Article |
2 Institute for Histology and Embryology, University of Vienna, A-1090 Vienna, Austria
Address correspondence to Thomas W. Grunt, Signaling Networks Program, Div. of Oncology, Dept. of Internal Medicine I, University of Vienna, Währinger Gürtel 18-20, A-1097 Vienna, Austria. Tel.: 43-1-40400-5487. Fax: 43-1-40400-5465. E-mail: thomas.grunt{at}akh-wien.ac.at
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Abstract |
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Key Words: c-erbB-3; epithelial polarity; heregulin; mammary epithelial cells; nuclear localization
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Introduction |
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Nontumorigenic MTSV1-7 cells represent milk derived, SV40 large T antigenimmortalized, nonmalignant human mammary epithelial cells that express cytokeratins 7, 8, 18, and 19 and reveal a morphology, which is indicative of their luminal origin (Bartek et al., 1991).
Here we demonstrate nuclear localization of c-erbB-3 in MTSV1-7 and in human breast cancer cells, which is due to an active nuclear localization signal (NLS) near the COOH terminus of the protein. Moreover, c-erbB-3 was found in the nucleoli of differentiated polarized MTSV1-7 and exported into the cytoplasm upon addition of exogenous HRG.
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Results |
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Table II demonstrates that all cell lines analyzed except BT20 contained c-erbB-3 within the surface membrane, albeit at varying intensities.
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Discussion |
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RTJ2 seemed to detect nuclear c-erbB-3 with higher affinity and specificity than the other antibodies tested. This might be due to a different conformation of nuclear c-erbB-3 when compared with nonnuclear c-erbB-3, thereby better exposing the particular epitope recognized by RTJ2. Therefore, RTJ2 was chosen for the majority of our immunofluorescence experiments. Our data indicate that RTJ2 does not cross-react with some unrelated nuclear bulk protein nor does it selectively detect nuclear c-erbB-3 only. First, nuclear localization of c-erbB-3 was also seen with additional polyclonal antibodies, which recognize unrelated epitopes (Fig. 1, A and B). Second, RTJ2 similarly labeled nonnuclear c-erbB-3 (Fig. 1 B, Fig. 2, BT483, and Fig. 5).
Nuclear staining of variable intensity was also seen in other nonmalignant and malignant human mammary epithelial cells and was found to be strictly dependent on the applied culture conditions. For instance, the nuclear export inhibitor LMB (Fukuda et al., 1997) caused a significant enrichment of c-erbB-3 in the nuclei of cell lines that otherwise revealed only low levels of nuclear staining (Fig. 2 A). A similar effect was observed when MCF-7 cells were transiently transfected with full-length c-erbB-3 tagged with the FLAG peptide followed by LMB treatment and immunofluorescent detection of the receptor using the M2 anti-FLAG antibody (Fig. 2 B). This is in accordance with data obtained by immunoprecipitation and Western blotting of C and N fractions from untreated and LMB-treated MCF-7 cells, which revealed a significant LMB-induced deprivation of c-erbB-3 in C and a concurrent enrichment in N (Fig. 3). Although we cannot entirely exclude that some c-erbB-3 found in the C fraction might come from leakage of the receptor from the nucleus during cell lysis and fractionation, several lines of evidence yet argue against this. First, analysis of the purity of the fractions revealed that there was virtually no cross-contamination of soluble proteins between C and N as indicated by the distribution of the nuclear protein histone H1 and the cytoplasmic marker pyruvate kinase (Fig. 3 B). Second, the data in Fig. 3 C demonstrate that "cytoplasmic" c-erbB-3 is primarily membrane bound in MCF-7 cells. Moreover, it is important to note that LMB did not affect the distribution of the membrane marker proteins calnexin and transferrin receptor between C and N, although it still caused cytoplasmic deprivation and nuclear accumulation of c-erbB-3, indicating that contaminating membrane components are not a major source for the presence of c-erbB-3 in N. Interestingly, the molecular mass of nuclear c-erbB-3 was 185 kD. Thus the nuclear species represents the full-length protein and not a truncated version of c-erbB-3.
