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J Biol Chem, Vol. 274, Issue 44, 31577-31582, October 29, 1999


Interaction and Functional Cooperation of PEBP2/CBF with Smads
SYNERGISTIC INDUCTION OF THE IMMUNOGLOBULIN GERMLINE Calpha PROMOTER*

Jun-ichi HanaiDagger §, Lin Feng Chen§, Tomohiko Kanno, Naoko Ohtani-Fujita, Woo Young Kim, Wei-Hui Guo, Takeshi ImamuraDagger , Yasuhiro IshidouDagger , Minoru FukuchiDagger , Meng-Jiao Shiparallel , Janet Stavnezerparallel **, Masahiro KawabataDagger , Kohei MiyazonoDagger ddager ddager , and Yoshiaki Ito §§

From the Dagger  Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 70-8455, Japan, the  Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, and the parallel  Department of Molecular Genetics and Microbiology, Graduate Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta ) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha  and beta  subunits. The alpha  subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha  subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-beta /activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2alpha C/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Calpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-beta /BMP signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Smad proteins are signal transducers for members of the transforming growth factor-beta (TGF-beta )1 superfamily, which includes TGF-beta s, activins, and bone morphogenetic proteins (BMPs) (1, 2). Smads are classified into three subgroups, i.e. receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads. Smad2 and Smad3 are R-Smads that transmit TGF-beta /activin signals, whereas Smad1, Smad5, and Smad8 act as R-Smads mediating BMP signals. Smad4 is the only Co-Smad identified in mammals. Upon ligand stimulation, R-Smads are phosphorylated by the serine/threonine kinase receptors, form complexes with Co-Smad, and translocate into the nucleus, where they cooperatively regulate the transcription of target genes with other transcription factors, including Xenopus FAST1 and its mammalian homologues (3-5) and also the c-Jun/c-Fos complex (6, 7). TGF-beta is a potent growth inhibitor for most cell types, including hematopoietic cells and lymphocytes. In addition, TGF-beta directs class switching to IgA in splenic B cells (8, 9). BMPs play important roles in early embryogenesis and in the induction of bone formation in vivo (10). It is thus important to identify and classify transcription factors that serve as nuclear targets of TGF-beta /BMP signals and regulate these biological events.

Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha  and beta  subunits (11, 12). Three mammalian alpha  subunits have been identified, termed PEBP2alpha A/CBFA1/AML3 (referred to as alpha A in this report), PEBP2alpha B/CBFA2/AML1 (alpha B), and PEBP2alpha C/CBFA3/AML2 (alpha C), whereas only a single beta  subunit (PEBP2beta /CBFB) with several spliced variants is present in mammals. The alpha  subunits of PEBP2, which contain the highly conserved Runt domain, are responsible for binding to DNA and transcription activity. In contrast, the beta  subunit does not bind to DNA by itself, but it enhances the DNA binding activity of the alpha  subunits by interacting via the Runt domain. PEBP2/CBF plays critical roles in growth and differentiation of cells in certain specific tissues, i.e. alpha A in bone formation (13-15) and alpha B in definitive hematopoiesis (16, 17); alpha C appears to be important in class switching to IgA because of its ability to activate the germline Ig Calpha promoter (18). Abnormalities of the PEBP2 genes are linked to human diseases. Mutations in one allele of the human PEBP2alpha A/CBFA1 gene cause human cleidocranial dysplasia syndrome (19, 20), whereas PEBP2alpha B/AML1 gene is frequently disrupted by chromosomal translocations in several types of human leukemia (11, 12).

PEBP2 has been shown to interact with several transcription factors and co-activators and support context-dependent transcription of target genes (21-23). Because BMPs and alpha A play critical roles in bone formation, and TGF-beta and alpha C in transcription of germline Ig alpha  transcripts required for IgA class switching, we examined the functional cooperation between the PEBP2alpha subunits and Smads. Our findings suggest that PEBP2alpha subunits and R-Smads cooperate to synergistically activate transcription in both the TGF-beta and BMP signaling pathways, thereby regulating the function of cells in specific tissues upon activation by TGF-beta -like factors.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- FLAG-pcDEF3 and 6Myc-pcDEF3 containing six tandem copies of the Myc-epitope tag were previously described (24, 25). The constructions of constitutively active forms of TGF-beta type I receptor (Tbeta R-I(TD)) and BMP-type IB receptor (BMPR-IB(QD)), Tbeta R-II, wild-type (WT) Smads, and Smad3(DE) were reported (24-26). The constructions of alpha A, alpha B, alpha C, and beta 2 have been described elsewhere (27-29).2 Deletion constructs of alpha C were prepared by a polymerase chain reaction-based approach. For construction of the isolated Ig Calpha /TGF-beta -responsive element (Tbeta RE) promoter reporter construct ((Tbeta RE)3-Lux) and its mutants, three tandemly repeated Tbeta REs (WT or mutant versions) of the Ig Calpha promoter were fused to the heterologous c-Fos (30) and luciferase reporters. All of the polymerase chain reaction products were sequenced.

