Download Heregulins Implicated in Cellular Functions Other Than Receptor

Heregulins Implicated in Cellular Functions Other
Than Receptor Activation
Madlaina Breuleux, Fabrice Schoumacher, Daniel Rehn, Willy Küng,
Heinz Mueller, and Urs Eppenberger
Molecular Tumorbiology Unit, Department of Research, Stiftung Tumorbank and
University Clinics Medical School, Basel, Switzerland
Abstract
Introduction
Heregulins (HRG) are known as soluble secreted growth
factors that, on binding and activating ErbB3 and ErbB4
cell surface receptors, are involved in cell proliferation,
metastasis, survival, and differentiation in normal and
malignant tissues. Previous studies have shown that
some HRG1 splice variants are translocated to the
nucleus. By investigating the subcellular localization of
HRGA1-241, nuclear translocation and accumulation in
nuclear dot-like structures was shown in breast cancer
cells. This subcellular distribution pattern depends on
the presence of at least one of two nuclear localization
sequences and on two domains on the HRG construct
that were found to be necessary for nuclear dot
formation. Focusing on the nuclear function of HRG,
a mammary gland cDNA library was screened with the
mature form of HRGA in a yeast two-hybrid system, and
coimmunoprecipitation of endogenous HRG was done.
The data reveal positive interactions of HRGA1-241 with
nuclear factors implicated in different biological
functions, including transcriptional control as
exemplified by interaction with the transcriptional
repressor histone deacetylase 2. In addition, HRGA1-241
showed transcriptional repression activity in a reporter
gene assay. Furthermore, a potential of HRG proteins to
form homodimers was reported and the HRG sequence
responsible for dimerization was identified. These
observations strongly support the notion that HRG1
splice variants have multifunctional properties, including
previously unknown regulatory functions within the
nucleus that are different from the activation of ErbB
receptor signaling. (Mol Cancer Res 2006;4(1):27– 37)
The heregulin (HRG) family of growth factors plays an
important role in regulating cell proliferation, metastasis,
differentiation, and survival of various normal and neoplastic
tissues (1). All human HRGs originate from four genes,
HRG1, HRG2, HRG3 , and HRG4 , respectively (2-4).
Alternative RNA splicing results in 15 HRG isoforms, which
vary in their mosaic-like composition of different functional
domains. Most HRGs are soluble, secreted 44-kDa glycoproteins originating from transmembrane precursors, undergoing typical glycosylation and trafficking (5). The secreted
extracellular region of HRGs contains a C2-type immunoglobulin (Ig) – like domain (exons 3 and 4), which binds to
extracellular matrix proteins containing glycosaminoglycan
chains (6), and an epidermal growth factor (EGF) – like
domain required for ErbB receptor binding and activation
(1, 7). However, the very NH2-terminal region (exons 2 and 3)
exhibits also a nuclear localization signal (8).
Secreted HRG proteins act as ligands for some members of
the ErbB family of class I receptor tyrosine kinases consisting
of EGF receptor, ErbB2 (HER-2/neu), ErbB3, and ErbB4.
The expression levels of ErbB1 to ErbB4 have an effect on
HRG response; furthermore, the precise signaling cascades
and biological responses evoked by HRG proteins and their
receptors are cell type specific (9). This finding is important to
define the tumorigenic role of ErbB receptors and HRGs
in various cancers. Analysis of cellular expression patterns of
HRGs and their transmembrane receptors indicate that the
signaling can be paracrine or autocrine in nature (10).
Up-regulation of HRG was shown to be sufficient for the
development of mammary tumors independently of estrogen
stimulation and ErbB2 overexpression (11). Moreover, inhibition of HRG expression suppressed the aggressive phenotype
by decreasing ErbB activation and reducing matrix metalloproteinase-9 activity (12). These data show a direct causal
role of HRG in the induction of tumorigenicity. Therefore,
HRG could be a key promoter of breast cancer tumorigenicity
and metastasis independently of ErbB2 overexpression.
HRGs do not only act by initiating surface receptormediated signaling but may also be involved in alternative
signaling pathways. In this respect, receptor-bound HRG1h1
was shown to be transported to the nucleus (13), independent of
nuclear receptor translocation. Nuclear HRG1h1 modulated the
activity of c-myc, a critical regulator of cell cycle progression,
differentiation, and malignant transformation (13), as well as
stimulated cancer cell proliferation in vitro (8). Another HRG
splice variant, HRG1h3, lacks the transmembrane domain and
the cytoplasmic tail and is not secreted; however, the nuclear
Received 2/16/05; revised 11/21/05; accepted 12/19/05.
Grant support: Swiss National Science Foundation grants SNF 3100-059819.99
and SNF 3100-49505.96 (U. Eppenberger).
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.
Note: M. Breuleux is currently at Novartis Institutes for Biomedical Research,
Oncology, Basel, Switzerland. F. Schoumacher and D. Rehn are currently at
F. Hoffmann-La Roche AG, Basel, Switzerland. W. Küng is currently at the
Department of Research, Medical Oncology, University Hospital Basel, Basel,
Switzerland. H. Mueller is currently at the Institute of Biochemistry and Genetics,
University of Basel, Basel, Switzerland. U. Eppenberger is currently at Stiftung
Tumorbank Basel, Riehen, Switzerland.
Requests for reprints: Madlaina Breuleux, Novartis Pharma AG, Klybeckstrasse
125, WKL-125.12.59, 4002 Basel, Switzerland. Phone: 41-61-696-25-17;
Fax: 41-61-696-63-81. E-mail: [email protected]
Copyright D 2006 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-05-0016
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Breuleux et al.
localization sequence (NLS) in its NH2 terminus suggests that it
accumulates in the nuclear compartment (8). Importantly,
nuclear localization of the HRG precursor was shown in
papillary thyroid carcinomas but not in normal thyroid tissue
(14) and was not associated with the expression of ErbB
receptors. Nuclear staining for HRG has also been observed in
medulloblastomas (15). Furthermore, HRG1a, HRG1h, and
HRG3 were recently shown to localize to the nucleus in ductal
carcinoma in situ of the breast (16). Several other growth
factors act not only as extracellular ligands for transmembrane
receptors but also as nuclear growth factors. Schwannomaderived growth factor and fibroblast growth factor-1 were
reported to depend on a NLS to achieve mitogenic activity
(17, 18). Heparin-binding EGF-like growth factor, a member
of the ErbB receptor ligand family, is also translocated to the
nucleus and plays a role in disease progression in various
cancers (19).
