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Originally published In Press as doi:10.1074/jbc.M603998200 on June 30, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25373-25380, September 1, 2006
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ErbB-4 s80 Intracellular Domain Abrogates ETO2-dependent Transcriptional Repression*

Bryan Linggi and Graham Carpenter1

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, April 26, 2006 , and in revised form, June 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ErbB-4 is cleaved by {alpha}- and {gamma}-secretases to release a soluble 80-kDa intracellular domain, termed s80, which translocates to the nucleus. s80 is present in the nucleus of normal and cancerous mammary cells and is predicted to have a role in cell differentiation. To further investigate the mechanism by which s80 may mediate differentiation, we tested whether s80 regulates Eto2, a transcriptional corepressor that is involved in erythrocyte differentiation and is also implicated in human breast cancer. Here we show that ligand binding to ErbB-4 causes s80 translocation to the nucleus, where it colocalizes and interacts with Eto2. Expression of s80 blocks Eto2-mediated transcriptional repression of a heterologous promoter. This effect on Eto2 does not require s80 kinase activity and is mediated by the carboxyl-terminal region of s80. Although other cell surface receptors regulate transcription by activating signal transduction cascades, these data present a novel mechanism of corepressor regulation and suggest a role for Eto2 in ErbB-4-dependent differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ErbB-4 (also known as HER4) is a receptor tyrosine kinase that regulates cell differentiation in the mammary gland as well as other cell types (1). ErbB-4 is activated by the binding of heregulins (also known as neuregulins), which provokes ErbB-4 homo- or heterodimerization (predominantly with ErbB-2). Dimerization allows tyrosine autophosphorylation of the ErbB-4 carboxyl-terminal region and activation of downstream signaling pathways, including the extracellular signal-regulated kinases (ERKs),2 phosphatidylinositol 3'-kinase, signal transducers and activators of transcription (STAT), and phospholipase C (PLC-{gamma}) (1).

ErbB-4 is sequentially cleaved by two proteases following ligand binding or 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. The first cleavage step involves tumor necrosis factor-{alpha}-converting enzyme, which cleaves within the extracellular juxtamembrane region between His-651 and Ser-652 (2), releasing the extracellular domain and leaving a membrane-bound intracellular domain (ICD), termed m80 (2). The m80 fragment is subsequently cleaved within its membrane region by a {gamma}-secretase complex to produce a soluble ICD termed s80. s80 is localized in both in the cytoplasm and in the nucleus (3).

The ability of ErbB-4 to regulate differentiation, particularly in the mammary gland, is attributed to s80 localization in the nucleus (4). ErbB-4 levels increase during mammary gland lactogenesis, and nuclear ErbB-4 is observed in lactating but not normal gland (4). Consistent with this role, genetic studies have shown that ErbB-4 is necessary for lactogenesis (4). Also, in cell culture, ErbB-4 activation provokes differentiation of breast cancer cells (5). Whether ErbB-4 or s80 confers a positive or negative prognosis in breast cancer patients is unclear (6); however, the cleavable ErbB-4 isoform, JMa, which is specifically overexpressed in breast cancers, increases cell growth (7). Also, breast cancer cells containing nuclear ErbB-4 are more differentiated than those without nuclear ErbB-4 (8). Further understanding of s80 function will help clarify the role of ErbB-4 in breast cancer and normal cell differentiation.

Eto2 (also known as MTG16 or CBFA2T3) is a nuclear transcriptional corepressor involved in breast cancer and normal cell differentiation. It is expressed in normal breast epithelial cells (9) and mouse mammary cell lines3 and is a candidate tumor suppressor due to it being a target of loss of heterozygosity at 16q24.3, which is common in breast cancer (9). In addition, Eto2 regulates differentiation during erythropoiesis by repressing genes important for cell differentiation (10, 11). However, little is known concerning how Eto2 function is regulated during cell growth and differentiation.

