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David A Jans - One of the best experts on this subject based on the ideXlab platform.
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Nuclear trafficking of the rabies virus interferon antagonist p protein is regulated by an importin binding Nuclear Localization Sequence in the c terminal domain
PLOS ONE, 2016Co-Authors: David A Jans, Kylie M Wagstaff, Caitlin Lorraine Rowe, Sibil Oksayan, Dominic J Glover, Gregory W MoseleyAbstract:Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although Nuclear import of P3 is known to be mediated by an importin (IMP)-recognised Nuclear Localization Sequence in the N-terminal region (N-NLS), the mechanisms underlying Nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in Nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro Nuclear transport assays to examine in detail the Nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on Nuclear export, suggesting that Nuclear exclusion of mutated P3 involves the P-CTD-localized Nuclear export Sequence (C-NES). However, assays using cells in which Nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 Nuclear accumulation and, importantly, prevent Nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent Nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular Localization, consistent with important roles in infection.
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the n terminal basic domain of the hiv 1 matrix protein does not contain a conventional Nuclear Localization Sequence but is required for dna binding and protein self association
Biochemistry, 2008Co-Authors: Anna C Hearps, Kylie M Wagstaff, Sabine C Piller, David A JansAbstract:The HIV p17 or matrix (MA) protein has long been implicated in the process of Nuclear import of the HIV genome and thus the ability of the virus to infect nondividing cells such as macrophages. While it has been demonstrated that MA is not absolutely required for this process, debate continues to surround the subcellular targeting properties of MA and its potential contribution to Nuclear import of the HIV cDNA. Through the use of in vitro techniques we have determined that, despite the ability of MA to interact with importins, the full-length protein fails to enter the nucleus of cells. While MA does contain a region of basic amino acids within its N-terminus which can confer Nuclear accumulation of a fusion protein, we show that this is due to Nuclear retention mediated by DNA binding and does not represent facilitated import. Importantly, we show that the 26 KK residues of MA, previously thought to be part of a Nuclear Localization Sequence, are absolutely required for a number of MA's functions including its ability to bind DNA and RNA and its propensity to form high-order multimers/protein aggregates. The results presented here indicate that the N-terminal basic domain of MA does not appear likely to play a role in HIV cDNA Nuclear import; rather this region appears to be a crucial structural and functional motif whose integrity is required for a number of other roles performed by MA during viral infection.
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Structural basis for the specificity of bipartite Nuclear Localization Sequence binding by importin-alpha.
The Journal of biological chemistry, 2003Co-Authors: Marcos R.m. Fontes, David A Jans, Trazel Teh, Ross I. Brinkworth, Bostjan KobeAbstract:Importin-alpha is the Nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite Nuclear Localization Sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse Sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker Sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus Sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel Nuclear proteins.
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novel low molecular weight microtubule associated protein 2 isoforms contain a functional Nuclear Localization Sequence
Journal of Biological Chemistry, 1999Co-Authors: Kate L Loveland, Daniella Herszfeld, Brendan Chu, Emily Rames, Elizabeth Christy, Lyndall J Briggs, Rushdi Shakri, David Morrits De Kretser, David A JansAbstract:Abstract Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly Nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected Localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a Nuclear Localization Sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-β-galactosidase fusion protein and an in vitro Nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both ∼70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.
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enhancement of polylysine mediated transferrinfection by Nuclear Localization Sequences polylysine does not function as a Nuclear Localization Sequence
Human Gene Therapy, 1999Co-Authors: Chee Kai Chan, David A JansAbstract:Polylysine (pLy) has been used successfully as a DNA carrier in receptor-mediated gene transfer, enhancement of transfection having been proposed to be in part through efficient Nuclear targeting stemming from the resemblance of pLy to the Nuclear Localization Sequence (NLS) from simian virus SV40 large tumor antigen (T-ag). In this study we test whether pLy carrying covalently attached peptides comprising the T-ag NLS (the pLyP101 derivative) can enhance transferrin-pLy-mediated transfection ("transferrinfection"). Unlike pLy itself or a pLy derivative (pLyP101T) carrying cross-linked T-ag NLS mutant peptides, pLyP101 significantly enhanced transferrinfection of a beta-galactosidase-expressing reporter plasmid. The basis of this was shown to be the ability of the pLyP101-plasmid DNA complex to be recognized with high affinity by the NLS-binding importin subunits, in contrast to pLyP101T- and pLy-plasmid complexes. Confocal laser scanning microscopy was used to determine the Nuclear import kinetics of flu...
