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Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan,1 Graduate Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan,2 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
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ABSTRACT |
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INTRODUCTION |
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Two GCM homologues called GCMa and b have been identified 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 trophoblast 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 tissues, 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 motif, at the N terminus (1, 36). Recent crystallographic analysis of the GCM motif has revealed that it is a zinc-containing domain of ß sheets interacting with the major groove of its cognate DNA element, 5'-ATGCGGGT-3' (14). Transactivation domain has been identified in the carboxyl terminus of GCM proteins (1, 38). In terms of physiological function, Drosophila GCM1 regulates expression of repo (reverse polarity) and pnt (pointed) genes, the principal mediators of glial differentiation, 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 endogenous 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 posttranslationally cleaved into a surface (SU) subunit and a transmembrane (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 generally 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 activation and CBP is involved in this pathway by directly interacting 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.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Coimmunoprecipitation and pull-down assay.
To study the interaction between GCMa and CBP, 293T cells were cotransfected with the indicated combinations 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 phenylmethylsulfonyl fluoride. Approximately 180 µg of cell lysate was immunoprecipitated with GCMa antibody and protein A-conjugated agarose beads (Roche, Mannheim, 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 Na3VO4 at 30°C for 1 h, followed by incubation with 1 µg 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). Recombinant Flag-CBP and GCMa-Flag proteins were purified from Sf9 cells infected with recombinant baculovirus strains using the anti-FLAG M2-conjugated agarose 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 mouse CBP cDNA fragments were cloned into the pGEX6P-1 vector (Amersham 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 µg of the indicated cell lysate was incubated with 2.5 µg 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 x 106 BeWo cells were treated with or without 50 µM 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 containing 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'-CCTGTCTCTCAGTTGCAAGATAATTGC-3' for syncytin and 5'-AAAAGCGGGGAGAAAGTAGG-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 ß-actin transcripts were analyzed by Northern blotting with full-length syncytin and ß-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 expressed in BL21(DE3). Affinity purification of MBP-GCMa proteins was performed with maltose agarose beads (NEB) according to the manufacturer's instructions. For in vitro acetylation of GCMa, 1.5 µg of MBP or MBP-GCMa protein was incubated with 150 ng of the purified recombinant Flag-CBP proteins in a 30 µl 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 a 32P-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 expression 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 immunoprecipitation and Ac-K MAb for immunoblotting. To study the effect of acetylation 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 µCi 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 µg 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 µg of GST or the indicated GST-GCMa fusion protein was incubated with 0.4 µg of Flag-CBP under the same reaction conditions as described 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.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBP is involved in the PKA-upregulated GCMa activity. Examination of the protein sequence of GCMa by PROSITE (25) and NetPhos (6) suggested several PKA consensus phosphorylation sites in GCMa. Indeed, GCMa was phosphorylated by the catalytic subunit of PKA in vitro (see Fig. S1A to D in the supplemental material). However, transient-expression 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 forskolin-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 endogenous 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), suggesting that CBP is an important regulator for GCMa-mediated transcriptional activation.
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-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 assays. 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. However, 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 placental cells, previously established stable BeWo cells expressing 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 different sources (Fig. 3A). As a control, this interaction was not detected using an unrelated antibody against the GAL4 DNA-binding domain, Gal4 antibody, for immunoprecipitation (Fig. 3A).
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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-interacting domain(s) of GCMa, GST pull-down assays were performed by incubating a series of GST-GCMa fusion proteins with cell lysate of 293T cells transfected with pCBP-HA, followed 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-terminal TAD. Likewise, to map the GCMa-interacting domain(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.
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-amino group of lysine residues in substrate proteins, we now tested whether ubiquitination of GCMa could be counteracted upon GCMa acetylation by CBP. 293T cells were cotransfected with different combinations of 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 presence 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 potential 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 GCMa.
