p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter*

In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complexin vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a.a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1–1097 and 1678–2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

The Ets family of transcription factors includes a large number of proteins that perform diverse functions in the cell including the serum stimulation of the c-fos promoter (Elk-1/SAP-1 (1)), activation of herpes simplex virus immediate early promoters (GA-binding protein ␣ and ␤ (2)), regulation of immunoglobulin light chain enhancers (Pu.1/Spi-1 (3)), erythroid differentiation (4), and Drosophila development (5). Constitutively active mutant Ets proteins (v-Ets-1 and -2) are involved in cellular transformation (6,7). A characteristic feature of this class of proteins is a highly conserved 85-amino acid (a.a.) 1 DNA binding domain termed the Ets domain which contains a helix-turn-helix motif. The Ets domain binds to a GGAA purine-rich core sequence found in the promoters and enhancers of viral and cellular genes. Outside the DNA binding domain, the Ets family proteins share limited homology (8 -10).
Ets-1 and Ets-2 are ubiquitous proteins that share significant homology (11). The transactivation and the DNA binding (Ets) domains in both Ets-1 and -2 map to the N-and the C-terminal regions, respectively (12)(13)(14). The homologous regions include the Ets domain which is 95% conserved between Ets-1 and Ets-2 (11) and the Pointed domain located in the transactivation domain (15). The Pointed domain consists of approximately a 100-a.a. region that is conserved within a subgroup of Ets factors including Drosophila Ets factor Pointed P2 and Ets-1 and Ets-2 (15). The Pointed domain contains a MAP kinase phosphorylation site, and Pointed P2 is a target of Ras/MAP kinase signaling pathways in Drosophila (5). Ets-1 and Ets-2 are targets of the Ras signaling pathway (16), and Ras-mediated activation of Ets-1 and -2 transactivation activity requires phosphorylation of Ets-2 threonine 72 and the corresponding Ets-1 threonine 38, which are also conserved in Pointed P2 and Yan (a Drosophila repressor (17)(18)(19)). The conserved MAP kinase site (threonine 72) in Ets-2 is phosphorylated by MAP kinase (17). Ets-binding sites, as well as AP-1-binding sites, are often found in the promoters of Ras-induced genes (20), and Ets-2 and AP-1 cooperate in gene expression (21)(22)(23).
p300 and the CREB-binding protein (CBP) are two highly homologous, conserved nuclear phosphoproteins that function as transcriptional coactivators by bridging a very large number of DNA-bound transcription factors with basal transcription complex (24 -26). p300/CBP also binds to a number of proteins that are not transcription factors including viral oncogene products (27,28), SRC1 (29), Cdk2 (30), and a protein containing enzymatic activity as histone acetyltransferase, p300/CBPassociated factor (31). Recent studies show that p300/CBP also has enzyme activity as a histone acetyltransferase, linking chromatin remodeling with transcription (32). It has been suggested that the amount of p300/CBP in cells may be ratelimiting and that different transcription factors may compete for rate-limiting amounts of these coactivators and thus provide mechanisms for cross-talk in the regulation of gene expression (33,34). Targeted gene disruption studies have confirmed that p300 function is essential for normal embryonic cellular proliferation, morphogenesis, and development with double knockouts resulting in 100% embryonic lethality (35). Normal levels of CBP in these mice did not substitute for the p300 functions suggesting that the double knock-out phenotype may be either due to gene dosage effect or the loss of specific functions provided by p300. Likewise, haploinsufficiency of CBP gives rise to severe developmental abnormalities characteristic of the Rubinstein-Taybi syndrome, including mental retardation, craniofacial abnormalities, skeletal abnormalities, and increased cancer incidence (36). These studies suggest that both proteins are required for embryonic development.
Stromelysin is an important member of a family of matrix metalloproteases (MMPs) which degrade extracellular matrix during a variety of normal and pathological processes. In this paper, we show that two important members of the Ets family, Ets-1 and Ets-2, recruit p300/CBP in the activation of the stromelysin promoter, and this recruiting involves multiple protein-protein interactions. Consistent with these multiple interactions, the N-and the C-terminal halves of p300/CBP independently can coactivate Ets-2 to stimulate the stromelysin promoter. Other Ets family transcription factors do not cooperate with p300/CBP in the stimulation of the stromelysin promoter. We also show that mutation of the Ets-2 MAP kinase phosphorylation site, important for Ras-mediated regulation of Ets-2, does not affect its ability to bind to p300/CBP or its ability to cooperate with p300/CBP in the transcriptional activation.

EXPERIMENTAL PROCEDURES
Plasmids-pSK200 is a promoter-reporter plasmid that contains 542 bp upstream from the cap site of the human stromelysin gene fused to the CAT reporter gene. pSK201 and pSK202 are two mutant versions of pSK200 in which the AP-1 site, located between Ϫ64 and Ϫ71, is mutated (pSK201) or the two palindromic Ets sites deleted (pSK202) (37). E18pal is a promoter-reporter plasmid in which two palindromic Ets sites were cloned upstream of a c-fos minimal promoter followed by the CAT gene. c-fos minimal promoter contains 56 bp upstream of the cap site of the c-fos gene (16). Plasmids pFNEts-1 and pFNEts-2 are two expression plasmids that express mouse ets-1 and ets-2, respectively (17). 2 The Ets-1 and Ets-2 protein coding sequences in these plasmids are tagged with FLAG epitope followed by the SV40 large T nuclear localization signal (NLS) at their N-terminal end. These plasmids are in pcDNA3 background. Plasmid pFNEts-2A72 is a derivative of pFNEts-2 in which the MAP kinase substrate Thr at 72 residue is changed to Ala (T72A mutation (17)). Plasmid p300CHA expresses hemagglutinin epitope-tagged p300 from the CMV promoter (27). Plasmids GST-p300N, GST-p300M, and GST-p300C contain a.a. 1-596, 744 -1571, and 1572-2370, respectively, fused in frame with glutathione S-transferase coding sequences and kind gifts of Dr. D. Livingston of Harvard Medical School (27). p300N, p300M, and p300C are plasmids in which p300 cDNA sequences corresponding a.a. 1-746, 743-1572, and 1572-2414 respectively, cloned downstream of the T7 phage promoter in Bluescript SK. Full-length and truncated murine CBP coding sequences are expressed from a CMV promoter-based expression plasmid (pCMV2N3-T (38)) in which the coding sequences are tagged at their N-terminal end with two copies of SV40 nuclear localization signals followed by three copies of HA epitope (38). These plasmids are kind gifts of Dr. A. Harel-Bellan (CNRS, Villejuif, France).