Thus, both ErbB-4 and Eto2 are described as having a role in both breast cancer and differentiation; however, the mechanisms by which this occurs are unknown. Therefore, we tested the hypothesis that ErbB-4 regulates Eto2 function. This was examined by assessing the ability of these proteins to interact and determining whether ErbB-4 can regulate Eto2-mediated transcriptional repression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Antibodies ErbB-4 (sc-283, Santa Cruz Biotechnology) or FLAG-M2-agarose (A2220, Sigma) were used for immunoprecipitations. Membranes were blotted with antibodies to ErbB-4 (rabbit polyclonal from M. Kraus, University of Alabama-Birmingham), FLAG (F3165, Sigma), Myc (Santa Cruz Biotechnology, sc-40), tubulin (Santa Cruz Biotechnology, sc-8035), PLC-{gamma}1 (Santa Cruz Biotechnology, sc-81), phosphotyrosine (Santa Cruz Biotechnology, PY99), and GAL4 (Santa Cruz Biotechnology, sc-577). For immunofluorescence, Myc (Santa Cruz Biotechnology, sc-40) or V5 antibody (Invitrogen) was used followed by anti-mouse TRITC antibody (Zymed Laboratories Inc.). Propidium iodide was purchased from Sigma.

Cell Culture, Transfection, and Treatments—Cos7 cells were maintained in Dulbecco's modified eagle medium with 10% fetal calf serum. HC11 cells were maintained as described previously (12). Cells were transfected with either Lipofectamine 2000 (Invitrogen) or Polyfect (Qiagen). Cells were treated with either TPA (100 ng/ml, Sigma) or heregulin 1beta (Hrg1beta 50 ng/ml, R&D Systems) for 1 h.

Plasmids—FLAG-s80-cyt2 was created by PCR amplifying the region corresponding to residues 676–1292 of GFP-s80-Cyt2 (3) and inserting it into the BglII and SalI of pCMV-FLAG2 (Sigma). FLAG-s80-Cyt1 was created by exchanging the KpnI to PstI region with the corresponding region from pCDNA3 ErbB-4-JmA/Cyt1 (provided by Klaus Elenius, University of Turku, Finland). FLAG-s80-K751R was created by using the QuikChange II kit (Stratagene) to change nucleotides AAG to AAA at residue 751–753 of ErbB-4 using FLAG-s80-Cyt2 as a template. FLAG-s80KD was created by amplifying the regions corresponding to residues 676–988, with the addition of the restriction sites BglII and SalI, and inserting the regions into BglII and SalI of pCMV-FLAG2. Similarly, FLAG-s80CT was created by amplifying the regions corresponding to residues 989–1292 (does not contain the Cyt1 region). Unless otherwise noted, the Cyt2 isoform of s80 was used. Myc-Eto2 and Gal4-Eto2 were provided by S. Hiebert (13). Human V5-ETO2 (MTG16) was provided by N. Sacchi (14).

Immunoprecipitation—Whole cell lysates for immunoprecipitation were prepared with TGH buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES pH 7.2, 10 mM NaCl) containing protease and phosphatase inhibitors (Sigma mammalian protease inhibitor mixture, phosphatase inhibitor mixture I, and phosphatase inhibitor mixture II). The lysates were sonicated and centrifuged (13,000 rpm, 4 min) to remove insoluble material. To obtain nuclear and cytoplasmic lysates, cells were scraped in phosphate-buffered saline and centrifuged (1000 x g for 5 min), and the pellet was resuspended in Buffer B (10 mM HEPES pH 7.2, 1.5 mM MgCl2, 10 mM KCl, plus inhibitors as above) including 1% Igepal, vortexed, and then centrifuged (400 x g for 5 min). The supernatant was saved as the cytoplasmic fraction; the pellet was washed in Buffer B and then centrifuged (400 x g for 5 min). The pellet was then resuspended TGH buffer plus inhibitors and sonicated to release nuclear proteins. Both the cytoplasmic and the nuclear fractions were then centrifuged (13,000 rpm for 10 min), and the pellet was discarded. For phosphotyrosine analysis of Myc-Eto2, cells were lysed in radioimmune precipitation buffer (0.1% Igepal, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate in phosphate-buffered saline + inhibitors as above), sonicated, and centrifuged. The protein levels in the lysates were quantitated using the DC protein assay kit (Bio-Rad). For fractionation experiments, three times the amount of protein was used in immunoprecipitation for the cytoplasmic fraction relative to the nuclear fraction to account for the different sizes of these cellular compartments. These lysates were precipitated with the indicated antibodies for 1–2 h followed by the addition of protein-A-Sepharose (Zymed Laboratories Inc.) for 30 min. The antibody-Sepharose complexes were washed three times in TGH buffer plus inhibitors. Complexes and lysates (equal to 5% of the volume immunoprecipitated) were incubated (70 °C for 10 min) in 4x LDS sample buffer (Invitrogen), separated on a 4–12% gel (Invitrogen Novex Midi gel), transferred to a polyvinylidene fluoride membrane (Millipore), and immunoblotted with the indicated primary antibodies followed by the addition of secondary horseradish peroxidase antibodies and detection by enhanced chemiluminescence.