Cell Culture and cDNA Transfection-- COS7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. A20.3 B lymphoma cells (18, 31) were cultured in RPMI 1640 with 10% fetal bovine serum, 50 µM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and antibiotics. P19 murine embryonal carcinoma cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with 10% fetal bovine serum and antibiotics (32, 33). For transient transfection, cells were transfected using FuGENE6 (Roche Molecular Biochemicals).

Immunoprecipitation and Immunoblotting-- COS7 cells were transiently transfected with expression constructs for PEBP2alpha subunits, Smads and constitutively active forms of type I receptors. Cells were then washed, scraped, and solubilized (25). Immunoprecipitation and immunoblotting using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) were performed as described (25).

Glutathione S-transferase (GST) Pull-down Assay-- A GST pull-down assay was performed as described previously (22). GST-fusion proteins containing the full-length Smad3 or the Mad homology (MH)1 or MH2 domain of Smad3 were expressed and purified as described (32). In vitro transcription and translation of C-terminal deletion constructs of alpha C were done using the TNT system (Promega) in the presence of [35S]methionine. GST-Smad3 (full-length), Smad3 (MH1), Smad3 (MH2), or GST bound to glutathione-Sepharose was mixed with alpha C proteins in 500 µl of Tris-buffered saline, pH 7.4, containing 0.5% Nonidet P-40 for 1 h and washed vigorously three times with 1 ml of the same buffer. After boiling in the SDS-sample buffer, they were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Luciferase Assay-- A20.3 B lymphocytes were transfected with the germline Ig Calpha promoter (18) together with the expression constructs for alpha C, Smads, and Tbeta R-I(TD). P19 murine embryonal carcinoma cells were transfected with WT or mutant versions of (Tbeta RE)3-Lux together with alpha C, Smads, and Tbeta R-I(TD). Firefly and Renilla luciferase activities were assayed with the dual luciferase assay system (Promega) using Lumat LB 9507 (EG&G Berthold). Firefly luciferase activity was normalized with respect to the Renilla luciferase activity.

Electrophoretic Mobility Shift Assay (EMSA)-- EMSA was performed as described (27) with minor modifications. Briefly, COS7 cells were transfected with a mixture of expression plasmids encoding Tbeta R-I, Tbeta R-II, Smads, alpha C, and beta 2. Whole-cell extracts were prepared, mixed in vitro in combinations as indicated if necessary, and used for EMSA with a 32P-labeled Tbeta RE.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of alpha  Subunits of PEBP2 with R-Smads in Vivo-- We first tested complex formation between alpha A and R-Smads activated by BMPs. alpha A interacted with Smad1 and Smad5, which were activated by, BMPR-IB(QD), a constitutively active form of BMPR-IB (Fig. 1A). Smad4 was co-immunoprecipitated with R-Smads activated by the receptors. Importantly, alpha A also interacted with Smad2 and Smad3 activated by Tbeta R-I(TD), an active Tbeta R-I.


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  		Fig. 1.   All three mammalian alpha  subunits of PEBP2 interact with R-Smads. A, COS7 cells were transfected with the indicated combinations of cDNAs encoding FLAG-tagged Smads, 6Myc-tagged Smad4, 6Myc-alpha A, and constitutively active forms of type I receptors (C.A. R-I). Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by immunoblotting (Blot) using anti-Myc antibody. alpha A and Smad4 co-immunoprecipitated with R-Smads are indicated. Expression levels of 6Myc-Smad4, 6Myc-alpha A, and FLAG-R-Smads are shown. B, COS7 cells were transfected with the indicated combinations of FLAG-Smad1 or -Smad3, 6Myc-alpha A, -alpha B, or -alpha C, and constitutively active forms of type I receptors. Complex formation between PEBP2alpha subunits and Smads was detected by anti-FLAG immunoprecipitation followed by anti-Myc immunoblotting. Expression of 6Myc-PEBP2alpha subunits and FLAG-Smads is indicated.