Coimmunoprecipitation Assays-To identify the Ets-2⅐CBP complex in live cells, proliferating human 293 cells were pelleted and lysed on ice in a lysis buffer (27) for 40 min, and the protein was quantitated by the Bradford method (39). The cell lysate, equivalent to 2 mg of protein, was concentrated by the addition of 5 volumes of ice-cold acetone for 30 min on ice and then centrifuged for 30 min at 15,000 rpm. The pellet was dissolved in 500 l of the lysis buffer containing 0.3% Nonidet P-40, and it was then incubated with 40 l of the anti Ets-2 rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-351) overnight at 4°C. 50 l of the protein A-agarose beads were then added, and the incubation was continued for another 2 h at 4°C. The beads were then pelleted, washed three times with lysis buffer containing 0.3% Nonidet P-40, subjected to SDS-8% PAGE, and electrophoresed in Tris glycine buffer for 5 h as described (40). The immunoprecipitates were then assayed in Western blots using an anti-CBP rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-583). For the negative control experiment, 2 mg of the protein from the above lysate was immunoprecipitated using an anti-PKR polyclonal antibody raised in our laboratory. For direct immunoprecipitation, cell lysate corresponding to 500 g of the protein was incubated with anti-CBP antibody, and the immunoprecipitated polypeptides were subjected to SDS-PAGE followed by Western blot as described above. For the detection of the complexes containing various forms of Ets and the truncated forms of CBP, 100-cm dishes of 293 cells were transfected with 15 g of each of the plasmids indicated in the figure legends using the calcium phosphate precipitation method, and cell extracts were subjected to immunoprecipitations followed by Western blots as described above with appropriate antibodies.
GST Pull-down and Yeast Two-hybrid Assays-The construction of the GST-Ets2 plasmid expressing GST full-length fusion protein was described previously (41). Other GST fusion plasmids expressing GSTtruncated Ets-2 fusion proteins, as well as full-length Ets-1 proteins, were constructed by a polymerase chain reaction-mediated cloning approach using pSVK3-Ets2 and pSG5-Ets-1 expression plasmids as templates. 3 p300-Ets-2 interaction in yeast cells was assayed using the Matchmaker Two-hybrid system of CLONTECH (catalog number 1604-1), following the manufacturer's instructions. Briefly, DNA fragments coding for the p300 coding segments from a.a. 328 to 1000 and 962 to 1575 were fused in frame with Gal-4 DNA binding domain in the DNA binding domain plasmid pGBT9 (p300-1 and p300-2 respectively). cDNAs coding for the WT Ets-2, a mutant Ets-2 containing the T72A mutation and the AAAAA mutation (both created by using the Stratagene QuickChange site-directed mutagenesis kit), were cloned into the GAL-4 activation domain plasmid pACT-2. Yeast strain CG1945 was cotransformed with the two-hybrid vectors expressing segments of p300 and Ets-2 proteins, and the cells were selected for growth on SD medium lacking leucine, tryptophan, and histidine. The colonies were then amplified and qualitatively assayed for ␤-galactosidase activity using o-nitrophenyl ␤-D-galactopyranoside as a substrate (42).

p300/CBP Cooperates with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter and a Synthetic Promoter Containing Palindromic Ets-binding Sites
Transcription of the human stromelysin gene is driven by AP1/Fos and Ets-2 transcription factors and regulated by a variety of factors including 12-O-tetradecanoylphorbol-13-acetate, cytokines, growth factors, and protooncogenes (37,(43)(44)(45)(46). The AP1/Fos and Ets elements of the stromelysin promoter map between Ϫ64 and Ϫ71 and Ϫ201 and Ϫ218 with respect to cap site, respectively ( Fig. 1A) (37,46). The Ets site in this promoter consists of two copies of a motif similar to the polyoma virus enhancer A-binding protein-3 (PEA-3) site that binds to the Ets family transcription factors Ets-1 and -2 and stimulates transcription (44). To determine whether p300 would cooperate with Ets-2 in the activation of the stromelysin promoter, HeLa cells were cotransfected with a stromelysin promoter-reporter construct that contained 542 bp upstream from the cap site fused to CAT reporter gene (pSK200) and expression vectors encoding p300 (p300CHA (27)) and mouse ets-2 (pFNEts-2WT (16)), in combinations as shown in Fig. 1B. These 542-bp promoter sequences were shown to be sufficient for the basal, 12-O-tetradecanoylphorbol-13-acetate, and Ets-2-stimulated transcription of this promoter (46). CAT activity was assayed 48 h after transfection as described (40). By itself, p300 or Ets-2 did not stimulate the stromelysin promoter significantly (less than 2-fold stimulation; Fig. 1B). However, cotransfection of p300 and ets-2 expression vectors together stimulated the stromelysin promoter by about 20-fold, indicating that p300 cooperates with Ets-2 in the activation of the stromelysin promoter. To determine whether the Ets-2 activation of the stromelysin promoter is mediated by the Ets-binding site in the promoter, mutant promoters with mutations in the AP-1 site (pSK201) or with a deletion of the two Ets sites (pSK202) were studied using same methods described above. Together, Ets-2 and p300 stimulated the AP-1 mutant promoter about 8-fold (Fig. 1B, pSK201). In contrast, a mutant promoter lacking the two Ets-2 sites was inactive in these assays (Fig. 1B, pSK202). These results suggest that the two Ets-2 sites can respond to transcriptional superactivation in the presence of exogenously provided Ets-2 and p300. It is interesting that the promoter mutant lacking the AP-1 site was not efficiently induced by p300 in the presence of Ets-2.
To study further the cooperative activity of Ets-2 and p300, we tested a synthetic promoter-reporter construct in which two palindromic Ets sites were placed upstream of the c-fos minimal promoter containing Ϫ56 to ϩ109 bp of the c-fos promoter fused to CAT reporter gene (Fig. 1A, E18pal) (16). By itself, p300 did not stimulate E18pal significantly (about 2-fold), whereas Ets-2 stimulated this construct by about 6-fold. In contrast, the two effectors together stimulated E18pal by about 17-fold (Fig. 1B, E18pal). These results provide evidence that Ets-2 and p300 can cooperate in the transcriptional activation of Ets-2 site containing promoters even in the absence of other upstream elements. CBP also cooperated with Ets-2 and stimulated the E18pal and the stromelysin promoter by about 8 -10-fold (data not shown; also see We considered it possible that the dramatic increase in the reporter activity by p300 and Ets-1/Ets-2 proteins together in the promoter-reporter assays, described above, may be due to a significant increase in the levels of Ets-1/Ets-2 as a result of stimulation of the CMV promoter of the Ets expression vectors by p300/CBP. We found that in human cells, using identical transfection assay conditions described above, p300 increased the levels of Ets-1 and Ets-2 only marginally (1.5-fold, Fig. 1C; data for Ets-1 not shown). U2OS cells were used in this experiment because of their higher transfection efficiency; p300/CBP cooperated with Ets-1/Ets2 in the stimulation of the stromelysin promoter in these cells as efficiently as HeLa cells (data not shown). Thus we conclude that p300/CBP increases the transcriptional activation activity of Ets-1 and Ets-2 and stimulates the Ets-2 site containing promoters.
