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Results: 1 to 6 of 6

Publication Record


Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.
Zienkiewicz J, Armitage A, Hawiger J
(2013) J Am Heart Assoc 2: e000386
MeSH Terms: Active Transport, Cell Nucleus, Cell Nucleus, Cell-Penetrating Peptides, Cells, Cultured, Humans, NF-kappa B p50 Subunit, Nuclear Localization Signals, alpha Karyopherins
Show Abstract · Added March 27, 2014
BACKGROUND - We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin β. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined.
METHODS AND RESULTS - We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α-mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high-affinity binding. The 2:1 stoichiometry of the N50-Imp α5 interaction (KD1 = 73 nmol/L, KD2 = 140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3-dimensional (3-D) docking models and comparative structural analysis, we identified a structural component of the Imp α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 also displayed rapid stimulus-induced turnover, which could influence its availability for nuclear transport during the inflammatory response.
CONCLUSIONS - These results provide direct evidence that N50 peptide selectively targets Imp α5, encouraging further refinement of NLS-derived peptides as new tools to modulate inflammatory disorders.
0 Communities
1 Members
0 Resources
8 MeSH Terms
STRADalpha regulates LKB1 localization by blocking access to importin-alpha, and by association with Crm1 and exportin-7.
Dorfman J, Macara IG
(2008) Mol Biol Cell 19: 1614-26
MeSH Terms: Active Transport, Cell Nucleus, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Amino Acid Sequence, Base Sequence, Carrier Proteins, Cell Line, DNA, Complementary, HeLa Cells, Humans, Karyopherins, Molecular Sequence Data, Protein Binding, Protein-Serine-Threonine Kinases, RNA, Small Interfering, Receptors, Cytoplasmic and Nuclear, Recombinant Proteins, Transfection, alpha Karyopherins, ran GTP-Binding Protein
Show Abstract · Added March 5, 2014
LKB1, a serine/threonine kinase, regulates cell polarity, metabolism, and cell growth. The activity and cellular distribution of LKB1 are determined by cofactors, STRADalpha and MO25. STRADalpha induces relocalization of LKB1 from the nucleus to the cytoplasm and stimulates its catalytic activity. MO25 stabilizes the STRADalpha/LKB1 interaction. We investigated the mechanism of nucleocytoplasmic transport of LKB1 in response to its cofactors. Although LKB1 is imported into the nucleus by importin-alpha/beta, STRADalpha and MO25 passively diffuse between the nucleus and the cytoplasm. STRADalpha induces nucleocytoplasmic shuttling of LKB1. STRADalpha facilitates nuclear export of LKB1 by serving as an adaptor between LKB1 and exportins CRM1 and exportin7. STRADalpha inhibits import of LKB1 by competing with importin-alpha for binding to LKB1. MO25 stabilizes the LKB1-STRADalpha complex but it does not facilitate its nucleocytoplasmic shuttling. Strikingly, the STRADbeta, isoform which differs from STRADalpha in the N- and C-terminal domains that are responsible for interaction with export receptors, does not efficiently relocalize LKB1 from the nucleus to the cytoplasm. These results identify a multifactored mechanism to control LKB1 localization, and they suggest that the STRADbeta-LKB1 complex might possess unique functions in the nucleus.
0 Communities
1 Members
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20 MeSH Terms
Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry.
Shin I, Rotty J, Wu FY, Arteaga CL
(2005) J Biol Chem 280: 6055-63
MeSH Terms: Active Transport, Cell Nucleus, Animals, Cell Cycle Proteins, Cell Line, Cell Nucleus, Cyclin-Dependent Kinase Inhibitor p27, Cytosol, G1 Phase, Humans, Mice, Models, Biological, Phosphorylation, Phosphoserine, Phosphothreonine, Protein Binding, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Time Factors, Tumor Suppressor Proteins, alpha Karyopherins
Show Abstract · Added March 5, 2014
We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.
0 Communities
1 Members
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21 MeSH Terms
Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor.
Liu D, Liu XY, Robinson D, Burnett C, Jackson C, Seele L, Veach RA, Downs S, Collins RD, Ballard DW, Hawiger J
(2004) J Biol Chem 279: 19239-46
MeSH Terms: Active Transport, Cell Nucleus, Adaptor Proteins, Vesicular Transport, Amino Acid Sequence, Animals, Cell Death, Cell Membrane Permeability, Cytokines, Enterotoxins, Liver, Mice, NF-kappa B, Peptides, T-Lymphocytes, alpha Karyopherins
Show Abstract · Added December 10, 2013
Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin alpha/karyopherin alpha 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor kappa B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses.
1 Communities
2 Members
0 Resources
14 MeSH Terms
Phosphorylation regulates the interaction between Gln3p and the nuclear import factor Srp1p.
Carvalho J, Bertram PG, Wente SR, Zheng XF
(2001) J Biol Chem 276: 25359-65
MeSH Terms: Amino Acid Transport Systems, Carrier Proteins, Cell Nucleus, Cytoplasm, DNA-Binding Proteins, Drosophila Proteins, Electrophoresis, Polyacrylamide Gel, Fluorescent Antibody Technique, Fluorescent Dyes, Fungal Proteins, Indoles, Karyopherins, Mutagenesis, Site-Directed, Nitrogen, Nuclear Proteins, Phosphorylation, Receptor Protein-Tyrosine Kinases, Receptors, Cytoplasmic and Nuclear, Repressor Proteins, Saccharomyces cerevisiae Proteins, Sirolimus, Temperature, Transcription Factors, Yeasts, alpha Karyopherins, ras GTPase-Activating Proteins
Show Abstract · Added March 21, 2014
Gln3p is a GATA-type transcription activator of nitrogen catabolite repressible (NCR) genes. Gln3p was recently found to be hyperphosphorylated in a TOR-dependent manner and resides in the cytoplasm in high quality nitrogen. In contrast, during nitrogen starvation or rapamycin treatment, Gln3p becomes rapidly dephosphorylated and accumulates in the nucleus, thereby activating nitrogen catabolite repression genes. However, a detailed mechanistic understanding is lacking for the regulation of Gln3p nucleocytoplasmic distribution. In this study, we applied a functional genomics approach to identify the nuclear transport factors for Gln3p. We found that yeast karyopherin alpha/Srp1p and Crm1p are required for the nuclear import and export of Gln3p, respectively. Similarly, the Ran GTPase pathway is also involved in the nuclear translocation of Gln3p. Finally, we show that Srp1p preferentially interacts with the hypophosphorylated versus the hyperphosphorylated Gln3p. These findings define a possible mechanism for regulated nucleocytoplasmic transport of Gln3p by phosphorylation in vivo.
0 Communities
1 Members
0 Resources
26 MeSH Terms
GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.
Murphy R, Watkins JL, Wente SR
(1996) Mol Biol Cell 7: 1921-37
MeSH Terms: Amino Acid Sequence, Fungal Proteins, GTPase-Activating Proteins, Gene Expression Regulation, Fungal, Genes, Lethal, Membrane Proteins, Molecular Sequence Data, Mutagenesis, Nuclear Envelope, Nuclear Matrix-Associated Proteins, Nuclear Pore Complex Proteins, Nuclear Proteins, Nucleocytoplasmic Transport Proteins, Peptides, Poly A, Proteins, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Sequence Homology, Amino Acid, alpha Karyopherins
Show Abstract · Added March 21, 2014
To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.
0 Communities
1 Members
0 Resources
23 MeSH Terms