Overlapping binding sites for importin b1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

Anette Szczepny, Kylie M Wagstaff, Manisha Dias, Kasia Anna Gajewska, Chunxiao Wang, Rebecca G Davies, Gurpreet Kaur, Jennifer Dung Ly Huynh, Katherine A L Loveland, David A Jans

Research output: Contribution to journalArticleResearchpeer-review

Abstract

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impbeta1 (importin beta1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impbeta1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impbeta1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impbeta1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impbeta1 for binding to Gli1, through the fact that the sites for SuFu and Impbeta1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impbeta1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.
Original languageEnglish
Pages (from-to)469 - 476
Number of pages8
JournalBiochemical Journal
Volume461
Issue number3
DOIs
Publication statusPublished - 2014

Cite this

@article{d3052d447b11433fa122bade837a3bd0,
title = "Overlapping binding sites for importin b1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization",
abstract = "A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impbeta1 (importin beta1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impbeta1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impbeta1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impbeta1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impbeta1 for binding to Gli1, through the fact that the sites for SuFu and Impbeta1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impbeta1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.",
author = "Anette Szczepny and Wagstaff, {Kylie M} and Manisha Dias and Gajewska, {Kasia Anna} and Chunxiao Wang and Davies, {Rebecca G} and Gurpreet Kaur and {Ly Huynh}, {Jennifer Dung} and Loveland, {Katherine A L} and Jans, {David A}",
year = "2014",
doi = "10.1042/BJ20130709",
language = "English",
volume = "461",
pages = "469 -- 476",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press",
number = "3",

}

Overlapping binding sites for importin b1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization. / Szczepny, Anette; Wagstaff, Kylie M; Dias, Manisha; Gajewska, Kasia Anna; Wang, Chunxiao; Davies, Rebecca G; Kaur, Gurpreet; Ly Huynh, Jennifer Dung; Loveland, Katherine A L; Jans, David A.

In: Biochemical Journal, Vol. 461, No. 3, 2014, p. 469 - 476.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Overlapping binding sites for importin b1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

AU - Szczepny, Anette

AU - Wagstaff, Kylie M

AU - Dias, Manisha

AU - Gajewska, Kasia Anna

AU - Wang, Chunxiao

AU - Davies, Rebecca G

AU - Kaur, Gurpreet

AU - Ly Huynh, Jennifer Dung

AU - Loveland, Katherine A L

AU - Jans, David A

PY - 2014

Y1 - 2014

N2 - A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impbeta1 (importin beta1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impbeta1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impbeta1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impbeta1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impbeta1 for binding to Gli1, through the fact that the sites for SuFu and Impbeta1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impbeta1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

AB - A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impbeta1 (importin beta1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impbeta1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impbeta1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impbeta1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impbeta1 for binding to Gli1, through the fact that the sites for SuFu and Impbeta1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impbeta1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

UR - http://www.biochemj.org/bj/461/0469/4610469.pdf

U2 - 10.1042/BJ20130709

DO - 10.1042/BJ20130709

M3 - Article

VL - 461

SP - 469

EP - 476

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 3

ER -