We therefore examined whether the other PEBP2alpha subunits associate with different R-Smads. Smad3 activated by Tbeta R-I(TD) formed complexes not only with alpha A but also with alpha B and alpha C (Fig. 1B). Smad1 activated by BMPR-IB(QD) also formed complexes with alpha A, alpha B, and alpha C. Other R-Smads, i.e. Smad2 activated by Tbeta R-I(TD) and Smad5 activated by BMPR-IB(QD), also interacted with all three alpha  subunits (data not shown). alpha B and alpha C formed complexes with Smad1 and Smad3 equally well, whereas alpha A associated more strongly with Smad1 and Smad5 than with Smad3 (Fig. 1, A and B). These results indicate that all three mammalian PEBP2alpha subunits can form complexes with R-Smads.

Interaction of alpha C with Smad3-- Because alpha C is predominantly induced by TGF-beta in B lymphocytes and is critical for the induction of the promoter for germline Ig Calpha transcripts upon TGF-beta stimulation (18), complex formation between alpha C and Smad3 was studied in detail. The alpha C/Smad3 complex was observed in the presence and absence of Tbeta R-I(TD) (Fig. 2A), and Smad4 interacted with Smad3 upon stimulation by Tbeta R-I(TD). The mode of interaction between alpha C and Smad3 was studied by GST pull-down assays using deletion constructs of these proteins. When a series of C-terminally truncated constructs of alpha C was examined, deletion of a C-terminal region (alpha C (1-283; see Fig. 2B)) corresponding to a part of the transcriptional activation domain (AD) identified in alpha B (27) resulted in a reduction of association with GST-Smad3, and interaction became undetectable by deletion of transcriptional AD (alpha C (1-234)) (Fig. 2B). Smads have highly conserved MH1 and MH2 domains in their N- and C-terminal regions, respectively (1, 2). A GST pull-down assay revealed that the MH2 domain bound to alpha C (Fig. 2C). In addition, the MH1 domain weakly interacted with alpha C, but the exact location in alpha C where MH1 interacts could not be determined unequivocally because of the weakness of the interaction.


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  		Fig. 2.   Interaction between alpha C and Smad3. A, COS7 cells were transfected with the indicated combinations of cDNAs encoding FLAG-tagged Smad3, 6Myc-Smad4, 6Myc-alpha C, and Tbeta R-I(TD)-HA. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting using anti-Myc antibody. alpha C and Smad4 co-immunoprecipitated (IP) with Smad3 are indicated. Expression levels of Smad4, alpha C, and Smad3 were confirmed. B, regions in alpha C proteins essential for their interaction were determined by GST pull-down assay. The structure of alpha C is shown in the upper panel. GST-Smad3 and GST alone were incubated with a series of C-terminal deletion constructs of alpha C. 10% of [35S]methionine-labeled proteins used for the assay were applied as controls (Input). C, the MH1 and MH2 domains of Smad3 were examined for the interaction with alpha C proteins by GST pull-down assay. GST-Smad3 (MH1), GST-Smad3 (MH2), and GST alone were incubated with C-terminal deletion constructs of alpha C. 25% of [35S]methionine-labeled proteins were applied as controls (Input).

Transcriptional Activation of the Germline Ig Calpha Promoter-- We next studied the functional consequence of R-Smad/PEBP2alpha interaction using the mouse Ig Calpha promoter. The promoter for mouse germline Ig Calpha transcripts has been shown to contain a TGF-beta -responsive element, Tbeta RE (34), in which two PEBP2alpha binding sites have recently been identified (18). The human germline Ig Calpha promoter was also shown to contain PEBP2alpha binding sites in its Tbeta RE (35). In addition, two potential Smad binding motifs (36-38) are found in the Tbeta RE (Fig. 3A). Moreover, an additional PEBP2alpha binding site and one Smad binding motif are observed between the Tbeta RE and the transcription initiation site. To determine the functional importance of these binding motifs, nucleotide mutations were introduced into the promoter, and a transcriptional response assay was performed using A20.3 B lymphocytes. As previously reported (18), TGF-beta activates the promoter, which is further enhanced by the presence of alpha C. Mutations in the Smad binding motifs in the Tbeta RE (Tbeta RE-mS) and those in the PEBP2alpha binding sites (Tbeta RE-mP) result in dramatic decreases in transcriptional activity (Fig. 3B). A complete loss of response was observed in the mSP mutant with mutations in all PEBP2alpha and Smad binding motifs, indicating that both of these binding motifs are essential for transcriptional activation.


