MAP kinase subcellular localization controls both pattern and proliferation in the developingDrosophilawing
Daniel R. MarendaAlysia D. Vrailas‐MortimerAloma B. RodriguesSummer CookMaureen A. PowersJames A. LorenzenLizabeth A. PerkinsKevin Moses
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Abstract:
Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK.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
Importin
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Caspase-3 is a key mediator of apoptosis.During apoptosis,caspase-3 is cleaved and activated by the protease,and enters into the nucleus,resulting in biochemical and morphological changes in apoptosis.However,the molecular mechanism of nucleus-entering of active caspase-3 is still unknown.Identifying the subcellular localization of caspase-3 molecule is important for understanding the process.In this study,we constructed various truncated caspase-3 mutant constructs that were fused with GFP,observed the cellular localization of these mutant proteins,and determined the subcellular localization of caspase-3 molecule.The results show that caspase-3 lacks obvious nuclear localization signal(NLS),but bears an obvious nuclear export signal(NES),which is located at the C-terminus of its small subunit including residues 220～245.
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Caspase-9
Caspase 2
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NLS
Nuclear export signal
Importin
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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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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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Importin
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We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway.
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Vpr is a conserved HIV-1 auxiliary protein that localizes to the nuclear region of cells. Vpr is also present in virions, and it is directed into the assembling virus when coexpressed with Gag. Each of these two localization activities may be important for Vpr function, and we recently identified regions of Vpr that are critical for virion incorporation. In this study we analyzed the Vpr domains involved in subcellular localization. Immunofluorescence staining of transfected cells showed that wild-type Vpr localized exclusively to the nuclear region. Mutations in the N-terminal domain that were designed to disrupt a predicted alpha-helical structure resulted in aberrant localization, while conservative substitutions showed a wild-type pattern. A region in the central portion of the protein also has the potential for helical structure, and mutagenesis of two conserved amino acids in this domain (A59, H71) impaired localization, while substitution of a third (Q65) did not. In contrast, neither the conserved Gly and Cys at positions 75-76 nor the C-terminal basic residues (R87, K95) were necessary for nuclear localization. In addition, two-residue insertions within and between the two putative helices disrupted localization but insertion in the C-terminal region did not. Thus, Vpr's subcellular localization function depends on the two putative helical domains but is independent of the conserved Gly-Cys motif and of specific C-terminal basic residues.
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C-terminus
Conserved sequence
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Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.
Nuclear export signal
NLS
Splicing factor
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The breast and ovarian cancer susceptibility gene BRCA1 has been recently cloned and revealed an open reading frame of 1863 amino acids, but a lack of significant homology to any known protein in the database has led to few clues about its functions. One of the first steps to investigate the function of BRCA1 was to define its subcellular localization. Several reports have led to contradictory findings that include: nuclear localization in normal cells and cytoplasmic in breast and ovarian cancer cells; nuclear in both normal and cancer cells; cytoplasmic and secreted to the extracellular space; present in tube-like invaginations of the nucleus; and colocalizing with the centrosome. As is apparent, the subcellular localization has been the most controversial aspect of BRCA1 biology and is a key point to uncover its functions. In this paper we review the published data on subcellular localization of BRCA1 with special emphasis on the antibodies and techniques used. We conclude that there is now overwhelming evidence to support a nuclear localization for BRCA1, both in normal and cancer cells. In addition, several BRCA1-interacting proteins have been isolated and they are preferentially located in the nucleus. Evidence supporting a physiological function for BRCA1 during DNA repair and transcriptional activation is also discussed.
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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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Circovirus
Coding region
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