Showing papers on "Importin published in 2017"
TL;DR: A model whereby RAPGEF5 activates the nuclear GTPases, Rap1a/b, to facilitate the nuclear transport of β-catenin, defining a parallel nuclear transport pathway to Ran is proposed and suggested new targets for modulating Wnt signaling in disease states are suggested.
64 citations
TL;DR: The first nonpeptide moiety that recruits intracellular transport machinery for nuclear targeting is reported, and it is reported that proteins too large to passively diffuse through nuclear pores were readily imported into the nucleus through this boronate-mediated pathway.
Abstract: Active intracellular transport is a central mechanism in cell biology, directed by a limited set of naturally occurring signaling peptides. Here, we report the first nonpeptide moiety that recruits intracellular transport machinery for nuclear targeting. Proteins synthetically modified with a simple aromatic boronate motif are actively trafficked to the nucleus via the importin α/β pathway. Significantly, proteins too large to passively diffuse through nuclear pores were readily imported into the nucleus through this boronate-mediated pathway. The use of this simple motif to provide active intracellular targeting provides a promising strategy for directing subcellular localization for therapeutic and fundamental applications.
55 citations
TL;DR: It is reported that nutritional stress induces nuclear translocation of ACSS2 (acyl-CoA synthetase short-chain family member 2) and a novel biologic role is revealed in recycling of nuclear acetate for histone acetylation to promote lysosomal and autophagy-related gene expression and counteract nutritional stress.
Abstract: Overcoming metabolic stress is a critical step in tumorigenesis. Acetyl coenzyme A (acetyl-CoA) converted from glucose or acetate is a substrate used for histone acetylation to regulate gene expression. However, how acetyl-CoA is produced under nutritional stress conditions is unclear. Herein we report that nutritional stress induces nuclear translocation of ACSS2 (acyl-CoA synthetase short-chain family member 2). This translocation is mediated by AMP-activated protein kinase (AMPK)-dependent ACSS2 Ser659 phosphorylation and subsequent exposure of the nuclear localization signal of ACSS2 to KPNA1/importin α5 for binding. In the nucleus, ACSS2 forms a complex with TFEB (transcription factor EB) and utilizes the acetate generated from histone deacetylation to locally produce acetyl-CoA for histone acetylation in the promoter regions of TFEB target genes. Knock-in of nuclear translocation-deficient or inactive ACSS2 mutants in glioblastoma cells abrogates glucose deprivation-induced lysosomal biogene...
43 citations
TL;DR: One of the unique PPE family member proteins of M. tuberculosis, PPE2, can limit nitric oxide production by inhibiting inos gene transcription and has a leucine zipper DNA-binding motif and a functional nuclear localization signal.
Abstract: Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is one of the most successful pathogens of humans. It has evolved several adaptive skills and evasion mechanisms to hijack the immunologically educated host to suit its intracellular lifestyle. Here, we show that one of the unique PPE family member proteins of M. tuberculosis, PPE2, can limit nitric oxide (NO) production by inhibiting inos gene transcription. PPE2 protein has a leucine zipper DNA-binding motif and a functional nuclear localization signal. PPE2 was translocated into the macrophage nucleus via the classical importin α/β pathway where it interacted with a GATA-binding site overlapping with the TATA box of inos promoter and inhibited NO production. PPE2 prolonged intracellular survival of a surrogate bacterium M. smegmatis in vitro as well as in vivo. This information are likely to improve our knowledge of host-pathogen interactions during M. tuberculosis infection which is crucial for designing effective anti-TB therapeutics.
41 citations
TL;DR: It is proposed importin α3 evolved to recognize topologically complex NLSs that lie next to bulky domains or are masked by quaternary structures, which is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation.