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  		Fig. 3.   Participation of both PEBP2 and Smad proteins in activation of the Ig Calpha promoter by TGF-beta . A, Smad binding motifs (indicated as S1, S2, and S3) and PEBP2 binding sequences (indicated as P1, P2, and P3) in the WT Ig Calpha promoter (-130 to approximately +14) and nucleotide mutations (indicated by large capital boldface letters) were introduced in Tbeta RE-mS, Tbeta RE-mP and mSP mutant reporters are shown. B, A20.3 B lymphocytes were transfected with the WT or a mutant Ig Calpha promoter reporter construct (0.45 µg) and expression plasmids encoding Tbeta R-I(TD) (0.45 µg) and alpha C (0.45 µg) in the indicated combinations, and the luciferase activity was determined. C, A20.3 cells were transfected with the WT Ig Calpha reporter (0.45 µg) and expression plasmids encoding dominant-negative Smad3, Smad3(DE) (0.45 µg or 1.5 µg), Tbeta R-I(TD) (0.45 µg), and alpha C (0.45 µg) in the indicated combinations. D, A20.3 cells were transfected with the WT Ig Calpha reporter (0.45 µg) and expression plasmids encoding Smad2 (0.45 µg), Smad3 (0.45 µg), Smad4 (1.8 µg), and alpha C (0.45 µg) in combinations as indicated.

A dominant negative form of Smad3, Smad3(DE), which prevents the activation of both Smad2 and Smad3 by Tbeta R-I(TD) (26), inhibited the transcription induced by Tbeta R-I(TD) and alpha C (Fig. 3C). This finding suggests that transcription may be induced by the endogenous R-Smads activated by Tbeta R-I(TD). Moreover, co-transfection of Smad3 with alpha C strongly induced transcription from the Ig Calpha promoter (Fig. 3D) but not from the Ig Calpha promoter containing mutations in the Tbeta RE, as shown in Fig. 3A (data not shown). Interestingly, Smad2 did not significantly induce the transcription, probably because Smad2 is unable to bind to the Smad binding motifs (36-39).

Transcriptional Activation through Tbeta RE by alpha C and Smad3/4-- To further study the roles of alpha C and Smad3/4 in activating transcription, three tandemly repeated Tbeta REs (WT or mutant versions) of the Ig Calpha promoter were fused to the heterologous c-Fos promoter, and transcriptional activity was determined using transfected P19 embryonal carcinoma cells, which have very low levels of endogenous PEBP2alpha activity (32, 33). Similar to the results obtained with the natural Ig Calpha promoter using A20.3 B lymphocytes, transcriptional activity of (Tbeta RE-WT)3-Lux was mildly induced by Smad3 and -4, whereas the addition of Smad3/4 and alpha C in cells activated by Tbeta R-I(TD) greatly induced transcription (Fig. 4A). In contrast, mutant versions of (Tbeta RE)3-Lux, i.e. (Tbeta RE-mP)3-Lux and (Tbeta RE-mS)3-Lux, which have mutations in the two PEBP2 binding sites and two Smad binding motifs, respectively, did not respond to Tbeta R-I(TD), Smad3/4, or alpha C, indicating that both of these binding motifs are essential for transcriptional activation by the Smad3/alpha C complex.


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  		Fig. 4.   The AD of alpha C is important for the transcriptional activation in concert with Smad3/4 and Tbeta R-I(TD). A, (Tbeta RE-WT)3, (Tbeta RE-mP)3, and (Tbeta RE-mS)3 luciferase reporters have three copies of Tbeta RE from the WT, Tbeta RE-mP, and Tbeta RE-mS reporters, respectively (shown in Fig. 3A). P19 embryonic carcinoma cells were transfected with reporters (0.2 µg) and expression plasmids encoding Tbeta R-I(TD) (0.2 µg), Smad3 (0.1 µg), Smad4 (0.3 µg), and alpha C (0.1 µg) in the indicated combinations, and the luciferase activity was determined. B, P19 cells were transfected with (Tbeta RE-WT)3 luciferase reporter (0.2 µg) and expression plasmids encoding Tbeta R-I(TD) (0.2 µg), Smad3 (0.1 µg), Smad4 (0.3 µg), and a series of alpha C deletion constructs (0.3 µg) as indicated.