Previous studies have shown that Ets-2 activates the human stromelysin promoter (37), whereas the rat stromelysin promoter is activated by both Ets-1 and Ets-2 (44). Both promoters contain palindromic Ets binding sites. Therefore, it was of interest to determine whether p300/CBP would cooperate with Ets-1 to activate the human stromelysin promoter. Ets-1 did not stimulate the stromelysin promoter significantly. In contrast, Ets-1 and p300 together stimulated the stromelysin promoter by about 25-fold (Fig. 1B). In the case of E18pal, p300 did not stimulate the promoter significantly, whereas it was stimulated by Ets-1 by about 5-fold. When the E18pal was tested in the combined presence of Ets-1 and p300, it was superactivated by about 38-fold (data not shown).
FIG. 1. Ets-1 and Ets-2 cooperate with p300 in the transcriptional activation of human stromelysin promoter and a synthetic promoter containing palindromic Ets-2-binding sites. A, structure of the human stromelysin promoter-reporter constructs (pSK200, pSK201, and pSK202) and the synthetic promoter-reporter construct containing palindromic Ets-2-binding sites (E18pal). B, activation of the promoter-reporter constructs shown in A by p300, Ets-1, and Ets-2 in HeLa cells. HeLa cells were transfected with 2 g of E18pal, 3 g of pSK200, pSK201, and pSK202, 3 g of a plasmid expressing mouse Ets-1 (pFNEts-1) or Ets-2 (pFNEts-2), and 6 g of a plasmid expressing p300 (p300CHA) in combinations as indicated using calcium phosphate transfection method. DNA concentration was maintained at 15 g/transfection by the addition of a CMV promoter-based empty vector DNA. CAT activities present in the lysates were quantitated using equal quantities of protein (40). To control transfection efficiency, a promoter-reporter construct (50 ng) in which luciferase gene was transcribed from the CMV promoter was included in each transfection. Average values obtained from three independent experiments after normalizing for transfection efficiency are shown. CAT activities for E18pal assays were quantitated by incubating the samples for 30 min, whereas the CAT activities for the stromelysin promoter were quantitated by incubating the samples for 6 h with a second addition of the substrate after 3 h of incubation. These assay conditions apply to all CAT assays reported in this paper. CAT activities were quantitated using a PhosphorImager. C, determination of Ets-2 levels in cells transiently transfected with pFNEts-2 and p300CHA. Approximately 10 6 U2OS (human osteosarcoma) cells were transfected with 3 g of pFNEts-2 and 6 g of p300CHA in a total of 15 g/transfection as indicated. All lanes also included 2 g of E18pal. Transfection conditions were identical to those described above. 48 h after transfection, the cell lysates corresponding to 50 g of protein were subjected to SDS-8% PAGE, transferred to a nitrocellulose membrane, and probed with anti-FLAG monoclonal antibody. The blot was then developed by using an ECL kit from Amersham Pharmacia Biotech. The lane corresponding to pFNEts-2A72 relates to experiments shown in Fig. 8.

Ets-2 and p300/CBP Exist as a Complex in Vivo
Because p300/CBP can cooperate with Ets-2 in the transcriptional activation of Ets-binding site containing promoters, we reasoned that Ets-2 and p300/CBP might interact and exist as a complex in vivo. To determine this, proliferating human 293 cells were lysed in a lysis buffer, and the cell lysate equivalent to 2 mg of protein was first immunoprecipitated with a polyclonal antibody directed against the C-terminal region of Ets-2. The immunoprecipitated proteins were then separated on an SDS-8% polyacrylamide gel (Fig. 2, lane 2). As a negative control, an equal amount of protein from the cell lysate was immunoprecipitated with a polyclonal antibody directed against the cellular double-stranded RNA-activated protein kinase (eIF-2 ␣-kinase, also called PKR (47)), and the immunoprecipitated proteins were loaded on the same gel (lane 3). Five hundred g of protein from the same lysate was also immunoprecipitated with a polyclonal antibody which recognizes the N-terminal region of human CBP and the immunoprecipitated proteins were separated as above (lane 4). In lane 1, 60 g of the protein from the same lysate was loaded directly. The gel was electrophoretically transferred to a nitrocellulose membrane and probed with an anti-CBP antibody. As shown in Fig.  2, anti-CBP antibody recognizes a band in all lanes except in lane 3, which corresponds to a 300-kDa protein. This band is absent in anti-PKR immunoprecipitates (lane 3). The molecular weight of the protein that was brought down by anti-Ets-2 antibody in immunoprecipitations and which reacted with anti-CBP antibody in Western blot is similar to that reacted with anti-CBP antibody (lanes 1 and 4). Thus we conclude that Ets-2 and CBP exist as a complex in vivo.

Ets-1 and Ets-2 Bind to Two Regions in p300/CBP in Vitro and in Vivo
To determine the regions of p300/CBP that bind to Ets-2 and to determine whether Ets-1 also binds to p300/CBP in similar manner, we employed GST pull-down, coimmunoprecipitation, and yeast two-hybrid assays. Fragments of p300 from a.a. 1 to 596 (GST-p300N), 744 to 1571 (GST-p300M), and 1572 to 2370 (GST-p300C, Fig. 3A) were expressed as GST fusion proteins. Fig. 3B shows a Coomassie Blue-stained gel of the affinity purified GST-p300 fusion proteins; all three fusion proteins were overexpressed in Escherichia coli. Ets-1 and Ets-2 were labeled with [ 35 S]methionine in a coupled transcription/translation system and were then incubated with equal quantities of GST fusion proteins immobilized on agarose beads. Agarose beads containing GST and luciferase labeled with [ 35 S]methionine in vitro, as described above, were used as the negative controls. Quantitation of the radiolabeled bands indicated that about 6 and 8% of the input Ets-2 were bound to the N-and the C-terminal regions of p300, respectively (Fig. 3C). Similarly, about 4% of the Ets-1 bound to the N-and the C-terminal regions of p300. In contrast, binding of the radiolabeled luciferase to GST-p300M was negligible. Similarly, negligible amounts of Ets-1 and Ets-2 bound to beads containing only GST.