Although cell surface labeling for c-erbB-3 was rather weak in immunofluorescence microscopy, flow cytometry clearly demonstrated that c-erbB-3 can be detected in the plasma membrane. Despite general paucity of reports on the subcellular distribution of c-erbB receptors, there is one paper (Xie and Hung, 1994) describing partial nuclear localization of low levels of c-neu, the rat homologue of human c-erbB-2, in rat DHFR/G8 cells. Another study (Srinivasan et al., 2000) demonstrated nuclear localization of c-erbB-4 in primary human breast cancers, which might be caused by a potential NLS within the c-erbB-4 amino acid sequence. These data are supported by a very recent report from Ni et al. (2001), who demonstrated that the intracellular domain of c-erbB-4 gets cleaved by -secretase. The truncated cytoplasmic fragment then translocates into the nucleus (Ni et al., 2001). In addition, Lin et al. (2001) reported nuclear localization of c-erbB-1 in several malignant cell lines and presented evidence suggesting that nuclear c-erbB-1 might function as a transcription factor. In contrast to c-erbB-4 but in accordance with our data on c-erbB-3 nuclear c-erbB-1 was found to be the full-length receptor and not a truncated version (Lin et al., 2001; Bourguignon et al., 2002).
Although the list of nuclear localization of transmembrane receptors is considerably long and includes the interleukin-1 receptor (Curtis et al., 1990), the NGF receptor (Rakowicz-Szulczynska et al., 1988), the growth hormone receptor (Lobie et al., 1994), and several endocytosis-related proteins (Vecchi et al., 2001), one of the most well-known examples is the nuclear translocation of the FGF receptor in response to various stimuli (Maher, 1996; Stachowiak et al., 1996). The lack of a clearly understood potential pathway for nuclear import of full-length transmembrane growth factor receptors has raised some concerns about the relevance of nuclear growth factor receptors (Oksvold et al., 2002). However, a recent report demonstrated physical interaction of the FGF receptor with the nuclear import factor importin ß (Reilly and Maher, 2001), suggesting that nuclear import of the FGF receptor occurs via the importin ß pathway. Although we have no clear indication about potential nuclear import pathways for c-erbB-3, we consider a similar pathway at least possible.
Furthermore, it is evident that several polypeptide growth factors can enter the cell nucleus (Keresztes and Boonstra, 1999). Correspondingly, FGF-3 was found to localize to the cell nucleus, and an NLS was identified in the protein (Antoine et al., 1997). Using the PSORT II software (http://psort.nibb.ac.jp), we found a potential NLS near the NH2 terminus (NLS-1) and the COOH terminus (NLS-2) of the c-erbB-3 sequence. Reinhardt's method (Reinhardt and Hubbard, 1998) for cytoplasmic/nuclear discrimination predicted nuclear localization for c-erbB-3 with a reliability of 70%. Therefore, we constructed EGFPmutant erbB-3 fusion proteins and found that a tetra arginine repeat near the COOH terminus of c-erbB-3 (NLS-2) was able to target EGFP to the nucleus, whereas a similar potential NLS (NLS-1) near the NH2 terminus of c-erbB-3 was inactive (Fig. 6). Furthermore, transfer of NLS-2 to CPK, an exclusively cytoplasmic protein (Frangioni and Neel, 1993), resulted in efficient nuclear targeting of the fusion protein (Fig. 7). These data clearly indicate that NLS-2 itself is sufficient to confer nuclear targeting to heterologous proteins different from c-erbB-3. Besides that, the c-erbB-3 sequence (Kraus et al., 1989) contains several leucine-rich domains, which fulfill the formal requirements for CRM1-dependent nuclear export signals, and we observed that the CRM1 inhibitor LMB (Fukuda et al., 1997) can trap endogenous c-erbB-3 in the nuclei of several different malignant and nonmalignant cell lines of mammary epithelial origin, supporting the idea that nuclear localization represents a general property of c-erbB-3 and is not restricted to a specific cell line. Additionally, we were able to demonstrate LMB-induced nuclear accumulation of a transiently transfected c-erbB-3FLAG fusion protein. In the past, nuclear localization of c-erbB-3 might have been overlooked in many cell lines due to prominent cytoplasmic staining caused by active nuclear export. The use of LMB allowed to selectively stop this export and thus enabled visualization of c-erbB-3 in the nuclei of several cell lines. In the absence of LMB, nuclear staining for c-erbB-3 was most intense in BT20 and MDA468 cells and could not be further enhanced by LMB, indicating that nuclear export pathways are not very active in these cells.