To determine the domain(s) in alpha C critical in the transcriptional activation in concert with Smads, a series of C-terminal deletions of alpha C was tested for transcription activity. alpha C mutants containing the transcriptional AD increased transcriptional activation in the presence of Tbeta R-I(TD) and Smad3/4; however, deletion of one-half of the AD resulted in a significant decrease in transcriptional response; complete loss of response was obtained with the mutants lacking the entire AD (Fig. 4B). This result indicates that the physical interaction between Smad3 and alpha C may be critical for the transcriptional activation through Tbeta RE (see Fig. 2B).

DNA Binding of the alpha C, beta 2, and Smad3 Complex-- The formation of DNA-binding complexes containing alpha C and Smad3 on the germline Calpha Tbeta RE DNA was studied by EMSA. The beta  subunit of PEBP2 (beta 2 isoform) was included in this assay to enhance the DNA binding of PEBP2. Smad3 activated by Tbeta R-I(TD) and alpha C independently formed DNA-binding complexes, which could be detected as slowly migrating complexes in EMSA (Fig. 5A, lanes 2 and 3, and B, lanes 3 and 4). In the presence of activated Smad3 and alpha C/beta 2, a more slowly migrating complex was formed both in vitro and in vivo (Fig. 5A, lanes 4 and 13, and B, lane 5). These complexes were super-shifted in the presence of corresponding antibodies to the epitope tags or an antibody to the beta  subunit, indicating that alpha C/beta 2 and Smad3 can concomitantly bind to DNA as a multimeric Smad3/alpha C/beta 2 complex.


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  		Fig. 5.   The presence of Smad3 and alpha C/beta 2 in the same complex bound to the Ig Calpha -Tbeta RE. A, lanes 1-12, COS7 cells were separately transfected with a mixture of expression plasmids encoding Tbeta R-I(TD) (0.4 µg), Tbeta R-II (0.2 µg), and FLAG-Smad3 (0.8 µg) or that containing 6Myc-alpha C (0.2 µg) and beta 2 (0.2 µg), or the cells were mock-transfected with an empty plasmid. Whole-cell extracts were mixed in vitro in combinations as indicated and used for EMSA with a 32P-labeled Tbeta RE. The total amount of extract was kept constant using the mock extract. For lanes 13-15, a whole-cell extract obtained from cells co-transfected with all of the above expression plasmids was used. The positions of Smad3, alpha C/beta 2, and Smad3/alpha C/beta 2 complexes are indicated on the left. Complexes super-shifted (SS) by the addition of anti-FLAG (F), anti-beta (beta ), or anti-Myc (M) antibody are indicated on the right. B, COS7 cells were transfected with expression plasmids encoding Tbeta R-I(TD) (0.4 µg), Tbeta R-II (0.2 µg), FLAG-Smad3 (0.8 µg), 6Myc-alpha C (0.2 µg), and beta 2 (0.2 µg) in the indicated combinations. Whole-cell extracts were subjected to EMSA using the 32P-labeled wild-type Tbeta RE or 32P-labeled oligonucleotides in which mutations (M) were introduced as indicated. The mutations correspond to those in Tbeta RE-mP and Tbeta RE-mS in Fig. 3A.

Mutations in the Smad binding motifs S1 or S2 resulted in the decrease or loss, respectively, of Smad3 and Smad3/alpha C/beta 2 bindings, but the binding of alpha C/beta 2 still remained (Fig. 5B). When the PEBP2alpha sites were mutated, a mutation in P2, but not in P1, disrupted the bindings of alpha C/beta 2 and Smad3/alpha C/beta 2, but binding of Smad3 was still detected. The Smad binding motifs and PEBP2alpha binding sites thus appear to be specific and sufficient for the binding of corresponding proteins, but both are required for the binding of the Smad3/alpha C/beta 2 complex to the Tbeta RE and for activation of the promoter by alpha C and Smad3.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The findings shown in the present study revealed that PEBP2alpha subunits and R-Smads specific for both TGF-beta and BMP signaling pathways form complexes together with Smad4 and that the complex formation appears to be critical for efficient transcriptional activation of target genes, including the germline Ig Calpha promoter. Our findings suggest that PEBP2 may function as a nuclear target of TGF-beta /BMP signaling pathways and that the biological effects of TGF-beta /BMP may be regulated by cooperation between Smads and PEBP2.