Immunofluorescence—Transfected cells were transferred into 4-well chamber slides (Labtek) the day after transfection. The next day, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized in phosphate-buffered saline/0.2% Triton X-100 for 5 min, and blocked in 5% bovine serum albumin/phosphate-buffered saline/0.1% Tween. Myc-Eto2 was detected using Myc antibody and human V5-tagged ETO2 with V5 antibody followed by anti-mouse TRITC secondary antibody. The slides were mounted with mounting medium (Vector Laboratories), and images were obtained under x40 magnification with additional digital zoom on a Zeiss LSM510 confocal microscope with a pinhole size set to 1 Airy unit.

Reporter Assays—Cos7 or HC11 cells were transfected in 6-well plates with 0.01 µg pCMV-Renilla, 0.29 µg Gal-TK-luciferase (firefly), 0.3 µg GAL4-Eto2, the indicated expression plasmids for ErbB-4-s80, and empty pCMV-FLAG2 to a total of 1.5 µg of DNA. Two days later, a Dual Luciferase assay kit (Promega) was used to calculate luciferase activity, which was adjusted to control for transfection efficiency by dividing the luciferase value by the Renilla value of the sample. Fold repression is calculated by dividing the control value by the sample value. Total lysates or nuclear fractions were analyzed by SDS-PAGE as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ErbB-4 is cleaved following stimulation of cells with either TPA or heregulin (3), releasing the s80 intracellular domain into the cytoplasm and nucleus. To determine whether s80 generated from ErbB-4 colocalizes with Eto2, a nuclear corepressor, Cos7 cells were transfected with both ErbB-4-GFP and murine Myc-Eto2 and treated with TPA or heregulin, and ErbB-4 and Eto2 localization was visualized using confocal microscopy. Without treatment, ErbB-4-GFP was localized to the plasma membrane (and Golgi), and Myc-Eto2 was nuclear (Fig. 1). Following treatment with either TPA or heregulin, ErbB-4-GFP, likely in the form of s80 (3), was present in the cytoplasm and nucleus (Fig. 1). s80 and Eto2 were both present in many regions of the nucleus, showing an overlap between s80 (GFP) and Myc-Eto2 (Fig. 1, red) signals. This demonstrates that ErbB-4 stimulation allows s80 to transport to the nucleus and colocalize with the corepressor Eto2. Given that the GFP tag that is used in the above assays has weak dimerization properties (15), the localization of GFP-tagged and untagged s80 was compared and found to be indistinguishable (data not shown).


Figure 1
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  		FIGURE 1.
ErbB-4-GFP colocalizes with Eto2 in the nucleus following ligand or TPA stimulation. Cos7 cells were transfected with ErbB-4-GFP and Myc-ETO2 and treated with or without TPA or heregulin for 60 min. Cells were fixed and stained with Myc antibody. Localization of ErbB-4-GFP (green) and Myc-Eto2 (red) was visualized by confocal microscopy. Colocalization of ErbB-4-GFP and Myc-ETO2 is indicated by the yellow color in the Merge panels. The bar represents 10 µm.