Due to the nuclear localization of HRG proteins and their
molecular diversity, questions have arisen concerning the
functional multiplicity of HRG isoforms in the various tissues.
Recently, it has been shown that NRG1h3 localizes to two
known intranuclear structures, nucleoli and SC35-positive
nuclear speckles (20), independent of the receptor-binding
domain or the previously predicted NLS.
In our study, we have characterized the nuclear localization
of HRGa1-241, a HRG1 isoform, which shows sequence
homology with NRG1h3, in breast cancer cells. Assessing the
structural requirements of HRGa1-241 for nuclear translocation
and specific subnuclear localization with a more detailed
analysis using different HRGa1-241 deletion variants, a second
putative NLS within the Ig-like domain of HRGa1-241 was
revealed. Moreover, two sequences responsible for subnuclear
dot formation were identified. Focusing on the nuclear function
of HRG, a mammary gland cDNA library was screened in a
yeast two-hybrid assay to isolate potential nuclear protein
interaction partners of HRGa1-241. Positive interactions of
HRG with several nuclear proteins were shown, including the
interaction with the transcriptional repressor histone deacetylase
2 (HDAC2), which was confirmed by coimmunoprecipitation
of endogenous HRG in breast cancer cells. Furthermore, a
transcriptional repression activity of HRGa1-241 was revealed
using a reporter gene assay. In addition, a potential of HRG
proteins to form homodimers was reported and the HRG
sequence responsible for dimerization was identified. These
results strongly support the hypothesis that HRG proteins
modulate cellular functions by nuclear mechanisms independent of receptor activation.
Results
Nuclear Expression of HRG in Breast Cancer Cells
To determine whether endogenous HRG was expressed in
the nucleus of breast cancer cell lines, nuclear and cytoplasmic
protein fractions were prepared and analyzed by Western blot
(Fig. 1). All cell lines tested expressed full-length HRGa
protein (44 kDa) in the nucleus as well as in the cytoplasm;
however, the relative expression levels differed for each cell
line (Fig. 1, top). In addition, MDA-MB-231 cells showed
nuclear expression of shorter HRG forms (f30 kDa), probably
FIGURE 1. Endogenous HRG expression in breast cancer cell lines.
Nuclear (N ) and cytoplasmic (C ) protein fractions were subjected to
immunoblotting using a polyclonal anti-HRG antibody. Poly(ADP-ribose)
polymerase (PARP ) and MEK-1 detection are used as a control for the
purity of the nuclear and the cytoplasmic fraction, respectively. Full-length
HRG (44 kDa) as well as either the secreted extracellular domain of HRG
or shorter intracellular HRG isoforms (f30 kDa) can be found in the
nuclear fraction of the different tumor cell lines at different expression
levels.
either nonsecreted isoforms lacking part of the EGF-like
domain (e.g., HRG1h3 and HRG1g) or the secreted extracellular domain of HRG (Fig. 1, top middle). The purity of nuclear
and cytoplasmic protein fractions was confirmed using antibodies against the nuclear protein poly(ADP-ribose) polymerase and the cytoplasmic protein MEK-1, respectively (Fig. 1,
bottom). These data clearly confirm the nuclear localization of
endogenous HRG in breast cancer cell lines, consistent with
previous reports on breast cancer biopsies (16).
Nuclear Import and Nuclear Distribution Are Dependent
on Different HRG Domains
To show the nuclear translocation of HRGa1-241, MCF-7
breast cancer cells were transfected with pEGFP/HRGa1-241
and analyzed 24 hours after transfection. Transfection of the
control plasmid pEGFP-C1 resulted in uniform distribution of
the green fluorescent protein (GFP) throughout the entire cell
(Fig. 2A). Nuclear accumulation of GFP was not observed,
indicating that GFP alone is not imported into the nucleus.
Fusing the sequence of a minimal nuclear localization signal
of the SV40 large T antigen to pEGFP-C1 led to the efficient
nuclear import of the fusion protein with diffuse nuclear
staining (Fig. 2B). Strikingly, the GFP/HRGa1-241 accumulated
predominantly in the nucleus and formed discrete nuclear dots
(Fig. 2C).
To determine which HRGa1-241 domains are responsible for
the nuclear import and subnuclear localization into dot-like
structures, the HRGa sequence in the pEGFP/HRGa1-241 was
progressively deleted from both the 5V and the 3V ends
(Fig. 3). Fusion proteins with deletions in the HRGa1-241
COOH terminus revealed two different localization patterns.
The fusion proteins of pEGFP/HRGa1-205DXhoI, pEGFP/
HRGa1-187DXmnI, and pEGFP/HRGa1-147DBbsI localized in
dot-like structures in the nucleus, comparable with the
expression of wild-type GFP/HRGa1-241 (Fig. 2C). In contrast,
GFP/HRGa1-26DSacII fusion proteins were imported into
the nucleus, but without the formation of distinct nuclear
dots (Fig. 2D), comparable with the localization pattern of
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Intracellular Functions of HRG Protein
the NLS-coupled GFP (Fig. 2B). HRGa1-52DSpeI and
HRGa1-113DBclI showed the same localization pattern as
GFP/HRGa1-26DSacII (data not shown). Transfection of
pEGFP/HRGa constructs with progressive deletions in the NH2
terminus of HRGa1-241 revealed three different cellular localization patterns. Transfection with pEGFP/DSacIIHRGa28-241
resulted in a wild-type localization pattern (Fig. 2E), whereas
cells transfected with pEGFP/DSpeIHRGa53-241 showed a
nuclear accumulation of this fusion protein without dot formation (Fig. 2F). GFP/DBclIHRGa114-241, DBbsIHRGa150-241,
and DXmnIHRGa180-241 fusion proteins were distributed
throughout the cell similar to the distribution pattern of GFP
alone (Fig. 2G). These results clearly show that the domains
responsible for nuclear import are distinct from those involved
in dot formation, with both being localized at the NH2 terminus
of HRGa1-241.
Nuclear Import and Localization Do Not Depend on the
EGF-Like Domain of HRGa
Categorization of the GFP/HRGa fusion proteins according to their capacity to be imported into the nucleus and/or
FIGURE 2. Subcellular localization of NH2- and COOH-terminally
deleted HRGa1-241 fusion proteins after transfection into MCF-7 cells.
A. Cellular distribution of GFP alone as control for cytoplasmic
distribution. B. Cellular and nuclear localization of GFP fused to the
NLS of the SV40 large T antigen as a control for nuclear accumulation.
