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Originally published In Press as doi:10.1074/jbc.M400036200 on January 27, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14909-14916, April 9, 2004
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Concerted Activation of ETS Protein ER81 by p160 Coactivators, the Acetyltransferase p300 and the Receptor Tyrosine Kinase HER2/Neu*

Apollina Goel and Ralf Janknecht{ddagger}

From the Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905

Received for publication, January 5, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activator of thyroid and retinoic acid receptor (ACTR) is overexpressed in ~60% of primary human breast tumors and belongs to the p160 steroid receptor coactivator family. In this study, we identified a novel interaction partner of ACTR, the ETS transcription factor ER81 that is also heavily implicated in mammary tumor formation. ACTR and related p160 family members (steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein-1 (GRIP-1)) augment ER81-mediated transcription. Although ACTR and GRIP-1 can acetylate ER81, this posttranslational modification of ER81 is not required for its stimulation by ACTR or GRIP-1. In addition, ACTR collaborates with the p300 coactivator, a joint interaction partner of ACTR and ER81, to stimulate ER81 function and the ability of p300 to acetylate ER81 is indispensable for this collaboration. Furthermore, the receptor tyrosine kinase HER2/Neu, an oncoprotein particularly found overexpressed in breast tumors, cooperates with both ACTR and p300 to stimulate ER81-mediated transcription. Thus, oncogenic HER2/Neu and ACTR may synergize to orchestrate mammary tumorigenesis through the dysregulation of the transcription factor ER81 and its target genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The p160 steroid receptor coactivator (SRC)1 family gained much attention as pivotal coactivators for nuclear hormone receptors. Not only do SRCs interact with nuclear hormone receptors, they also facilitate the recruitment of other coactivators including the acetyltransferases CBP, p300, and P/CAF and the arginine methyltransferases CARM1 and PRMT1. Additionally, SRCs may acetylate histones on their own, all of which promotes transcription of nuclear hormone receptor target genes (1, 2). Three homologous members of the SRC coactivator family exist: SRC-1/NCoA-1 (nuclear coactivator-1); SRC-2/NCoA-2/TIF-2 (transcriptional intermediary factor-2)/GRIP-1; and SRC-3/ACTR. ACTR has also been called p300/CBP cointegrator-associated protein (p/CIP), receptor-associated coactivator-3 (RAC-3), amplified in breast cancer 1 (AIB1), and thyroid hormone receptor activator molecule-1 (TRAM-1) (3–8). Knock-out studies of ACTR have indicated that this coactivator has unique physiological functions that all relate to steroid and thyroid hormone action such as mammary gland development, female reproductive function, puberty, and somatic growth (9–11).

Recent studies have uncovered that the function of SRCs is not restricted toward nuclear hormone receptors but that they also regulate other transcription factors like MEF-2C (12), TEF-4 (13), AP-1 (14), serum response factor (15), nuclear factor {kappa}B (16), STATs (5), and p53 (17). Moreover, ACTR mRNA has been shown to be overexpressed in ~60% of primary human breast tumors, which is the result of transcriptional up-regulation or gene amplification (7, 18–20). Similarly, amplification and up-regulation of the ACTR gene were observed in ovarian, pancreatic, and gastric cancers (7, 21, 22). These studies suggest that ACTR overexpression may pleiotropically contribute to tumor formation, especially within the breast, as a cofactor of a variety of different transcription factors.

We wondered whether the PEA3 subfamily of ETS transcription factors might utilize ACTR as a coactivator, because this group of transcription factors is specifically overexpressed in HER2/Neu-induced mouse mammary tumors (23, 24) as well as in HER2/Neu-positive human breast tumors and many metastatic human breast cancer cell lines (25, 26). The PEA3 group of transcription factors is comprised of PEA3/E1AF, ER81/ETV1 and ERM/ETV5 (27), all of which share highly homologous transactivation domains and an evolutionary extremely conserved ETS domain, an 85 amino acid-long signature motif mediating DNA binding (28, 29).