The above experiments were repeated in which full-length Ets-1 and Ets-2 proteins were expressed in E. coli as GST fusion proteins. Fig. 3D shows the Coomassie Blue-stained gel of the affinity purified GST-Ets-1 and GST-Ets-2 fusion proteins. Fragments of p300 corresponding to a.a. 1-746 (p300N), 743-1572 (p300M), and 1572-2414 (p300C) were radiolabeled in vitro and assayed with equal quantities of GST fusion proteins in GST pull-down assays. Considerable amounts of p300N (10%) and p300C (15%) were recovered when GST fusion proteins containing Ets-1 or Ets-2 were used (Fig. 3E). In contrast, less than 2% of the input p300M was detected for Ets-1 and Ets-2. Negligible amounts of p300 bound to GST beads without attached proteins.
To determine whether Ets-2 binds to the N-and the Cterminal regions of p300/CBP in vivo, we cotransfected human 293 cells with expression vectors encoding mouse ets-2 (pFN-Ets-2), the N-terminal a.a. 1-1097 (pCBP1-1097), and the Cterminal a.a. 1678 -2442 (pCBP1678 -2442) regions of CBP. CBP expressed from these expression vectors contain a nuclear localization signal (NLS) followed by three copies of influenza HA epitope at their N-terminal ends (38). As a control, ets-2 expression plasmid was cotransfected with the empty vector containing only the NLS and the three copies of HA epitope (pCMV2N3-T). The cell extracts containing equal quantities of protein were immunoprecipitated with an anti-Ets-2 polyclonal antibody, and the immunoprecipitates were analyzed by Western blot using an anti-HA monoclonal antibody (12CA5). Strong signals were detected in lanes corresponding to cell extracts prepared from cells cotransfected with pFNEts-2 and CBP expression vectors expressing a.a. 1-1097 (Fig. 3F) and 1678 -2442 (Fig. 3G). This band was not detected in cell extracts prepared from cells cotransfected with pFNEts-2 and the empty vector.
Also, in yeast two-hybrid assays, ets-2 cloned into yeast activation domain plasmid pACT-2 interacted with a region of p300 from a.a. 328 to 1000 but not from a.a. 962 to 1575 (data not shown; also see Fig. 7C). Amino acids 1-328 and 1575-2414 could not be tested in yeast two-hybrid assays as these two regions displayed significant transcriptional activation activities when cloned into DNA binding domain plasmids (48) (49). Taken together, the above data suggest that a.a. 338 -452 and 1678 -2370 of p300/CBP independently are able to form complexes with Ets-1/Ets-2.

In Vitro Binding Studies Identify Two p300-binding Sites in Ets-2
To determine whether the transactivation (TA) or the DNA binding (DB) domains or both bind to p300/CBP, GST pulldown assays were carried out using GST-Ets fusion proteins containing the TA (GST-Ets-2-(1-290)) or the DB domains p300N (a.a. 1-746), p300M (a.a. 743-1572), and p300C (a.a. 1572-2414). This experiment was repeated three times, and an autoradiogram of a typical experiment is shown in Fig. 4A, and quantitation of the bound radioactivity is shown in Fig. 4B. These results suggest the transactivation domain of Ets-2 binds to both the N-and the C-terminal regions of p300 as efficiently as the full-length Ets-2 protein. In contrast, the DB domain binds to only the C-terminal region of p300. Thus the TA and the DB domains of Ets-2 interact with the N-and the C-terminal regions of p300/CBP in a specific manner.

The N-and the C-terminal Halves of p300/CBP Independently Cooperate with Ets-2 in the Stimulation of the Stromelysin Promoter
Other studies have shown that the N-and the C-terminal regions of p300/CBP contain strong transcriptional activation activity (48). Because our studies suggest the N-and the Cterminal regions independently can bind to Ets-2, and because these two regions also contain the transcriptional activation domains, it seemed possible that the N-and the C-terminal regions of p300/CBP independently can cooperate with Ets-2 in the transcriptional activation of the stromelysin promoter. To test this possibility, HeLa cells were cotransfected with pSK200 containing the stromelysin promoter-reporter plasmid, the expression vectors expressing ets-2, and the full-length CBP, and the N-and the C-terminal regions of CBP (a.a. 1-1097 and 1678 -2442, respectively). CAT activities in the transfected cell lysates were then determined. As shown in Fig.  5, in the presence of Ets-2, the N-and the C-terminal halves of CBP independently stimulated the stromelysin promoter by about 8 -10-fold which was comparable to that observed for the full-length CBP. The N-and the C-terminal regions of p300 also behaved similarly in these assays (data not shown). These results suggest that in transient assays, the N-and the Cterminal halves of p300/CBP independently are able to cooperate with Ets-2 in the stimulation of the stromelysin promoter as efficiently as the full-length p300/CBP, and only one of the two Ets-binding sites of p300/CBP is sufficient to coactivate Ets-2.

Other Ets Family Transcription Factors Do Not Cooperate with p300/CBP in the Transcriptional Activation of the Stromelysin Promoter
Although Ets family transcription factors consist of a large number of transcription factors related by virtue of their Ets domain, the target genes that are activated by these proteins are dependent on the specificity of interaction between these proteins and their binding sequences. Ets family proteins bind to a 10-bp DNA sequence with an invariant core motif (C/ A)GGA(A/T). The specificity of the interaction is determined by the flanking sequences surrounding this core motif (8 -10). We determined whether other Ets family transcription factors such as PEA3 and Erg would cooperate with p300/CBP in the transcriptional activation of the stromelysin promoter. HeLa cells were cotransfected with the stromelysin or the E18pal promoter-reporter constructs with expression vectors expressing mouse ets-2 (pFNEts-2WT), PEA-3 (pCMV-PEA-3), and erg-2 (pSG5-Erg-2) in the presence or absence of an expression vector expressing p300 (p300CHA). Basal activity of E18pal was stimulated by all three Ets proteins by about 4 -6-fold, and this activity was further increased to about 12-16-fold in the presence of p300 suggesting that p300 is capable of coactivating PEA-3 and Erg-2 in these assays (Fig. 6). These results also suggest that the expression vectors used in this assay produce functional PEA-3 and Erg-2. Surprisingly, under these conditions, p300 did not cooperate with these two Ets proteins to  -p300N), a.a. 744 to 1571 (GST-p300M), and 1572 to 2370 (GST-p300C) and full-length Ets-1 (GST-Ets-1) and Ets-2 (GST-Ets-2) were expressed in E. coli, and approximately 60 pmol of each of these proteins was immobilized on glutathione-agarose beads and incubated with in vitro synthesized [ 35 S]methionine-labeled p300, Ets-1, Ets-2, or luciferase (negative control) as appropriate. Bound proteins were analyzed on SDS-8% PAGE and visualized by autoradiography as described (40). The GST fusion proteins were quantitated by running bovine serum albumin as standard in the same SDS gels. stimulate the stromelysin promoter. Consistent with previous results (Fig. 1B), the stromelysin promoter activity increased by about 20-fold in the presence of Ets-2 and p300. Thus, it appears that in the context of the stromelysin promoter, the Ets-2-binding sites show strict specificity with respect to transcriptional cooperativity between p300/CBP and the Ets family transcription factors.