C. Nuclear dot-like structure formation by wild-type HRGa1-241 as
well as by HRGa1-148DBbs I, HRGa1-187DXmn I, and HRGa1-205DXho I
(data not shown). D. Nuclear retention of HRGa1-26DSac II as
well as HRGa1-52DSpe I and HRGa1-113 DBcl I (data not shown).
E. Accumulation of DSac II-HRGa28-241 in nuclear dots. F. Accumulation
of DSpeI-HRGa53-241 in the nucleus. G. Cytoplasmic distribution
of DBcl I-HRGa114-241 as well as DBbs I-HRGa150-241 and DXmnIHRGa188-241 (data not shown). Bar, 10 Am.
FIGURE 3. Nuclear import and subnuclear localization of HRG
constructs. All HRGa1-241 fusion proteins were assessed for their capacity
to be imported into the nucleus and/or to be localized in nuclear dot-like
structures.
to be confined to dot-like structures is shown in Fig. 3. Two
putative nuclear localization signals that are required for
nuclear import or nuclear retention of the fusion proteins
were found in HRGa1-241 (Fig. 4A). The NH2-terminal NLS
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is located at amino acid positions 12 to 18 and was described
by others as a putative NLS for HRG proteins (8). The
second putative NLS is located within the Ig-like domain,
showing no homology to one of the three types of NLS
described earlier (21). Either of the two NLS is sufficient for
nuclear import. In contrast, for nuclear dot formation, both
of the two dot-forming sequences found in HRGa1-241 are
required (Fig. 4A). The first of these domains is encoded in
exon 2/3 (amino acids 28-52) corresponding to a stretch of
25 amino acids NH2-terminal of the Ig-like domain, and the
second is in exon 4/5 (amino acids 114-147), which
corresponds to the 34 – amino acid spacer region located
between the Ig-like domain and the EGF-like domain.
Deletion of one of these two domains resulted in the loss
of subnuclear dot formation (Fig. 3).
Thus, the minimal requirements for HRGa1-241 to be
imported into the nucleus and to form nuclear dot-like
structures are the presence of either one of the two NLS and
both dot-forming sequences located on both sides of the Ig-like
domain. To confirm this notion, we constructed a minimal HRG
fusion protein (pEGFP/DSacIIHRGa28-147DBbsI) consisting of
the two domains responsible for nuclear dot formation on either
side of the Ig-like domain and the Ig-like domain containing
just one of the two NLS (Fig. 4B). As predicted, the
intracellular distribution of this fusion protein was comparable
with that of wild-type HRGa1-241 (Fig. 4C). This finding
clearly indicates that the EGF-like domain is not involved in
directing HRGa1-241 into the nucleus.
FIGURE 5. Relative strength of protein-protein interactions with
HRGa1-241. A. Schematic representation of HRG constructs. Gray
boxes, Ig-like domain and EGF-like domain; black boxes, two putative
NLS; hatched boxes, two domains necessary for nuclear dot formation;
black bars, HRGa1-241 subfragments, each showing its respective position
in the protein and its length. DFS, dot-forming sequences. B to D.
HRGa1-241 (black column ) and its deletion variants (dotted column, Ig-like
domain; hatched column, EGF-like domain; squared column, minimal
sequence) were cotransformed with the respective clones and the
h-galactosidase units were expressed as relative values compared with
an internal positive control for protein interaction. As a background control,
the empty yeast vector pGBKT7 was cotransformed in AH109 yeast
together with the different clones to look for HRGa1-241 independent
activation of the h-galactosidase reporter gene (white column ). All
experiments were done three times in duplicates. B. Cullin-1. C. MDGI.
D. G/T mismatch-specific TDG. E. hUBC9.
FIGURE 4. Nuclear dot formation by HRGa1-241 does not require the
EGF-like domain. A. Two putative NLS (gray boxes ) and two regions
(black boxes ) required for the localization in nuclear dots. B. Minimal
HRGa1-241 sequence required for nuclear import and dot formation.
C. MCF-7 cells transfected with the minimal HRGa1-241 sequence fused to
GFP. Hatched boxes, GFP tags; light gray boxes, HRGa1-241 sequences;
dark gray circles, cysteine positions. Bar, 10 Am.
HRGa1-241 Interacts with Known Nuclear Proteins
To further evaluate the potential role of intracellular and
nuclear HRGa1-241, a yeast two-hybrid screen was done to
detect novel protein interaction partners. A human mammary
gland cDNA library fused to the GAL4 activation domain,
containing 3.5 105 independent clones, was screened with
HRGa1-241 fused to the GAL4-DNA-binding domain (DBD;
ref. 22). Three different reporter genes (ADE2, HIS3, and lacZ)
were used to detect protein interactions in the GAL4-responsive
yeast strain AH109, each under the control of distinct GAL4
upstream activating sequences and TATA boxes. HRGa1-241
was unable to activate reporter gene expression by itself (data
not shown). Of a total of 1,013 positive clones, which were
picked and back-transformed either with or without HRGa1-241
to confirm the need for HRGa1-241 interaction to activate
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Intracellular Functions of HRG Protein
the reporter genes, 360 clones remained positive for their
interaction with HRGa1-241 and were used for further
sequencing analysis. Thirty-one different proteins implicated
in multiple cellular functions were identified and further tested
for domain specificity of their interaction with HRGa1-241 (data
not shown). To identify HRG domains needed for interaction,
deletion variants of HRGa1-241 were constructed either coding
for the Ig-like domain (amino acids 1-128), the EGF-like
domain (amino acids 112-241), or the minimal sequence shown
to be sufficient for nuclear import and subnuclear dot formation
(amino acids 21-150; Fig. 5A).
A summary of the key interacting proteins is presented in
Table 1. The interaction of HRGa1-241 with the nuclear proteins
RS cyclophilin, serine/arginine nuclear matrix protein, and
RNA helicase, proteins required for pre-mRNA splicing, is
supported by previous data showing that NRG1h3 was found to
associate with specific intranuclear compartments implicated in
RNA splicing (20). Therefore, these findings serve as a control
for the robustness of the data obtained by the yeast two-hybrid
screen.