Smads have been reported to interact with various DNA-binding proteins as well as the transcriptional coactivator p300/CBP and co-repressor TGIF (40, 41). Because members of the TGF-beta superfamily have pleiotropic functions, interaction with various transcription factors may be required for Smads to exhibit specific effects in certain cell types. Many of these interacting partners, including c-Jun and the vitamin D receptor (6, 42), preferentially interact with Smad3, but Xenopus FAST1 and murine FAST2 have been shown to associate with Smad2 as well (3, 4, 39). Recently, a homeodomain transcription factor Hoxc-8 has been shown to bind to Smad1 (43). PEBP2 is conspicuous compared with these factors, because all three mammalian alpha  subunits of PEBP2 interact with all R-Smads tested in the present study. Recently, SIP1 has been shown to interact with all R-Smads; in contrast to the PEBP2 alpha  subunits, however, SIP1 is a transcriptional repressor, and interaction with R-Smads may lead to relief of repression of target genes by SIP1 (44).

Smad3 interacts with alpha C mainly through the MH2 domain, whereas the MH1 domain binds weakly to alpha C. Analysis by C-terminal deletion of alpha C revealed that the C-terminal region, including the transcriptional AD of alpha C, is required for efficient interaction with the MH2 domain of Smad3.

PEBP2 is a context-dependent transcription factor, requiring interacting partners for transcriptional activation, including Ets-1 (21, 22). In the germline Ig Calpha promoter, both PEBP2 and Smad binding sites are essential for transcriptional activation. In contrast, FAST1 binds to the Mix.2 gene promoter with high affinity, and therefore direct binding of Smads to DNA may be less important than in the Ig Calpha promoter (39). Thus, in certain other promoters to which PEBP2 binds with a high affinity together with other transcription factors, direct DNA binding of Smads may not be critical for cooperative transcriptional activation by PEBP2 and Smads.

Our present study revealed that PEBP2alpha subunits interact with R-Smads activated by TGF-beta /activin, as well as with those activated by BMPs, and that functional cooperation between alpha C and Smad3 is required for transcription driven by the germline Calpha promoter. Germline Ig alpha  transcripts are required for IgA class switching (45). Because members of the TGF-beta superfamily exhibit a wide variety of biological effects, it will be very important to examine whether PEBP2 is involved in these biological events as a nuclear target of Smads.

    ACKNOWLEDGEMENTS

We are grateful to Y. Udagawa and A. Nishihara for valuable discussions and help and to Y. Inada and Y. Yuuki for technical assistance.

    FOOTNOTES

* This work was supported by Grant-in-aid for Priority Areas on Cancer Research 09253220 (to Y. I.) and Grant-in-aid on Immune Disease Research 08282105 (to K. M.) from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These two authors contributed equally to the work.

Supported by National Institutes of Health Grant RO1 AI23283.

Supported by the Research for the Future Program of the Japan Society for the Promotion of Science. To whom correspondence may be addressed. Tel. and Fax: 81-3-3918-0342; E-mail: miyazono-ind@umin.ac.jp.

 To whom correspondence may also be addressed. Tel.: 81-75-751-4028; Fax: 81-75-752-3232; E-mail: yito@virus.kyoto-u.ac.jp.

2 Y.-W. Zhang and Y. Ito, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; BMP, bone morphogenetic protein; R-Smad, receptor-regulated Smad; Co-Smad, common-partner Smad; PEBP2, polyomavirus enhancer binding protein 2; CBF, core binding factor; Ig Calpha , immunoglobulin Calpha ; Tbeta R-I, TGF-beta type I receptor; BMPR-IB, BMP type IB receptor; WT, wild-type; Tbeta RE, TGF-beta -responsive element; GST, glutathione S-transferase; MH, Mad homology; EMSA, electrophoretic mobility shift assay; AD, activation domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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