Nuclear Translocation of IFN-γ Is an Intrinsic Requirement for Its Biologic Activity and Can Be Driven by a Heterologous Nuclear Localization Sequence

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.

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SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m110.203729 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10297-10304 ◽

Cited By ~ 31

Author(s):

Guochang Huang ◽

Andrew J. Kaufman ◽

Y. Ramanathan ◽

Bhuvanesh Singh

Keyword(s):

Nuclear Localization ◽

Essential Role ◽

Nuclear Translocation ◽

Nuclear Localization Sequence ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Transgenic Expression ◽

Localization Sequence ◽

Efficient Transfer

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.

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Inhibition of Nuclear Translocation of Transcription Factor NF-κB by a Synthetic Peptide Containing a Cell Membrane-permeable Motif and Nuclear Localization Sequence

Journal of Biological Chemistry ◽

10.1074/jbc.270.24.14255 ◽

1995 ◽

Vol 270 (24) ◽

pp. 14255-14258 ◽

Cited By ~ 683

Author(s):

Yao-Zhong Lin ◽

SongYi Yao ◽

Ruth Ann Veach ◽

Troy R. Torgerson ◽

Jacek Hawiger

Keyword(s):

Transcription Factor ◽

Cell Membrane ◽

Nuclear Localization ◽

Synthetic Peptide ◽

Nuclear Translocation ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

A Cell

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Identification of a putative nuclear localization sequence within ANG II AT1A receptor associated with nuclear activation

AJP Cell Physiology ◽

10.1152/ajpcell.00337.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1398-C1408 ◽

Cited By ~ 40

Author(s):

Thomas A. Morinelli ◽

John R. Raymond ◽

Aleksander Baldys ◽

Qing Yang ◽

Mi-hye Lee ◽

...

Keyword(s):

Nuclear Localization ◽

Intracellular Signaling ◽

Nuclear Translocation ◽

Receptor Expression ◽

Nuclear Localization Sequence ◽

Ang Ii ◽

Wild Type ◽

Nuclear Activation ◽

Localization Sequence ◽

Carboxy Terminal

Angiotensin II (ANG II) type 1 (AT1) receptors, similar to other G protein-coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy-terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxy-terminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild-type AT1A receptor-green fluorescent protein (AT1AR/GFP) construct or a site-directed mutation of a putative nuclear localization sequence (NLS) [K307Q]AT1AR/GFP (KQ/AT1AR/GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II-induced internalization of the wild-type/GFP construct and of the KQ/AT1AR/GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT1AR/GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II (100 nM) stimulation, whereas the KQ/AT1AR/GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti-GFP antibody confirmed these observations. Nuclear localization of the wild-type receptor correlated with increase transcription for both EGR-1 and PTGS-2 genes while the nuclear-deficient KQ/AT1AR/GFP mutant demonstrated increases for only the EGR-1 gene. These results suggest that a NLS (KKFKKY; aa307–312) is located within the cytoplasmic tail of the AT1A receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX-2 gene PTGS-2.

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The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

International Journal of Molecular Sciences ◽

10.3390/ijms21197428 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7428

Author(s):

José L. Neira ◽

Bruno Rizzuti ◽

Ana Jiménez-Alesanco ◽

Olga Abián ◽

Adrián Velázquez-Campoy ◽

...

Keyword(s):

Nuclear Localization ◽

Protein Transport ◽

Nuclear Protein ◽

Nuclear Translocation ◽

Intrinsically Disordered Protein ◽

Titration Calorimetry ◽

Nuclear Localization Sequence ◽

Intrinsically Disordered ◽

Localization Sequence ◽

High Flexibility

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

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A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin

Biomolecules ◽

10.3390/biom10091313 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1313 ◽

Cited By ~ 2

Author(s):

José L. Neira ◽

Bruno Rizzuti ◽

Ana Jiménez-Alesanco ◽

Martina Palomino-Schätzlein ◽

Olga Abián ◽

...

Keyword(s):

Nuclear Localization ◽

Protein Transport ◽

Nuclear Translocation ◽

Nuclear Overhauser Effect ◽

Intrinsically Disordered Protein ◽

Random Coil ◽

Disordered Proteins ◽

Nuclear Localization Sequence ◽

Intrinsically Disordered ◽

Localization Sequence

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

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The sal3+ Gene Encodes an Importin-β Implicated in the Nuclear Import of Cdc25 in Schizosaccharomyces pombe

Genetics ◽

10.1093/genetics/162.2.689 ◽

2002 ◽

Vol 162 (2) ◽

pp. 689-703 ◽

Cited By ~ 2

Author(s):

Gordon Chua ◽

Carol Lingner ◽

Corey Frazer ◽

Paul G Young

Keyword(s):

Schizosaccharomyces Pombe ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Translocation ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

G1 Arrest ◽

Mitotic Entry ◽

Localization Sequence ◽

Mutant Phenotypes

Abstract In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the sal3+ gene product as an importin-β homolog that participates in the nuclear import of Cdc25. Loss of sal3+ results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.

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