Role of LXXLL Sequence in Ets-1 and Ets-2 in Their
Interactions with p300/CBP Recent studies suggest that protein-protein interactions between steroid receptor family members such as SRC-1/p160 and p300/CBP are mediated by a short sequence motif LXXLL (where L is leucine and X is any amino acid) (50). A sequence, LLELL, that fits the consensus LXXLL sequence is conserved in loop 1 of the Ets domain in all known Ets family transcription factors with the exception of PEA-3 (8), raising the possibility that this sequence may mediate interaction of Ets family proteins with p300/CBP. In Ets-2, this sequence motif maps in the DB domain between a.a. 368 and 374. Therefore, we deter-mined whether a mutation of this sequence affects its interaction with p300/CBP. Because both the TA and the DB domains of Ets-2 independently interact with p300/CBP, we compared the ability of the WT DB domain and the DB domain containing mutations in LLELL sequence to interact with the p300 Cterminal region in vitro in GST pull-down assays.
The LLELL motif in an expression plasmid coding for the WT human Ets-2 was changed to AAAAA (Ets-2AAAAA; see "Experimental Procedures"). The GST fusion constructs coding for the DNA binding domain (a.a. 334 -469) of the WT or the AAAAA mutant were then overexpressed in E. coli. Data presented in Fig. 7A (Coomassie Blue-stained gel) indicate that all three fusion proteins are overexpressed in E. coli. Equal amounts of GST-fusion proteins were then assayed for their capacity to bind to in vitro synthesized radiolabeled p300N and the p300C proteins. In agreement with data shown in Fig. 3,

FIG. 5. Cooperation between Ets-2 and the N-and the C-terminal regions of CBP in the transcriptional stimulation of the stromelysin promoter.
HeLa cells were transfected with 3 g of the stromelysin promoter-reporter plasmid (pSK200) along with 3 g of pFNEts-2 and 6 g each of the expression plasmids encoding full-length CBP (pCBP-FL), a.a. 1-1097 (pCBP1-1097), and a.a. 1678 -2442 (pCBP1678-2442) as indicated. CAT activities in the lysates were quantitated as described in the legend to Fig. 1. Average values obtained from three independent experiments with error bars are shown.
the GST fusion protein containing the WT Ets-2 interacted with both the p300N and the p300C proteins. The WT DB domain interacted only with the C-terminal region of p300. The mutant DB domain, in which the LLELL was changed to AAAAA, also interacted with p300C as efficiently as the WT DB domain. Thus we conclude that the LLELL motif does not play a role in Ets-2 interaction with p300.
We also compared the p300/CBP-binding capacities of the WT and the Ets-2 AAAAA mutant in yeast and human cells. ETS-2 cDNA with AAAAA mutation was cloned in frame with activation domain plasmid of the yeast two-hybrid system. Yeast cells were cotransformed with the activation domain fusion plasmid containing WT or the mutant forms of ets-2 genes (Ets-2 AAAAA), the DNA binding domain fusion plasmids Gal-4-p300-1 and Gal4-p300-2 containing a.a. 328 -1000 and a.a. 962-1575 of p300, respectively. The ␤-galactosidase activities generated in the transformants were quantitated (42). As shown in Fig. 7C, plasmid coding for the Ets-2AAAAA mutant protein generated about 80% of WT ␤-galactosidase activity (Ets-2A72 data in Fig. 7C relate to the experiments shown in Fig. 8 and will be discussed below). Coimmunopre- The LLELL sequence was mutated to AAAAA in the context of full-length Ets-2 using a site-directed mutagenesis procedure, and the DNA sequences corresponding to a.a. 334 -469 were then cloned in frame into a GST plasmid. GST fusion proteins were overexpressed as described under "Experimental Procedures" and fractionated on a SDS-10% PAGE. Note that WT Ets-2 in this gel comigrates with an E. coli protein. B, GST pull-down assays showing the interaction between the N-and the C-terminal regions of p300 and the full-length Ets-2, Ets-2 DB domain (a.a. 334 -469) from WT, and the Ets-2 mutant containing AAAAA mutation. Lanes Input contain 1/10 of the radiolabeled proteins used in binding experiments. Binding assays were repeated three times, and the results of a typical experiment are shown. C, yeast two-hybrid assays showing interaction of full-length WT Ets-2 (human) and full-length Ets-2 containing AAAAA mutation with p300 fragments from a.a. 328 to 1000 (p300-1) and 962 to 1575 (p300-2). Also included in this experiment is an Ets-2 mutant in which the MAP kinase substrate Thr-72 mutated to Ala (see Fig. 8). Yeast cells were cotransformed with Gal-4 fusion plasmids containing p300 fragments, and the activation domain plasmids containing various Ets mutants and the ␤-galactosidase activity in the transformants were quantitated (42). Average values and error bars from three independent experiments are shown. D, transactivation of E18pal by the human WT Ets-2 and Ets-2 containing AAAAA mutation in the presence and absence of p300. Transactivation assays were carried out exactly as described in legend to Fig. 1 except that the Ets plasmids contained human Ets-2 tagged with FLAG epitope similar to that described for mouse Ets-2.

FIG. 8. Interaction of Ets-2 T72A mutant protein with p300/ CBP and coactivation of the WT and T72A mutant Ets-2 by p300.
