scholarly journals Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis

2003 ◽  
Vol 23 (8) ◽  
pp. 2790-2799 ◽  
Author(s):  
Daniela Barilà ◽  
Alessandra Rufini ◽  
Ivano Condò ◽  
Natascia Ventura ◽  
Karel Dorey ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Death Receptor ◽  
Kinase Domain ◽  
Actin Binding ◽  
Caspase Cleavage ◽  
Nonreceptor Tyrosine Kinase ◽  
Nuclear Localization Signals ◽  
Induced Apoptosis ◽  
Export Signal

ABSTRACT The nonreceptor tyrosine kinase c-Abl may contribute to the regulation of apoptosis. c-Abl activity is induced in the nucleus upon DNA damage, and its activation is required for execution of the apoptotic program. Recently, activation of nuclear c-Abl during death receptor-induced apoptosis has been reported; however, the mechanism remains largely obscure. Here we show that c-Abl is cleaved by caspases during tumor necrosis factor- and Fas receptor-induced apoptosis. Cleavage at the very C-terminal region of c-Abl occurs mainly in the cytoplasmic compartment and generates a 120-kDa fragment that lacks the nuclear export signal and the actin-binding region but retains the intact kinase domain, the three nuclear localization signals, and the DNA-binding domain. Upon caspase cleavage, the 120-kDa fragment accumulates in the nucleus. Transient-transfection experiments show that cleavage of c-Abl may affect the efficiency of Fas-induced cell death. These data reveal a novel mechanism by which caspases can recruit c-Abl to the nuclear compartment and to the mammalian apoptotic program.

A functional study of nucleocytoplasmic transport signals of the EhNCABP166 protein from Entamoeba histolytica

Parasitology ◽  
2012 ◽  
Vol 139 (13) ◽  
pp. 1697-1710 ◽  
Author(s):  
R. URIBE ◽  
J. ALMARAZ BARRERA MA DE ◽  
M. ROBLES-FLORES ◽  
G. MENDOZA HERNÁNDEZ ◽  
A. GONZÁLEZ-ROBLES ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Domain ◽  
Nucleocytoplasmic Transport ◽  
Actin Binding ◽  
Functional Study ◽  
Nuclear Localization Signals ◽  
Export Signal

SUMMARYEhNCABP166 is an Entamoeba histolytica actin-binding protein that localizes to the nucleus and cytoplasm. Bioinformatic analysis of the EhNCABP166 amino acid sequence shows the presence of 3 bipartite nuclear localization signals (NLS) and a nuclear export signal (NES). The present study aimed to investigate the functionality of these signals in 3 ways. First, we fused each potential NLS to a cytoplasmic domain of ehFLN to determine whether the localization of this domain could be altered by the presence of the NLSs. Furthermore, the localization of each domain of EhNCABP166 was determined. Similarly, we generated mutations in the first block of bipartite signals from the domains that contained these signals. Additionally, we added an NES to 2 constructs that were then evaluated. We confirmed the intranuclear localization of EhNCABP166 using transmission electron microscopy. Fusion of each NLS resulted in shuttling of the cytoplasmic domain to the nucleus. With the exception of 2 domains, all of the evaluated domains localized within the nucleus. A mutation in the first block of bipartite signals affected the localization of the domains containing an NLS. The addition of an NES shifted the localization of these domains to the cytoplasm. The results presented here establish EhNCABP166 as a protein containing functional nuclear localization signals and a nuclear export signal.

scholarly journals OsWRKY62 and OsWRKY76 interact with Importin α1s for Negative Regulation of Defensive Responses in Nucleus

2021 ◽  
Author(s):  
Xiaohui Xu ◽  
Han Wang ◽  
Jiqin Liu ◽  
Shuying Han ◽  
Miaomiao Lin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Defense Responses ◽  
Nuclear Localization Signals ◽  
Defensive Responses ◽  
Export Signal ◽  
Positively Charged

Abstract Background: OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation?Results: In this study, we characterized they interacted with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαDIBB1a lacking the auto-inhibitory importin β-binding domain. OsIMαDIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal. Similarly, we found that OsIMαDIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1 with an extra nuclear export signal compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae.Conclusion: These results indicated that OsWRKY62 localization is a consequence of competition binding between rice importins and exportins. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

scholarly journals Novel Nuclear Export Signal-Interacting Protein, NESI, Critical for the Assembly of Hepatitis Delta Virus