Interestingly, both PEA3 and ER81 are downstream targets of HER2/Neu (25, 30), a receptor tyrosine kinase that is overexpressed in ~30% of all breast cancers where its overexpression correlates with the aggressiveness and lethality of the disease (31–33). In case of ER81, the molecular details of its activation by HER2/Neu have been elucidated and include the stimulation of mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase that both phosphorylate and thereby stimulate ER81-mediated transcription (30, 34–36). In addition, HER2/Neu induces the phosphorylation of the coactivator p300 and concomitantly stimulates the acetyltransferase activity of p300 and thus acetylation of ER81 by p300, which is required for maximal transcriptional activity of ER81 (37). Finally, HER2/Neu-induced activation of MAPK can result in the phosphorylation of ACTR, affecting its coactivator activity (38). Accordingly, HER2/Neu, ACTR, and ER81 might be poised to synergize in the process of transforming normal breast cells to malignant ones. Here, we present evidence that indeed HER2/Neu leads to the stimulation of gene transcription mediated by ER81 and its newly identified coactivator, ACTR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The mammalian expression vectors for ER81 and ER81-K33R/K116R (37), SRC-1 (39), ACTR (3), p300-HA, and p300{Delta}HAT-HA (lacking amino acids 1430–1504) (40), p300{Delta}SRC (lacking amino acids 2042–2157) (41), FLAG-P/CAF (42), HER2/Neu-V664E (43), GST-p300-HAT (37), GST-GRIP-11158–1432 (12), HA-CARM1 (44), and HA-PRMT1 (44) were as reported. HA-tagged GRIP-1, GRIP-1{Delta}AD1, GRIP-1{Delta}AD2, and GRIP-1{Delta}AD1+{Delta}AD2 have been described previously (44). Myc-tagged ACTR and truncations thereof were cloned into the pCS3+-Myc6 vector, whereas Myc-tagged ER81 constructs were as described previously (37). The histone acetyltransferase (HAT) domain of ACTR (amino acids 1086–1292) was cloned into pGEX-2T-His6-PL2 to produce a respective GST fusion protein.

Acetylation Assays—In vitro, acetylation assays were performed in 50 mM HEPES, pH 7.4, 10 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM DTT in the presence of 0.25 µCi of [14C]acetyl coenzyme A (57 mCi/mmol, Amersham Biosciences) (37). The enzyme source was the HAT domain of p300 (amino acids 1195–1681) (37), GRIP-1 (amino acids 1158–1432) (12), or ACTR (amino acids 1086–1292) fused to GST and purified from bacteria. Samples were separated by SDS-PAGE, and gels were dried and subjected to autoradiography.

GST Pull-down Assay—GST, GST-ER812–477, or various truncations of ER81 were expressed and purified as described previously (37). 50 µl of lysates from 293T cells transiently transfected with Myc6-ACTR (2.5 µg), HA-CARM1 (2.5 µg), or HA-PRMT1 (2.5 µg) expression plasmid were incubated for 2 h at 4 °C with approximately equimolar quantities of GST fusion proteins pre-bound to glutathione-agarose beads (Sigma) in 2.5 mM Tris-HCl, 7.5 mM Na4P2O7, 12.5 mM NaCl, 12.5 mM NaF, 1 mM DTT, and 0.25% Triton X-100, pH 7.1, containing protease inhibitor mixture. The beads were washed four times, boiled in sample buffer, and subjected to SDS-PAGE. Bound ACTR was detected by anti-Myc (9E10 mouse monoclonal antibody), and CARM1 or PRMT1 was detected by anti-HA (12CA5 mouse monoclonal antibody) Western blotting.

Isolation of RNA and RT-PCR—293T cells were transiently transfected with SRC-1 (0.1 µg), GRIP-1 (0.1 µg), ACTR (0.1 µg), p300 (0.1 µg), HER2/Neu-V664E (1 µg), and 0.2 µg of ER81 or various other ETS protein expression plasmids as indicated. Cytoplasmic RNA was extracted and employed for the Access RT-PCR kit (Promega) with matrix metalloproteinase-1 (MMP-1) and GAPDH primers (30, 37). Reaction products were separated on a 1.5% agarose gel and detected by ethidium bromide staining.

Immunoprecipitation—293T cells were transiently transfected with Myc-tagged ER812–477 (1.5 µg) and Myc6-ACTR2–1412 (2.5 µg), Myc6-ACTR2–1022 (2.5 µg), or HA-p300 (2.5 µg) expression plasmid. 36 h after transfection, the cells were lysed in radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.1, 30 mM Na4P2O7, 50 mM NaF, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with 10 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na3VO4, 10 mM sodium butyrate, and 0.2 mM DTT and centrifuged at 20,800 x g for 10 min at 4 °C. Lysates were pre-cleared with protein A-agarose beads (Repligen), and inputs were removed (1–3%). Immunoprecipitations were done using anti-acetyllysine antibodies (rabbit antiserum 06–933, Upstate Biotechnology) or control anti-HA antibody (12CA5) followed by incubation with protein A-agarose beads. The immunoprecipitated proteins were removed from protein A beads by heating at 100 °C for 5 min in radioimmune precipitation buffer containing 0.5% SDS and re-immunoprecipitated with anti-Myc (9E10) antibody. Subsequently, proteins were resolved by SDS-PAGE and revealed by anti-Myc Western blotting.