Epithelial cell polarity and subnuclear localization of c-erbB-3
In contrast to solid substrate culture, MTSV1-7 grown on permeable filters polarized and differentiated as determined by localization of E-cadherin and ZO-1 (unpublished data) and by the presence of microvilli (Fig. 5 C), which was accompanied by concentration of c-erbB-3 in the nucleoli, where ribosome biosynthesis occurs, including transcription of ribosomal genes and assembly of the ribosome subunits (Shaw and Jordan, 1995). Previous studies showed that growth factors like FGFs and parathyroid hormonerelated peptide can enter the nucleolus (Pederson, 1998). However, to our knowledge no transmembrane growth factor receptor has ever been observed within the nucleolus.
Effect of endogenous and exogenous HRG on the subcellular distribution of c-erbB-3
A previous report demonstrated that primary human mammary epithelial cells express large amounts of HRG (Aguilar et al., 1999). We examined immortalized nonmalignant MTSV1-7 and MCF10A human mammary epithelial cells for secretion of HRG and found it in one of them (MTSV1-7) (Fig. 4). HRG is the cognate ligand for c-erbB-3 and might affect its subcellular distribution in an autocrine and/or paracrine fashion. Therefore, we blocked secreted HRG with a neutralizing antibody, which caused nucleolar confinement of c-erbB-3 in MTSV1-7 grown on filters. Conversely, exogenous HRGß1 caused export of c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. In contrast to a previous report demonstrating that HRG contains a potential NLS and is transported into the nucleus of the breast cancer cell line SKBR-3 (Li et al., 1996), we found that neither endogenous nor exogenous HRG enters the nucleus of nonmalignant MTSV1-7 cells (Fig. 5 D). These data led us to suppose that HRG induces nucleolar and nuclear export of c-erbB-3 by anchoring it to nonnuclear structures, thus leading to a slow enrichment of c-erbB-3 in the cytoplasm and its deprivation in the nucle(ol)us. Although the exact mechanisms controlling the equilibrium between nucle(ol)ar and nonnucle(ol)ar c-erbB-3 are still unknown, it still appears that HRG is crucial in determining its balance by binding it outside the nucleus not allowing its reentry. Interestingly, four tumor cell lines (MCF-7, BT474, T47D, and BT483) spontaneously exhibited low levels of nuclear c-erbB-3, whereas two others (BT20 and MDA468) and two nonmalignant cell lines (MTSV1-7 and MCF10A) revealed strong nuclear staining, possibly reflecting different activation states of the c-erbB-3 pathway. Many breast cancer cells exhibit overexpression/hyperactivation of c-erbB-2, which is the major heterodimer partner of c-erbB-3 and plays a crucial role in controlling c-erbB-3 activation and maybe subcellular localization. Accordingly, MDA468 are negative for c-erbB-2 and thus devoid of c-erbB-2/-3 heterodimers. On the other hand, BT20 cells overexpress c-erbB-1, which competes with c-erbB-3 for heterodimerization with c-erbB-2. It is thus very likely that these cells are also very poor in active c-erbB-2/-3 heterodimers. Strikingly, c-erbB-2 was invariably expressed in the cytoplasm and the membrane, irrespective of stage of malignancy, the level of baseline expression, and the culture conditions (unpublished data), indicating that the subcellular distribution of c-erbB-3 is specific for this particular receptor.
Although the ultimate mechanisms of nucleocytoplasmic shuttling of c-erbB-3 and its precise role in the nucleus remain to be elucidated, our data open up new perspectives on the functions of c-erbB proteins. Together, c-erbBs may not only represent membrane-anchored signal transducers with crucial roles in malignant transformation and progression, but as constituents of nucleolar structures they might also regulate ribosome biosynthesis.
