LYRIC/AEG-1 Is Targeted to Different Subcellular Compartments by Ubiquitinylation and Intrinsic Nuclear Localization Signals
Hayley J. ThirkettleJoanne GirlingAnne Y. WarrenIan G. MillsKanagasabai SahadevanHing Y. LeungFreddie C. HamdyHayley C. WhitakerDavid E. Neal
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Abstract:
LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.Keywords:
NLS
Tissue microarray
Immunoprecipitation
Proximity ligation assay
Objective: To structurally analyse and functionally identify the nuclear localization signal(NLS) in BRD7 and then study its effect on the subcellular localization of BRD7. Methods: Bioinformatics was performed to predict and anslyse the nuclear localization signal sequences(NLSs) in BRD7, then green fluorescent protein(GFP) direct fluorescence and indirect immunofluorescence assays were used to identify the function of the NLSs and the effect on the subcellular localization of BRD7. Results: The region from amino acid 65 to 96 in BRD7 was characteristic of putative nuclear localization signal sequence and contained three clusters of base amino acid residues. It was viewed to consist of two bipartite nuclear targeting sequences, NLS1 and NLS2, which were tightly linked and extremely overlapped. It was also shown that both the entire NLS and the two bipartite nuclear targeting sequences, NLS1 and NLS2, could respectively determine the nuclear import of GFP, which supported that the region from aa 65 to 96 in BRD7 was a functional nuclear localization signal and the deletion of a cluster of base residues was insufficient to demolish the function of the NLS. The most important was that wild BRD7 localized in nucleus, whereas NLS-deleted BRD7 shifted the nuclear localization to be mostly in cytoplasm. Conclusion: The amino acid region from 65 to 96 is a functional NLS in BRD7 and it is an essential motif affecting BRD7 nuclear distribution.
NLS
Nuclear export signal
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NLS
Nuclear export signal
Importin
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Abstract During animal development, transcription factor activities are modulated by several means, including subcellular localization. The Hox cofactor Extradenticle (Exd) has a dynamic subcellular localization, such that Exd is cytoplasmic by default, but is nuclear when complexed with another homeodomain protein, Homothorax (Hth). These observations raise the question of whether dimerization with Hth simply induces Exd's nuclear localization or, alternatively, if Hth is also necessary for Exd activity. To address this question, we analyzed the nuclear transport signals in Exd, including a divergent nuclear export signal (NES) and two nuclear localization signals (NLSs). We show that, although these signals are weak compared to canonical signals, they balance each other in Exd. We also provide evidence that Exd contains an NLS mask that contributes to its cytoplasmic localization. With these signals characterized, we generated forms of Exd that are nuclear localized in the absence of Hth. Surprisingly, although these Exd forms are functional, they do not phenocopy Hth overexpression. These findings suggest that Hth is required for Exd activity, not simply for inducing its nuclear localization.
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Nuclear export signal
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LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.
NLS
Tissue microarray
Immunoprecipitation
Proximity ligation assay
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In order to analyse whether a C-terminal polybasic sequence represents a nuclear localization signal (NLS) we obtained several truncated and mutant forms of protein of regerating liver (PRL)-3 and evaluated their subcellular localization as compared to the wild-type form. Our results invalidate the hypothesis that this is an NLS. We also analysed the influence of the C- and N-terminal residues on the phosphatase activity of PRL-3. Our results provide in vitro evidence that the C-terminal CAAX motif, besides directing the protein farnesylation, plays an additional regulatory role by inhibiting the catalytic efficiency of PRL-3. Taking into account the results we obtained, as well as reported data, we propose a hypothetical molecular mechanism for the nucleocytoplasmic localization and transfer of PRL-3.
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To identify nuclear localization signal sequence (NLS) of proline-rich nuclear receptor coregulator protein 1 (PNRC1), vectors expressing green fluorescence protein(GFP)-tagged PNRC1 and GFP-tagged PNRC1 mutant with the putative NLS(aa 94-101, PKKRRKKK) deletion were generated then transfected into mammalian cells. The subcellular localization of PNRC1 and putative NLS deleted PNRC1 were examined by confocal microscope. In addition, recombinants expressing GFP-tagged putative NLS and GFP-tagged cytoplasm protein fused to putative NLS were constructsed, the subcellular localization of these NLS fusion proteins were examined in the transfected cells accordingly. The results demonstrated the native PNRC1 localized to the nucleus as expected, whereas the PNRC1 mutant with the putative NLS deletion primarily localized in the cytoplasm. The putative NLS of PNRC1 was found to be able to drive GFP and other cytoplasm protein into nucleus when it was fused to these proteins. We conclude that the putative NLS within PNRC1 indeed has a potent activity to mediate protein nuclear transportation.
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To construct the NLS(ING1)-GFP vector, transfer it into MRC-5 cells and establish a cell model expressing NLS (ING1)-GFP fusion protein.Firstly, cDNA fragment of nuclear locating sequence (NLS) of inhibitor of growth-1 gene (ING1) was gained by RT-PCR and inserted into multi-clone site of pEGFP-C1 to construct the NLS (ING1)-GFP expression vector. Then the vector was used to transfect the MRC-5 cells to observe the subcellular signal localization of green fluorescence protein (GFP).We successfully constructed the expressing vector of NLS (ING1)-GFP fusion protein. After transferring the fusion expressing vector into MRC-5 cells, we observed that green fluorescence signal located in the cell nucleus. However, the green fluorescence signal located in the cytoplasm in MRC-5 cells transfected with pEGFP-C1 control only expressing GFP.In living cells, physiologically p33 ING1b locates absolutely in nucleus. The p33(ING1b) NLS plays a decisive role in the transporting process of subcellular localization.
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Abstract Background 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. Results 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 ( 242 HGKRRR 247 ) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102 LCNCALEELRL 112 ) and another to the DNA binding domain (DBD), (NES2: 275 LWEFIRDILI 284 ). Moreover, analysis of a putative NLS located in the DBD ( 316 GQKKKNSN 323 ) 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. Conclusions 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.
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Nuclear export signal
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Duck circovirus (DuCV) possess a circular, single-stranded DNA genome that requires the replication protein (Rep) for its replication.Based on the viral genotype, there are two categories of Rep proteins: Rep1 and Rep2.To characterize the nuclear localization signals (NLSs) conferring the nuclear localization of the Rep proteins, defi ned coding regions of the rep gene of two genotypes of DuCV were cloned and co-expressed with the red fl uorescent protein DsRed2.Th e results showed that deleting the putative N-terminal NLS located at amino acid residues 10-37 of Rep1 and Rep2 abrogated nuclear translocation, while deleting the putative C-terminal NLS located at residues 244-274 of Rep1 did not signifi cantly alter its subcellular localization, confi rming that only the NLS located at residues 10-37 in the N-termini of the Rep proteins had nuclear targeting activity.
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Coding region
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