Having done this detailed analysis of the positively interacting clones, a subset of these candidates was chosen for
further studies, focusing on potential nuclear interaction partners of HRG. HRGa1-241 was found to interact strongly and in a
domain-specific manner with Cullin-1 (Genbank AF62536),
mammary-derived growth inhibitor (MDGI; Genbank Y10255),
G/T mismatch-specific thymine DNA glycosylase (TDG;
Genbank HS51166), and human ubiquitin-conjugating enzyme
9 (hUBC9; Genbank HSUBC9ENZ), which are involved
in diverse biological processes. A liquid h-galactosidase assay
was applied to quantitate the relative strength of positive protein interactions with HRGa1-241 and its deletion variants
(Fig. 5B-E). To compare the results of different assays, all
values were normalized to a mean number of h-galactosidase
activity obtained in a simultaneously performed internal positive control for protein interaction.
Cullin-1 revealed a clear interaction specificity for the EGFlike domain of HRGa1-241 (Fig. 5B). Although MDGI was able
to slightly activate the h-galactosidase reporter gene without the
need of a positive interaction with HRGa1-241, the interaction
with HRGa1-241 and with the EGF-like domain showed
enhanced activity (Fig. 5C), indicating specific protein
interaction with parts of HRGa1-241. G/T mismatch-specific
TDG interacted specifically with the Ig-like domain of
HRGa1-241, whereas the interaction with the EGF-like domain
was comparable with the background activation of the reporter
gene (Fig. 5D). Although hUBC9 was not able to activate the
reporter gene without HRGa1-241 interaction, no clear domain
specificity for either one of the deletion variants of HRG was
detected; hUBC9 seems to interact with both the Ig-like domain
and the EGF-like domain. The interaction was much stronger
if both of the dot-forming domains were present in the construct
(Fig. 5E).
Coimmunoprecipitation experiments with the candidate
proteins and HRGa1-241 were done to confirm the data
obtained with the yeast two-hybrid analysis. Cos-7 cells were
transiently cotransfected with pcDNA3.1 plasmids, containing
either the respective full-length cDNA of the clones identified
in the two-hybrid screen fused to a hemagglutinin (HA) tag
or the HRGa1-241 fused to a myc tag. As a negative control,
pcDNA3.1/myc was cotransfected with pcDNA3.1/HA (data
not shown). Cullin-1 (Fig. 6A), MDGI (Fig. 6B), and G/T
mismatch-specific TDG (Fig. 6C) coprecipitated together
with HRGa1-241. Only hUBC9 did not show any interaction
with HRGa1-241 (Fig. 6D) with this experimental approach.
Hence, most of these results confirm the interaction of
HRGa1-241 with nuclear proteins found in the yeast twohybrid screen.
HRG Forms Homodimers via Its NH2 Terminus
Homodimerization of proteins could be one mechanism
inducing nuclear dot formation. Furthermore, the activity of
HRG might be regulated via homodimerization. Therefore, we
used the yeast two-hybrid system to test HRG dimerization.
The respective coding sequences of HRGa1-241 and its deletion
variants were subcloned in-frame into yeast two-hybrid vectors
containing the GAL4-DBD (bait vectors) and the GAL4
activation domain (prey vectors), respectively. For direct testing
of the interaction between HRG fusion proteins, each bait
plasmid was paired with each prey plasmid and the interaction
was assessed qualitatively (ADE2 and HIS3) and quantitatively
(lacZ) by determining reporter gene activity in cotransfected
AH109 cells. All HRGa1-241 fusion proteins containing the
Ig-like domain were found to form homodimers, whereas
interactions with the EGF-like domain of HRGa1-241 did not
take place (data not shown).
Table 1. Result of Yeast Two-Hybrid Screen
Protein Name
MDGI
hUBC9
RING1 and YY1 binding protein
RS cyclophilin
RNA helicase
HDAC2
ZNF237
Cullin-1
G/T mismatch-specific TDG
Serine/arginine nuclear matrix protein
p53-binding protein
Times Found in
Yeast Two-Hybrid Screen
14
6
6
4
4
4
2
1
1
1
1
Protein Function
Mammary gland differentiation; transcriptional control
SUMOylation
Transcriptional control
Pre-mRNA processing
Pre-mRNA processing
Transcriptional control
Transcriptional control
SCF complex (ubiquitin ligase complex)
DNA mismatch repair; transcriptional control
Pre-mRNA processing; transcriptional control
Transcriptional control
NOTE: Nuclear proteins found in the yeast two-hybrid screen to interact with HRGa1-241.
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corepressor (23), we did a coimmunoprecipitation analysis
in MDA-MB-231 cells and were able to coprecipitate HDAC2
with HRG. These results show clear evidence for an interaction of endogenous HRG with one of the proteins found in
the yeast two-hybrid screen in a natural setting (Fig. 7A);
furthermore, these data support a transcriptional regulation
function of HRG.
Hence, a transcriptional study was done using a plasmid
containing the luciferase reporter gene either under the
control of a constitutively active GAL4 promoter
[pGAL4(5 UAS)-SV40-Luc] or with a minimal GAL4
FIGURE 6. Association of proteins with HRGa1-241. Cos-7 cells were
either cotransfected with myc-tagged HRGa1-241 and the respective HAtagged Cullin-1 (A), MDGI (B), G/T mismatch-specific TDG (C), and
hUBC9 (D) or cotransfected with different myc-tagged and HA-tagged
HRG domains (E-H). To look at HRG dimerization, cotransfections were
done with the following combinations: (E) myc-tagged HRGa1-241, HAtagged HRGa1-241, (F) myc-tagged HRGa1-241, HA tagged Ig-like domain
of HRGa1-128, (G) myc-tagged minimal sequence of HRGa (amino acids
21-150), HA-tagged HRGa1-241. H. Myc-tagged HRGa1-241 was cotransfected with HA-tagged EGF-like domain (amino acids 121-241; lanes 1
and 2) and with HA-tagged EGF-like domain coupled to a NLS (lanes 3
and 4). Blots are overexposed to show that no interaction takes place
between HRGa1-241 and the EGF-like domain. The lysates were
precipitated with a monoclonal anti-myc antibody. The supernatants
(Sup ) and the immunoprecipitates (IP ) were subjected to immunoblotting
using a polyclonal anti-HA antibody. Right, molecular size markers.
Asterisks, expected sizes of the proteins.
Coimmunoprecipitation was done confirming HRGa1-241
homodimerization via its NH2 terminus (Fig. 6E-G) but not via
the EGF-like domain (Fig. 6H, lanes 1 and 2). However, the
construct containing the EGF-like domain is homologous to
the HRG protein encoded by pEGFP/DBclIHRGa114-241, which
does not contain a NLS and was shown not to be translocated
to the nucleus. Cloning a NLS from the SV40 large T antigen
to the HA-tagged EGF-like domain (Fig. 6H, lanes 3 and 4)
did not lead to coimmunoprecipitation with HRGa1-241, further
confirming that the dimerization of HRGa1-241 is independent
of the EGF-like domain.