Coimmunoprecipitations—293T cells were transiently transfected with vectors for FLAG3-ER81 (1.5 µg) and 2.5 µg of full-length or various truncations of Myc6-ACTR and lysed in 2.5 mM Tris-HCl, 7.5 mM Na4P2O7, 12.5 mM NaCl, 12.5 mM NaF, and 0.25% Triton X-100, pH 7.1, supplemented with 10 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na3VO4, 10 mM sodium butyrate, and 0.2 mM DTT. Precipitations were then performed with protein A-agarose beads (Repligen) and anti-FLAG (M2, Sigma) antibodies, and coimmunoprecipitated ACTR proteins were detected by anti-Myc (9E10) Western blotting. For coimmunoprecipitation of endogenous proteins, anti-ER81 antibodies (anti-ETV1 C20, Santa Cruz Biotechnology) or control anti-GAL4 antibodies (DBD, Santa Cruz Biotechnology) were used followed by Western blotting with anti-ACTR antibodies (clone AX15, number 5-490, Upstate Biotechnology).

Luciferase Assays—CV-1 cells were seeded in 6-cm dishes and transiently transfected by the calcium phosphate coprecipitation method as described previously (45). 1 µg of human MMP-1 promoter (-525 to +15) luciferase reporter plasmid was transfected with various combinations of the indicated mammalian expression plasmids. Whole cell lysates were prepared and assayed for luciferase activity essentially as described previously (46).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SRCs Are Coactivators of the PEA3 Subfamily of ETS Proteins—To analyze whether the p160 family of SRCs can affect the function of the ETS protein ER81, we studied ER81-mediated gene transcription with a luciferase reporter construct driven by the promoter of the MMP-1 gene, an established target gene of ER81 (30, 47). As reported earlier, ER81 alone induces the MMP-1 promoter by 6-fold (Fig. 1A). In contrast, none of the three SRCs (SRC-1, GRIP-1, and ACTR) alone was able to significantly stimulate the MMP-1 promoter, but when coexpressed with ER81, all three p160 coactivators potentiated ER81-mediated transcription by ~9-fold, suggesting that p160 proteins are bona fide coactivators of ER81.



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  		FIG. 1.
SRCs stimulate the PEA3 subfamily of transcription factors. A, CV-1 cells were cotransfected with MMP-1 luciferase reporter, CMV-ER812–477 (0.5 µg), SRC-1 (0.25 µg), GRIP-1 (0.25 µg), or ACTR (0.25 µg) as indicated. The resulting relative luciferase activities are depicted. B, RT-PCR analyses of endogenous MMP-1 mRNA production, and as a control GAPDH, in 293T cells transfected with CMV-ER812–477 (0.2 µg), p160 coactivators (0.1 µg), and p300 (0.1 µg) as indicated. C, transient transfections of CV-1 cells with MMP-1 luciferase reporter, 0.25 µg of ACTR, and 0.5 µg of expression plasmids for ER81, PEA3, ERM, Elk1, Sap1a, or ER71. D, corresponding RT-PCR analyses of 293T cells transfected with 0.1 µg of ACTR and 0.2 µg of various ETS protein expression plasmids.

 
To further prove that p160 proteins are coactivators of ER81, we examined their role in the activation of the endogenous MMP-1 gene (Fig. 1B). No MMP-1 expression was detectable by RT-PCR when ER81 or SRCs were expressed alone; however, SRC-1, GRIP-1, and ACTR were able to elicit MMP-1 mRNA production in the presence of ER81. As a control, GAPDH levels were found to be similar under all of the conditions tested. Also, the degree of MMP-1 activation by SRCs and ER81 was comparable to the one reported previously (37) for ER81 and the coactivator p300 (Fig. 1B) that physically binds to ER81 and stimulates its function (48). Because ACTR is implicated in breast tumorigenesis (7), as is ER81 (27), and because among the three p160 SRCs ACTR seemingly had the greatest impact on the ER81-dependent activation of the endogenous MMP-1 gene, we elected to conduct further experiments primarily with ACTR.

We were interested to study whether ACTR specifically coactivates ER81 or also other ETS proteins, particularly the ER81 homologous proteins PEA3 and ERM. As shown in Fig. 1C, the effect of ACTR on the activity of PEA3 and ERM was indistinguishable from ER81. In contrast, the ETS protein ER71, which was previously shown to activate the MMP-1 promoter (49), was not stimulated by ACTR. Furthermore, two other ETS proteins, the ternary complex factors Elk1 and Sap1a (50, 51), which on their own activated the MMP-1 promoter by ~5-fold, were barely stimulated by ACTR. Accordingly, ACTR also synergized with PEA3 or ERM but not ER71, Elk1, or Sap1a to induce the endogenous MMP-1 gene (Fig. 1D). These results indicate that ACTR is a cofactor of not only ER81 but also of the related ETS proteins PEA3 and ERM.