A, interaction of GSTp300N (a.a. 1-596) and GST-p300M (a.a. 744 -1571) fusion proteins with in vitro synthesized human WT and Ets-2 T72A mutant proteins. GST-p300 proteins were synthesized as described above and incubated with in vitro synthesized radiolabeled Ets-2 proteins, and the bound proteins were analyzed on SDS-8% PAGE. Lanes 1 and 4 contain 1/10th of the radiolabeled proteins used for binding assay. B, interaction of the WT Ets-2, the T72A, and the Ets-2AAAAA mutant proteins with CBP in vivo in human cells. Human 293 cells were cotransfected with expression vectors encoding human Ets-2WT, Ets-2 AAAAA, and Ets-2 Ala-72 mutant proteins and a.a. 1 to 1890 of CBP. The Ets proteins were tagged with FLAG epitope at their N-terminal ends, whereas CBP was tagged with three copies of the HA epitope at its N-terminal end. The lysates were immunoprecipitated from transfected cells using anti-FLAG monoclonal antibody, transferred to a nitrocellulose membrane, and probed with anti-HA antibody 12CA5. The vector lane corresponds to an experiment in which the Ets expression vectors were cotransfected with a plasmid in which the FLAG epitope is expressed from the CMV promoter. C, coactivation of the WT and T72A mutant by p300. HeLa cells were cotransfected with E18pal, dlFosCAT, and expression vectors expressing mouse WT (pFNEts-2 (17)), a T72A mutant Ets-2 protein (pFNEts-2A72), and p300 (p300CHA) at concentrations indicated in legend to Fig. 1. Where appropriate assays included a plasmid expressing the MEK-1 mutant (pMEK-1) at a concentration of 3 g per assay. CAT activities were quantitated as described in legend to Fig. 1. Experiments were repeated three times, and average values and error bars are shown. Each E18pal transfection assay was carried out in parallel with dlFosCAT plasmid transfection. Values obtained for dlFosCAT were subtracted from the values obtained for E18pal before calculating the fold increase. cipitation followed by Western blot assays similar to those described in Fig. 3F indicated that in 293 cells a CBP fragment containing a.a. 1-1890 bound to Ets-2AAAAA mutant as efficiently as the WT-Ets-2 protein (Fig. 8B; see figure legends for further details). Although in these experiments we did not study the interactions of the DB domain with the C-terminal region of p300 separately, they nonetheless suggest that the mutation of the LLELL residues in the context of full-length Ets-2 does not affect its overall interaction with p300/CBP in vivo.
Next, we examined whether Ets-2 mutant with AAAAA mutation would cooperate with p300 in the transcriptional activation of E18pal reporter construct. The Western blot assays similar to those shown in Fig. 1C revealed that in human cells, the stability of the Ets-2 AAAAA mutant protein was comparable to that of the WT Ets-2 protein (data not shown). In transient assays in HeLa cells, similar to those described in Fig. 1, we found that the WT human Ets-2 transactivated the E18pal by about 6-fold, and in the presence of p300 this activation increased to 12-fold (Fig. 7D). In contrast, the Ets-2AAAA mutant was found to be severely defective for its capacity to stimulate E18pal. This residual stimulation did not increase significantly in the presence p300. Because the Ets-2 AAAAA mutant failed to stimulate the Ets-2 site containing promoter, we could not unambiguously determine whether p300/CBP would coactivate this mutant Ets-2.

p300/CBP Binds to and Coactivates a Mutant Ets-2 Nonphosphorylatable by MAP Kinase
Interaction of Ets-2 T72A Mutant with p300/CBP-Previous studies have shown that the efficient activation of Ets-2 transcriptional activation activity by Ras requires phosphorylation of the conserved MAP kinase site threonine 72, and mutant Ets-2 in which Thr-72 is mutated to Ala is not efficiently activated by Ras (17). It was of interest to determine whether p300/CBP would bind to and coactivate this Ets-2 mutant. Western blot data presented in Fig. 1C show that in transient assays, the Ets-2 T72A mutant is as stable as the WT Ets-2 protein.
Equal quantities of GST fusion proteins corresponding to p300 fragments a.a. 1-596 (GST-p300N) and 743-1572 (GST-p300M; see Fig. 3B) were assayed in GST pull-down assays with in vitro radiolabeled WT and T72A Ets proteins. As shown in Fig. 8A, the amounts of radiolabeled WT and the T72A mutant Ets proteins recovered from the agarose beads containing GST-p300N were comparable. In agreement with previous data, GST-p300M did not bind to WT or the mutant Ets proteins. Our data presented in Figs. 4 and 7 have indicated that the DB (Ets) domain does not bind to the N-terminal region of p300/CBP. Thus, the binding of the Ets-2 observed in this experiment is soley due to an interaction between the TA domain of Ets-2 and the N-terminal region of p300/CBP. These results indicate that the phosphorylation of Thr-72 in the Pointed domain is not critical for the interaction of the TA domain with the N-terminal region of p300/CBP. We have not investigated whether T72A mutation of Ets-2 affects its interaction with the C-terminal region of p300/CBP.
We also compared the p300/CBP binding capacities of the WT and the Ets-2 T72A mutant in yeast cells. Human Ets-2 cDNA with T72A mutation was cloned in frame with activation domain plasmid of the yeast two-hybrid system. Yeast cells were cotransformed with activation domain fusion plasmid containing WT or T72A forms of Ets-2 genes and the Gal-4-p300-1 fusion plasmids (p300-1; a.a. 328 -1000), and the ␤-galactosidase activity was quantitated. As shown in Fig. 7C, the plasmid coding for the T72A Ets-2 mutant protein (Ets-2A72) generated about 80% of WT ␤-galactosidase activity. These results are in agreement with results obtained in GST pulldown assays (Fig. 8A) and confirm that the T72A mutation of Ets-2 did not significantly affect its capacity to bind to the N-terminal region of p300/CBP.
To determine whether interaction of Ets-2 with p300 is influenced by the phosphorylation of Ets-2 at Thr-72 in human cells, we carried out the coimmunoprecipitation assays followed by Western blots similar to those described in Fig. 3F. Data presented in Fig. 8B indicated that both WT and the mutant Ets-2 proteins coimmunoprecipitated the epitope-tagged CBP fragment a.a. 1-1890 from transfected 293 cell lysates at comparable levels. Because the CBP expression vector used in this experiment contained most of the CBP coding sequences, these results did not allow us to separate the Ets-2 interaction with N-terminal region of p300/CBP from the Ets-2 interaction with the C-terminal region. Nevertheless, they suggest that the mutation of the conserved MAP kinase phosphorylation site did not affect its overall interaction with p300/CBP in human cells.
