scholarly journals A Novel Heterodimerization Domain, CRM1, and 14-3-3 Control Subcellular Localization of the MondoA-Mlx Heterocomplex

2002 ◽  
Vol 22 (24) ◽  
pp. 8514-8526 ◽  
Author(s):  
Alanna L. Eilers ◽  
Eleanor Sundwall ◽  
Monica Lin ◽  
April A. Sullivan ◽  
Donald E. Ayer
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
N Terminus ◽  
Dimerization Interface

ABSTRACT Among members of the bHLHZip family of transcriptional regulators, MondoA and Mlx have the unique property of cytoplasmic localization. We have proposed that MondoA-Mlx heterodimers accumulate in the nucleus in response to extracellular cues. Our previous work implicated heterodimerization between MondoA and Mlx and a conserved domain in the N terminus of MondoA as important determinants of MondoA-Mlx subcellular localization. MondoA and Mlx share sequence similarity in their bHLHZip domains and C termini. Here we show that for both MondoA and Mlx, this C-terminal domain has cytoplasmic localization activity that is required by the protein monomers to accumulate in the cytoplasm. This C-terminal domain is also a novel dimerization interface that functions independently of the leucine zipper to mediate heterotypic interactions between MondoA and Mlx. Dimerization between MondoA and Mlx inactivates the cytoplasmic localization activity of their C termini and is necessary for the heterocomplex to accumulate in the nucleus. MondoA-Mlx heterodimers, while poised for nuclear entry, are retained in the cytoplasm by conserved domains in the N terminus of MondoA. Mondo conserved regions (MCRs) II and III contribute to cytoplasmic localization of MondoA-Mlx by functioning as a CRM1-dependent nuclear export signal and as a novel binding site for 14-3-3 family members, respectively. We propose that the nuclear accumulation of MondoA and Mlx is a two-step process. First, heterodimerization abolishes the cytoplasmic localization activity of their C termini. Second, an extracellular signal(s) must overcome the cytoplasmic localization function imparted by CRM1 and 14-3-3 binding to the N terminus of MondoA.

scholarly journals Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

1999 ◽  
Vol 19 (10) ◽  
pp. 7088-7095 ◽  
Author(s):  
Edward W. Harhaj ◽  
Shao-Cong Sun
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Nuclear Factor Κb ◽  
Nuclear Accumulation ◽  
Physical Interaction ◽  
Cytoplasmic Localization ◽  
Heterologous Proteins ◽  
Export Signal

ABSTRACT Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-κB exhibits strong cytoplasmic localization activity even in the absence of IκBα inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-κB.

scholarly journals MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

2000 ◽  
Vol 20 (23) ◽  
pp. 8845-8854 ◽  
Author(s):  
Andrew N. Billin ◽  
Alanna L. Eilers ◽  
Kathryn L. Coulter ◽  
Jennifer S. Logan ◽  
Donald E. Ayer
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Functional Characterization ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

scholarly journals Visualization of Myc/Max/Mad Family Dimers and the Competition for Dimerization in Living Cells

2004 ◽  
Vol 24 (10) ◽  
pp. 4294-4308 ◽  
Author(s):  
Asya V. Grinberg ◽  
Chang-Deng Hu ◽  
Tom K. Kerppola
Keyword(s):  
Nuclear Export ◽  
Leucine Zipper ◽  
Nuclear Export Signal ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Cytoplasmic Localization ◽  
Amino Acid Residues ◽  
Helix Loop Helix ◽  
Cell Growth And Proliferation ◽  
Export Signal

ABSTRACT Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

2004 ◽  
Vol 36 (12) ◽  
pp. 817-823 ◽  
Author(s):  
Qing-Ming Wang ◽  
Guo-Cai Fan ◽  
Ji-Zhong Chen ◽  
Hui-Peng Chen ◽  
Fu-Chu He
Keyword(s):  
Tumor Cells ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chicken Anemia Virus ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Normal Cells ◽  
Localization Signal ◽  
Anemia Virus ◽  
Export Signal

Abstract Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

scholarly journals A Nuclear Export Signal and Phosphorylation Regulate Dok1 Subcellular Localization and Functions