ER81 Forms Complexes with ACTR—Because ER81-mediated transcription of the MMP-1 gene was enhanced by ACTR, we investigated whether ACTR forms complexes with ER81. To this end, lysates from HeLa cells were challenged with no antibody, anti-GAL4 antibodies, or anti-ER81 antibodies. Resulting immunoprecipitates were resolved by SDS-PAGE and the presence of any coimmunoprecipitated ACTR probed by anti-ACTR Western blotting. Indeed, ACTR coimmunoprecipitated with ER81 (Fig. 2A), whereas no ACTR was detectable in mock and anti-GAL4 immunoprecipitates, indicating that ER81 and ACTR do interact in vivo. To further ascertain that ER81 interacts with ACTR, pull-down assays were performed with either GST or GST-ER81. As expected, ACTR specifically interacted with GST-ER81 (Fig. 2B). In contrast, two arginine methyltransferases, CARM1 and PRMT1, which bind to SRCs (52, 53), did not interact with GST-ER81. Altogether, these results indicate that ER81 and ACTR can form complexes in vitro and in vivo.



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  		FIG. 2.
ACTR interacts with ER81. A, coimmunoprecipitation of endogenous ACTR and ER81 in HeLa cells. Immunoprecipitations were performed with anti-ER81, control anti-GAL4, or no antibodies, and ACTR was subsequently detected by anti-ACTR Western blotting. The bottom panels show input levels of ER81 and ACTR. B, GST pull-downs of Myc6-ACTR, HA-CARM1, or HA-PRMT1. Protein binding to GST or GST-ER812–477 was revealed by anti-Myc (ACTR) or anti-HA Western blotting (CARM1 and PRMT1).

 
A Central Region of ER81 Interacts with the N terminus of ACTR—For localizing the ER81 interaction domain within ACTR, coimmunoprecipitation assays were performed utilizing 293T cells transfected with FLAG-tagged ER81 and various truncations of Myc-tagged ACTR. Cell lysates were immunoprecipitated with anti-FLAG antibody, and the presence of ACTR was visualized by anti-Myc Western blotting. As shown in Fig. 3A, full-length ACTR (ACTR2–1412), ACTR2–1022, and ACTR2–810 coimmunoprecipitated with ER81, whereas ACTR506–1412 and ACTR810–1412 did not. ACTR2–810 was further divided into ACTR2–457 and ACTR459–810, and both of these molecules did not bind to ER81 (Fig. 3A), indicating that the N-terminal 810 amino acids of ACTR are required for its interaction with ER81.



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  		FIG. 3.
Mapping of interaction domains in ACTR and ER81. A, the top panel shows a schematic representation of ACTR. RID, receptor-interacting domain; CID, CBP/p300-interacting domain. The middle panels show anti-FLAG immunoprecipitates of 293T cells transfected with FLAG-tagged ER81 and the indicated Myc-tagged ACTR constructs. Coimmunoprecipitated ACTR molecules were detected by anti-Myc Western blotting. The bottom panels show input levels of the various ACTR truncations. B, the top panel schematically depicts ER81. AD, activation domain; NRD, negative regulatory domain; ETS, DNA binding domain. GST-ER81 fusion proteins were pre-bound to glutathione-agarose and incubated with Myc-tagged ACTR. Bound ACTR was revealed by anti-Myc Western blotting. The bottom panel shows a Coomassie Blue-stained protein gel of GST and the various GST-ER81 proteins utilized.

 
To map the ER81 amino acids interacting with ACTR, GST pull-down assays were performed (Fig. 3B). Whole cell lysates of 293T cells transfected with Myc-tagged ACTR were incubated with GST or ER81 portions fused to GST, and bound ACTR was revealed by anti-Myc Western blotting. Similar to full-length ER81 (ER812–477), the C-terminal half of ER81 (amino acids 249–477) interacted with ACTR, whereas the N-terminal half of ER81 (amino acids 1–249) did not. Subdividing ER81249–477 indicated that ACTR interacted with ER81 amino acids 249–383 but not amino acids 383–477. Thus, a central region of ER81 consisting of parts of its negative regulatory and DNA binding domains but excluding any of its activation domains (see scheme of ER81 in Fig. 3B) interacts with ACTR.

Acetylation of ER81 by ACTR—ER81 is functionally regulated through acetylation via the homologous coactivators p300 and CBP as well as another coactivator/acetyltransferase, P/CAF (37). Two members of the SRC family, SRC-1 and ACTR, are known to possess weak intrinsic HAT activity (3, 39). Therefore, it was imperative to assess whether the ability of ACTR to coactivate ER81 also entails acetylation of ER81 by ACTR.