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Materials and methods |
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LMB treatments
For immunofluorescence, cells were seeded at 104/cm2 into 8-well glass chamber slides (Nunc) and allowed to settle for 24 h. Media were replaced, and 20 ng/ml LMB (a gift from Dr. B. Wolff-Winiski, Novartis, Vienna, Austria) in 0.1% methanol or solvent alone were added to the cells and incubated for 24 h. c-erbB-3FLAGtransfected MCF-7 cells were treated for 24 h with 5 ng/ml LMB or solvent.
For fractionation experiments, MCF-7 cells were seeded into five 500-cm2 plates and grown to 80% confluence. 5 ng/ml LMB was added to three of the plates, whereas the remaining plates were treated with 0.1% methanol for 24 h. These conditions were found previously to yield optimal nuclear localization of c-erbB-3 in MCF-7 cells, whereas toxicity was kept at a minimum (unpublished data).
Flow cytometry
MCF-7, MDA468, T47D, and BT20 cells were rinsed with PBS, briefly trypsinized, and washed with FACS® buffer (1% BSA, 0.1% NaN3 in PBS). All subsequent incubations and washings were performed in this buffer. Labeling with 10 µg/ml of mouse monoclonal antic-erbB-3 antibody (SGP1; Neomarkers) or irrelevant control mIgG (Dako) was performed for 30 min on ice. Washed cells were then labeled for 30 min on ice with goat antimouse Ig-Alexa488 (1:200; Molecular Probes). Analysis was performed with a FACScan® (Becton Dickinson) equipped with CellQuest software. For life gating, propidium iodide (0.5 µg/ml; Sigma-Aldrich) was added to the cold cells immediately before measurement, and 5 x 103 nonpermeable cells per sample were measured. Results were expressed as mean fluorescence intensity.
Nuclear and cytoplasmic fractions
Control and LMB-treated MCF-7 were washed twice with PBS, harvested by scraping and centrifuged (1,850 g, 10 min, 4°C), suspended in 5 packed cell vol hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, protease inhibitors [1 tablet Complete® EDTAfree/50 ml, Roche]), recentrifuged, resuspended in 3 packed cell vol hypotonic buffer, incubated on ice (10 min), homogenized (Dounce, type B pestle, 25 strokes), and spun (3,300 g, 15 min, 4°C). The supernatant was designated S1 and saved. The nuclear pellet was washed twice with 10 ml hypotonic buffer and suspended in 0.5 packed nuclear vol low salt buffer (20 mM Hepes, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 1 mM EGTA, 0.2 mM PMSF, 0.5 mM DTT, 0.2% Triton X-100, 1 tablet Complete® Mini EDTAfree/10 ml). Then, 0.5 packed nuclear vol high salt buffer (low salt buffer with 1.2 M KCl) were added, and nuclei were homogenized (25 strokes), extracted on a shaker (30 min, 4°C), and spun (25,000 g, 30 min, 4°C). 1 U/ml 2-macroglobulin (Roche), which is unable to pass a 10-kD-cutoff dialysis membrane, was added to the supernatant and dialyzed (dialysis buffer [DB]; 20 mM Hepes, pH 7.9, 10% glycerol, 300 mM KCl, 0.5 mM EGTA, 0.2 mM PMSF, 0.2% Triton X-100; 1 h, 4°C) in a slide-a-lyzer cassette (MWCO 10000; Pierce Chemical Co.). The dialyzed nuclear extract was recentrifuged (25,000 g, 20 min, 4°C), aliquoted, and stored at -80°C.
Cytoplasmic extracts.
Saved supernatant S1 was mixed with 0.11 vol 10x cytoplasmic extract buffer (300 mM Hepes, pH 7.9, 30 mM MgCl2, 1.4 M KCl, 2% Triton X-100), spun (100,000 g, 1 h, 4°C; S100 fraction), and 2-macroglobulin was added before dialysis. The dialyzed fraction was centrifuged (25,000 g, 20 min, 4°C) and stored as above.
For some experiments a simplified protocol was used.
