Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism
The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells.
To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112) and another to the DNA binding domain (DBD), (NES2: 275LWEFIRDILI284). Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions.
These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.
KeywordsGreen Fluorescent Protein Nuclear Localization Signal Green Fluorescent Protein Fusion Putative Nuclear Localization Signal Nuclear Localization Signal Sequence
(Nuclear Localization Sequence)
(Nuclear Export Sequence)
- SAR domain
(Serine and Aspartic acid Rich domain)
(mammary epithelial cell)
The human ETS (E26 Transformation-Specific) protein family is a diverse group of 27 known transcription factors that regulate such varied cellular processes as differentiation and apoptosis, but also appear to induce oncogenesis when mutated or aberrantly expressed [1, 2, 3, 4]. In particular, aberrant ETS protein activity and/or expression has been implicated in human mammary epithelial cell (MEC) transformation . The ER81 ETS protein, for example, is activated in human breast cancer cells by the oncoprotein HER-2, resulting in over-expression of the prosurvival telomerase reverse transcriptase (hTERT) gene . In addition, ETS-1 mRNA overexpression appears to be a strong independent predictor of poor prognosis in primary human breast cancers . Furthermore, ETS-2 overexpression can inhibit expression of the tumor-suppressor gene BRCA1, the downregulation of which is clearly linked to familial breast cancer [7, 8].
Overexpression of one ETS protein in particular, the epithelium-specific ETS factor ESE-1, is implicated in human mammary transformation. ESE-1 mRNA is overexpressed in primary human ductal carcinomas in situ (DCIS), and the genomic ESE-1 locus (1q32.1) is commonly amplified in primary human breast cancer cells [9, 10, 11]. In addition, we have shown that ESE-1 expression confers a transformed phenotype to the nontransformed MCF-12A and MCF-10A human MECs, including enhanced invasiveness and motility, anchorage independent growth, epidermal growth factor-independent proliferation, and formation of disorganized structures in three-dimensional cultures on matrigel [12, 13, 14]. A later study screening a collection cDNAs associated with breast cancer independently identified ESE-1 as a factor that promotes motility and induces formation of disorganized structures on matrigel in MCF-10A cells .
While previous publications have established ESE-1's transcription factor function, we have reported that ESE-1 initiates transformation of MECs via a novel non-nuclear, non-transcriptional mechanism . We have shown that a 40-amino acid (AA) serine and aspartic acid rich (SAR) domain within the ESE-1 is both necessary and sufficient to mediate ESE-1 transforming function and that enforced nuclear localization of full-length ESE-1 or of the SAR domain alone, abrogates ESE-1 ability to initiate transformation . These results imply that ESE-1 contains an endogenous nuclear export signal that is required for ESE-1-mediated initiation of MEC transformation via a cytoplasmic mechanism. In addition to transformation initiating function that requires cytoplasmic localization of ESE-1, we have reported that ESE-1 is required for the maintenance of transformed phenotype in breast cancer cell lines. We have shown that shRNA-mediated downregulation of ESE-1 protein levels in MCF7 and ZR-75-1 breast cancer cell lines results in decreased anchorage independent growth, and that in these cells lines, as well as in T47D, ESE-1 is localized to the nucleus . Thus, nuclear function of ESE-1 is required for the maintenance of transformed phenotype. Together these reports establish that nuclear-cytoplasmic shuttling of ESE-1 is essential for transformation initiation in benign MECs as well as for the maintenance of transformed phenotype in breast cancer cells, and imply that ESE-1 contains both nuclear export (NES) as well as nuclear localization (NLS) signals.
In the current report we use fusion between green fluorescent protein (GFP) and specific ESE-1 motifs to map functional ESE-1 NES and NLS sequences and to define the role of these motifs in ESE-1 transforming function. We localize the functional ESE-1 NLS to a six AA basic motif within the ESE-1 A/T Hook domain and we demonstrate that, unlike in other ETS proteins [17, 18, 19], in-frame deletion of the ESE-1 DBD does not abrogate ESE-1 nuclear localization. Using both gain-of-function and loss-of-function approaches, we identify a single NES within the ESE-1 DBD that is required for ESE-1-mediated initiation of MCF-12A cell transformation. Furthermore, we sequentially mutagenize 11-14 AAs blocks in the SAR domain to establish that while each of the SAR mutants partially retains transformation function in MCF-12A cells, an intact SAR domain is required for its full transforming activity. Finally, we identify ESE-1 region 216-228 within the SAR domain as the site of interaction with anti-ESE-1 antibody mAB405, what suggests that this region is surface exposed and thus likely to mediate protein-protein interactions. In summary, these data represent a paradigm shift in our understanding of the specific subcellular functions of ETS transcription factors, by revealing a novel NES2 and providing insights into SAR domain-dependent cytoplasmic mechanism by which ESE-1 initiates MEC transformation.