2006 ◽  
Vol 26 (11) ◽  
pp. 4288-4301 ◽  
Author(s):  
Yamei Niu ◽  
François Roy ◽  
Frédéric Saltel ◽  
Charlotte Andrieu-Soler ◽  
Wen Dong ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Nuclear Accumulation ◽  
Serum Starvation ◽  
Cytoplasmic Localization ◽  
Export Signal

ABSTRACT Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKβ. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

scholarly journals Characterization of localization and export signals of bovine torovirus nucleocapsid protein responsible for extensive nuclear and nucleolar accumulation and their importance for virus growth

2020 ◽  
Author(s):  
Makoto Ujike ◽  
Yukako Kawachi ◽  
Yui Matsunaga ◽  
Yuka Etho ◽  
Hideki Asanuma ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Rna Viruses ◽  
Nuclear Export Signal ◽  
Economic Loss ◽  
Nuclear Accumulation ◽  
Structural Protein ◽  
Nucleocytoplasmic Trafficking ◽  
Virus Growth ◽  
N Proteins

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N-proteins, but little is known about ToV N-proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N-protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToN fused with EGFP revealed that clusters of arginine (R) residues comprise nuclear/nucleolar localization signals (NLS/NoLS), and the C-terminal region served as a chromosomal maintenance 1 (CRM1)-independent nuclear export signal (NES). Moreover, recombinant viruses with mutations in the NLS/NoLS, but retaining nuclear accumulation, were successfully rescued and showed slightly reduced growth ability, while the virus that lost the NLS/NoLS-mediated nuclear accumulation of BToN was not rescued. These results indicate that BToN uniquely accumulates mainly in nuclear compartments during infection, regulated by an R-rich NLS/NoLS and a CRM1-independent NES, and that the BToN-accumulation in the nuclear compartment driven by NLS/NoLS is important for virus growth. IMPORTANCE ToVs are diarrhea-causing pathogens detected in many species, including humans. BToV has spread worldwide, leading to economic loss, and there is currently no treatment or vaccine available. Positive-stranded RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN predominantly accumulated in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. Understanding its unique features, BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.

scholarly journals Regulation of the Nucleocytoplasmic Distribution of Snf1-Gal83 Protein Kinase

2006 ◽  
Vol 5 (12) ◽  
pp. 1950-1956 ◽  
Author(s):  
Kristina Hedbacker ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Glucose Limitation ◽  
C Terminus ◽  
N Terminus ◽  
Glucose Phosphorylation ◽  
Nucleocytoplasmic Distribution ◽  
Necessary And Sufficient

ABSTRACT Snf1 protein kinase containing the β subunit Gal83 is localized in the cytoplasm during growth of Saccharomyces cerevisiae cells in abundant glucose and accumulates in the nucleus in response to glucose limitation. Nuclear localization of Snf1-Gal83 requires activation of the Snf1 catalytic subunit and depends on Gal83, but in the snf1Δ mutant, Gal83 exhibits glucose-regulated nuclear accumulation. We show here that the N terminus of Gal83, which is divergent from those of the other β subunits, is necessary and sufficient for Snf1-independent, glucose-regulated localization. We identify a leucine-rich nuclear export signal in the N terminus and show that export depends on the Crm1 export receptor. We present evidence that catalytically inactive Snf1 promotes the cytoplasmic retention of Gal83 in glucose-grown cells through its interaction with the C terminus of Gal83; cytoplasmic localization of inactive Snf1-Gal83 maintains accessibility to the Snf1-activating kinases. Finally, we characterize the effects of glucose phosphorylation on localization. These studies define roles for Snf1 and Gal83 in determining the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase.

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

scholarly journals Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Xiaojuan Li ◽  
Fanxiu Zhu
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Kaposi's Sarcoma ◽  
Nuclear Export Signal ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

scholarly journals Distinct Subcellular Localization Patterns Contribute to Functional Specificity of the Cln2 and Cln3 Cyclins of Saccharomyces cerevisiae

2000 ◽  
Vol 20 (2) ◽  
pp. 542-555 ◽  
Author(s):  
Mary E. Miller ◽  
Frederick R. Cross
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Cytoplasmic Localization ◽  
Cyclin Dependent Kinase ◽  
Functional Profile ◽  
Functional Specificity ◽  
Biochemical Fractionation ◽  
Nuclear Events

ABSTRACT The G1 cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G1 cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.

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