Mary Shannon Moore - One of the best experts on this subject based on the ideXlab platform.
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the Nuclear import of rcc1 requires a specific Nuclear Localization Sequence receptor karyopherin α3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:Abstract RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopusovarian cytosol nor a mixture of recombinant import factors (karyopherin α2, karyopherin β1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathioneS-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin α3 (Kapα3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapα1 and Kapα2, would not. Accordingly, in vitrobinding studies revealed that only Kapα3 showed significant binding to RCC1 (unlike Kapα1 and Kapα2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapα3, we found that the Nuclear import of pA-RCC1 also required both karyopherin β1 and Ran.
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The Nuclear import of RCC1 requires a specific Nuclear Localization Sequence receptor, karyopherin alpha3/Qip.
The Journal of biological chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
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the Nuclear import of rcc1 requires a specific Nuclear Localization Sequence receptor karyopherin alpha3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
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The Nuclear Import of RCC1 Requires a Specific Nuclear Localization Sequence Receptor, Karyopherin α3/Qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
Bradford Talcott - One of the best experts on this subject based on the ideXlab platform.
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the Nuclear import of rcc1 requires a specific Nuclear Localization Sequence receptor karyopherin α3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:Abstract RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopusovarian cytosol nor a mixture of recombinant import factors (karyopherin α2, karyopherin β1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathioneS-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin α3 (Kapα3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapα1 and Kapα2, would not. Accordingly, in vitrobinding studies revealed that only Kapα3 showed significant binding to RCC1 (unlike Kapα1 and Kapα2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapα3, we found that the Nuclear import of pA-RCC1 also required both karyopherin β1 and Ran.
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The Nuclear import of RCC1 requires a specific Nuclear Localization Sequence receptor, karyopherin alpha3/Qip.
The Journal of biological chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
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the Nuclear import of rcc1 requires a specific Nuclear Localization Sequence receptor karyopherin alpha3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
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The Nuclear Import of RCC1 Requires a Specific Nuclear Localization Sequence Receptor, Karyopherin α3/Qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the Nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical Nuclear Localization Sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant Nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the Nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
Howard M. Johnson - One of the best experts on this subject based on the ideXlab platform.
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The IFNAR1 subunit of the type I IFN receptor complex contains a functional Nuclear Localization Sequence
FEBS letters, 2004Co-Authors: Prem S. Subramaniam, Howard M. JohnsonAbstract:A Nuclear Localization Sequence (NLS) in the type II interferon (IFN) IFNγ, which is responsible for the Nuclear translocation of both the ligand and the α-subunit (IFNGR1) of the receptor complex, has previously been characterized and its role in signaling examined in detail. We have now identified an NLS in the type I IFN receptor (IFNAR) common subunit IFNAR1 from humans and show that the human IFNAR1 subunit can translocate to the nucleus following human IFNβ stimulation. An NLS in human IFNAR1 is located in the extracellular domain of IFNAR1 within the Sequence 382RKIIEKKT (numbered for the precursor form). Nuclear import by the NLS functions in a conventional fashion requiring cytosolic import factors, is energy-dependent and inhibited by the prototypical NLS of the SV40 large T-antigen. These studies provide a mechanism for Nuclear import of IFNAR1, as well as for type I IFN ligands, and a starting point for studying an alternate role for IFNAR1 in Nuclear signaling within the type I IFN system.
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The role of IFNγ Nuclear Localization Sequence in intracellular function
Journal of Cell Science, 2003Co-Authors: C.m. Iqbal Ahmed, Prem S. Subramaniam, Marjorie A. Burkhart, Mustafa G. Mujtaba, Howard M. JohnsonAbstract:Intracellularly expressed interferon γ (IFNγ) has been reported to possess biological activity similar to that of IFNγ added to cells. This study addresses the mechanisms for such similar biological effects. Adenoviral vectors were used to express a non-secreted form of human IFNγ or a non-secreted mutant form in which a previously demonstrated Nuclear Localization Sequence (NLS), 128 KTGKRKR 134 , was replaced with alanines at K and R positions. With the vector expressing non-secreted wild-type IFNγ, biological responses normally associated with extracellular IFNγ, such as antiviral activity and MHC class I upregulation, were observed, although the mutant IFNγ did not possess biological activity. Intracellular human IFNγ possessed biological activity in mouse L cells, which do not recognize extracellularly added human IFNγ. Thus, the biological activity was not due to leakage of IFNγ to the surroundings and subsequent interaction with the receptor on the cell surface. Biological function was associated with activation of STAT1α and Nuclear translocation of IFNγ, IFNGR1 and STAT1α. Immunoprecipitation of cellular extracts with antibody to the Nuclear transporter NPI-1 showed the formation of a complex with IFNγ-IFNGR1-STAT1α. To provide the physiological basis for these effects we show that extracellularly added IFNγ possesses intracellular signaling activity that is NLS dependent, as suggested by our previous studies, and that this activity occurs via the receptor-mediated endocytosis of IFNγ. The data are consistent with previous observations that the NLS of extracellularly added IFNγ plays a role in IFNγ signaling.