ESE-1 contains a single, basic amino acid-rich NLS that maps to the A/T hook domain
Previous reports have shown that basic AA-rich sequences in the DBDs of several different ETS proteins, including ETS-1 (376GKRKNKPK383), ELK-1 (47GLRKNKTN54), and ER71 (275GERKRKPG282), mediate the nuclear localization of these proteins [17, 18, 19]. In murine Elf3, internal deletion or site-specific mutation of the 318KKK320 sequence, within the context of the entire DBD, resulted in the localization to both the nucleus and the cytoplasm . Considering these data, we tested whether a similar putative NLS sequence, 316GQKKKNSN323 (ESE-1 NLS6) in the ESE-1 DBD also shows NLS function, using the GFP fusion strategy described above (Figure 1D). Transient expression of GFP-NLS6-SAR in MCF-12A cells revealed diffuse cytoplasmic and nuclear fluorescence (Figure 1D) that was indistinguishable from that of GFP-SAR (Figure 1C, panel 6) and , indicating that ESE-1 NLS6 is insufficient to mediate nuclear localization. To test whether the ESE-1 NLS6 is necessary to mediate nuclear localization, we generated an additional construct in which the ESE-1 DBD was deleted in-frame from the previously described pEGFP-ESE-1 expression plasmid, containing the full-length ESE-1 protein, to generate pEGFP-ESE-1ΔDBD (Figure 1E). Transient transfection in MCF-12A cells revealed exclusive nuclear GFP-ESE-1ΔDBD localization, thus demonstrating that in the human ortholog of ESE-1, the DBD is not required for ESE-1 nuclear localization. Together with the data shown in Figures 1C & Figure 1D, these findings indicate that, unlike previously examined ETS proteins, the ETS DBD does not play a role in ESE-1 nuclear localization.
ESE-1 contains two separate CRM1-dependent NES motifs
Site-specific mutation of ESE-1 NES2 inhibits GFP-ESE-1-induced MCF-12A cell transformation
Stable expression of the GFP-NES1-SAR protein is sufficient to transform MCF-12A cells
An intact SAR domain is required for optimal transforming activity
The SAR domain contains the epitope for anti-ESE-1 mAb405
ETS family proteins have been shown to function in the nucleus as regulators of gene transcription [1, 2, 3, 4]. However, despite previous documentation of ESE-1 transcription factor function [12, 13, 20, 27, 28, 29, 30], we have proposed a novel nontranscriptional, cytoplasmic model whereby ESE-1, functioning via its SAR domain, initiates mammary epithelial cell transformation . For ESE-1 to mediate transformation from a cytoplasmic location, ESE-1 must contain a functional nuclear export sequence. In this report, we used molecular and pharmacological methods to define functional NLS and NES sequences within human ESE-1 and to characterize the critical role of nuclear export of ESE-1 in its transforming function. Furthermore, we demonstrated that cytoplasmically-restricted SAR domain is sufficient to initiate MEC transformation and that full transforming activity requires an intact SAR domain.
ESE-1 has been documented to operate as a nuclear activator of promoter function in transient transfection reporter assays [28, 29, 30]. Indeed, transient transfection of GFP-ESE-1 into several different cell lines, including HeLa cervical carcinoma and T47D and SKBR-3 breast cancer cells, demonstrates nuclear localization of this fusion protein (data not shown). In this report we use progressive truncations in GFP-fusion gain-of-function studies to map ESE-1 nuclear localizing activity to a basic, six AA sequence (242HGKRKR247) located within the A/T Hook domain, but outside of the ESE-1 DBD (Figures 1A, B, & 1C). We confirmed that the DBD does not contain an NLS sequence required for nuclear localization of ESE-1, using a loss-of-function deletion study of the ESE-1 DBD, demonstrating that DBD deletion does not impair ESE-1 nuclear import (Figure 1E). Furthermore, we have previously reported that in-frame deletion of the ESE-1 A/T Hook domain (AAs 236-267), which includes the functional ESE-1 NLS identified here, completely inhibits ESE-1 nuclear import . Indeed, Elf3, the murine ortholog of ESE-1, has been shown to contain a functional NLS located at an equivalent position and, in contrast to ESE-1, an additional NLS in its DBD . Interestingly, while both Elf3 NLS motifs function autonomously in fluorescent protein-fusion assays, they appear to target to different subnuclear regions. However, since neither of the Elf3 NLS motifs has been individually mutated or deleted in the context of full-length Elf3, the requirement of either NLS in Elf3 nuclear targeting remains unknown. Nevertheless, our data confirm that the functional ESE-1 NLS resides within the A/T hook domain and that ESE-1 DBD is neither necessary nor sufficient to mediate ESE-1 nuclear localization. This finding is surprising in light of previous reports demonstrating an essential role for the highly conserved ETS DBD in ETS factor nuclear localization [17, 18, 19]. Finally, amino acid comparison analyses performed by us and others  reveal that the ESE-1 NLS discovered here is not present in any other ETS factor, including other members of the ESE subfamily.
