A functional bipartite nuclear localisation signal in the cytokine interleukin‐5

Jans, David A.; Briggs, Lyndall J.; Gustin, Sonja E.; Jans, Patricia; Ford, Sally C.; Young, Ian G.

doi:10.1016/s0014-5793(97)00293-7

Cited by 40 publications

(39 citation statements)

References 41 publications

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“…These results were not attributable to partial denaturation of the RB-Bip-␤-Gal preparation as indicated by the results for the ␤-galactosidase ELISA (see also "Materials and Methods"), which is routinely used to standardize results for equivalent amounts of native protein (30). We have obtained very similar results for an IL-5 bipartite NLS containing ␤-galactosidase fusion protein (27), so that it seems reasonable to conclude that the results for RB-Bip-␤-Gal do not represent an artifact of either the RB-Bip-␤-Gal protein preparation or the ELISA-based binding assay. NLS masking within the ␤-galactosidase tetramer can also not be the basis of the differences in B max , since the four subunits of ␤-galactosidase are structurally identical (see Ref.…”

Section: Affinity Of Rb-bip-␤-gal For the Nls-binding Importin 58/97supporting

confidence: 66%

“…In the case of microinjection, HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (26,27,30). Reticulocyte lysate (Promega) was used as the source of cytosol for the in vitro assay (27,29,33). Image analysis of CLSM files using the NIH Image public domain software and curve fitting were performed as described (26,33).…”

Section: Methodsmentioning

confidence: 99%

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Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo andin Vitro

Efthymiadis¹,

Shao²,

Hübner³

et al. 1997

Journal of Biological Chemistry

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110

198

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The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860 -877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used ␤-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target ␤-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTP␥S. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.All passive and active transport into and out of the nucleus occurs through the nuclear pore complex (NPC) 1 (1-3). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (4) to be targeted to the nucleus. NLSs are defined as the sequences sufficient and necessary for nuclear import of their respective proteins (5-7) and are generally functional in targeting heterologous, normally cytoplasmic proteins to the nucleus. NLS-dependent protein transport can be divided into two steps. The first is energy-independent and involves recognition and targeting of the NLS-containing protein to the NPC by a heterodimeric protein complex consisting of importin 58/97 (8, 9) or ␣/␤ (10, 11). The second, energy-dependent step is the translocation of the NLS-bearing protein through the NPC into the nucleus (12, 13) and requires GTP hydrolysis mediated by the GTP-binding protein Ran/TC4 (14 -16) and the interacting factor p10/NTF2 (17-19). We were interested in the nuclear import kinetics of tumor suppressor proteins, one of which is the retino...

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Section: Affinity Of Rb-bip-␤-gal For the Nls-binding Importin 58/97supporting

confidence: 66%

Section: Methodsmentioning

confidence: 99%

Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo andin Vitro

Efthymiadis¹,

Shao²,

Hübner³

et al. 1997

Journal of Biological Chemistry

Self Cite

110

198

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“…It could be blocked by an excess of either unlabeled PTHrP 1-108 (Fig. 2) or unlabeled PTHrP 1-34 (not shown), which is responsible for PTHrP binding to the PTHrP receptor, indicating that the uptake was receptor-mediated and thus comparable with that of other nuclear localizing ligands such as interleukin-5 (35) and growth hormone (36).…”

Section: Pthrp Localizes In the Nucleus After Endocytosis By Intactmentioning

confidence: 92%

“…2). The precise details of the pathway by which PTHrP is able to localize in the nucleus subsequent to receptor-mediated endocytosis are incomplete at this stage, but the speed of nuclear/ nucleolar accumulation (detectable within 35 min) implies that a lysosomal pathway is unlikely to be involved, as is the case for receptor-mediated uptake/nuclear localization of growth hormone (36), interleukin-5 (35), and other polypeptide ligands (49,50). In analogous fashion to growth hormone and interleukin-5, it seems possible that PTHrP is internalized, rapidly escapes from the endosomal vesicle by an as yet undetermined mechanism (see Ref.…”

Section: Nls Binding Parameters Of Pthrp Derivatives As Measured Usinmentioning

confidence: 99%

Importin β Recognizes Parathyroid Hormone-related Protein with High Affinity and Mediates Its Nuclear Import in the Absence of Importin α