First, we assessed whether ACTR could acetylate ER81 in vitro. To this end, because the HAT domain of ACTR resides between residues 1092 and 1292 (3), we used GST-ACTR1086–1292 and a similar region of GRIP-1 (amino acids 1158–1432) fused to GST as enzymes to acetylate histones and ER81. As reported previously (3), the HAT domain of ACTR selectively acetylated histones H3 and H4 (Fig. 4A). In addition, the HAT domain of ACTR acetylated ER81 (Fig. 4B). Similarly, GST-GRIP-11158–1432 acetylated histones H3 and H4 as well as ER81, which to our knowledge is the first demonstration that GRIP-1 possesses HAT activity. No acetylation of GST by either GRIP-1 or ACTR was observable (data not shown). Thus, ACTR and GRIP-1 are capable of acetylating ER81 in vitro. However, the degree of this in vitro acetylation of ER81 by SRCs was much less than acetylation mediated by p300 (Fig. 4B), indicating that ER81 may only be substoichiometrically and inefficiently acetylated by SRCs.



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  		FIG. 4.
Acetylation of ER81 by ACTR and GRIP-1. A, in vitro acetylation of histones with HAT domains of ACTR and GRIP-1 fused to GST. The reaction products were subjected to SDS-PAGE and revealed by autoradiography (upper panel). Coomassie Blue-stained histones are shown in the bottom panel. B, in vitro acetylation of GST-ER812–477 with HAT domains of ACTR, GRIP-1, and p300. C, 293T cells were transfected with Myc-ER81 (1.5 µg) and ACTR2–1412 (2.5 µg), ACTR2–1022 (2.5 µg), or p300 (2.5 µg) as indicated. Cell lysates were immunoprecipitated with anti-acetyllysine antibodies or control anti-GAL4 antibodies followed by anti-Myc Western blotting to reveal ER81. D, CV-1 cells were transiently transfected with CMV-ER812–477 (0.5 µg) and 0.25 µg of ACTR2–1412, ACTR2–1172, ACTR2–810, ACTR506–1412, or ACTR810–1412. Resultant MMP-1 reporter luciferase activities are shown. E, analogous to D with 0.8 µg of GRIP-1 constructs instead of ACTR.

 
To explore whether ACTR acetylates ER81 in vivo, 293T cells were transfected with Myc-tagged ER81 and either full-length ACTR2–1412 or the C-terminal truncation ACTR2–1022. Immunoprecipitations then were done with either control anti-GAL4 antibodies or anti-acetyllysine antibodies, and the presence of any acetylated ER81 in the immunoprecipitates revealed by anti-Myc Western blotting. As shown in Fig. 4C, coexpression of ACTR2–1412 and ER81 resulted in in vivo acetylation of ER81, whereas expression of ACTR1–1022 that is devoid of a HAT domain (3) did not. Again, p300 proved to be significantly more efficient than ACTR in eliciting in vivo acetylation of ER81 (Fig. 4C). Altogether, these results indicate that ACTR is capable, albeit inefficiently, to acetylate ER81 both in vitro and in vivo.

Furthermore, the HAT-deficient ACTR2–1172 protein was as efficient as full-length ACTR2–1412 in stimulating the MMP-1 promoter in the presence of ER81 (Fig. 4D), proving that ACTR-mediated acetylation of ER81 does not contribute to the regulation of ER81 transcriptional activity. In stark contrast to ACTR2–1412, ACTR2–810 that still forms complexes with ER81 was completely incompetent to stimulate ER81 activity, demonstrating that ACTR amino acids 811–1172 are required for the observed coactivator function (Fig. 4D). ACTR506–1412 and ACTR810–1412, both lacking a functional ER81-interacting domain but with an intact C terminus, showed no effect on ER81-mediated transactivation, indicating that ACTR needs complex formation with ER81 to coactivate ER81-dependent transcription.

ACTR contains two C-terminal activation domains, AD1 and AD2 (54). The observed equivalent activation of ER81 by ACTR2–1172 (devoid of an intact AD2 domain) and by full-length ACTR2–1412 indicates that the AD2 domain does not play a significant role in the ability of ACTR to stimulate ER81 function. On the other hand, because ACTR2–810 is devoid of both the AD1 and AD2 domains and is unable to functionally collaborate with ER81, the AD1 domain appears to be crucial for the coactivator activity of ACTR toward ER81. Consistently, deletion of the AD1 ({Delta}AD1) domain but not the AD2 domain ({Delta}AD2) of GRIP-1 abolished the ability of GRIP-1 to stimulate ER81-dependent MMP-1 promoter activation (Fig. 4E).