Extensive evidence supports nuclear-cytoplasmic shuttling as a regulatory mechanism for ETS protein function [32, 33, 34, 35]. A common regulatory mechanism involves MAPK signaling cascades, which trigger nuclear export of ETS repressors such as NET (new ETS), YAN, ERF (ETS-2 Repressor Factor) and TEL (Translocation ETS Leukemia) and thus release ETS-mediated gene repression. For example, the ETS DBD of the ternary complex factor NET contains a functional, CRM1- dependent NES (7LWQFLLQLLL16) that appears to be highly-conserved within the DBDs of most ETS proteins , including ESE-1 (i.e. NES2: 275LWEFIRDILI284). Activation of the c-Jun N terminal kinase kinase (JNKK) pathway mediates nuclear exclusion of NET, relieving transcriptional repression induced by NET. Moreover, site-specific mutation of the NET NES traps NET in the nucleus, resulting in increased NET repressor function . These data point to a crucial regulatory role for the NET NES.
In this report, we identify two ESE-1 NES signals, NES1 and NES2, but we demonstrate that only one, NES2, plays a critical role in the nuclear export and transforming function of intact ESE-1 protein. NES1 is located in the ESE-1 Pointed domain but appears to mediate nuclear export, in a CRM1-dependent manner, only when outside of the context of full length ESE-1 protein (Figure 2C). In addition, comparative analysis of ETS factors Pointed domain sequences reveals that most other ETS factors, including ESE-2 and ESE-3, do not conserve the NES1 motif. In contrast, NES2 appears to be well conserved in the DBD region of most ETS proteins, suggesting a conserved function of this motif in the ETS family (analysis not shown). Here we show that inactivating mutations in the ESE-1 NES2 completely inhibit GFP-ESE-1 transforming function (Figures 3C & 1D), indicating that GFP-ESE-1 nuclear export plays an essential role in GFP-ESE-1-mediated transformation. An alternative to this conclusion is that mutation of DBD-embedded NES2 disrupts ETS DBD-DNA binding and that it is this disruption, rather than the inhibition of NES2 function, that impairs GFP-ESE-1 transforming activity. However, crystallographic structural data for the DNA-bound ESE-1 DBD indicate that NES2 is localized to a DBD subregion that does not make direct contact with target DNA, except for leucine 275 . This finding is consistent with our previously published data showing that the domains of ESE-1 that are required for transcription factor function (e.g. the ESE-1 TAD) are not necessary to initiate transformation in benign MECs, whereas the SAR domain alone is sufficient in this type of transformation assay (Figure 4) . As noted above, the ESE-1 NES2 is similar in sequence and location to the functional MAPK-regulated NES motifs in NET and ERF. However, we have been unable to identify any specific kinase(s) that regulate ESE-1 subcellular localization. Specifically, co-transfection studies using constitutively active forms of JNK, MAPK, ERK and v-SRC protein kinases revealed that none of these kinases enhanced cytoplasmic shuttling of transiently co-expressed nuclear GFP-ESE-1 (data not shown). Taken together, our data suggest that basal ESE-1 subcellular localization represents the summed influences of NES and NLS functions.
Our data precisely define NLS and NES signals in the human version of ESE-1 that play a pivotal role in regulating its subcellular localization and its ensuing transforming function. Furthermore, we report that transformation of human MECs requires an intact SAR domain that can be targeted exclusively to the cytoplasm, and that the SAR motif is accessible for protein and/or ligand interactions. This report is important, since it provides critical mechanistic details of ESE-1 function, and it significantly expands our understanding of the role of ETS factors in mammary cell transformation.
Mammalian cell culture
All cell lines were acquired from the American Type Culture Collection (Manassas, VA) and were maintained as described in .
