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Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif

PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is foun...

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Autores principales: Anguissola, Sergio, McCormack, William J., Morrin, Michelle A., Higgins, Wayne J., Fox, Denise M., Worrall, D. Margaret
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196545/
https://www.ncbi.nlm.nih.gov/pubmed/22028839
http://dx.doi.org/10.1371/journal.pone.0026234
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author Anguissola, Sergio
McCormack, William J.
Morrin, Michelle A.
Higgins, Wayne J.
Fox, Denise M.
Worrall, D. Margaret
author_facet Anguissola, Sergio
McCormack, William J.
Morrin, Michelle A.
Higgins, Wayne J.
Fox, Denise M.
Worrall, D. Margaret
author_sort Anguissola, Sergio
collection PubMed
description PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-β family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a novel NLS and mechanism for serpinF1 nuclear import, which may be critical for anti-angiogenic and neurotrophic function.
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spelling pubmed-31965452011-10-25 Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif Anguissola, Sergio McCormack, William J. Morrin, Michelle A. Higgins, Wayne J. Fox, Denise M. Worrall, D. Margaret PLoS One Research Article PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-β family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a novel NLS and mechanism for serpinF1 nuclear import, which may be critical for anti-angiogenic and neurotrophic function. Public Library of Science 2011-10-18 /pmc/articles/PMC3196545/ /pubmed/22028839 http://dx.doi.org/10.1371/journal.pone.0026234 Text en Anguissola et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Anguissola, Sergio
McCormack, William J.
Morrin, Michelle A.
Higgins, Wayne J.
Fox, Denise M.
Worrall, D. Margaret
Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title_full Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title_fullStr Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title_full_unstemmed Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title_short Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif
title_sort pigment epithelium-derived factor (pedf) interacts with transportin sr2, and active nuclear import is facilitated by a novel nuclear localization motif
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196545/
https://www.ncbi.nlm.nih.gov/pubmed/22028839
http://dx.doi.org/10.1371/journal.pone.0026234
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