The supernatant S1 was divided into 2 aliquots, and cytoplasmic extract buffer with or without Triton X-100 was added before centrifugation at 100,000 g. The supernatants were subjected to protein assay according to Bradford and used directly for Western blotting. The membrane pellet from the fraction without Triton X-100 was dissolved by boiling (5 min, 95°C) in 400 µl Laemmli buffer, and insoluble components were removed by centrifugation before 10 µl of this solution was used for Western blotting.
Immunoprecipitation
Concentration of nuclear and cytoplasmic protein (400 µg each) from LMB-treated or untreated MCF-7 cells was balanced with DB, which was supplemented with 1 tablet Complete® Mini EDTAfree/10 ml (DBC). Gamma bind plus sepharose beads (Amersham Pharmacia Biotech) were washed and resuspended to original vol with DBC. 10 µl beads, 4 µg antic-erbB-3 (SGP1; Neomarkers) or murine IgG1 (Dako), and respective fraction (400 µg protein) were mixed, incubated on a shaker (1 h, 4°C), washed five times (200 µl DBC), spun (10,000 g, 1 min, 4°C), resuspended in Laemmli buffer, boiled (5 min), and stored frozen.
Western blotting
Proteins were separated by 7.5% SDS-PAGE, and gels were equilibrated for 30 min in blotting buffer (20% methanol, 380 mM glycine, 50 mM Tris, 0.1% SDS). Proteins were blotted onto polyvinylidene difluoride membranes (New England Nuclear), which were then blocked for 1 h in 5% BSA/PBS at room temperature (RT), washed with PBS containing 0.1% Tween 20 (PBST), and immunostained for 3 h at RT in 1% BSA/PBS containing the primary antibody: polyclonal antic-erbB-3 (0.2 µg/ml, C17; Santa Cruz Biotechnology, Inc.), polyclonal anti-CPK (cross-reactive with human pyruvate kinase) (1:4,000; Convance), monoclonal antihistone H1 (0.2 µg/ml; Neomarkers), monoclonal anti-calnexin (1:400; Affinity BioReagents, Inc.), or monoclonal antitransferrin receptor (1 µg/ml; Neomarkers). Washed blots were labeled for 1 h with sheep antimouse or donkey antirabbit Ig F(ab')2-HRP conjugate (1:10,000 in 1% BSA/PBST; Amersham Pharmacia Biotech), washed extensively in PBST, immersed in ECL detection reagent (New England Nuclear), and exposed to X-Omat AR film (Eastman Kodak Co.).
For the detection of secreted HRG, MTSV1-7 and MCF10A cells were grown in serum-free MEGM. Conditioned culture supernatants were then recovered and 10-fold concentrated with Centricon 10 concentrators (Millipore). 10 µl Laemmli buffer were added to 10 µl of the concentrates and boiled (5 min), separated by 10% SDS-PAGE, blotted, and immunostained with 1 µg/ml rabbit anti-HRG (Ab-2; Neomarkers) and donkey antirabbit Ig F(ab')2-HRP conjugate (1:10,000; Amersham Pharmacia Biotech) as above.
Immunofluorescence (c-erbB, FLAG, and HRG)
Washed cells (PBS) were fixed in 2% paraformaldehyde (PFA) (5 min, RT), permeabilized in methanol/acetone (1:1, 3 min, -20°C), air dried, and stored at -80°C. Before use, cells were rehydrated in PBS (5 min, RT), blocked in 5% sheep serum/PBS (30 min, RT), and washed again. Antibodies (in 5% sheep serum/PBS, 1 h, RT) were against c-erbB-2 (10 µg/ml mouse monoclonal mAB-3; Oncogene Research Products), c-erbB-3 (10 µg/ml mouse monoclonal RTJ2; Neomarkers) (5 µg/ml rabbit polyclonal C17; Santa Cruz Biotechnology, Inc.) (10 µg/ml rabbit polyclonal Ab-9; Neomarkers), FLAG (20 µg/ml, mouse monoclonal M2; Sigma-Aldrich), and HRGß (10 µg/ml goat polyclonal C18; Santa Cruz Biotechnology, Inc.). Secondary antibodies were goat antimouse or goat antirabbit Ig-Alexa488 or donkey antigoat Ig-Alexa546 (1:200 in 5% sheep serum/PBS, 30 min, RT; Molecular Probes). Washed cells were embedded in Prolong Antifade (Molecular Probes) and evaluated in an LSM400 confocal microscope (ZEISS) with a thickness of the optical sections of 0.8 µm.