2005 ◽  
Vol 79 (13) ◽  
pp. 8113-8120 ◽  
Author(s):  
Yun-Hsin Wang ◽  
Shin C. Chang ◽  
Cheng Huang ◽  
Ya-Ping Li ◽  
Chia-Huei Lee ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Hepatitis Delta Virus ◽  
Critical Role ◽  
Specific Interaction ◽  
Nuclear Export Signal ◽  
Actin Binding ◽  
Hepatitis Delta ◽  
Interacting Protein ◽  
Export Signal ◽  
Delta Virus

ABSTRACT The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.

scholarly journals Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins

2003 ◽  
Vol 373 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Jose A. RODRIGUEZ ◽  
Simone W. SPAN ◽  
Frank A. E. KRUYT ◽  
Giuseppe GIACCONE
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Cowpox Virus ◽  
Amino Acid Region ◽  
Localization Signal ◽  
Induced Apoptosis ◽  
Export Signal

The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments.

scholarly journals Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1

Blood ◽  
2011 ◽  
Vol 118 (11) ◽  
pp. 3096-3106 ◽  
Author(s):  
Ramesh Balusu ◽  
Warren Fiskus ◽  
Rekha Rao ◽  
Daniel G. Chong ◽  
Srilatha Nalluri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Chaperone ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
S Phase ◽  
Wild Type ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis ◽  
Export Signal

Abstract Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

scholarly journals An Evolutionarily Conserved Nuclear Export Signal Facilitates Cytoplasmic Localization of the Tbx5 Transcription Factor

2007 ◽  
Vol 28 (5) ◽  
pp. 1553-1564 ◽  
Author(s):  
Andre Kulisz ◽  
Hans-Georg Simon
Keyword(s):  
Transcription Factor ◽  
Nuclear Export ◽  
Cardiac Development ◽  
Nuclear Export Signal ◽  
Direct Interaction ◽  
Site Directed Mutagenesis ◽  
Nuclear Localization Signals ◽  
T Box ◽  
Evolutionarily Conserved ◽  
Export Signal

ABSTRACT During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.

scholarly journals Sox10 Is an Active Nucleocytoplasmic Shuttle Protein, and Shuttling Is Crucial for Sox10-Mediated Transactivation

2002 ◽  
Vol 22 (16) ◽  
pp. 5826-5834 ◽  
Author(s):  
Stephan Rehberg ◽  
Peter Lischka ◽  
Gabi Glaser ◽  
Thomas Stamminger ◽  
Michael Wegner ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Export ◽  
Target Genes ◽  
Nuclear Export Signal ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Nucleocytoplasmic Shuttling ◽  
Nucleocytoplasmic Shuttle ◽  
Nuclear Localization Signals ◽  
Export Signal

ABSTRACT Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence

1998 ◽  
Vol 111 (21) ◽  
pp. 3129-3143 ◽  
Author(s):  
R.L. Shirley ◽  
M.J. Lelivelt ◽  
L.R. Schenkman ◽  
J.N. Dahlseid ◽  
M.R. Culbertson
Keyword(s):  
Protein Function ◽  
Nuclear Export ◽  
Mrna Decay ◽  
Signal Sequence ◽  
Nuclear Export Signal ◽  
Reporter Protein ◽  
Nuclear Localization Signals ◽  
Sequence Elements ◽  
Nonsense Mediated Mrna Decay ◽  
Export Signal

In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd- phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd-. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd- phenotype even when export is restored.

scholarly journals Determination of the Functional Domain Organization of the Importin α Nuclear Import Factor

1998 ◽  
Vol 143 (2) ◽  
pp. 309-318 ◽  
Author(s):  
Andrea Herold ◽  
Ray Truant ◽  
Heather Wiegand ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
T Antigen ◽  
Functional Domain ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Export Signal

Although importin α (Imp α) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin β nuclear import factor, little is known about the functional domains present in Imp α, with the exception that importin β binding is known to map close to the Imp α NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp α COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp α nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp α nuclear export. Analysis of sequences within Imp α that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp α. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp α may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp α with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

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