 
Nuclear corepressors are regulated by extracellular signals that are transmitted to the nucleus via signaling cascades (16). Treatment of cells expressing ErbB-4 with either TPA or heregulin would, in addition to causing ErbB-4 cleavage and s80 formation, also activate either protein kinase C or signaling cascades downstream of ErbB-3 or ErbB-1 (1). To determine whether formation of s80 alone is sufficient for colocalization with Eto2, cells were co-transfected with GFP-s80 and Myc-Eto2, and their respective localization was examined in the absence of heregulin or TPA. In these cells, GFP-s80 and Myc-Eto2 colocalized in the nucleus similar to that observed with cells expressing ErbB-4-GFP after treatment with TPA or heregulin (Fig. 2A). These data reveal that s80 expression alone, in the absence of other signals, leads to colocalization of s80 and Eto2 and suggests that s80 might regulate Eto2 function through a direct mechanism.

In the mouse, ErbB-4 and Eto2 are expressed in many tissues, including the breast, brain, and heart (17, 18). In view of the known functions of ErbB-4 and Eto2 in human breast physiology and cancer, we tested whether human ETO2 (also known as CBFA2T3 or MTG16), which shares 86% identity with mouse Eto (17), also colocalizes with s80 in the nucleus. Cells were transfected with GFP-s80 and V5-tagged ETO2 and then stained with V5 antibody. In these experiments, GFP-s80 and V5-ETO2 colocalized in the nucleus (Fig. 2B), similar to that observed with murine Eto2 (Fig. 2A). This indicates that s80 can colocalize with either mouse or human Eto2.

Since s80 and Eto2 colocalize in the nucleus, we next tested whether s80 interacts with Myc-Eto2. Cells expressing both s80 and Myc-ETO2 were separated into nuclear and cytoplasmic fractions and precipitated with an antibody to an epitope in the carboxyl terminus of ErbB-4. Although only 5–10% of s80 was present in the nuclear fraction, it coprecipitated with Myc-Eto2 (Fig. 3A). s80 also interacted with human V5-ETO2, indicating that, similar to the colocalization experiments, this interaction is conserved. (Fig. 3B). To verify specificity of the antibody, Myc-Eto2 was expressed in the absence of s80 and did not precipitate with the ErbB-4 antibody (Fig. 3, A and B). Together, these results show that s80 interacts with Eto2 in the nucleus.

s80 contains an active tyrosine kinase domain that autophosphorylates its carboxyl-terminal region (CT) (19)(Fig. 4A). This phosphorylated CT can serve as a docking site for both phosphotyrosine binding domain-containing proteins and SH2 domain-containing proteins in the context of full-length ErbB-1 (1). s80 can also phosphorylate substrates, including Mdm2 (20). To determine whether kinase activity or a phosphorylated CT is required for s80 to interact with Eto2, the mutant s80 protein, s80K751R, which lacks both tyrosine kinase activity and phosphorylated CT tyrosine residues (due to the lack of kinase activity), was utilized (20).

To characterize this mutant, we first tested whether s80K751R enters the nucleus since nuclear localization is required for the interaction between s80 and Eto2 (Fig. 3). Cells were transfected with GFP-s80K751R and wild type GFP-s80, and the localization was examined. Similar to GFP-s80, GFP-s80K751R localized to the cytoplasm and nucleus, indicating that neither kinase activity nor CT phosphorylation is necessary for nuclear localization (Fig. 4B).

Next, s80K751R was tested for its ability to interact with Eto2. Cells were transfected with s80K751R (or wild type s80 as a positive control) and Eto2, separated into nuclear and cytoplasmic fractions, precipitated with ErbB-4 antibodies, and analyzed by immunoblotting for Eto2. In these experiments, s80K751R and wild type s80 were associated with similar amounts of Eto2 (Fig. 5A). Additionally, Eto2 was not phosphorylated in cells expressing wild type s80 (data not shown). Thus, neither kinase activity nor CT phosphorylation is required for the interaction between s80 and Eto2, and Eto2 is not a substrate for s80 kinase activity.