Coactivation of T72A Ets-2 Mutant by p300/CBP-Next, we determined whether coactivation of Ets-2 by p300 is dependent on the phosphorylation of Ets-2 at Thr-72 by MAP kinase. To ensure that the Thr-72 of WT Ets-2 is phosphorylated by MAP kinase, we included a mutant of MAP kinase kinase (MEK-1) which constitutively phosphorylates and activates MAP kinase (51). An expression plasmid encoding this MEK-1 mutant when included in transfection assays is expected to express the mutant MEK protein and phosphorylate the Thr-72 of the WT Ets-2 but not the Ala-72 of the mutant Ets-2 expressed from the transiently transfected Ets-2 expression plasmids.
The capacity of the WT and T72A mutant Ets-2 to cooperate with p300 in the transcriptional activation of E18pal in the presence and absence of MEK-1 was assayed as described above. HeLa cells were cotransfected with E18pal and expression vectors encoding WT mouse ets-2 (pFNEts-2) or Ets-2 T72A mutant (pFNEts-2A72), p300, and MEK-1 mutant in appropriate combinations, and the CAT activities obtained from the lysates are presented in a bar diagram shown in Fig.  8C. Western blot data presented in Fig. 1C showed that the T72A Ets-2 produced from pFNEts-2A72 is stable in transfection assays and that p300 does not increase the levels of this mutant Ets-2 significantly. Because we used a constitutively active MEK-1 mutant in these assays, which may have some general transcriptional stimulatory effect in the cell, we also assayed in parallel the empty vector (dlFosCAT (16)) and subtracted these values from those obtained for E18pal, i.e. each transfection was carried out in duplicate with one set containing E18pal reporter plasmid and the second set containing dlFosCAT reporter plasmid. The value obtained for the empty vector was subtracted from the value obtained for E18pal in a matching set, and the fold increase was then determined. The results show that in the presence of MEK-1, the WT Ets-2 and p300 together stimulated the activity of the E18pal by about 85-fold when compared with basal activity (E18pal without any activator; Fig. 8C). This activity was dramatically higher than that observed for the E18pal reporter in the presence of p300, MEK-1, and without Ets-2 (22-fold, lane 7). Basal activity of the E18pal was increased by about 5-fold by the WT Ets-2 (lane 4), whereas the T72A mutant Ets-2 did not increase the basal activity of E18pal significantly (1.5-fold, lane 9). This is consistent with previous reports showing that T72A Ets-2 mutant is severely defective for transcription activation activity (17). MEK-1 increased the Ets-2 Ala-72-mediated stimulation of E18pal only marginally (compare lane 10 with lane 9). Together, p300 and Ets-2A72 increased the E18pal activity by about 7-fold (compare lane 11 with lane 9). A combination of Ets-2 A72, p300, and MEK-1 together increased the E18pal activity by about 63-fold (lane 12). This fold increase in activity is much higher than all the controls, including that obtained for the E18pal in the presence of p300 and MEK-1 (22- fold; lane 7). Thus, we conclude that in the presence of MEK-1, p300/CBP was able to coactivate both the WT and the mutant Ets-2 in which the MAP kinase substrate Thr-72 is mutated to Ala. DISCUSSION In this paper, we have shown that Ets-2 and p300/CBP exist as a complex in human cells under physiological conditions (Fig. 3). By using a variety of approaches, we have identified two Ets-1/Ets-2-binding sites on p300/CBP. One binding region maps between a.a. 328 and 596, and the second region maps between a.a. 1678 and 2370 (Figs. 3 and 7C). These two regions contain cysteine/histidine-rich regions I and III, respectively. These results are consistent with the recent data published by Yang et al. (52) who showed that Ets-1 interacts with a.a. 313 to 452 and 1449 to 1892 of p300/CBP. Surprisingly, we did not detect interactions between Ets-1/Ets-2 and p300/CBP in a region between a.a. 744 and 1571 which contains the bromodomain and the second C/H region. It is interesting to note that the Ets-1/Ets-2-binding regions on p300/CBP also interact with many cellular transcription factors and viral oncogenes. For example, the N-terminal Ets-1/Ets-2-binding regions of p300/ CBP interact with a large number of transcription factors including CREB/activating transcription factor (53), c-Jun (34,54), c-Myb (55), Stat1 (56), Stat2 (57), hypoxia-inducible factor-1␣ (58), and sterol regulatory element-binding protein-2 (59). The C-terminal Ets-1/Ets-2-binding region also overlaps with binding sites for several transcription factors including c-Jun and JunB (60), YY1 (61), MyoD (48,62), and p53 (63).
Currently, we do not know whether two different transcription factors compete for a single site on p300/CBP. If such a competition exists, one can speculate the interaction of Ets-1/2 with p300/CBP may exclude the interaction of other transcription factors that compete for the same site. Thus, Ets-1 or Ets-2 when present in high concentrations may interfere in p300/ CBP interacting with other transcription factors that bind to the same region. Also, availability of two Ets-1/Ets-2-binding sites in p300/CBP may allow these proteins to compete more efficiently for limiting amounts of p300/CBP and divert this coactivator to pathways in which Ets proteins are intimately involved. Recent studies suggest that certain cellular activators compete for the limiting amounts of p300/CBP resulting in the inhibition of activity of one transcription factor by another. Such a competition is believed to be a molecular basis of reciprocal antagonism between AP1 and (i) retinoic acid receptors (34) and (ii) p53 (63) and signal transducers and activators and transcriptions (57). It is likely that several other reciprocal antagonisms may be discovered in the future.
We attempted to address the question of whether both p300/ CBP binding regions are essential for coactivation of Ets-1/ Ets-2. Surprisingly, we found that the N-and the C-terminal regions independently are capable of coactivating Ets-2 in the transcriptional stimulation of the stromelysin promoter. This suggests that only one of the two Ets-1/Ets-2-binding sites is adequate for the transcriptional coactivation of Ets-2. These results are consistent with the properties of p300/CBP. Both the N-and the C-terminal regions of p300/CBP contain strong transcriptional activation domains, and both these regions bind to TBP ((48, 64) 4 and transcription factor IIB (65)). Thus, it is conceivable that these truncated p300/CBP molecules are capable of bridging the promoter-bound Ets-1/Ets-2 and the basal transcription complex and stimulating transcriptional initia-tion. Based on these data, it would appear that the Ets-1/Ets-2 interacting regions on p300/CBP are functionally redundant. However, these studies need to be extended to cellular genes in the chromosomal context to understand the significance of multiple interactions between Ets-1/Ets-2 and p300/CBP.
