Potential Nuclear Export Signals Research Articles

The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation.

Inactivation of DNA mismatch repair propels colorectal cancer (CRC) tumorigenesis. CRCs exhibiting elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) show reduced nuclear MutS homolog 3 (MSH3) expression with surrounding inflammation and portend poor patient outcomes. MSH3 reversibly exits from the nucleus to the cytosol in response to the proinflammatory cytokine interleukin-6 (IL-6), suggesting that MSH3 may be a shuttling protein. In this study, we manipulated three putative nuclear localization (NLS1 to -3) and two potential nuclear export signals (NES1 and -2) within MSH3. We found that both NLS1 and NLS2 possess nuclear import function, with NLS1 responsible for nuclear localization within full-length MSH3. We also found that NES1 and NES2 work synergistically to maximize nuclear export, with both being required for IL-6-induced MSH3 export. We examined a 27-bp deletion (Δ27bp) within the polymorphic exon 1 that occurs frequently in human CRC cells and neighbors NLS1. With oxidative stress, MSH3 with this deletion (Δ27bp MSH3) localizes to the cytoplasm, suggesting that NLS1 function in Δ27bp MSH3 is compromised. Overall, MSH3's shuttling in response to inflammation enables accumulation in the cytoplasm; reduced nuclear MSH3 increases EMAST and DNA damage. We suggest that polymorphic sequences adjacent to NLS1 may enhance cytosolic retention, which has clinical implications for inflammation-associated neoplastic processes.

Amino Acid Exchanges in the Putative Nuclear Export Signal of Adenovirus Type 5 L4-100K Severely Reduce Viral Progeny due to Effects on Hexon Biogenesis

The adenovirus type 5 nonstructural L4-100K protein is indispensable for efficient lytic infection. During the late phase, L4-100K promotes selective translation of viral late transcripts and mediates the trimerization of the major capsid protein hexon. In the present study, the role of a potential nuclear export signal in L4-100K was investigated. Intriguingly, amino acid substitutions in this sequence resulted in severely diminished progeny virus production, seemingly by precluding proper hexon biogenesis.

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP 2 reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP 2 indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.

Nucleocytoplasmic shuttling of the HSV-2 serine/threonine kinase Us3

The alphaherpesvirus serine/threonine kinase Us3 plays diverse roles in virus multiplication and modifies both nuclear and cytoplasmic substrates. We recently reported that treatment of HSV-2 Us3-transfected and HSV-2-infected cells with leptomycin B, an inhibitor of nuclear export mediated by interaction of chromosomal regional maintenance protein (CRM1) with leucine rich nuclear export signals (NESs), resulted in nuclear trapping of Us3. Here, we utilized fluorescence loss in photobleaching to monitor nuclear export of HSV-2 Us3 and confirm that this process proceeds solely via a CRM1-mediated mechanism. Analysis of deletion derivatives of HSV-2 Us3 fused to a nuclear export reporter protein implicated the involvement of NES-like sequences in nuclear export. However, nuclear trapping of HSV-2 Us3 proteins carrying mutations in these potential NESs was not observed, indicating that these sequences are not functional in the context of full-length protein. Our analyses also revealed previously unidentified regions of HSV-2 Us3 that contribute to its kinase activity.

Nucleocytoplasmic Shuttling of Dysbindin-1, a Schizophrenia-related Protein, Regulates Synapsin I Expression

Dysbindin-1 is a 50-kDa coiled-coil-containing protein encoded by the gene DTNBP1 (dystrobrevin-binding protein 1), a candidate genetic factor for schizophrenia. Genetic variations in this gene confer a susceptibility to schizophrenia through a decreased expression of dysbindin-1. It was reported that dysbindin-1 regulates the expression of presynaptic proteins and the release of neurotransmitters. However, the precise functions of dysbindin-1 are largely unknown. Here, we show that dysbindin-1 is a novel nucleocytoplasmic shuttling protein and translocated to the nucleus upon treatment with leptomycin B, an inhibitor of exportin-1/CRM1-mediated nuclear export. Dysbindin-1 harbors a functional nuclear export signal necessary for its nuclear export, and the nucleocytoplasmic shuttling of dysbindin-1 affects its regulation of synapsin I expression. In brains of sandy mice, a dysbindin-1-null strain that displays abnormal behaviors related to schizophrenia, the protein and mRNA levels of synapsin I are decreased. These findings demonstrate that the nucleocytoplasmic shuttling of dysbindin-1 regulates synapsin I expression and thus may be involved in the pathogenesis of schizophrenia.