Immunofluorescence (CPK)
For detection of CPK fusion proteins, fixed cells were quenched in 50 mM glycine/PBS (5 min, RT). Blocking was performed with 10% goat serum in PBS containing 0.2% NP-40 (30 min, RT). All subsequent washing and incubation steps were performed as above in solutions containing 0.2% NP-40 to suppress background staining caused by cross-reactivity of the antiserum with endogenous (human) pyruvate kinase (Frangioni and Neel, 1993).
The following antibodies were used: crude rabbit antiserum against CPK (Convance, 1:500) and goat antirabbit Ig-Alexa488 (1:200; Molecular Probes).
Filter assay
MTSV1-7 cells were plated at 105 into 25 mm Anopore-Inserts (Nunc). Neutralizing anti-HRG (Ab-2, 10 µg/ml; Neomarkers) was added where indicated. After 48 h, cells were washed and exposed for the indicated times to 1 nM recombinant HRGß1 (Neomarkers). Filters were broken out at the end of the experiments, cut, and processed as above.
Immuno EM
MTSV1-7 grown in culture flasks were fixed with 2.5% PFA/0.5% glutaraldehyde in 0.1 M Soerensen buffer (SB; 30 min, 4°C) and washed 3 x 10 min with SB. Two batches of MTSV1-7 grown on filter were prepared: one treated with neutralizing anti-HRG and one with subsequent exposition to 1 nM HRGß1 for 24 h. Small pieces of filters were fixed with 4% PFA/0.5% glutaraldehyde in 0.1 M SB (30 min, 4°C) and washed three times with SB. Filters and MTSV1-7 in suspension were embedded in the hydrophilic resin LRWhite (London Resin Company). Ultrathin sections of MTSV1-7 cells were mounted on meshed gold grids; sections of the filters were mounted on Formvar-coated gold grids.
Grids were preincubated in PBS plus 1% BSA plus 1% normal goat serum, pH 7.4 (PBSB; 30 min, RT), incubated overnight with antic-erbB-3 (monoclonal RTJ2, 10 µg/ml in PBSB, and rabbit polyclonal Ab-9, 5 µg/ml in PBSB; 4°C), washed thoroughly in PBST, pH 7.4 and 8.2, labeled with 10 nm gold-conjugated goat antimouse or goat antirabbit IgG (1:40 in PBSB, pH 8.2; British BioCell), and washed in PBST, pH 8.2, and in distilled water. Contrast was enhanced with 2% aqueous uranyl acetate (30 min), and sections were evaluated with a Jeol EM1200 electron microscope equipped with a digital camera (BioScan 792; Gatan). For controls, the primary antibody was omitted. Signal distribution over cytoplasm and nucleus was quantified by counting gold particles per respective area in 10 randomly chosen cells per sample. The area was measured with the help of morphometry software (KS400; Kontron). For statistical comparison of datasets of RTJ2, Ab-9 and control series Student's t test was applied. Normal (Gaussian) distribution of datasets was checked using the R/s test.
EGFP fusion protein construction and analysis
A plasmid containing full-length c-erbB-3 was linearized by complete XbaI digestion, and the signal peptide was removed by partial SmaI digestion. pEGFP-C1 (CLONTECH Laboratories, Inc.) was digested with Ecl136II and XbaI before ligation.
Two COOH-terminal deletion mutants of the c-erbB-3 cDNA were constructed by cleaving the c-erbB-3 insert at the unique Ecl136II and XbaI or the EcoRI and XbaI restriction sites. The EcoRI and XbaI sites of c-erbB-3 and pEGFP-C1 were then filled in, respectively, and the COOH-terminally truncated c-erbB-3 inserts were ligated with the pEGFP-C1 vector. This yielded proteins with EGFP fused to amino acids 11,088 (Ecl136II) or 1-74 (EcoRI) of c-erbB-3.