Lam¹,

Briggs²,

Hu³

et al. 1999

Journal of Biological Chemistry

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200

215

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Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/ paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin ␤ and not by importin ␣. The sequence of PTHrP responsible for binding was mapped to amino acids 66 -94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of ϳ2 nM for importin ␤). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin ␤, together with the GTPbinding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin ␣, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin ␣ and perhaps also of NTF2.Parathyroid hormone-related protein (PTHrP) 1 is expressed in a range of tumors and is an endocrine agent in humoral hypercalcemia of malignancy. It is also expressed in many normal tissues where it exerts autocrine/paracrine or intracrine actions (1-6). The structural similarities to parathyroid hormone (PTH) at the amino terminus of PTHrP are sufficient to confer functions similar to those of PTH, mediated by the shared PTH/PTHrP receptor and its ability to activate adenylate cyclase. Although both PTH and PTHrP promote bone resorption and reduce renal calcium excretion (1, 7), roles in the regulation of placental calcium transport to the fetus (1, 7, 8), osteoclast inhibition (9, 10), and the regulation of cell growth and apoptosis (2, 11, 12) have been ascribed to distinct regions of PTHrP.We and others have recently shown that PTHrP is expressed in a cell cycle-dependent manner (13, 14) as well as being localized to the nucleus/nucleolus at G 1 (14). Regulation of the nuclear localization of PTHrP appears to be mediated through phosphorylation by the cyclin-dependent kinases p33 cdk2 and p34 cdc2 . 2 These observations are consistent with the idea that cell cycle-dependent regulation of nuclear localization of PTHrP is central to the control of growth and apoptosis (2, 4). Of significance in this regard is our observation 2 that within amino acids 61-93, PTHrP retains a putative CcN motif, originally described for the SV40 large tumor antigen (T-ag; Refs. 16 and 17), comprising consensus protein kinase CK2 (S 61 DDE and ET 78 KYE-"C") and con...

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“…The functional significance of nuclear PRL was demonstrated by the finding that a nuclear-targeted construct of PRL containing the simian virus 40 large T antigen nuclear localization sequence provided a necessary comitogenic stimulus for IL-2-driven growth (19). The nuclear retrotransport and potential action of peptide hormones and growth factors is widespread, as epidermal growth factor, insulin, growth hormone, platelet-derived growth factor, IL-5, and others have been noted within the nucleus (20)(21)(22)(23). However, the mechanism of nuclear function has remained uncertain.…”

mentioning

confidence: 99%

The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer

Rycyzyn

Clevenger

2002

Proc. Natl. Acad. Sci. U.S.A.

140

124

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The nuclear translocation of peptide hormones, such as the somatolactogenic hormone prolactin, after receptor internalization has been widely reported. Prolactin has been demonstrated to interact with cyclophilin B, a member of the immunophilin family of proteins. Cyclophilin B interaction with prolactin potentiated prolactin-induced proliferation, cell growth, and the nuclear retrotransport of prolactin. These effects could be abrogated by the removal of the peptidyl-prolyl isomerase activity of cyclophilin B. Our findings indicate that the intranuclear prolactin͞cyclophilin B complex acts as a transcriptional inducer by interacting directly with Stat5, resulting in the removal of the Stat-repressor protein inhibitor of activated Stat 3 (PIAS3), thereby enhancing Stat5 DNA-binding activity and prolactin-induced, Stat5-mediated gene expression.T he pleiotropic actions of the somatolactogenic hormone prolactin (PRL) are mediated by signaling through the prolactin receptor, a member of the type I family of cytokine receptors (1-3). Upon ligand binding and dimerization of the receptor, several signaling cascades are activated, including Ras-Raf, Fyn-Vav and Jak2-Stat5 (4-7). Activated Jak2 phosphorylates Stat5 on tyrosine residues, inducing Stat5 dimerization and translocation to the nucleus (8). Intranuclear Stat5 binds to consensus Stat5 response elements, resulting in the transactivation of numerous PRL-specific genes, of which ␤-casein is the most extensively studied (9). This Stat5 transcriptional activation can be cooperatively enhanced by the glucocorticoid receptor (GR) and C͞EBP␤ (10-12).While the above-mentioned pathways are all associated with PRL-induced signaling, activation of the PRL receptor is also associated with ligand internalization via an endosomal-like pathway across the endoplasmic reticulum (ER) and nuclear envelopes (13,14). The phenomenon of protein retrotransport was initially characterized through the study of retrotranslocated viral and bacterial proteins and peptides destined for presentation on the major histocompatibility complex (15-18). These studies revealed that protein retrotransport depends on transport through the protein-conducting channel formed by the Sec61 complex in the ER membrane. Electron microscopy studies employing colloidal gold-labeled PRL demonstrated that upon ligand internalization into endosomes, approximately 90% of the internalized PRL either remained within the endosome or was degraded. However, the remaining 10% was detected as passing from the endosome through multivesicular bodies and the Golgi͞ER to arrive in the nucleus within 2 h poststimulation (13). The functional significance of nuclear PRL was demonstrated by the finding that a nuclear-targeted construct of PRL containing the simian virus 40 large T antigen nuclear localization sequence provided a necessary comitogenic stimulus for IL-2-driven growth (19). The nuclear retrotransport and potential action of peptide hormones and growth factors is widespread, as epidermal growth factor, insulin, grow...

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A functional bipartite nuclear localisation signal in the cytokine interleukin‐5

Cited by 40 publications

References 41 publications

Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo andin Vitro

Kinetic Characterization of the Human Retinoblastoma Protein Bipartite Nuclear Localization Sequence (NLS) in Vivo andin Vitro

Importin β Recognizes Parathyroid Hormone-related Protein with High Affinity and Mediates Its Nuclear Import in the Absence of Importin α

The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer

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