GFP fusion vectors
All GFP-SAR fusion constructs were generated using the previously described PCR-based strategy  and subcloned into the pEGFP-C3 parental expression plasmid (Invitrogen Inc., Carlsbad, CA). Specific sense primers for GFP-SAR fusions were: 5'- cgggaattc atcccaagcacgggaagcggaaacga TCCCCTGGCAGCTCTG (GFP-NLS1-SAR); 5'- ccggaattc ataagaagggggatcccaagcacgggaagcggaaacga TCCCCTGGCAGC (GFP-NLS2-SAR); 5'- cgggaattc atcgaaagctgagcaaagagtactgggactgtctcgagggcaagaagagcaagcac TCCCCTGGCAGCTCTGAC (GFP-NLS3-SAR); 5'- cgggaattc ataagcacgggaagcggaaacga TCCCCTGGCAGCTCTG (GFP-NLS4-SAR); 5'- cgggaattc atcacgggaagcggaa acga TCCCCTGGCAGCTCTG (GFP-NLS5-SAR); 5'- cgggaattc atggccaaaagaaaaagaacagcaac TCCCCTGGCAGCTCTG (GFP-NLS6-SAR). 5'- cgggaattc atctctgcaattgtgcccttgaggagctgcgtctg TCCCCTGGCAGCTCTG (GFP-NES1-SAR); 5'- cgggaattc atctctgcaattgtgccgccgaggaggcccgtctg TCCCCTGGCAGCTCTG (GFP-NES1Mut-SAR); 5'- cgggaattc atctgtgggagttcatccgggacatcctcatc TCCCCTGGCAGC (GFP-NES2-SAR); 5'- cgggaattc atctgtgggagttcgcccgggacgccctcatc TCCCCTGGCAGC (GFP-NES2Mut-SAR); Bold sequences show EcoR I restriction sites, capital letters represent SAR coding sequence and NES/NLS sequences are underlined. Generation of GFP-ESE-1ΔDBD and GFP-ESE-1 NES2Mut was accomplished using a two-step PCR overlap extension strategy [38, 39]. In both cases, the 5' ESE-1 sense primer  was used with the following antisense primers to generate the 5' segment of each construct, respectively: 5'-GGCAAAAACTCAAGCGGCTGGAAG (GFP-ESE-1ΔDBD antisense) and 5'-GATGAGGGCGTCCCGGGCGAACTCCCACAG (ESE-1 NES2Mut antisense). To generate 3' overlap segments, the 3' ESE-1 antisense primer  was used with the following respective sense primers: 5'-AGTTTTTGCCGTGGGTGCCTCTG (GFP-ESE-1ΔDBD sense) and 5'- CTGTGGGAGTTCGCCCGGGACGCCCTCATCCACCCGGAGCTCAACGAG (ESE-1 NES2Mut sense). The resulting PCR overlap extension products were ligated into the pEGFP-C3 plasmid as described previously . Similar PCR strategy, followed by ligation into pEGFP-C3 plasmid, was used to generate GFP-SAR-myc Box 2 and GFP-SAR-myc Box 3 constructs. In both cases, 5' SAR sense primer  was used with the following respective antisense primers to generate the 5' segments of each construct: 5'- gtcttcctcgctgatcagtttctgctc ACCAGTCCCTGCGGTGGAG (GFP-SAR-myc Box 2 antisense), 5'- gtcttcctcgctgatcagtttctgc TCACTTCCACCGGAGTCTGAGG (GFP-SAR-myc Box 3 antisense). Whereas, the 3' SAR antisense primer  was used with the following respective sense primers to generate the 3' segment of each construct: 5'- ctgatcagcgaggaagacctgttg GACGTGGACCTGGATCCCACTG (GFP-SAR-myc Box 2 sense), 5'- ctgatcagcgaggaagacctgttg AGCGATGGTTTTCGTGACTGC (GFP-SAR-myc Box 3 sense). To produce the GFP-SAR-myc Box 1 construct, the following sense primer was used in a PCR with the 3' SAR antisense primer : 5'- ccggaattcatctgatcagcgaggaagacctgttg GCTTCTCGGAGCTCCCACTCC. Similarly, the GFP-SAR-myc Box 4 sequence was amplified using the following antisense primer in a PCR with the 5' SAR sense primer : 5'- ccggaattcatgtcttcctcgctgatcagtttctgctc GGGGAAGAGCTTGCCATC. Both sequences were ligated into the pEGFP-C3 EcoR I site, to produce the GFP-SAR-myc Box 1 and GFP-SAR-myc Box 4 constructs, respectively. For each primer used in generation of GFP-SAR-myc Box mutants, capital letters show SAR domain coding sequence, and italicized text shows myc epitope sequence. To generate the pEGFP-PEA3 and pEGFP-ETS-2 expression plasmids, the full-length human PEA3 and ETS-2 coding sequences were amplified by RT-PCR from T47D human breast cell line whole cell RNA. The respective primer pairs used in these amplifications were as follows: 5'-cgagatct ccggaattc atATG GAGCGGAGGATGAAAGCCGG vs. 5'-cgagatct ccggaattc atCTA GTAAGAGTAGCCACCCTTG (PEA3) and 5'-cgagatct ccggaattc atATG AATGATTTCGGAATCAAG vs. 5'-cgagatct ccggaattc atTCA GTCCTTCGTGTCGGGC (ETS-2). In each case, restriction sites are in bold, and start and stop codons are underlined. Each full-length coding sequence was then ligated into the pEGFP-C3 plasmid as described . The absence of mutations in each expression construct was confirmed by DNA sequencing.