In addition, the COOH terminus of c-erbB-3 was fused to EGFP using the Ecl136II site of c-erbB-3, the filled-in HindIII site of pEGFP-C1 and the XbaI sites of both vector and insert, yielding a construct containing amino acids 1,0891,323 of c-erbB-3 downstream of EGFP. Site-specific mutagenesis (R1184S/R1185G) of the latter construct was done with the Quick-Change kit (Stratagene) according to the manufacturer's protocol using the following oligonucleotides: 5'-GGTGGACTGTGCCTTCCGCTCCGGTTCATGTATTC-3' and 5'-GAATACATGAACCGGAGCGGAAGGCACAGTCCACC-3'. Presence of the double mutation was verified by sequencing.
MTSV1-7 cells were seeded and transfected using Effectene (QIAGEN) adhering to manufacturer's protocols. 24 h after transfection, cells were washed, fixed with 2% PFA as above, embedded in Mowiol/DABCO, and evaluated by confocal microscopy as described above.
FLAG fusion protein construction and analysis
A vector containing full-length c-erbB-3 (supplied by F. Wouters, European Molecular Biology Laboratory, Heidelberg, Germany) was used in order to fuse the FLAG peptide to the COOH terminus of c-erbB-3. The sequence coding for the FLAG peptide was supplied using the following oligonucleotides: 5'-CGACTACAAGGACGACGATGACAAGTGAGCTCGC-3' and 5'-GGCCGCGAGCTCACTTGTCATCGTCGTCCTTGTAGTCGAT-3', which were dissolved at 200 µM in 10 mM Tris/HCl, 150 mM NaCl, pH 7.5, and mixed in equal volumes. Annealing was performed by heating the mixture to 94°C and cooling it down to 25°C over a period of 1 h before ligation to c-erbB-3. MCF-7 cells were seeded and transfected using Fugene6 (Roche) according to the supplied protocol. 1 d after transfection, cells were subjected to LMB treatment. Immunofluorescence microscopy was performed as described above.
CPK fusion protein construction and analysis
The p3PK vector, which was used to create fusion proteins of CPK with various human c-erbB-3 fragments, and a positive control plasmid for nuclear localization containing CPK fused to the heterologous NLS of human lamin C (p3PK-NLS-Lamin C) were supplied by Dr. J. V. Frangioni (Beth Israel Deaconess Medical Center, Boston, MA). The sequence coding for NLS-2 of c-erbB-3 (RRRRHSP*) was cloned using the following oligonucleotides: 5'-GATCCCGGAGGAGAAGGCACAGTCCATGAG-3' and 5'-AATTCTCATGGACTGTGCCTTCTCCTCCGG-3', which were annealed as above. Using standard procedures, this dsDNA was then ligated to p3PK, which had been digested with EcoRI and BamHI in order to yield a CPK-NLS-2 fusion protein.
Another construct was produced by digesting p3PK with BamHI, filling in, redigesting with XbaI, and dephosphorylation. The insert (COOH-terminal end of c-erbB-3, amino acids 9151,323) was prepared by digesting c-erbB-3 with BglII, filling in, and redigesting with XbaI. Ligation was performed as above, yielding a construct of CPK fused to the COOH terminus (amino acids 9151,323) of c-erbB-3 (CPK-erbB3CT). Transfection of MTSV1-7 cells with Effectene and immunofluorescence microscopy were performed as described above.
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Footnotes |
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* Abbreviations used in this paper: C, cytoplasmic-enriched; CRM, chromatin region maintenance; CPK, chicken pyruvate kinase; EGFP, enhanced green fluorescent protein; HRG, heregulin; LMB, leptomycin B; N, nuclear-enriched; NLS, nuclear localization signal; PFA, paraformaldehyde.
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Acknowledgments |
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This work was supported by a grant from the Austrian Science Fund (FWF, P13622-GEN) and the Austrian National Bank (ONB, 7503) to T.W. Grunt.
Submitted: 10 September 2001
Revised: 12 February 2002
Accepted: 17 April 2002
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References |
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