ACTR and p300 Collaborate to Enhance ER81 Function—We next wished to assess whether the essential coactivator p300 (55, 56), which can interact with ACTR (3) and ER81 (48), collaborates with ACTR to activate ER81. As expected, whereas relative MMP-1 promoter luciferase activity with ER81, p300, or ACTR alone was 8, 2, or 1.5, respectively (Fig. 5A), p300 and ACTR individually elevated these levels in the presence of ER81 to 15 and 38, respectively. Importantly, ER81, p300, and ACTR altogether synergized by raising luciferase levels to 73. Similarly, ACTR, p300, and ER81 synergized to induce the endogenous MMP-1 gene (see Fig. 7B). In contrast, P/CAF, which coactivates ER81 function similarly as p300 (37) and also interacts with ACTR (3), did not significantly collaborate with ACTR, whereas P/CAF synergized with p300 to stimulate ER81 (Fig. 5A). Furthermore, neither of the ACTR-interacting proteins CARM1 and PRMT1 (54) was able to stimulate ER81-dependent MMP-1 promoter activity in the absence and presence of ACTR (Fig. 5B). Thus, ACTR specifically synergizes with p300 to activate the transcription factor ER81, whereas three other ACTR-binding proteins, P/CAF, CARM1, and PRMT1, had no impact on ACTR-mediated stimulation of ER81 function.



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  		FIG. 5.
ACTR synergizes with p300 but not P/CAF, CARM1, or PRMT1. Transient transfections in CV-1 cells were performed with the MMP-1 luciferase reporter plasmid, CMV-ER812–477 (0.5 µg), Myc6-ACTR (0.25 µg), and (A) HA-p300 (0.3 µg) or FLAG-P/CAF (0.3 µg), or (B), HA-CARM1 (0.4 µg) or HA-PRMT1 (0.4 µg).

 



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  		FIG. 7.
Collaboration among HER2/Neu, p300, and ACTR in activating ER81. A, MMP-1 luciferase activities in CV-1 cells transiently transfected with CMV-ER812–477 (0.2 µg), HA-p300 (0.3 µg), Myc6-ACTR (0.25 µg), and HER2/Neu-V664E (0.25 µg). B, RT-PCR analysis of endogenous MMP-1 gene activation was performed using cytosolic RNA from 293T cells transiently transfected with CMV-ER812–477 (0.2 µg), p300 (0.1 µg), ACTR (0.1 µg), and HER2/Neu-V664E (1 µg). Expression of GAPDH was evaluated as an internal control.

 
To further investigate the mechanism underlying the synergy between ER81, ACTR, and p300, we utilized p300{Delta}SRC, a p300 molecule containing a deletion (amino acids 2042–2157) in its SRC interaction domain (41), and p300{Delta}HAT in which amino acids 1430–1504 were deleted, resulting in the destruction of the HAT domain (40). Expression levels of p300, p300{Delta}SRC, and p300{Delta}HAT were found to be similar (data not shown). No ER81 stimulation was observable as reported previously (37) with p300{Delta}HAT, whereas p300 and p300{Delta}SRC were equally able to stimulate ER81-dependent MMP-1 promoter activation (Fig. 6A), indicating that p300 does not require the SRC interaction domain to activate ER81. Similar to p300{Delta}HAT, however, p300{Delta}SRC was unable to collaborate with ACTR in activating ER81-dependent transcription (Fig. 6A). These results demonstrate that both its HAT activity as well as its SRC interaction domain are required for p300 to synergize with ACTR in the stimulation of ER81 function.



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  		FIG. 6.
HAT activity of p300 contributes to its synergy with ACTR. A, CV-1 cells were transiently transfected with MMP-1 luciferase reporter and 0.5 µg of CMV-ER812–477, 0.3 µg of p300, p300{Delta}HAT, or p300{Delta}SRC, and 0.25 µg of Myc6-ACTR as shown. B, a similar comparison between wild-type CMV-ER81 and its K33R/K116R mutant.

 
Acetylation of ER81 at lysine residues 33 and 116 by p300 is instrumental for maximal ER81 transcriptional activity (37). Therefore, we also compared wild-type ER81 to its K33R/K116R mutant. As expected, ER81-K33R/K116R was not stimulated by p300, whereas ACTR stimulated this mutant equally well as wild-type ER81 (Fig. 6B). However, in contrast to wild-type ER81, additional expression of p300 did not enhance transcription mediated by ER81-K33R/K116R and ACTR. These results indicate that acetylation of ER81 on Lys-33 and Lys-116 is required for the synergistic activation of ER81 by p300 and ACTR but not for the collaboration between ER81 and ACTR.

