phosphorylation status of
GCMa, thereby facilitating an interaction between GCMa and
CBP. Concomitantly, the level of GCMa-CBP complex is in-
creased and so does the level of stabilized acetylated GCMa
protein, which further activates syncytin gene expression and
promotes placental cell fusion.
We thank Hsou-min Li for critical reading of the manuscript.
This study was supported by a grants to H.C. from the National
Science Council (94-2311-B-001-035) and Academia Sinica of Taiwan.
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