Our observations that the TA and the DB domains of Ets-2 can independently interact with the N-and the C-terminal regions of p300/CBP further increase the complexity of Ets-1/ Ets-2 interactions with this family of coactivators. The Ets-2 TA domain (a.a. 1-290) is able to bind to both the N-and the C-terminal regions of p300. However, the Ets domain binds only to the C-terminal region. It is possible that the nature of the Ets binding domains in the N-and the C-terminal regions of p300/CBP are significantly different. A detailed mutational analysis of Ets-2-binding sites on p300/CBP as well as the a.a. sequences of Ets-2 that interact with different domains in p300/CBP will be necessary to understand further the Ets-1/ Ets-2-p300/CBP interactions.
Also interesting is the fact that the N-and the C-terminal regions (a.a. 1-1097 and 1678 -2442, respectively) used in these studies do not contain the region responsible for the histone acetylation activity of p300/CBP as this enzyme activity maps between a.a. 1195 and 1673 on CBP (66). The mutant pCBP1-1097 also lacks the p300/CBP-associated factor and RNA helicase A binding regions (31) (67). RNA helicase is believed to bridge CBP and RNA polymerase II (67). Both p300/CBP and p300/CBP-associated factor are reported to acetylate histones and augment transcription (68). Other studies have shown that a truncated CBP containing only the N-terminal 1-714 a.a. can stimulate phospho-CREB and activating transcription factor activity (69). Similarly, the N-terminal half of CBP was shown to cooperate with p67 SRF , a serum response element-binding protein, in the transcriptional stimulation through SRE (38). Currently, it is not known how a mutant of p300/CBP that lacks the ability to acetylate histones or to bind to RNA polymerase II is able to cooperate with various transcription factors and stimulate transcription. It is conceivable that transcription of transiently introduced templates does not require histone acetylation, whereas transcriptional activation of the chromosomal genes may be linked to p300/CBP-associated histone acetylation activity.
We have also shown that a single signature motif LLELL reported to be important in receptor-coactivator interactions, located in the DNA binding domain of Ets-2, does not appear to play in the interactions with p300/CBP. This motif is present in the first helix turn helix region (H1) of Ets-2 which is directly involved in DNA binding (10,70). The fact that the N-terminal region of Ets-2 can interact with the C-terminal region of p300/CBP also supports this suggestion. Both the N-terminal region of Ets-2 and the C-terminal region of p300/CBP lack such sequences (the N-terminal region of p300/CBP contain two copies of this motif (50)). However, we have discovered that the Ets-2 mutant carrying this mutation is severely defective for transactivation consistent with an important role for this region in DNA binding (10,70).
Surprisingly, we found that two other Ets family transcription factors PEA-3 and Erg-2 did not cooperate with p300 in the stimulation of the stromelysin promoter, although they cooperated with p300/CBP to superactivate E18pal. Other studies have shown that the PEA-3 expression vector used in this study synthesizes PEA-3 protein and binds to the E18pal (17), and our Erg-2 expression vector stimulates the minimal promoter containing the Erg-binding sites efficiently (71). Therefore, the lack of superactivation of the stromelysin promoter by these transcription factors in the presence of p300/CBP may not be due to their inability to interact with p300/CBP but may be due to other reasons. For example, the superactivation of the stromelysin promoter requires functional synergy between p300/CBP, Ets-1/Ets-2, and AP-1 during formation of active transcription complex. Although PEA3 and Erg may interact with p300/CBP, their interaction with Ets-binding sites on the stromelysin promoter may not provide appropriate proteinprotein interactions with AP-1 and other stromelysin promoter-specific transcription factors to generate active transcription complex. For example, studies have shown that Erg can bind to Ets sites on the stromelysin promoter and competitively inhibit promoter activation by Ets-2 (72). Formation of an active transcription complex on a native promoter is a complex process in which promoter-specific transcription factors interact with selected members of TBP-associated factors and general transcription factors in an orderly fashion to initiate transcription (reviewed in Ref. 73). E18pal reporter construct used in this study consists of a synthetic promoter in which the palindromic Ets-binding sites are placed upstream of the c-fos minimal promoter. Thus, it is possible that E18pal would allow proteinprotein interactions between PEA-3 and Erg-2 and p300 and basal transcription complex to stimulate transcription, whereas with the stromelysin promoter being a natural promoter such interactions would not occur. Clearly, further studies are needed to resolve these complex issues.
In these studies, we have also shown that the Ets-2 mutant in which the MAP kinase-phosphorylatable Thr residue mutated to Ala binds to p300/CBP as efficiently as the WT Ets-2. Thus interactions between the TA domain of Ets-2 and p300/ CBP is not dependent on the phosphorylation of the Thr-72 residue of the Pointed domain. It is strikingly in contrast to the interactions observed between CREB and p300/CBP. In the case of CREB, phosphorylation of Ser-133 is absolutely required for it to bind to CBP (53). It is also noteworthy that phosphorylation of Ets-2 at this site is not required for its coactivation by p300/CBP. Our transfection assays included a constitutively active MEK-1 mutant and thus, in the transfection assays, the phosphorylation of the Thr-72 residue of the WT Ets-2 would occur but not the Ala-72 residue of the mutant. Consistent with a previous report (17), the capacity of the T72A Ets-2 mutant to activate the E18pal is dramatically reduced. However, in the presence of MEK-1 and p300/CBP, the residual transactivation activity of the T72A Ets-2 mutant is dramatically increased. Thus our results suggest that p300/CBP may play a role in regulating the basal transcriptional activation activity of Ets-2. Such a role may be important in the regulation of expression of the Ets-2-driven genes under conditions where the transcriptional activation activity of Ets-2 may be low in the cell such as during serum starvation where Ets proteins are not phosphorylated by MAP kinase.
Stromelysin is an important member of a family of matrix metalloproteases (MMPs) which act on extracellular matrix during tissue remodeling, growth, and morphogenesis (74). For example, in chronic inflammation such as rheumatoid arthritis, macrophages produce stromelysin-1 (MMP3) and collagenase I (MMP1) and contribute directly to joint destruction (75). Both these MMPs require Ets-1/Ets-2 and AP1 for their activation. Stromelysin I has been shown to be important in the mammary gland development and branching morphogenesis (76). Metastatic tumor cells express matrix metalloproteases including stromelysin at high levels, a process that correlates with the metastatic potential of the tumor cells (74,77). Studies have shown that in addition to stromelysin, Ets-2 is also important in the activation of the type I collagenase promoter (78). Thus, p300 may play a role in the regulated expression of these different MMP genes and contribute directly in processes where MMPs are implicated.