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The role of IFNgamma Nuclear Localization Sequence in intracellular function.
Journal of cell science, 2003Co-Authors: C.m. Iqbal Ahmed, Prem S. Subramaniam, Marjorie A. Burkhart, Mustafa G. Mujtaba, Howard M. JohnsonAbstract:Intracellularly expressed interferon gamma (IFNgamma) has been reported to possess biological activity similar to that of IFNgamma added to cells. This study addresses the mechanisms for such similar biological effects. Adenoviral vectors were used to express a non-secreted form of human IFNgamma or a non-secreted mutant form in which a previously demonstrated Nuclear Localization Sequence (NLS), 128KTGKRKR134, was replaced with alanines at K and R positions. With the vector expressing non-secreted wild-type IFNgamma, biological responses normally associated with extracellular IFNgamma, such as antiviral activity and MHC class I upregulation, were observed, although the mutant IFNgamma did not possess biological activity. Intracellular human IFNgamma possessed biological activity in mouse L cells, which do not recognize extracellularly added human IFNgamma. Thus, the biological activity was not due to leakage of IFNgamma to the surroundings and subsequent interaction with the receptor on the cell surface. Biological function was associated with activation of STAT1alpha and Nuclear translocation of IFNgamma, IFNGR1 and STAT1alpha. Immunoprecipitation of cellular extracts with antibody to the Nuclear transporter NPI-1 showed the formation of a complex with IFNgamma-IFNGR1-STAT1alpha. To provide the physiological basis for these effects we show that extracellularly added IFNgamma possesses intracellular signaling activity that is NLS dependent, as suggested by our previous studies, and that this activity occurs via the receptor-mediated endocytosis of IFNgamma. The data are consistent with previous observations that the NLS of extracellularly added IFNgamma plays a role in IFNgamma signaling.
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Nuclear Translocation of IFN-γ Is an Intrinsic Requirement for Its Biologic Activity and Can Be Driven by a Heterologous Nuclear Localization Sequence
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 2001Co-Authors: Prem S. Subramaniam, Marino M. Green, Joseph Larkin, Barbara A. Torres, Howard M. JohnsonAbstract:We have previously identified a Nuclear Localization Sequence (NLS) in interferon-γ (IFN-γ). This NLS functions intracellularly by forming a complex with its transcription factor Stat1α and the nuc...
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the cooh terminal Nuclear Localization Sequence of interferon gamma regulates stat1 alpha Nuclear translocation at an intracellular site
Journal of Cell Science, 2000Co-Authors: Prem S. Subramaniam, Joseph Larkin, Mustafa G. Mujtaba, Mark R Walter, Howard M. JohnsonAbstract:We have recently shown that the Nuclear Localization of IFN gamma is mediated by a polybasic Nuclear Localization Sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the Nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95–133) containing the NLS blocked the induction of STAT1 alpha Nuclear translocation. The antibodies had no effect on Nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1–123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K(d) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1–123) to initiate the Nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha Nuclear Localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K(d) approx. 3 × 10(−8) M(−1) has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the Nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha Nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95–133), that contains the NLS motif, induced Nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha Nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha Nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent Nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate Nuclear translocation STAT1 alpha.
Peter Aleström - One of the best experts on this subject based on the ideXlab platform.
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the Nuclear Localization Sequence of the sv40 t antigen promotes transgene uptake and expression in zebrafish embryo nuclei
Transgenic Research, 1996Co-Authors: Philippe Collas, Harald Husebye, Peter AleströmAbstract:We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen Nuclear Localization Sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 103 NLS-bound pCMVL copies. With 104 copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p<0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days: The specificity of NLS on luciferase expression was tested by using a Nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of NLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.
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The Nuclear Localization Sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei.
Transgenic research, 1996Co-Authors: Philippe Collas, Harald Husebye, Peter AleströmAbstract:We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen Nuclear Localization Sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 103 NLS-bound pCMVL copies. With 104 copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p