Abstract: Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3. This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Here we identify the mechanisms of importin α3 selectivity for RCC1. We find this isoform binds RCC1 with one order of magnitude higher affinity than the generic importin α1, although the two isoforms share an identical NLS-binding groove. Importin α3 uses its greater conformational flexibility to wedge the RCC1 β-propeller flanking the NLS against its lateral surface, preventing steric clashes with its Armadillo-core. Removing the β-propeller, or inserting a linker between NLS and β-propeller, disrupts specificity for importin α3, demonstrating the structural context rather than NLS sequence determines selectivity for isoform 3. We propose importin α3 evolved to recognize topologically complex NLSs that lie next to bulky domains or are masked by quaternary structures. Importin α3 facilitates the nuclear transport of the Ran guanine nucleotide exchange factor RCC1. Here the authors reveal the molecular basis for the selectivity of RCC1 for importin α3 vs the generic importin α1 and discuss the evolution of importin α isoforms.
40 citations
TL;DR: Experiments conducted in the presence of inhibitors of the classicalnuclear import pathway indicated that due to overexpression of importin &agr;, classical nuclear import in glioma is impaired leading to aberrant NP intracellular trafficking and nuclear import.
38 citations
TL;DR: This work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta and speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.
Abstract: Summary
Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.
35 citations
TL;DR: Mechanistic evidence of how Importin-&agr;/-&bgr; regulates the NuMA functioning required for assembly of higher-order microtubule structures is provided, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.
Abstract: Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-β and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-β-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-β inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-β on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-β regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.
31 citations
TL;DR: New tools for evaluating the host cell response to RV infections in real time are provided and it is suggested that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.
Abstract: The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/β, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes.
30 citations
TL;DR: The data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF‐κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad‐spectrum antiviral therapies.
Abstract: In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon-β (IFNB1) induction. Using a comprehensive microscopy-based high-content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A-interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF-κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown-a hub protein highly targeted by several viruses-decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus-mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF-κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad-spectrum antiviral therapies.
26 citations
TL;DR: Several recent studies are highlighted which suggest novel roles of small GTPases, importins and sorting nexin proteins in the nuclear translocation of GPCRs via vesicular transport pathways.
Abstract: The G-protein coupled receptor (GPCR) signaling was long believed to involve activation of receptor exclusively at the cell surface, followed by its binding to heterotrimeric G-proteins and arrestins to trigger various intracellular signaling cascades, and termination of signaling by internalization of the receptor. It is now accepted that many GPCRs continue to signal after internalization in the endosomes. Since the breakthrough discoveries of nuclear binding sites for their ligands in 1980s, several GPCRs have been detected at cell nuclei. But mechanisms of nuclear localization of GPCRs, many of whom contain putative nuclear localization signals, remain poorly understood to date. Nevertheless, it is known that subcellular trafficking of GPCRs is regulated by members of Ras superfamily of small GTPases, most notably by Rab and Arf GTPases. In this commentary, we highlight several recent studies which suggest novel roles of small GTPases, importins and sorting nexin proteins in the nuclear translocation of GPCRs via vesicular transport pathways. Taken together with increasing evidence for in vivo functionality of the nuclear GPCRs, better understanding of their trafficking will provide valuable clues in cell biology.
TL;DR: The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP4K2 in addition to its reported cytosolic functions, and supports the notion of a nuclear phosphoinositide system in plants.
Abstract: The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence-tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha-importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa-d) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha-importin isoforms in cytosolic split-ubiquitin-based yeast-two-hybrid tests, in dot blot experiments, and in immuno-pull-downs. A 27-amino acid-fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides.
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TL;DR: This study investigated the effect of inhibiting nuclear import via Karyopherin β1 on cancer cell motility and inflammatory signaling using siRNA and the novel small molecule, Inhibitor of Nuclear Import-43, INI-43 and found it led to reduced migration and invasion of cervical cancer cells.