Figure 2
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  		FIGURE 2.
s80 colocalizes with Eto2 in the nucleus. A, Cos7 cells were transfected with GFP-s80 and Myc-Eto2, fixed, and stained with Myc antibody. Localization of GFP-s80 (green) and Myc-Eto2 (red) was visualized by confocal microscopy. Colocalization of GFP-s80 and Myc-Eto2 is indicated by the yellow color in the merge panels. B, Cos7 cells were transfected with GFP-s80 and V5-ETO2, fixed, and stained with V5 antibody. Localization of GFP-s80 (green) and V5-ETO2 (red) was visualized by confocal microscopy. Colocalization of GFP-s80 and V5-ETO2 is indicated by the yellow color in the merge panels. The bar represents 10 µm.

 
In an effort to determine how s80 might regulate Eto2, we mapped the region of s80 required to interact with Eto2. s80 retains three distinct regions of ErbB-4: a short juxtamembrane sequence, an active tyrosine kinase domain, and a CT region (Fig. 4A). Due to alternative splicing, the CT is present as either a Cyt1 isoform, containing a 16-residue sequence, including a phosphatidylinositol 3'-kinase binding motif, or a Cyt2 isoform, which lacks this sequence (21) (Fig. 4A). Also, a putative nuclear localization sequence (NLS) within the juxtamembrane region of ErbB-4 is reported to be required for s80 nuclear localization (22).

First, we tested whether Eto2 differentially interacts with the Cyt1 and Cyt2 isoforms of s80. Cells expressing each isoform and Myc-Eto2 were fractionated, precipitated with ErbB-4 antibodies, and immunoblotted for Myc-Eto2. We found that Eto2 was associated with similar levels of s80Cyt1 and s80Cyt2 (Fig. 5A), demonstrating that both isoforms can associate with Eto2.


Figure 3
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  		FIGURE 3.
s80 interacts with Eto2 in the nucleus. A, Cos7 cells were co-transfected with s80 and Myc-Eto2. Cells were separated into nuclear (N) and cytoplasmic (C) fractions and precipitated with ErbB-4 antibody. Myc-Eto2 was detected with Myc antibody, and V5-ETO2 was detected with V5 antibody. Lysates were blotted for Myc-Eto2 (nuclear) and PLC-{gamma} (cytoplasmic) to validate fractionation. The plus or minus sign indicates the presence or absence of transfected s80. Lysates indicates pre-IP lysates equal to 5% of IP input. B, Cos7 cells were co-transfected with s80 and V5-ETO2. Cells were separated into nuclear and cytoplasmic fractions and precipitated with ErbB-4 antibody. The plus or minus sign indicates the presence or absence of transfected s80. Lysates indicates pre-IP lysates equal to 5% of IP volume.

 
Next, s80 mutants composed of either the juxtamembrane and kinase domain (s80KD) or the CT region (s80CT) were created (Fig. 4A). A FLAG epitope was added to the amino terminus of each construct to facilitate detection. While characterizing these mutants, we found that a larger percentage of the total amount of s80KD is in the nucleus when compared with the percentage of wild type s80 in the nucleus (Fig. 5B). Also, although the s80CT domain lacks the NLS in the juxtamembrane region, it is still localized in the nucleus as measured by either cell fractionation (Fig. 5B) or cell fluorescence (Ref. 3 and data not shown). This suggests that, in the absence of the putative NLS, s80CT utilizes another NLS or enters the nucleus via non-NLS mediated transport. These mutants were then tested for their ability to interact with Eto2. In these assays, both wild type s80 and s80CT interacted with Eto2 in the nucleus and, although it had the highest amount of protein in the nucleus of all s80 mutants tested, s80KD did not associate with Eto2 (Fig. 5B). Thus, the CT region of s80 is sufficient to interact with Eto2.