HRG Interacts with HDAC2 and Shows Potential
Transcriptional Repression Activity
Many proteins implicated in transcriptional control are able
to form homodimers. The homodimerization of HRG and its
specific interaction with nuclear proteins suggest, therefore, a
role for HRG in transcriptional control. To assess the interaction of endogenous HRG with HDAC2, a protein shown
to interact with HRGa1-241 in the yeast two-hybrid screen
(Table 1) and which is a well-known enzymatic transcriptional
FIGURE 7. HRGa1-241 interacts with HDAC2 and shows transcriptional
repression in a luciferase reporter gene assay. A. In vivo interaction of
HRGa with HDAC2 shown by coimmunoprecipitation. B and C. Luciferase
reporter gene was either under the control of a constitutively active GAL4
promoter in the pGAL4(5UAS)-SV40-Luc plasmid (B) or under the
control of a minimal GAL4 promoter in the pGK-1 plasmid (C). Luciferase
activity was measured either alone (black columns , background activation), in combination with overexpressed HDAC2 (dotted columns ), or in
the presence of HRGa1-241 coupled to the GAL4-DBD without (squared
columns ) or with (hatched columns ) HDAC2. When no HRGa1-241 was
transfected, the plasmid containing the GAL4-DBD was cotransfected
with the respective luciferase reporter gene plasmids with or without
HDAC2.
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Intracellular Functions of HRG Protein
promoter (pGK-1). We transfected the DBD of GAL4 or
HRGa1-241 coupled to the DBD of GAL4 together with either
one of the luciferase plasmids and could show that HRGa1-241
was able to repress luciferase expression independently of the
promoter status (Fig. 7B and C). Moreover, we transfected Cos7 cells with a plasmid expressing HDAC2. HDAC2 was
transfected either alone or in combination with HRGa1-241;
however, it did not potentiate the transcriptional repression seen
with HRGa1-241, which may already show maximal repression
potential in this reporter gene assay. Our results clearly suggest
that HRG has transcriptional control activity.
Discussion
Mitogenic growth factors are generally cell surface –
associated or secreted proteins, which produce effects by
binding to cell surface receptor tyrosine kinases. Evidence is
mounting, however, that growth factors and members of the
type I family of transmembrane receptors have also an
important role directly within the nucleus. At present, there
are few hypotheses suggesting nuclear functions of growth
factors or their receptors (24).
Nuclear Translocation of HRGa1-241
NRG1h3 was shown recently to localize to the nuclei of
human breast cancer cells in a receptor-independent way,
supporting the idea that secretion and subsequent cell surface
receptor binding of HRG proteins are not a prerequisite for
nuclear localization and that nonsecreted ligands may have
highly specific functions in defined nuclear compartments, such
as the nucleoli and SC35-positive nuclear speckles (20). Further
evidence for a nuclear function of HRG proteins during
malignancy arises from studies showing that HRG is expressed
in the nucleus of breast as well as thyroid cancer biopsies and
medulloblastomas (14-16), whereas no nuclear HRG was found
in normal tissues. We have shown that full-length HRG and
either extracellular HRG or shorter intracellular isoforms of HRG
are present in the nucleus of different breast cancer
cell lines. Furthermore, we have shown clear evidence for
HRGa1-241 being translocated to the nucleus, where it is
localized in dot-like structures. Several of our HRGa1-241 deletion constructs (such as HRGa1-205DXhoI, HRGa1-187DXmnI,
and HRGa1-148DBbsI) lacked a portion of the EGF-like domain,
shown to be necessary and sufficient for receptor binding and
activation (7), yet they were imported into the nucleus and able to
form nuclear dot-like structures. Thus, it is conceivable that the
nuclear translocation of our constructs did not depend on
ErbB3 and ErbB4 binding and activation, confirming the data
of Golding et al. (20), showing nuclear localization of
NRG1h3 in a receptor-independent way. Our demonstration
that HRGa1-241 is translocated to the nucleus suggests that
novel mechanisms of action of HRG different from receptor
binding and activation might exist, whereby HRG may also
act as an intracrine growth factor, potentially extending the
biological functions of HRG proteins.
HRGa1-241 Contains Two NLS
Certain proteins are transported actively and selectively into
the nucleus if they contain a nuclear localization signal or are
associated with NLS-containing proteins. In HRGa1-241, two
short peptides were defined as active nuclear targeting
sequences. The first NLS (KGKKKER) is located at the NH2
terminus of the mature (secreted) HRG, whereas the second
NLS is located within a region of 16 amino acids in the Ig-like
domain of HRG and resembles the consensus NLS sequence
K-R/K-X-R/K (25). Our HRG constructs transfected into breast
cancer cells gave rise to proteins smaller than the diffusion size
limit for the nuclear envelope (f40-60 kDa). Therefore,
nuclear import of HRG may occur by passive diffusion with
nuclear retention or binding to nuclear proteins in a subnuclear
compartment causing the dot-like structures. However, because
small HRGa1-241 constructs lacking both NLS did not enter the
nucleus, nuclear accumulation of HRGa1-241 is not diffusion
dependent and requires at least one NLS, indicating an active
nuclear import.
Subnuclear Dot Formation
We show here that HRGa1-241 exhibits a pattern of subnuclear
dot formation similar to Sp100 and PML proteins (26) and
similar to neuregulin-1 (20). The Sp100 and PML proteins
were shown to be covalently modified by the SUMO-1 protein,
which was partly required for dot formation (27). A consensus
sequence for SUMOylation, (I/L)KXE, was proposed (28) and
comparison of this consensus sequence with the sequences of
both dot-forming domains in HRGa1-241 revealed that amino
acids 28 to 52 contain an amino acid stretch very similar to the
SUMOylation consensus sequence (data not shown). These data
would support the findings of Golding et al., which show that the
first 79 amino acids of NRG1h3 were necessary and sufficient
to direct the protein to nucleoli and nuclear speckles (20).
However, we further showed that an additional domain (amino
acids 113-148) is also required for dot formation. The nature of
this second domain remains unclear because we were not able to
identify a known consensus sequence in this domain. Thus, the
mechanism of concerted action of both domains for dot formation remains to be elucidated. Nuclear dot-associated proteins
were reported earlier to play a role in cell transformation and
growth control or regulation of differentiation (29). Although
NRG1h3 was shown to associate with nucleoli and SC35positive nuclear speckles, two nuclear compartments involved in
ribosome synthesis, transcriptional control, and RNA splicing
(20), the molecular composition and the biological function of
the nuclear dots containing HRGa1-241 is not clear yet.