Abstract: // Tamara Stelma 1 and Virna D. Leaner 1 1 Division of Medical Biochemistry and Structural Biology, SAMRC Gynaecology Cancer Research Centre, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa Correspondence to: Virna D. Leaner, email: Virna.Leaner@uct.ac.za Keywords: karyopherin/importin, cervical cancer, chemotherapeutic, nuclear import, inflammation Received: November 18, 2016 Accepted: February 07, 2017 Published: March 02, 2017 ABSTRACT Karyopherin β1 is a nuclear import protein involved in the transport of proteins containing a nuclear localisation sequence. Elevated Karyopherin β1 expression has been reported in cancer and transformed cells and is essential for cancer cell proliferation and survival. Transcription factors such as NFĸB and AP-1 contain a nuclear localisation sequence and initiate the expression of multiple factors associated with inflammation and cancer cell biology. Our study investigated the effect of inhibiting nuclear import via Karyopherin β1 on cancer cell motility and inflammatory signaling using siRNA and the novel small molecule, Inhibitor of Nuclear Import-43, INI-43. Inhibition of Karyopherin β1 led to reduced migration and invasion of cervical cancer cells. Karyopherin β1 is essential for the translocation of NFĸB into the nucleus as nuclear import inhibition caused its cytoplasmic retention and decreased transcriptional activity. A similar decrease was seen in AP-1 transcriptional activity upon Karyopherin β1 inhibition. Consequently reduced interleukin-6, interleukin-1 beta, tumour necrosis factor alpha and granulocyte macrophage colony stimulating factor expression, target genes of NFkB and AP-1, was observed. Migration studies inhibiting individual transcription factors suggested that INI-43 may affect a combination of signaling events. Our study provides further evidence that inhibiting KPNB1 has anti-cancer effects and shows promise as a chemotherapeutic target.
TL;DR: Experimental results on transport of nuclear transport factor 2 (NTF2), importin β and mRNA particles, and putative regulatory functions of importinβ for the NPC transport mechanism and the RNA helicase Dbp5 for mRNA export kinetics are discussed.
TL;DR: The authors show that the importin RanBP6 acts as a tumor suppressor in Glioblastoma and regulates EGFR signalling through promoting translocation of STAT3 to the nuclei and repressing EGFR transcription.
Abstract: Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.
TL;DR: It is established that the HIV-1 Tat:NLS/CPP is able to form a stable and direct interaction with the classical nuclear import receptor importin-α and it is shown for the first time that the interface is the same as host factors such as Ku70 and Ku80, rather than other virus proteins such as Ebola VP24 that bind on the outer surface of importin -α.
Abstract: HIV-1 has caused 35 million deaths globally, and approximately the same number is currently living with HIV-1. The trans-activator of transcription (Tat) protein of HIV-1 plays an important regulatory function in the virus life cycle, responsible for regulating the reverse transcription of the viral genome RNA. Tat is found in the nucleus of infected cells, but can also invade uninfected neighbouring cells. Regions within Tat responsible for these cellular localisations are overlapping and include a nuclear localisation signal (NLS) spanning 48GRKKRR, and a cell penetrating peptide (CPP) signal spanning 48GRKKRRQRRRAPQN. However, the mechanism by which this NLS/CPP region mediates interaction with the nuclear import receptors remains to be resolved structurally. Here, we establish that the HIV-1 Tat:NLS/CPP is able to form a stable and direct interaction with the classical nuclear import receptor importin-α and using x-ray crystallography, we have determined the molecular interface and binding determinants to a resolution of 2.0 A. We show for the first time that the interface is the same as host factors such as Ku70 and Ku80, rather than other virus proteins such as Ebola VP24 that bind on the outer surface of importin-α.
TL;DR: This study proposes NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.
Abstract: The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.
TL;DR: This review summarizes the latest research on activity-dependent movement and nuclear import of postsynaptic proteins that modulate neuronal plasticity and focuses on the mechanism of active transport as well as the role of importins in mediatingnuclear import of the post Synapse to nucleus proteins.