Figure 4
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  		FIGURE 4.
A, schematic of s80 proteins and mutants. B, GFP-s80 K751R translocates to the nucleus. Cos7 cells were transfected with GFP-s80, GFP-s80 K751R, or GFP-PLC-g1 and fixed, and the nucleus was stained with propidium iodide (PI). The bar represents 10 µm. DIC, differential interference contrast.

 
Although Eto2 functions as a non-DNA binding transcriptional corepressor in hematopoietic cells (10, 11), the target genes regulated by Eto2 in the mammary gland are not known. Therefore, to address the possible functional consequence of the s80 interaction with Eto2, we tested whether s80 could alter GAL4-Eto2-dependent repression of a heterologous promoter. Cells were transfected with a luciferase reporter containing multimerized binding sites for GAL4 and empty vector, GAL4-Eto2, or GAL4-Eto2 and s80. Transfection of GAL4-Eto2 reduced transcription (as measured by luciferase activity) by ~20-fold (13). When s80 was co-transfected, GAL4-Eto2-mediated repression was significantly reduced in a dose-dependent manner (Fig. 6A). s80 also reduced GAL4-Eto2-mediated repression in HC11 cells, a normal mouse mammary gland cell line (data not shown). As a control, we note that s80 did not have a significant effect on luciferase activity in the absence of GAL4-Eto2 (data not shown). In addition, the overall level of Gal4-Eto2 expression did not change, indicating that this regulation is not due to a reduction of total Gal4-Eto2 levels (Fig. 6A). Previous studies using GAL4 transfection assays with the ICDs of ErbB-4 and ErbB-1 (epidermal growth factor receptor) have shown that only the CT fragments regulate transcription, whereas the intact ICDs (corresponding to ErbB-4 s80) do not (3, 23). Therefore, s80 inhibition of Eto2-mediated repression is unique in that the intact ICD can regulate transcription.


Figure 5
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  		FIGURE 5.
s80 carboxyl-terminal region interacts with Eto2. A, Cos7 cells were transfected with Myc-Eto2 and the indicated s80 construct. Cells were separated into nuclear (N) and cytoplasmic (C) fractions, and s80 was precipitated with an ErbB-4 antibody. Lysate indicates pre-IP lysates equal to 5% of IP input and were blotted for Myc-Eto2 (nuclear) and tubulin (cytoplasmic) to validate fractionation. Note that the image was spliced to correct for the misaligned gel. B, Cos7 cells were transfected with Myc-Eto2 and the indicated s80 construct. Cells were fractionated into nuclear and cytoplasmic fractions and precipitated with FLAG antibody. Lysate indicates pre-IP lysates equal to 5% of IP volume. The asterisk indicates the size of s80 or s80 mutants. The blot was probed for Myc followed by FLAG; therefore the band from Myc is seen on the FLAG blot (upper asterisk). Lanes that were spliced together are indicated by vertical white lines. WB, Western blot.

 
Although kinase activity is not required for s80 to interact with Eto2 (Fig. 5A), it remained possible that s80 kinase activity is necessary to block Eto2-mediated repression. To test this, s80K751R was co-transfected with GAL4-Eto2, and the luciferase values were measured. In these experiments, s80K751R reduced Gal4-Eto2-mediated repression to levels equivalent to the effect of wild type s80 (Fig. 6B), indicating that kinase activity is dispensable for s80 inhibition of Eto2-mediated repression.

In view of the fact that the CT of s80 is sufficient for the interaction between s80 and Eto2 and kinase activity is not required for s80 reduction of Gal4-Eto2 repression, we next tested whether the CT is sufficient to reduce Gal4-Eto2-mediated repression. FLAG-s80, FLAG-s80KD, and FLAG-s80CT were co-transfected with Gal4-Eto2 into cells, and the luciferase values were measured. In this assay, both wild type s80 and s80CT reduced Gal4-Eto2-mediated repression, whereas s80KD was severely impaired (Fig. 6C). Thus, s80CT is sufficient to interact with Eto2 and to functionally reduce Gal4-Eto2-mediated repression. The kinase domain is not necessary for these functions, thereby identifying a novel, kinase-independent mechanism of Eto2 inhibition.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we show that ErbB-4 regulates Eto2 function. The mechanism involves a nuclear interaction between s80 and Eto2 that is sufficient to reduce Eto2-mediated repression. The evidence that this mechanism involves a nuclear interaction between s80 and Eto2 is that both proteins colocalize (Fig. 2) and interact (Fig. 3) in the nucleus. Also, s80 does not require the kinase domain to block Eto2-mediated repression (Fig. 6, B and C), eliminating the possibility that ErbB-4 regulates Eto2 by phosphorylating and activating a signaling cascade.