HRGa1-241 Interacts with Nuclear Proteins
At present, it is not known whether the mechanism
underlying formation of HRG nuclear dots involves the posttranslational modification of HRGs and/or the interaction of
HRGs with other proteins. In our attempt to isolate genes
encoding nuclear proteins interacting with HRGa1-241, we
have screened a mammary gland cDNA library using a yeast
two-hybrid system and obtained several candidate substrates
(Table 1). This is the first study showing proteins interacting
with HRG, which do not belong to the ErbB family of type I
receptor tyrosine kinases.
Cullin-1 is a member of the SCF protein complex, an E3
ubiquitin protein ligase controlling the G1-S transition of the
eukaryotic cell cycle (30), being expressed in the cytoplasm as
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Breuleux et al.
well as in nuclear dots during interphase. Constitutive
activation of nuclear factor-nB is observed in several cancers,
including breast cancer. It was shown that HRG, but not its
associated receptor ErbB2, plays a major role in constitutive
nuclear factor-nB activation (31), thereby increasing the
expression of proinvasive, prometastatic, and antiapoptotic
genes in cancer cells, which leads to invasive and drug-resistant
growth of breast cancer. Activation of nuclear factor-nB
requires the degradation of its inhibitor InBa, which is
catalyzed by the SCF complex containing Cullin-1 (32). It is
tempting to speculate that a direct interaction of intracellularly
expressed HRG with Cullin-1 might circumvent the need for
ErbB-dependent nuclear factor-nB activation.
MDGI was shown to display 95% homology with the
heart fatty acid – binding protein (33), a member of a family of
proteins thought to be involved in cell signaling, growth
inhibition, and differentiation (34). MDGI is a nuclear protein
that is maximally expressed in terminally differentiated
mammary tissue (35) and has inhibitory activity on the growth
of different breast cancer cell lines (36), reducing the
transcriptional expression of c-fos, c-myc, and c-ras and
suppressing the mitogenic effects of EGF (37). Nuclear
translocation of HRGh1 is correlated with c-myc induction
(13); however, further investigations are needed to understand
the functional effect of the protein interaction between MDGI
and HRGa1-241 on growth response and cell differentiation.
The G/T mismatch-specific TDG was originally cloned as a
base excision repair enzyme (38) but was also shown to act as
an activator (39) or repressor of transcriptional activity (40).
It is not clear yet what the functional implication of the protein
interaction of HRGa1-241 with TDG may be. However, we have
found that HRGa1-241 has a potential role in transcriptional
control supporting the physical interaction with TDG.
Another nuclear protein found to be a binding partner for
HRGa1-241 is hUBC9, a protein showing significant identity
with ubiquitin-conjugating enzymes required for cell cycle
progression (41). The structure of hUBC9, however, displays
significant differences with other ubiquitin-conjugating
enzymes, which reflects its specificity for SUMO1 rather than
for ubiquitin (42). In contrast to ubiquitination, SUMOylation
does not tag proteins for degradation but seems rather to
enhance their stability or modulate their subcellular compartmentalization (43). Furthermore, SUMOylation was correlated
to transcriptional regulation (44) as well as altering protein
activity and protein-protein interactions (45). As discussed
above, HRG indeed contains a putative SUMOylation site;
however, it remains to be defined if HRGa1-241 is indeed
SUMOylated by hUBC9 and if this potential SUMOylation is
necessary for the nuclear import, the formation of subnuclear
dots, and/or the transcriptional activity of HRGa1-241.
To further specify these protein interactions, we constructed
different HRG deletion variants and confirmed the ability of the
specific domain(s) of HRG to interact with the nuclear proteins
identified. Whereas Cullin-1 and MDGI showed very specific
interaction with the EGF-like domain of HRGa1-24, suggesting
that the interaction is probably not confined to the nucleus but
instead plays a functional role in the cytoplasm, G/T mismatchspecific TDG was able to interact specifically with the Ig-like
domain of HRGa1-241. However, MDGI and G/T mismatch-
specific TDG showed activation of the reporter gene with all
different deletion variants of HRGa1-241 as well as when
transformed together with the empty bait vector. Because the
activation of h-galactosidase was significantly stronger when
these proteins were cotransformed together with HRGa1-241, we
assume that reporter gene activation by the other constructs may
reflect background activation of the reporter gene and that the
interaction with HRGa1-241 is specific and not due to an artifact
resulting from the yeast two-hybrid system. Although hUBC9
was not able to activate the reporter gene without an interaction
with HRGa1-241, this clone seemed to interact with both the
Ig-like domain and the EGF-like domain. Interestingly, the
interaction strength was much higher when both dot-forming
sequences were present (HRGa1-241, minimal sequence),
including the putative SUMOylation site. We assume that
different parts of HRGa1-241 are necessary for the interaction
with hUBC9, which act synergistically to enhance the proteinprotein interaction.
Performing a coimmunoprecipitation assay based on transient overexpression of proteins in Cos-7 cells supported the
findings from the yeast two-hybrid screen, whereby HRGa1-241
was interacting specifically with Cullin-1, MDGI, and G/T
mismatch-specific TDG. However, we could not confirm the
interaction of hUBC9 with HRGa1-241 with this approach. The
reason might be that other factors are required for the formation
of this complex or that the interaction of these two proteins
must be triggered by an external signal. Another possibility
might be that other intracellular substrates are titrating hUBC9
away from HRGa1-241.
HRGs Are Able to Form Dimers and Exhibit Transcriptional Repression Activity
Investigation of the dimerization potential is an important
prerequisite for understanding how nuclear dots can be formed,
because some proteins being part of nuclear dots have been
shown to form homodimers. Dimerization can occur by means of
a specific domain having the capability to self-interact and target
the protein to discrete nuclear substructures (28). However,
Sp100 has been shown to contain a self-aggregation domain that
exists as separate entity besides the domain responsible for dot
formation (46). To test the homomeric interaction potential of
HRGa1-241, we have used the yeast two-hybrid assay and did
coimmunoprecipitation experiments. Our results indicated that
the minimal sequence shown to be sufficient for subnuclear
dot formation of HRGa1-241 is capable of mediating HRG
dimerization. The EGF-like domain, in contrast, completely
lacked dimerization activity even when fused to a NLS. We do
not know yet if different domains of HRGa1-241 are implicated
in dimerization and subnuclear dot formation or if the same
domains are responsible for both functions. Further mutational
analyses have to be done to address this and further define the
role of dimerization on HRG function.