TL;DR: It is proposed that mutations within NLS and NES motifs affect nuclear shuttling activity, and that TR mislocalization contributes to the development of some types of cancer and Resistance to Thyroid Hormone syndrome.
Abstract: The thyroid hormone receptors, TRα1 and TRβ1, are members of the nuclear receptor superfamily that forms one of the most abundant classes of transcription factors in multicellular organisms. Although primarily localized to the nucleus, TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm. The fine balance between nuclear import and export of TRs has emerged as a critical control point for modulating thyroid hormone-responsive gene expression. Mutagenesis studies have defined two nuclear localization signal (NLS) motifs that direct nuclear import of TRα1: NLS-1 in the hinge domain and NLS-2 in the N-terminal A/B domain. Three nuclear export signal (NES) motifs reside in the ligand-binding domain. A combined approach of shRNA-mediated knockdown and coimmunoprecipitation assays revealed that nuclear entry of TRα1 is facilitated by importin 7, likely through interactions with NLS-2, and importin β1 and the adapter importin α1 interacting with both NLS-1 and NLS-2. Interestingly, TRβ1 lacks NLS-2 and nuclear import depends solely on the importin α1/β1 heterodimer. Heterokaryon and fluorescence recovery after photobleaching shuttling assays identified multiple exportins that play a role in nuclear export of TRα1, including CRM1 (exportin 1), and exportins 4, 5, and 7. Even single amino acid changes in TRs dramatically alter their intracellular distribution patterns. We conclude that mutations within NLS and NES motifs affect nuclear shuttling activity, and propose that TR mislocalization contributes to the development of some types of cancer and Resistance to Thyroid Hormone syndrome.
TL;DR: By using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins, which will improve understanding of UL2-mediated biological functions in HSv-1 infection cycles.
Abstract: As a crucial protein, the herpes simplex virus 1 (HSV-1) UL2 protein has been shown to take part in various stages of viral infection, nonetheless, its exact subcellular localization and transport molecular determinants are not well known thus far. In the present study, by using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins. Moreover, the nuclear transport of UL2 was characterized to be assisted by multiple transport pathways through Ran-, importin α1-, α5-, α7-, β1- and transportin-1 cellular transport receptors. Consequently, these results will improve understanding of UL2-mediated biological functions in HSV-1 infection cycles.
TL;DR: Data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin β and Hsp83, and a tetratricopeptide repeat repeat (TPR) domain at the N-terminal end of N up358 interacts with HSp83 and is indispensable for Met nuclear localization.
TL;DR: It is demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network and represents to the authors' knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.
Abstract: β-Dystroglycan (β-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of β-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/β mediated nuclear import pathway has already been described for β-DG, the intracellular trafficking route by which β-DG reaches the nucleus is unknown. In this study, we demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of β-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of β-DG at Tyr890 is a key stimulus for β-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that β-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.
TL;DR: The crystal structure of the nuclear import adaptor importin‐α1 bound to the nuclear localization signal (NLS) of EBNA‐LP that shows EB NA‐LP residues 44‐RRVRRR‐49 binding to the major NLS‐binding site at the P0‐P5 positions is reported.
Abstract: Epstein-Barr virus EBNA-LP protein is a transcriptional coactivator of EBNA2. Efficient nuclear localization of EBNA-LP is essential for cooperation with EBNA2. Here, we report the crystal structure of the nuclear import adaptor importin-α1 bound to the nuclear localization signal (NLS) of EBNA-LP that shows EBNA-LP residues 44-RRVRRR-49 binding to the major NLS-binding site at the P0-P5 positions. In contrast to previously characterized classical NLSs that invariably have a basic residue [either lysine (in the vast majority of cases) or arginine] at the P2 position, the EBNA-LP NLS is unique in that it has valine at the P2 position. The loss of the critical P2 lysine (or arginine) is compensated by arginine at the P0 position in the EBNA-LP NLS.