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 containing 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 encoding 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 recombinant GCMa proteins by CBP did not change the DNA-binding activity of GCMa (Fig. 5C), we concentrated on identifying CBP acetylation sites in the C terminus of GCMa, which 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 harboring 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, compared to wild-type Gal4-GCMa-Flag(300-436), mutations at lysine367, lysine406, or lysine409 residues, but not lysine349, resulted in reduced acetylation of mutant Gal4-GCMa-Flag(300-436) by CBP. Moreover, combined mutation of both lysine406 and lysine409 residues in Gal4-GCMa-Flag(300-436) (K2R) completely eliminated its acetylation by CBP (Fig. 7B). Similarly, combined mutation of lysine367, lysine406, and lysine409 residues (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 detected 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, lysine406 and lysine409 are 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 tandem 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 on GCMa-upregulated syncytin promoter activity was also stronger than that of CBPHAT– in JAR cells (Fig. 3C).
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GCMa ubiquitination is regulated by CBP-mediated acetylation of the lysine367, lysine406, and lysine409 residues in GCMa. We next investigated whether acetylation of lysine367, lysine406, and lysine409 by CBP has any effect on GCMa ubiquitination. We first assayed CBP-mediated acetylation of full-length wild-type and lysine-to-arginine mutant GCMa by transfecting 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 coexpressed, 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 compared to wild-type GCMa-Myc (Fig. 9A). These results suggested that lysine367, lysine406, and lysine409 are 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.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. Second, this forskolin-induced upregulation of GCMa transcriptional 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 experiments 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-upregulated GCMa transcriptional activity (see Fig. S1 in the supplemental material). Two possibilities may explain this observation. 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 investigated.
In order to ascertain whether PKA phosphorylation of GCMa directly enhances GCMa transcriptional activity, we investigated the possibility that PKA may regulate the interaction 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 phosphorylation status of GCMa to facilitate an interaction between 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), prolactin, GH-releasing hormone receptor, and thyrotropin ß-subunit 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 interaction 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 significant 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 between CBP and GCMa or Pit-1.
Protein acetylation regulates the biological activities of histone and nonhistone proteins. Acetylation occurs on all core histones (H3, H4, H2A, and H2B) at the evolutionarily conserved lysine residues located at the N terminus and consequently 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 variety 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 activation mediated by Gal4-GCMa-Flag(300-436) was significantly enhanced by CBP, but not by CBPHAT–, with a corresponding 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. Therefore, 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 lysine409 were identified as the major CBP acetylation sites but not the major ubiquitination sites because changing these lysine residues into arginines in GCMa-Myc-K3R significantly reduced its level of acetylation mediated by CBP but not ubiquitination (Fig. 9A and B). Moreover, 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. 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 ubiquitination. Whether similar mechanisms are applicable to the stabilization of acetylated GCMa is still an open question. However, 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 protect ubiquitination of other lysine residues (perhaps including the minor acetylation sites) in GCMa.
Previous studies have demonstrated a good correlation between increased cAMP synthesis and cell fusion in BeWo cells treated with forskolin (40). Moreover, addition of cAMP analogues 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 demonstrated that suppression of syncytin expression by antisense oligonucleotides resulted in a decrease in fusion and differentiation 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 receptors, 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 additional 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 increased and so does the level of stabilized acetylated GCMa protein, which further activates syncytin gene expression and promotes placental cell fusion.
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ACKNOWLEDGMENTS |
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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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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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REFERENCES |
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). In a separate set of experiments, 0.1 µg of pPKAcata was included in each transfection group (
). 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 µg of p(pGBS)4E1bCAT, 0.3 µg of pHA-GCMa, and 10 nM GL2 or CBP siRNA. At 24 h posttransfection, cells were mock-treated or treated with 30 µM 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 µg of pCBP-HA alone or together with 1 µg of pHA-GCMa or 1 µg of pHA-GCMa plus 0.1 µg 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 µg 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 
, pHA-GCMa plus pCBP-Flag; 
, 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 µg of pGCMa-Myc, 7 µg of pHA-Ub, 3.5 µg of pCBP-Flag, and 1 µg of pPKAcata. At 24 h posttransfection, cells were treated with 40 µM 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.
