In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Susan A. Kremer, Naz Erdeniz, Wynne Peterson-Nedry, Elizabeth A. Swanson, Marcel Wehrli

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.

Original languageEnglish (US)
Pages (from-to)110-123
Number of pages14
JournalDevelopmental Biology
Volume337
Issue number1
DOIs
StatePublished - Jan 1 2010

Fingerprint

Armadillos
Catenins
Drosophila
Amino Acids
Axin Protein
Alleles
Two-Hybrid System Techniques
Wnt Signaling Pathway
Diptera
Proteins

Keywords

  • Axin mutations
  • Cancer
  • Drosophila
  • In vivo analysis
  • Wnt/β-catenin signaling

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology
  • Molecular Biology

Cite this

In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin. / Kremer, Susan A.; Erdeniz, Naz; Peterson-Nedry, Wynne; Swanson, Elizabeth A.; Wehrli, Marcel.

In: Developmental Biology, Vol. 337, No. 1, 01.01.2010, p. 110-123.

Research output: Contribution to journalArticle

Kremer, Susan A. ; Erdeniz, Naz ; Peterson-Nedry, Wynne ; Swanson, Elizabeth A. ; Wehrli, Marcel. / In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin. In: Developmental Biology. 2010 ; Vol. 337, No. 1. pp. 110-123.
@article{d2a4c640a1cb4b85a2b31f96abff6ab7,
title = "In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin",
abstract = "Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.",
keywords = "Axin mutations, Cancer, Drosophila, In vivo analysis, Wnt/β-catenin signaling",
author = "Kremer, {Susan A.} and Naz Erdeniz and Wynne Peterson-Nedry and Swanson, {Elizabeth A.} and Marcel Wehrli",
year = "2010",
month = "1",
day = "1",
doi = "10.1016/j.ydbio.2009.10.016",
language = "English (US)",
volume = "337",
pages = "110--123",
journal = "Developmental Biology",
issn = "0012-1606",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

AU - Kremer, Susan A.

AU - Erdeniz, Naz

AU - Peterson-Nedry, Wynne

AU - Swanson, Elizabeth A.

AU - Wehrli, Marcel

PY - 2010/1/1

Y1 - 2010/1/1

N2 - Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.

AB - Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E, retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.

KW - Axin mutations

KW - Cancer

KW - Drosophila

KW - In vivo analysis

KW - Wnt/β-catenin signaling

UR - http://www.scopus.com/inward/record.url?scp=70450222904&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70450222904&partnerID=8YFLogxK

U2 - 10.1016/j.ydbio.2009.10.016

DO - 10.1016/j.ydbio.2009.10.016

M3 - Article

C2 - 19850033

AN - SCOPUS:70450222904

VL - 337

SP - 110

EP - 123

JO - Developmental Biology

JF - Developmental Biology

SN - 0012-1606

IS - 1

ER -