Several proteins shown to form nuclear dots or to dimerize
have either transcriptional transactivating or transrepressing
properties (47, 48) or are implicated in DNA repair (49) or
RNA splicing (50). HRG has been described to inhibit estrogen
receptor expression (51) and to modulate the activity of c-myc
after nuclear translocation (13). Because no DNA-binding site
has been described for HRGa1-241 thus far, HRGa1-241 may
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Intracellular Functions of HRG Protein
regulate transcription indirectly by recruiting cofactors essential
for transcriptional control. Indeed, in the yeast two-hybrid
screen, we found that HRGa1-241 is specifically interacting with
several proteins implicated in transcriptional regulation
(HDAC2, MDGI, G/T DNA mismatch glycosylase, ZNF237,
serine/arginine nuclear matrix protein, RING1 and YY1 binding
protein, and p53-binding protein). Looking in more detail at the
interaction of HRG with HDAC2, we were able to show
interaction of endogenous HRG with HDAC2 in MDA-MB-231
cells. These data strongly support the hypothesis that nuclear
HRG may be implicated in transcriptional control.
To assess the potential role of HRGa1-241 in transcriptional
regulation, we devised a cellular luciferase reporter gene assay
using two different constructs of the luciferase reporter gene
either under the control of a constitutively active GAL4
promoter [pGAL4(5 UAS)-SV40-Luc] or under the control
of a minimal GAL4 promoter (pGK-1). When Cos-7 cells
were cotransfected with GAL4-DBD-HRGa1-241 together with
either of the reporter gene constructs, we could show that
HRGa1-241 was able to repress transcription when targeted to
the promoter site of these constructs. When transfecting
HDAC2 together with the reporter gene plasmids, it was able
to repress luciferase expression; however, it failed to show
any further transcriptional repression when cotransformed
together with HRGa. These results suggest that the
transcriptional repression by HRGa alone may already be
maximal because of recruitment of endogenous cofactors by
HRGa. At the moment, it is unclear which target genes may
be regulated by HRG, but clearly testing for this activity
against physiologic target reporter genes would be very
informative.
In conclusion, we show nuclear localization of endogenous
HRG in breast cancer cell lines and define the sequences
necessary and sufficient for nuclear translocation and subsequent subnuclear dot formation by investigating the subcellular
localization of transfected HRGa1-241. In our attempt to search
for functional aspects of intracellular HRGa1-241, we show
interaction of HRGa1-241 with different nuclear proteins and
that these interactions are domain specific, either involving the
Ig-like domain or the EGF-like domain or both. Furthermore,
we report the ability of HRG proteins to form homodimers
and reveal the HRG sequence responsible for dimerization.
Additionally, we show interaction of endogenous HRG with
HDAC2 and transcriptional regulation activity of HRG in a
reporter gene assay. This is the first study showing interaction
of HRG with nuclear proteins and our data clearly support a
nuclear function of HRG in tumorigenesis.
Materials and Methods
Cell Lines
The hormone-dependent breast cancer cell line MCF-7 was
obtained from Mason Research Institute (Rockville, MD) and
grown in IMEM-ZO as described (52). Cos-7 cells were
cultured in DMEM supplemented with 10% FCS. The breast
cancer cell lines SKBR3 and MDA-MB-231 were obtained
from American Type Culture Collection (Rockville, MD).
T47D and BT474 cells were obtained from Dr. Nancy Hynes
(Friedrich Miescher Institute for Biomedical Research, Basel,
Switzerland).
Endogenous Nuclear HRG Expression
Nuclear cell extracts were prepared using the CelLytic
nuclear extraction kit (Sigma, St. Louis, MO) according to
the recommendations of the manufacturer. Briefly, 107 cells
were swelled in hypotonic lysis buffer followed by mechanical disruption. The cytoplasmic fraction was removed
and the nuclear proteins were released from the nuclei
by high-salt buffer. Western blots were done according
to standard enhanced chemiluminescence procedures (Amersham, Otelfingen, Switzerland) using polyclonal anti-HRG
antibodies (sc-347 and sc-348; Santa Cruz Biotechnologies,
Santa Cruz, CA). Nuclear and cytoplasmic protein fractions
were confirmed using antibodies against poly(ADP-ribose)
polymerase (Cell Signaling Technologies, Beverly, MA) and
MEK-1 (Zymed Laboratories, San Francisco, CA), respectively.
HRGa Vector Constructs
Total RNA was isolated from MDA-MB-231 cells with the
RNeasy Total RNA kit (Qiagen, Basel, Switzerland), primed
with oligo(dT) primers, and reverse transcribed with the FirstStrand cDNA Synthesis kit (Clontech, Mountain View, CA).
This reaction mixture was used as a template to amplify the
extracellular part of HRGa (HRGa1-241). The resulting PCR
product was cloned into the bacterial expression vector pCR2
(Invitrogen, Basel, Switzerland) by TA cloning and further
subcloned into pEGFP-C1 (Clontech) to obtain a GFP fusion
protein.
Construction of HRGa Deletion Mutants
COOH- and NH2-terminal sequential HRGa1-241 deletion
mutants were constructed. The restriction enzymes used are
listed from the 5V to the 3V end, SacII, SpeI, BclI, BbsI, and
XmnI and, for the COOH-terminal deletion, XhoI. The
COOH-terminal HRGa1-241 deletion constructs were obtained
by linearization of pEGFP/HRGa1-241 at the appropriate sites,
fill-in with T4 DNA polymerase, SmaI digestion in the
multiple cloning site of pEGFP-C1 3V to the HRGa1-241 insert,
and religation. The NH2-terminal HRGa1-241 deletions were
constructed to be in-frame with the GFP coding sequence after
digestion, fill-in, and religation. The pEGFP/NLS plasmid
was obtained by ligating a phosphorylated synthetic oligonucleotide linker (pN1 3V-TCGATATCCAAAGAAGAAGCGC A A G G T G C A - 5 V a n d p N 2 3 V- C C T T G C G C T TCTTCTTTGGATA-5V) into pEGFP-C1 digested with XhoI
and PstI.