These data present a novel mechanism to regulate Eto2 function. Others have shown that receptor tyrosine kinases can regulate corepressors by activating signaling cascades. For example, epidermal growth factor receptor, through activation of ERK, activates the thyroid hormone receptor by inhibiting nuclear corepressor/silencing mediator of retinoic acid and thyroid hormone receptor (NCoR/SMRT) (16), and in Drosophila, ErbB-1 (epidermal growth factor receptor) activates Notch by blocking the corepressor Groucho (24). Similarly, FMS-like tyrosine kinase 3 (Flt3) activates promyelocytic leukemia zinc finger (PLZF) transcription by inhibiting SMRT (25). In each of the above examples, corepressor function is reduced by intact receptor activating a signaling cascade, typically involving ERK and, ultimately, phosphorylation of the corepressor. In contrast, we find that ErbB-4 regulates transcription by undergoing ligand-mediated proteolysis and transport to the nucleus, where it interacts with Eto2 (Figs. 1, 3, and 6). A similar pathway has recently been described for the plasma membrane protein CKIP-1, which is cleaved by caspase-3 to release carboxyl-terminal fragments that regulate AP-1 activity (26). Although others have demonstrated a role for phosphorylation and/or export of corepressors, we find that Eto2 is not phosphorylated by s80 (data not shown), nor is the kinase domain necessary for inhibition of Eto2 repression (Fig. 6). Additionally, we did not observe Eto2 export from the nucleus (Figs. 1, 2, 3 and 5). Currently, studies are ongoing investigating whether s80 sterically or allosterically disrupts interactions between Eto2 and other corepressors.


Figure 6
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  		FIGURE 6.
s80 reduces Gal4-Eto2-mediated repression. A, Cos7 cells were transfected with GAL4-TK-luciferase and GAL4-Eto2 or FLAG-s80(Cyt2) or FLAG-s80 mutants as indicated. The plus sign indicates the presence of plasmid in transfection, and the minus sign indicates its absence. The amount of FLAG-s80 transfected is indicated in µg amounts. Fold repression is calculated by dividing the average of the control sample (without GAL4-Eto2 or s80) by the average of the test samples. Whole cell lysates were analyzed by SDS-PAGE and blotted for GAL4 or s80 (ErbB-4 antibody) in the panel below the graph. Lanes were spliced (white line) to eliminate intervening lanes. B, Cos7 cells were transfected with GAL4-TK-luciferase, GAL4-Eto2, and GFP-s80 (wt) or GFPs80K751R (KR) as indicated. The plus sign indicates the presence of plasmid in transfection, and the minus sign indicates its absence. C, Cos7 cells were transfected with GAL4-TK-luciferase and GAL4-Eto2 or FLAG-s80(Cyt2) or mutants as indicated. Nuclear lysates were analyzed by SDS-PAGE and immunoblotted with FLAG antibody in the panel next to the graph. Lanes were spliced (white line) to eliminate intervening lanes.

 
ErbB-4 regulation of Eto2 may be important for controlling differentiation in many cell types. Eto2 is expressed in multiple tissues (27), and loss of Eto2 function or expression may be involved in breast cancer progression or initiation (9). To date, however, its function has only been clearly described in erythropoietic progenitors, in which ErbB-4 is not expressed. In these cells, Eto2 is a key member of a multiprotein transcription complex including T-cell acute lymphocytic leukemia/stem cell leukemia (TAL1/SCL), HeLa E-Box-binding protein (HEB), LM02, mSin3a, and Gfi1-b, which together regulate the transcription of differentiation-specific genes (10, 11). Reducing the amount of Eto2 in these complexes increases gene transcription and, conversely, increasing the levels of Eto2 reduces transcription (10, 11).

