scholarly journals Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own

Development ◽  
1999 ◽  
Vol 126 (6) ◽  
pp. 1327-1335 ◽  
Author(s):  
R.T. Cox ◽  
L.M. Pai ◽  
J.R. Miller ◽  
S. Orsulic ◽  
J. Stein ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Current Model ◽  
Intracellular Localization ◽  
Beta Catenin ◽  
Wild Type ◽  
Binding Partners ◽  
Mutant Forms ◽  
Wnt Signal

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.

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 Mutational Analysis of Bovine Leukemia Virus Rex: Identification of a Dominant-Negative Inhibitor

2005 ◽  
Vol 79 (11) ◽  
pp. 7172-7181 ◽  
Author(s):  
Eun-A Choi ◽  
Thomas J. Hope
Keyword(s):  
Nuclear Localization ◽  
Bovine Leukemia Virus ◽  
Nuclear Export ◽  
Protein Localization ◽  
Leukemia Virus ◽  
Nuclear Export Signal ◽  
Dominant Negative ◽  
Nuclear Pore ◽  
Large Animal ◽  
Wild Type

ABSTRACT The Rex proteins of the delta-retroviruses act to facilitate the export of intron-containing viral RNAs. The Rex of bovine leukemia virus (BLV) is poorly characterized. To gain a better understanding of BLV Rex, we generated a reporter assay to measure BLV Rex function and used it to screen a series of point and deletion mutations. Using this approach, we were able to identify the nuclear export signal of BLV Rex. Further, we identified a dominant-negative form of BLV Rex. Protein localization analysis revealed that wild-type BLV Rex had a punctate nuclear localization and was associated with nuclear pores. In contrast, the dominant-negative BLV Rex mutation had a diffuse nuclear localization and no nuclear pore association. Overexpression of the dominant-negative BLV Rex altered the localization of the wild-type protein. This dominant-negative derivative of BLV Rex could be a useful tool to test the concept of intracellular immunization against viral infection in a large animal model.

scholarly journals Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

1998 ◽  
Vol 18 (10) ◽  
pp. 5952-5960 ◽  
Author(s):  
Dieter Näf ◽  
Gary M. Kupfer ◽  
Ahmed Suliman ◽  
Kathleen Lambert ◽  
Alan D. D’Andrea
Keyword(s):  
Nuclear Localization ◽  
Fanconi Anemia ◽  
Functional Activity ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Amino Terminal ◽  
Complementation Groups ◽  
Mutant Forms

ABSTRACT Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.

Myogenesis in Wilms' Tumors is Associated with Mutations of the WT1 Gene and Activation of Bcl-2 and the Wnt Signaling Pathway

2004 ◽  
Vol 7 (2) ◽  
pp. 125-137 ◽  
Author(s):  
Ryuji Fukuzawa ◽  
Rosemary W. Heathcott ◽  
Makoto Sano ◽  
Ian M. Morison ◽  
Kankatsu Yun ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Cytoplasmic Membrane ◽  
Wnt Signaling Pathway ◽  
Nuclear Accumulation ◽  
Beta Catenin ◽  
Wild Type ◽  
Molecular Events ◽  
Epithelial Development

Wilms tumors with WT1 mutations [ WT1(—)] have a stromal-predominant histology with varying extents of rhabdomyogenesis. These tumors also frequently have mutations in the beta-catenin gene (CTNNB1). We have investigated the molecular events that may explain the origins of rhabdomyogenesis in WT1(-) tumors. Of 35 Wilms tumors, we identified 12 with WT1 mutations, of which 9 carried CTNNB1 mutations. We compared WT1 wild-type tumors [ WT1(+)] with WT1(-) tumors for histological features, localization of beta-catenin, Bcl-2 expression, and apoptosis using an in-situ end-labeling technique. WT1(+) tumors showed triphasic and blast-emal- and epithelial predominant-histology. Expression of WT1, beta-catenin, and Bcl-2 recapitulated those of normal kidney epithelial development. Localization of beta-catenin was observed in the cytoplasm and cytoplasmic membrane of early glomerular epithelial structures. Bcl-2 is also expressed in condensing blastema and early glomerular epithelial structures which had little apoptosis. WT1(-) tumors, regardless of whether CTNNB1 mutations were detected or not, showed a stromal-rich phenotype with abundant expression of beta-catenin in the nucleus of the rhabdomyoblasts. Bcl-2 was expressed in rhabdomyoblasts, but not in blastemal cells undergoing apoptosis, suggesting that WT1 regulates Bcl-2 positively in the epithelial pathway, but negatively in the myogenic pathway. These data indicate that mutations in WT1 might alter the Wnt signaling pathway and Bcl-2 related-apoptosis. In WT1(-) tumors, the nuclear accumulation of beta-catenin and Bcl-2 expression are associated with rhabdomyogenesis, and dysregulation of Bcl-2 may be a mechanism by which the histogenesis (loss of blastemal component, muscle differentiation) may be explained.

scholarly journals A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Mohamed Hamed ◽  
Birgit Caspar ◽  
Sarah A. Port ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Wild Type ◽  
Leptomycin B ◽  
Nuclear Export Sequence ◽  
Binding Partners ◽  
Dependent Protein ◽  
Nuclear Export Receptor ◽  
Cytoplasmic Side

ABSTRACT Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

Plakoglobin and beta-catenin: protein interactions, regulation and biological roles

2000 ◽  
Vol 113 (18) ◽  
pp. 3127-3139 ◽  
Author(s):  
J. Zhurinsky ◽  
M. Shtutman ◽  
A. Ben-Ze'ev
Keyword(s):  
Wnt Signaling ◽  
Cancer Progression ◽  
Protein Interactions ◽  
Intramolecular Interactions ◽  
Beta Catenin ◽  
Functional Difference ◽  
Structural Protein ◽  
Unique Role ◽  
Protein Partners ◽  
Wnt Signal

Beta-catenin can play different roles in the cell, including one as a structural protein at cell-cell adherens junctions and another as a transcriptional activator mediating Wnt signal transduction. Plakoglobin (gamma)-catenin), a close homolog of beta-catenin, shares with beta-catenin common protein partners and can fulfill some of the same functions. The complexing of catenins with various protein partners is regulated by phosphorylation and by intramolecular interactions. The competition between different catenin partners for binding to catenins mediates the cross-talk between cadherin-based adhesion, catenin-dependent transcription and Wnt signaling. Although plakoglobin differs from beta-catenin in its functions and is unable to compensate for defects in Wnt signaling resulting from lack of beta-catenin, recent evidence suggests that plakoglobin plays a unique role in Wnt signaling that is different from that of beta-catenin. The functional difference between catenins is reflected in their differential involvement in embryonic development and cancer progression.

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

2001 ◽  
Vol 358 (1) ◽  
pp. 269-274 ◽  
Author(s):  
Per NØRGAARD ◽  
Jakob R. WINTHER
Keyword(s):  
Active Sites ◽  
Current Model ◽  
Secretory Proteins ◽  
Main Function ◽  
Wild Type ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Oxidative Refolding ◽  
Mutant Forms

Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

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