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

2011 ◽  
Vol 286 (12) ◽  
pp. 10297-10304 ◽  
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.

Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

1999 ◽  
Vol 112 (24) ◽  
pp. 4501-4512 ◽  
Author(s):  
Y.M. Yannoni ◽  
K. White
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Localization Sequence ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Localization Sequence ◽  
Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

2000 ◽  
Vol 113 (15) ◽  
pp. 2771-2781
Author(s):  
P.S. Subramaniam ◽  
J. Larkin ◽  
M.G. Mujtaba ◽  
M.R. Walter ◽  
H.M. Johnson
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Nuclear Localization Sequence ◽  
Receptor Complex ◽  
Ifn Gamma ◽  
High Affinity ◽  
Gamma Receptor ◽  
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.

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

2007 ◽  
Vol 292 (4) ◽  
pp. C1398-C1408 ◽  
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.

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

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.

scholarly journals The Unfolded Protein Response Transducer Ire1p Contains a Nuclear Localization Sequence Recognized by Multiple β Importins

2006 ◽  
Vol 17 (12) ◽  
pp. 5309-5323 ◽  
Author(s):  
Laurence Goffin ◽  
Sadanand Vodala ◽  
Christine Fraser ◽  
Joanne Ryan ◽  
Mark Timms ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Nuclear Localization ◽  
Nuclear Localization Sequence ◽  
Binding Studies ◽  
Linker Region ◽  
Unfolded Protein ◽  
Localization Sequence ◽  
The Unfolded Protein Response ◽  
Protein Response

The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of targeting green fluorescent protein to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one classical NLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin α (Kap60p) and a novel βNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin β homologues. Kinetic binding data suggest that binding to importin β proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization and also requires functional RanGAP and Ran GEF proteins. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.

scholarly journals The Effect of the Anticipated Nuclear Localization Sequence of ‘Candidatus Phytoplasma mali’ SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor

2021 ◽  
Vol 9 (8) ◽  
pp. 1756
Author(s):  
Alisa Strohmayer ◽  
Timothy Schwarz ◽  
Mario Braun ◽  
Gabi Krczal ◽  
Kajohn Boonrod
Keyword(s):  
Transcription Factors ◽  
Nuclear Localization ◽  
Nuclear Localization Sequence ◽  
Candidatus Phytoplasma Mali ◽  
Localization Sequence ◽  
Amino Acid Stretch ◽  
Proliferating Cell ◽  
Two Hybrid ◽  
Plant Nucleus

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

scholarly journals Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons

2015 ◽  
Vol 26 (5) ◽  
pp. 843-858 ◽  
Author(s):  
Lea Arnold ◽  
Sebastian Höckner ◽  
Wolfgang Seufert
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Budding Yeast ◽  
Cellular Mechanism ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
In Vivo Analysis ◽  
Spatiotemporal Control

In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.

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

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1313 ◽  
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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