TL;DR: Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners.
Abstract: DNA polymerase β (pol β) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol β residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol β(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol β. Together these data demonstrate that pol β contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.
TL;DR: ZoCDPK1 is operating through NAC TF mediated ABA-independent, cold non-responsive stress signaling pathway in ginger, corroborate with the results of gene expression and over-expression studies of ZoC DPK1.
Abstract: Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavour and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2‑hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence ZoCDPK1 is operating through NAC TF mediated ABA-independent, cold non-responsive stress signaling pathway in ginger.
TL;DR: It is shown that JAK1 has a classical nuclear localization signal toward the N-terminal region, which can be recognized by multiple importin α isoforms and is essential for the optimal fitness of ABC DLBCL cells in vitro.
Abstract: JAKs are non-receptor tyrosine kinases that are generally found in association with cytokine receptors. In the canonical pathway, roles of JAKs have well been established in activating STATs in response to cytokine stimulation to modulate gene transcription. In contrast, a noncanonical role of JAK2 has recently been discovered, in which JAK2 in the nucleus imparts the epigenetic regulation of gene transcription through phosphorylation of tyrosine 41 on the histone protein H3. Recent work further demonstrated that this noncanonical mechanism is conserved with JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL), a cancer type that is particularly difficult to treat and has poor prognosis. However, how JAK1 gains access to the nucleus to enable epigenetic regulation remains undefined. Here, we investigated this question and revealed that JAK1 has a classical nuclear localization signal toward the N-terminal region, which can be recognized by multiple importin α isoforms. Moreover, the nuclear import of JAK1 is independent of its kinase activity but is required for the optimal expansion of ABC DLBCL cells in vitroImplications: This study demonstrates that the nuclear import of JAK1 is essential for the optimal fitness of ABC DLBCL cells, and targeting JAK1 nuclear localization is a potential therapeutic strategy for ABC DLBCL. Mol Cancer Res; 15(3); 348-57. ©2016 AACR.
TL;DR: Importin-5 (IPO5), a member of the importin family of nuclear transport proteins, is identified as an intracellular binding partner of full-length interleukin-33 and physically interacts with FLIL33, showing that this interaction localizes to a cluster of charged amino acids but not to an adjacent segment in theFLIL33 N-terminal region.
TL;DR: The operation of the nuclear barrier in aging provides a fundamental mechanism for cellular protection against stress and promotes survival at the expense of growth via stress-sensitive transcriptional control.
Abstract: Aging-dependent cellular behaviors toward extrinsic stress are characterized by the confined localization of certain molecules to either nuclear or perinuclear regions Although most growth factors can activate downstream signaling in aging cells, they do not in fact have any impact on the cells because the signals cannot reach their genetic targets in the nucleus For the same reason, varying apoptotic stress factors cannot stimulate the apoptotic pathway in senescent cells Thus, the operation of a functional nuclear barrier in an aging-dependent manner has been investigated To elucidate the mechanism for this process, the housekeeping transcription factor Sp1 was identified as a general regulator of nucleocytoplasmic trafficking (NCT) genes, including various nucleoporins, importins, exportins and Ran GTPase cycle-related genes Interestingly, the posttranslational modification of Sp1 is readily influenced by extrinsic stress, including oxidative and metabolic stress The decrease in SP1 O-GlcNAcylation under oxidative stress or during replicative senescence makes it susceptible to proteosomal degradation, resulting in defective NCT functions and leading to nuclear barrier formation The operation of the nuclear barrier in aging provides a fundamental mechanism for cellular protection against stress and promotes survival at the expense of growth via stress-sensitive transcriptional control
TL;DR: It is shown for the first time that the nuclear localization ofCat L and its substrate Cux1 can be positively regulated by Snail NLS and importin β1, suggesting that Snail, Cat L and Cux 1 all utilize importinβ1 for nuclear import.
TL;DR: Findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.