MCF-12A cells were transfected with GFP-fusion expression plasmids and plated as described previously . Alternatively, stable MCF-12A transfectants were plated directly onto glass coverslips for confocal microscopy. For nuclear staining, some cover slips were stained with 300 nM 4',6-diamidino-2-phenylindole (DAPI) . In addition, some coverslips were incubated for 15 minutes at 37°C in PBS containing 10 ng/ml leptomycin B (Sigma-Aldrich, Inc., St. Louis, MO). Cell imaging and image acquisition were performed as described previously .
Stable cell lines
Stable MCF-12A cell expression of each GFP fusion protein was obtained as described in  and two or three independent stable transfectant populations were generated for each expression plasmid.
Soft agarose assays
Triplicate soft agarose cultures were prepared for each stable MCF-12A transfectant population, as described in . Each experiment was repeated as noted in the text. Representative colonies were imaged and quantitated as described in .
Whole cell RNA was prepared from individual stable transfectant populations using an RNA STAT-60 kit (Tel-test, Inc., Friendswood, TX). GFP fusion transcripts in each RNA sample were identified using a sense primer directed against a terminal portion of the GFP open-reading frame and an antisense primer specific for a transcribed but untranslated sequence immediately downstream of the DNA insertion site in the pEGFP-C3 plasmid. The Omniscript™ RT kit (Qiagen) was used for reverse transcription as described in . Some RNA samples were treated with RNAse A (Five Prime Three Prime, Inc., Boulder, CO) prior to reverse transcription. All RT-PCRs were analyzed by 1% agarose gel electrophoresis.
Cells were plated directly onto glass cover slips in a 12-well tissue culture plate and transfected with GFP-SAR constructs using Effectene (Qiagen). Two days post-plating, cells were fixed with 2% paraformaldehyde for 20-25 min at room temperature and washed with phosphate-buffered saline (PBS). Subsequently, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 minutes, followed by three washes in 100 mM glycine in PBS. Permeabilized cells were blocked in blocking buffer containing 0.5% Tween-20, 10% goat serum in PBS for 1-2 h. Cells were incubated with anti-ESE-1 monoclonal antibody mAB405  diluted 1:500 in the blocking buffer overnight at 4°C. After washes, cells were incubated for 1 h with Cy3-conjugated donkey anti-mouse IgG secondary antibody (Jackson Immunoresearch). Nuclei were counterstained using 300 nM DAPI (Invitrogen, #D-1306).
Alignments of SAR protein sequences
Amino acid sequences were retrieved from non-redundant protein sequences NCBI database using BLASTP 2.2.24+ program and the human SAR sequence (ESE-1 AAs 189-239) as a query (http://blast.ncbi.nlm.nih.gov/, ). For the purpose of calculation of percent identity to human sequence, conservative substitutions and gaps were considered as non-identical amino acids. When there was an insertion, percent identity was calculated with the number of amino acids in the longer protein as the denominator. For the purpose of calculation of percent conservation at a given AA position between human and the remaining species, conservative substitutions and gaps were considered as non-identical amino acids, while insertions were excluded from analysis.
We thank Dr. Heide Ford for critical review of this manuscript, Dr. Melissa Gonzales for providing the pEGFP-ESE-1ΔDBD plasmid, and Tammy Trudeau for help with isolation of mAB405 antibody. DNA sequencing was provided by Core Facility of the University of Colorado Cancer Center (NIH P30 CA 46934). Fluorescence microscopy equipment was provided by the University of Colorado Denver Light Microscopy Facility. Monoclonal antibody production was conducted in collaboration with Protein Production-Moab-Tissue Culture Core of the University of Colorado Cancer Center. This work was supported by DOD DAMD 17-00-1-0474 grant to JDP, NIH T32DK007446-28 support for JMP and by DOD DAMD 17-00-1-0476 grant to AGH.
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