Transient Expression of HRG Constructs in Mammalian
Cells
MCF-7 cells were used for transient expression analysis
of HRGa1-241 constructs. Transfections were carried out by
electroporation with the Gene Pulser II (Bio-Rad Technologies,
Reinach, Switzerland). Alternatively, MCF-7 cells were grown
on glass coverslips to 60% to 80% confluence and transfected
for 3 hours with 1 Ag plasmid DNA using Superfect reagent
(Qiagen). For coimmunoprecipitation studies, Cos-7 cells were
transfected with Fugene 6 transfection reagent (Roche Diagnostics, Rotkreuz, Switzerland).
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Breuleux et al.
Fluorescence Microscopy
Transfected cells were seeded on glass coverslips in six-well
plates and incubated for 24 hours. Cell nuclei transfected
with GFP fusion constructs were stained with either 2 Ag/mL
Hoechst 33342 dye (blue) or 5 Amol/L SYTO 17 red
fluorescent nucleic acid stain (Molecular Probes, Eugene,
OR). Alternatively, the cells were fixed in ice-cold methanol/
acetone (1:1, v/v) at 20jC and the coverslips were allowed to
dry. Coverslips were subsequently mounted with 90% glycerol
in 1 PBS onto microscope slides. Cells were observed with a
Zeiss Axioskop microscope. Image acquisition was done with a
CCD camera using the MacProbe program (Perceptive
Scientific Instruments Ltd., Suffolk, England).
Yeast Two-Hybrid Screen
The yeast two-hybrid assay was done using the Matchmaker
cloning system 3 according to the recommendations of the
manufacturer (Clontech). HRGa1-241 was subcloned into
pGBKT7 vector in-frame with the GAL4-DBD. This fusion
construct was used to screen, on histidine-free medium, a
human mammary gland cDNA library (Clontech) cloned into
the pACT2 vector in-frame with the GAL4 activation domain.
The GAL4-responsive AH109 yeast strain was used for the
screening and transformation was accomplished according to
the lithium acetate transformation protocol. Positive colonies
were reselected on adenine-free medium and the relative
strength of the interactions was assessed using a liquid hgalactosidase assay. Positive clones were rescued via transformation of DH5a bacteria and subsequent selection with
ampicillin and analyzed by DNA sequencing. To evaluate the
interaction of the positive clones with different domains of
HRGa1-241, the individual HRG deletion variants coding for the
Ig-like domain (amino acids 1-128), the EGF-like domain
(amino acids 121-241), or the minimal sequence (amino acids
21-150), necessary and sufficient for nuclear translocation and
subnuclear dot formation, have been cloned into the pGBKT7
and the pGADT7 vector.
Coimmunoprecipitation
The myc epitope tag and the HA epitope tag were subcloned
into the pcDNA3.1(+) vector (Invitrogen). HRGa1-241, its
deletion variants, and the full-length cDNA of some positive
clones were subsequently subcloned into the pcDNA3.1/myc
and the pcDNA3.1/HA vectors, respectively. Cos-7 cells were
transiently cotransfected with pcDNA3.1/myc containing a
HRG variant together with pcDNA3.1/HA containing either
the full-length cDNA of a positive clone or a HRG variant. All
transfections included 0.2 Ag p6RlacZ vector (53) to measure
transfection efficiency and to define the input amount of protein
for each immunoprecipitation. For immunoprecipitation,
cells were lysed 48 hours after transfection in NP40 lysis
buffer (150 mmol/L NaCl, 1% NP40, 50 mmol/L Tris, 1 mmol/L
EDTA) supplemented with protease inhibitors. Cell lysates were
incubated overnight with protein G-Sepharose 4 fast flow
(Amersham) preincubated with a monoclonal anti-myc antibody
9B11 (Cell Signaling). The supernatant was precipitated using
TCA and protein G-Sepharose was washed in immunoprecipitation buffer [20 mmol/L K+ HEPES (pH 7.4), 200 mmol/L
sucrose, 1 mmol/L EDTA, 100 mmol/L NaCl] supplemented
with protease inhibitors. Immunoprecipitated material was
solubilized by resuspending the washed beads in 2 SDS protein sample buffer containing h-mercaptoethanol. The immunoprecipitation efficiency was f50% to 80%. Western blots
were done according to standard enhanced chemiluminescence
procedures (Amersham) using a polyclonal anti-HA antibody
Y-11 (Santa Cruz). For coimmunoprecipitation of endogenous
HRG, cells were lysed in extraction buffer (50 mmol/L HEPES,
150 mmol/L NaCl, 25 mmol/L h-glycerophosphate, 25 mmol/L
NaF, 5 mmol/L EGTA, 1 mmol/L EDTA, 15 mmol/L PPI)
without detergent, supplemented with protease inhibitors.
Cell lysates were immunoprecipitated using a polyclonal antiHRG antibody (Santa Cruz), and Western blots were done
using a monoclonal anti-HDAC2 antibody (Upstate, Lake
Placid, NY).
Luciferase Reporter Gene Activation Assay
Cos-7 cells were seeded into six-well plates 24 hours
before transfection. All transfections included 0.2 Ag p6RlacZ
vector (53) for normalization of transfection efficiency.
Standard amounts of expression and reporter plasmids per
transfection in reporter gene activation assays were 1 Ag
GAL4-DBD expression vector pcDNA3.1/GAL4-DBD or
1 Ag HRG expression plasmid pcDNA3.1/GAL4-DBDHRGa1-241, 1 Ag GAL4-responsive luciferase reporter plasmid pGAL4(5 UAS)-SV40-Luc (under the control of a
constitutively active GAL4 promoter), or pGK-1 (under the
control of a minimal GAL4 promoter; ref. 54) and, optionally,
1 Ag pcDNA3.1/HA/HDAC2. Cell lysates were prepared 48
hours after transfection and subsequently assayed for luciferase and h-galactosidase activity. Luciferase values normalized to h-galactosidase activity are called relative luciferase
units. The data shown represent mean F SD of three independent experiments done.
Acknowledgments
We thank Gerhard Christofori, Heidi Lane, and Chris Benz for their mutual
interest and support in this work; Wilhelm Krek for the hUBC9 and Cullin-1
expression vectors; Nancy Hynes for the T47D and BT474 cells; Natasha Kralli
for the pGK-1 and pGAL4(5UAS)-SV40-Luc plasmids; and Francois David
and Heidi Bodmer for their technical assistance.
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Mol Cancer Res 2006;4(1). January 2006
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37
Heregulins Implicated in Cellular Functions Other Than
Receptor Activation
Madlaina Breuleux, Fabrice Schoumacher, Daniel Rehn, et al.
Mol Cancer Res 2006;4:27-37.
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