Impact of HER2/Neu on the Synergy between ER81 and ACTR—Because HER2/Neu can regulate ER81 (30), p300 (37), and ACTR (38), we wished to investigate how HER2/Neu affects the functional collaboration among ER81, p300, and ACTR. To this end, ER81, p300, ACTR, and HER2/Neu were cotransfected as indicated and resultant MMP-1 luciferase reporter gene activities were measured (Fig. 7A). As reported earlier, HER2/Neu, ER81, and p300 collaborated in the activation of the MMP-1 promoter. Importantly, whereas ACTR+ER81 and HER2/Neu+ER81 resulted in relative luciferase activities of 45 and 80, respectively, the combination of ACTR, ER81, and HER2/Neu resulted in a relative luciferase activity of 165, which was slightly further enhanced by coexpression of p300 to 188. More pronouncedly, maximal transcription of the endogenous MMP-1 gene was elicited by the combination of ACTR, p300, ER81, and HER2/Neu (Fig. 7B), indicating that indeed HER2/Neu can enhance the synergistic activation of ER81 by p300 and ACTR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that the p160 SRC family member ACTR not only forms complexes with the ETS transcription factor ER81 in vitro and in vivo but also stimulates ER81-dependent transcription. By these criteria, ACTR has been identified as a bona fide coactivator of ER81. To our knowledge and in contrast to the suppression of p53 transactivation by human ACTR (17), this is the first demonstration that ACTR is not an exclusive nuclear hormone receptor coactivator. Aside from ACTR, the two other members of the p160 SRC family, SRC-1 and GRIP-1, were also able to stimulate ER81-dependent transcription. Conversely, ACTR was not only a functional coactivator of ER81 but was also a functional coactivator of two other ER81-related ETS transcription factors, PEA3 and ERM. This finding suggests a large degree of promiscuity to exist in the coactivation of gene transcription mediated by SRCs and the PEA3 group of ETS transcription factors.

Functional dissection of ACTR revealed that the region of ACTR (amino acids 2–810) forming complexes with ER81 was insufficient to coactivate ER81-mediated transcription. Rather, ACTR amino acids 811–1172, which encompass the AD1 domain, were additionally required for stimulation of ER81. However, the AD2 domain of ACTR was dispensable for the stimulation of ER81 by ACTR, pointing at the fact that CARM1/PRMT1, which interact with the AD2 domain (2, 54), do not participate in this coactivation of ER81 by ACTR. Accordingly, CARM1/PRMT1 had no impact on ER81-dependent transcription in the absence and presence of ectopic ACTR (see Fig. 5B) and did not form complexes with ER81 (see Fig. 2B) or utilize ER81 as a substrate in in vitro methylation assays (data not shown).

The coactivator and acetyltransferase p300 synergized with ACTR in stimulating ER81 function. Importantly, acetylation of ER81 by p300 was indispensable for this synergy, as demonstrated by the inability of p300{Delta}HAT to synergize with ACTR in stimulating wild-type ER81 and by the inability of wild-type p300 to enhance ACTR-mediated stimulation of ER81-K33/116R, a mutant of ER81 that can no longer be acetylated by p300 (37). p300{Delta}SRC, a p300 molecule deficient in binding to SRCs, activated ER81-mediated transcription as efficiently as wild-type p300 (see Fig. 6A). However, p300{Delta}SRC was unable to synergize with ACTR, indicating that probably joint recruitment of p300 by both ER81 and ACTR is required for transcriptional synergy to occur. Although both ACTR and ER81 can also interact with P/CAF (3, 37), P/CAF as opposed to p300 did not synergize with ACTR to stimulate ER81-dependent transcription. At present, the reason for this difference between p300 and P/CAF remains unresolved.

Two members of the SRC family, SRC-1 and ACTR, are known to selectively acetylate two components of nucleosomes, histones H3 and H4 (3, 39). Here, we showed that GRIP-1 also possesses HAT activity of the same selectivity. Furthermore, we demonstrated that ER81 is acetylated by GRIP-1 and ACTR in vitro. However, this acetylation was much less efficient than in vitro acetylation by p300, and similar results were obtained for the in vivo acetylation of ER81 upon overexpression of ACTR compared with p300. Furthermore, ACTR acetylated multiple regions within ER81 (data not shown) in contrast to the restricted acetylation of Lys-33 and Lys-116 by p300, implicating that acetylation at any single lysine residue is of very low stoichiometry and therefore probably without physiological relevance. Moreover, both ACTR and GRIP-1 molecules lacking the AD2 domain and consequently also HAT activity were indistinguishable from respective full-length SRCs in their ability to coactivate ER81. Thus, the HAT activity of SRCs is not involved in coactivation of ER81, similarly as reported for SRC-1 and nuclear hormone receptors (39, 57).