Beyond regulating Eto2, other Eto2 family members might be regulated by s80 since s80 also interacts with both Eto and Mtgr1.4 Eto2 family members contain conserved Nervy homology domains 1–4, and each binds a similar set of corepressors (13, 17). Eto interacts with the DNA binding transcription factors PLZF and Gfi-1b (17). Eto and Mtgr1 are expressed in many tissues, and in some cases, are expressed in the same cell type (27). In gene deletion experiments, Eto was necessary for gut development (28) and Mtgr1 was necessary in the development of the small intestine (29). This suggests that although similar, each Eto family member has distinct roles and that in some instances, these processes might be regulated by ErbB-4.

Since the discovery that ErbB-4 is proteolytically cleaved and translocates to the nucleus, a number of s80-interacting proteins have been identified. Among these are Mdm2 (20) and Stat5 (22). Activation of the differentiation gene beta-casein requires STAT5 phosphorylation and s80 nuclear translocation (22). Two WW domain-containing proteins, Wwox and Yes-associated protein (YAP) (30), interact with PXXP motifs in the carboxyl terminus of s80. Wwox interacts with s80 in the cytoplasm (31), whereas YAP interacts with s80 in the nucleus, where together they synergize to activate transcription (30). Because Eto2 can be part of multiprotein transcriptional complexes, it is possible that proteins that interact with s80 might be part of the same complex or compete with one another for s80 binding. We did not observe an effect of Eto2 on beta-casein promoter activity,4 suggesting that the STAT5 and Eto2 pathways are independent. Further investigation is required to test the role of these proteins on ErbB-4 function.

Although ErbB-4 is not expressed in hematopoietic cells, including erythropoietic progenitors, ErbB-4 regulation of Eto2 may similarly regulate differentiation in other cell types. ErbB-4 and Eto2 are both expressed in the normal mammary gland, and changes in the levels of both proteins have been linked to breast cancer (6, 9). ErbB-4 is in the nucleus of the lactating mammary gland, and ErbB-4-deficient mice do not undergo lactogenesis (4). Eto2 is expressed in normal mammary gland cells and is underexpressed or deleted in many breast cancers (9). Thus, our data suggest that Eto2 may be a key target of s80 function in the mammary gland.

In conclusion, our data that ErbB-4 s80 regulates Eto2 present a novel mechanism to regulate corepressor function. In addition, it implies that ErbB-4 and Eto2 are linked in the same pathway to regulate cell differentiation and that this pathway might be deregulated in cancer.


   FOOTNOTES
 
* This work was supported by the Department of Defense Grant BC040357 (to B. L.), National Institutes of Health Grant CA97456 (to G. C.), and both the Vanderbilt Ingram Cancer Center Grant P30 CA6845 and the Vanderbilt Diabetes Center Grant P30 DK20593 for core resources. 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

1 To whom correspondence should be addressed: Vanderbilt University School of Medicine, Dept. of Biochemistry, 647 Light Hall, Nashville, TN 37232-0146. Tel.: 615-322-6678; Fax: 615-322-2931; E-mail: graham.carpenter{at}vanderbilt.edu.

2 The abbreviations used are: ERK, extracellular signal-regulated kinase; STAT, signal transducers and activators of transcription; PLC, phospholipase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; ICD, intracellular domain; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; CT, carboxyl-terminal region; NLS, nuclear localization sequence; IP, immunoprecipitation. Back

3 B. Linggi and G. Carpenter, unpublished observations. Back

4 B. Linggi and G. Carpenter, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. S. Hiebert for helpful discussions, reading the manuscript, and providing reagents and Dr. N. Sacchi and Dr. S. Earp III for providing reagents.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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