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

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

Journal of Cell Science ◽

10.1242/jcs.113.15.2771 ◽

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.

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Nab1, a Corepressor of NGFI-A (Egr-1), Contains an Active Transcriptional Repression Domain

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.512 ◽

1998 ◽

Vol 18 (1) ◽

pp. 512-524 ◽

Cited By ~ 73

Author(s):

Alexander H. Swirnoff ◽

Elizabeth D. Apel ◽

John Svaren ◽

Bradley R. Sevetson ◽

Drazen B. Zimonjic ◽

...

Keyword(s):

Nuclear Localization ◽

Transcriptional Repression ◽

Early Gene ◽

Nuclear Localization Sequence ◽

Distal Enhancer ◽

Genomic Locus ◽

Activation Domains ◽

Evolutionarily Conserved ◽

Localization Sequence ◽

Carboxy Terminal

ABSTRACT Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.

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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 ◽

10.1089/107999001753289569 ◽

2001 ◽

Vol 21 (11) ◽

pp. 951-959 ◽

Cited By ~ 31

Author(s):

Prem S. Subramaniam ◽

Marino M. Green ◽

Joseph Larkin ◽

Barbara A. Torres ◽

Howard M. Johnson

Keyword(s):

Nuclear Localization ◽

Nuclear Translocation ◽

Nuclear Localization Sequence ◽

Biologic Activity ◽

Localization Sequence ◽

Ifn Γ

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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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PTHrP Nuclear Localization and Carboxyl Terminus Sequences Modulate Dental and Mandibular Development in Part via the Action of p27

Endocrinology ◽

10.1210/en.2015-1555 ◽

2016 ◽

Vol 2016 (1) ◽

pp. 72-84 ◽

Cited By ~ 9

Author(s):

Wen Sun ◽

Jun Wu ◽

Linying Huang ◽

Hong Liu ◽

Rong Wang ◽

...

Keyword(s):

Nuclear Localization ◽

Alveolar Bone ◽

Bone Volume ◽

Carboxyl Terminus ◽

Nuclear Localization Sequence ◽

Wild Type ◽

Mineral Density ◽

C Terminus ◽

Localization Sequence ◽

Dentin Sialoprotein

Abstract To determine whether the action of the PTHrP nuclear localization sequence and C terminus is mediated through p27 in modulating dental and mandibular development, compound mutant mice, which are homozygous for both p27 deletion and the PTHrP1–84 knock-in mutation (p27−/−PthrpKI/KI), were generated. Their teeth and mandibular phenotypes were compared with those of p27−/−, PthrpKUK\ and wild-type mice. At 2 weeks of age, the mandibular mineral density, alveolar bone volume, osteoblast numbers, and dental volume, dentin sialoprotein-immunopo-sitive areas in the first molar were increased significantly in p27−/− mice and decreased dramatically in both PthrpKI/KI and p27−/− PthrpKI/KI mice compared with wild-type mice; however, these parameters were partly rescued in p27−/− PthrpKI/KI mice compared with PthrpKI/KI mice. These data demonstrate that the deletion of p27 in PthrpKI/KI mice can partially rescue defects in dental and mandibular development. Furthermore, we found that deletion of p27 in PthrpKI/KI mice partially corrected the dental and mandibular phenotype by modulating cell cyclin-regulating molecules and antioxidant enzymes. This study therefore indicates that the p27 pathway may function downstream in the action of PTHrP nuclear localization sequence to regulate dental and mandibular development. (Endocrinology 157: 1372–1384, 2016)

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Analysis of mutations in the putative nuclear localization sequence of interleukin-1β

Biochemical Journal ◽

10.1042/bj2800111 ◽

1991 ◽

Vol 280 (1) ◽

pp. 111-116 ◽

Cited By ~ 21

Author(s):

S Grenfell ◽

N Smithers ◽

S Witham ◽

A Shaw ◽

P Graber ◽

...

Keyword(s):

Nuclear Localization ◽

Interleukin 1 ◽

Simian Virus 40 ◽

T Antigen ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

Target Cells ◽

Wild Type ◽

Receptor Mediated Endocytosis ◽

Localization Sequence

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210→Ala), M50 (Lys211→Ala) and M51 (Pro208→Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

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Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2913 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2913-2920 ◽

Cited By ~ 29

Author(s):

Lawrence H. Milocco ◽

Jennifer A. Haslam ◽

Jonathan Rosen ◽

H. Martin Seidel

Keyword(s):

Tyrosine Phosphorylation ◽

Nuclear Translocation ◽

Regulatory Function ◽

Nuclear Localization Sequence ◽

Wild Type ◽

Localization Sequence ◽

Gene Regulatory ◽

First Time ◽

Insight Into

ABSTRACT The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity.

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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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Parathyroid Hormone-Related Protein Is a Mitogenic and a Survival Factor of Mesangial Cells from Male Mice: Role of Intracrine and Paracrine Pathways

Endocrinology ◽

10.1210/en.2012-1802 ◽

2013 ◽

Vol 154 (2) ◽

pp. 853-864 ◽

Cited By ~ 24

Author(s):

Mazène Hochane ◽

Denis Raison ◽

Catherine Coquard ◽

Olivier Imhoff ◽

Thierry Massfelder ◽

...

Keyword(s):

Parathyroid Hormone ◽

Nuclear Localization ◽

Signal Peptide ◽

Mesangial Cells ◽

Nuclear Localization Sequence ◽

Related Protein ◽

Wild Type ◽

Parathyroid Hormone Related Protein ◽

Proliferation And Apoptosis ◽

Localization Sequence

Glomerulonephritis is characterized by the proliferation and apoptosis of mesangial cells (MC). The parathyroid-hormone related protein (PTHrP) is a locally active cytokine that affects these phenomena in many cell types, through either paracrine or intracrine pathways. The aim of this study was to evaluate the effect of both PTHrP pathways on MC proliferation and apoptosis. In vitro studies were based on MC from male transgenic mice allowing PTHrP-gene excision by a CreLoxP system. MC were also transfected with different PTHrP constructs: wild type PTHrP, PTHrP devoid of its signal peptide, or of its nuclear localization sequence. The results showed that PTHrP deletion in MC reduced their proliferation even in the presence of serum and increased their apoptosis when serum-deprived. PTH1R activation by PTHrP(1–36) or PTH(1–34) had no effect on proliferation but improved MC survival. Transfection of MC with PTHrP devoid of its signal peptide significantly increased their proliferation and minimally reduced their apoptosis. Overexpression of PTHrP devoid of its nuclear localization sequence protected cells from apoptosis without changing their proliferation. Wild type PTHrP transfection conferred both mitogenic and survival effects, which seem independent of midregion and C-terminal PTHrP fragments. PTHrP-induced MC proliferation was associated with p27Kip1 down-regulation and c-Myc/E2F1 up-regulation. PTHrP increased MC survival through the activation of cAMP/protein kinase A and PI3-K/Akt pathways. These results reveal that PTHrP is a cytokine of multiple roles in MC, acting as a mitogenic factor only through an intracrine pathway, and reducing apoptosis mainly through the paracrine pathway. Thus, PTHrP appears as a probable actor in MC injuries.

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