Membrane localization of adenomatous polyposis coli protein at cellular protrusions: Targeting sequences and regulation by β-catenin

Manisha Sharma, Louie Leung, Mariana Brocardo, Jasmine Henderson, Cameron Flegg, Beric R. Henderson

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo-(binds KAP3A, B56α, and ASEF) and β-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of β-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of β-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of β-catenin reduced APC atmembrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with β-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only β-catenin stimulated APC anchorage at the membrane. Our findings identify β-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

LanguageEnglish
Pages17140-17149
Number of pages10
JournalJournal of Biological Chemistry
Volume281
Issue number25
DOIs
Publication statusPublished - 23 Jun 2006
Externally publishedYes

Fingerprint

Adenomatous Polyposis Coli Protein
Cell Surface Extensions
beta Catenin
Membranes
Protein Transport
Armadillos
Microtubules
Cell Movement

Cite this

Sharma, Manisha ; Leung, Louie ; Brocardo, Mariana ; Henderson, Jasmine ; Flegg, Cameron ; Henderson, Beric R. / Membrane localization of adenomatous polyposis coli protein at cellular protrusions : Targeting sequences and regulation by β-catenin. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 25. pp. 17140-17149.
@article{c8afc8d6383e45beb1e34f7939a121ef,
title = "Membrane localization of adenomatous polyposis coli protein at cellular protrusions: Targeting sequences and regulation by β-catenin",
abstract = "Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo-(binds KAP3A, B56α, and ASEF) and β-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of β-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of β-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of β-catenin reduced APC atmembrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with β-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only β-catenin stimulated APC anchorage at the membrane. Our findings identify β-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.",
author = "Manisha Sharma and Louie Leung and Mariana Brocardo and Jasmine Henderson and Cameron Flegg and Henderson, {Beric R.}",
year = "2006",
month = "6",
day = "23",
doi = "10.1074/jbc.M513027200",
language = "English",
volume = "281",
pages = "17140--17149",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "25",

}

Membrane localization of adenomatous polyposis coli protein at cellular protrusions : Targeting sequences and regulation by β-catenin. / Sharma, Manisha; Leung, Louie; Brocardo, Mariana; Henderson, Jasmine; Flegg, Cameron; Henderson, Beric R.

In: Journal of Biological Chemistry, Vol. 281, No. 25, 23.06.2006, p. 17140-17149.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Membrane localization of adenomatous polyposis coli protein at cellular protrusions

T2 - Journal of Biological Chemistry

AU - Sharma, Manisha

AU - Leung, Louie

AU - Brocardo, Mariana

AU - Henderson, Jasmine

AU - Flegg, Cameron

AU - Henderson, Beric R.

PY - 2006/6/23

Y1 - 2006/6/23

N2 - Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo-(binds KAP3A, B56α, and ASEF) and β-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of β-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of β-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of β-catenin reduced APC atmembrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with β-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only β-catenin stimulated APC anchorage at the membrane. Our findings identify β-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

AB - Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo-(binds KAP3A, B56α, and ASEF) and β-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of β-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of β-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of β-catenin reduced APC atmembrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with β-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only β-catenin stimulated APC anchorage at the membrane. Our findings identify β-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

UR - http://www.scopus.com/inward/record.url?scp=33745200754&partnerID=8YFLogxK

U2 - 10.1074/jbc.M513027200

DO - 10.1074/jbc.M513027200

M3 - Article

VL - 281

SP - 17140

EP - 17149

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 25

ER -