Regulation of nucleocytoplasmic localization of the atDjC6 chaperone protein

The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A-C) and three potential nuclear export signal (NES) sequences (X-Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-alpha. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-alpha. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.

Identification of functional nuclear export sequences in human topoisomerase IIα and β

Nuclear localization of topoisomerase IIα and β is important for normal cell function as well as being a determinant of tumour cell sensitivity to topoisomerase II-targeting chemotherapeutic agents. However, topoisomerase II is cytoplasmic under certain circumstances, indicating that it may undergo active nuclear export. We have examined the ability of Leu-rich potential nuclear export signal (NES) sequences present in human topoisomerase IIα and β to direct the export of a green fluorescent protein–glutathione- S-transferase fusion protein following microinjection into HeLa cell nuclei. Of 12 sequences tested, only one potential NES sequence from the comparable location in each isoform (αNES 1018–1028 and βNES 1034–1044) was active. Mutation of hydrophobic residues in αNES 1018–1028 and βNES 1034–1044 substantially reduced their nuclear export activity as did leptomycin B treatment of microinjected cells. Our results provide the first evidence of active nuclear export of topoisomerase II and suggest it is mediated by a CRM1-dependent pathway.

Molecular and phylogenetic characterization of Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled

Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP ( Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.

Control of STAT Localization

Signal transducer and activator of transcription (STAT) proteins become tyrosine phosphorylated and dimerize before transport to the nucleus, where they activate the transcription of specific genes. What signals are required for STAT localization into the nucleus, however, has remained a mystery. Two reports provide some insight into this process. Begitt et al . showed that interferon-γ (IFN-γ)-stimulated cells treated with the nuclear export inhibitor leptomycin B (LMB) had reduced cytoplasmic levels of STAT1, suggesting that a nuclear export signal (NES) might exist in STAT1. Potential NES motifs were examined, and a functional motif (as measured by fluorescence assays) was identifed at residues 302 to 314, within the fourth helix of the STAT1 coiled-coil domain. Disruption of the NES, in a STAT1 mutant, strongly reduced cytoplasmic accumulation of mutant STAT1 in response to IFNγ treatment. Nuclear accumulation of STAT1 did not result in increased transcription of a STAT1-responsive reporter gene, implying that the transcription-enhancing integrity of accumulated STAT1 was diminished. Although it was not proven, it was suggested that the weakened transcriptional activity may be due to dephosphorylation of Tyr 701 . Thus, after Tyr 701 phosphorylation and dimerization, STAT1 translocates to the nucleus to affect gene transcription, STAT1 becomes dephosphorylated, and an intact NES shuttles STAT1 back to the cytoplasm. Mowen and David also identify a NES motif in STAT1; however, it is located at residues 197 to 205. Nuclear translocation of STAT1 depended on the presence and kinase activity of Jak1. However, even when the EGF receptor phosphorylated STAT1, Jak1 was still necessary for the nuclear accumulation of STAT1. LMB-treated Jak1 -/- cells accumulated nuclear STAT1, suggesting that Jak1's role in STAT1 nuclear accumulation is most likely due to nuclear retention (and blocking export) rather than translocation to the nucleus. One caveat exists, that mutation of possible NES motifs may cryptically activate nonphysiological NESs. Mowen, K., and David, M. (2000) Regulation of STAT1 nuclear export by Jak1. Mol . Cell . Biol . 20 : 7273-7281. [Abstract] [Full Text] Begitt, A., Meyer, T., van Rossum, M., and Vinkemeier, U. (2000) Nucleocytoplasmic translocation of Stat is regulated by a leucine-rich export signal in the coiled-coil domain. Proc . Natl . Acad . Sci . USA 97 : 10418-10423. [Abstract] [Full Text]