Both ACTR, which is overexpressed in ~60% of primary human breast tumors (7), and HER2/Neu, which is found overexpressed in ~30% of all human breast cancer patients (33), play important roles in breast tumorigenesis. It even appears that ACTR overexpression is correlated with HER2/Neu positivity and absence of estrogen and progesterone receptors in breast tumors (58), implicating that ACTR functions via a transcription factor(s) other than nuclear hormone receptors in the process of malignant cell transformation in the breast. Up until now, the identity of such a transcription factor had been in question; however, our study has unveiled potential candidates with the related ETS proteins ER81, PEA3, and ERM. Ample evidence points to the involvement of these ETS transcription factors in breast tumorigenesis including their frequent overexpression in HER2/Neu-positive human and mouse mammary tumors (23–25) and their ability to function as downstream targets of HER2/Neu (25, 30). As such, HER2/Neu and ACTR overexpression may result in the synergistic stimulation of ER81- and p300-mediated transcription as observed in this study by various means (Fig. 8): (i) phosphorylation of ER81 upon HER2/Neu-triggered activation of the MAPK signaling pathway (30, 32); (ii) stimulation of p300 HAT activity by oncogenic HER2/Neu (37), leading to enhanced acetylation of ER81; (iii) HER2/Neu-mediated stimulation of ACTR via MAPKs resulting in stronger interaction with p300 (38); and (iv) ACTR-mediated stimulation of ER81, which does not involve acetylation of ER81 by ACTR.



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  		FIG. 8.
Model for how oncogenic HER2/Neu and ACTR contribute to breast cancer formation via ER81 and p300.

 
Tumorigenesis is a multistep process that requires the dysregulation of genes encoding key factors mediating growth factor independency, evasion from anti-proliferative signals and apoptosis, immortalization, or tumor invasion/metastasis (59). In concert with the oncogenic growth factor receptor HER2/Neu, the transcription factor ER81 has been found to dysregulate several target genes implicated in cancer onset and metastasis. For instance, ER81 stimulates the proteinases MMP-1 and matrilysin that are involved in tissue invasion and metastasis (30, 60). Also, ER81, in conjunction with HER2/Neu, enables cells to induce human telomerase reverse transcriptase expression thought to mediate immortalization (61). Moreover, HER2/Neu synergizes with ER81 to attenuate transforming growth factor-{beta} signaling by up-regulating Smad7, thereby evading apoptotic and anti-growth signals (62). Finally, ER81 as well as PEA3 can further activate HER2/Neu transcription, thereby establishing a positive feedback loop in which HER2/Neu stimulates ER81 and PEA3 via the MAPK signaling pathway and thereby its own expression (25, 63).

In conclusion, our study provides evidence for one mechanism of how joint overexpression of ACTR and HER2/Neu can lead to breast tumor formation through the synergistic activation of transcription factor complexes including p300 and ER81 (or PEA3 or ERM). As a corollary, our study strongly suggests to not only treat HER2/Neu-positive breast cancer patients with trastuzumab, an anti-HER2/Neu antibody delaying disease progression (64, 65), but to additionally inhibit ACTR and/or the PEA3 subfamily of ETS proteins with yet-to-be developed strategies to enhance therapeutic effectiveness.


   FOOTNOTES
 
* This work was supported by the Mayo Foundation, Grant CA085257 [GenBank] from the National Cancer Institute, and a grant from the Fraternal Order of Eagles' Cancer Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mayo Clinic, Guggenheim Bldg. 1501A, 200 First St., S. W., Rochester, MN 55905. Tel.: 507-266-4393; Fax: 507-284-1767; E-mail: janknecht.ralf{at}mayo.edu.

1 The abbreviations used are: SRC, steroid receptor coactivator; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; ACTR, activator of thyroid and retinoic acid receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRIP-1, glucocorticoid receptor-interacting protein-1; HA, hemagglutinin; GST, glutathione S-transferase; HAT, histone acetyltransferase; MAPK, mitogen-activated protein kinase; MMP-1, matrix metalloproteinase-1; DTT, dithiothreitol; RT, reverse transcription; CMV, cytomegalovirus; STAT, signal transducers and activators of transcription. Back


   ACKNOWLEDGMENTS
 
We thank Drs. W. Kraus, R. Evans, B. O'Malley, and G. Muscat for generously providing p300{Delta}SRC, ACTR, SRC-1, and GST-GRIP-11158–1432 plasmids, respectively. We are also indebted to Dr. M. Stallcup for providing CARM1 and various GRIP-1 deletion constructs.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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