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
N1 - Funding Information:
We are very grateful to Jackie Parker for editing, Drs. David Morton and Jan Christian for comments on the manuscript. We also acknowledge the Bloomington Stock Center and the Developmental Studies Hybridoma Bank (DSHB) for providing us with fly stocks and antibodies, respectively. We thank Drs. Jan Christian, Philip Copenhaver, Richard Maurer, Caroline Enns, Sarah Smolik, Mike Forte and David Morton for discussions and advice. Kaitlin Leonard and Jean Kitzman provided excellent technical assistance. This work was supported by NIH grant GM67029 to M.W.
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
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U2 - 10.1016/j.ydbio.2009.10.016
DO - 10.1016/j.ydbio.2009.10.016
M3 - Article
C2 - 19850033
AN - SCOPUS:70450222904
SN - 0012-1606
VL - 337
SP - 110
EP - 123
JO - Developmental Biology
JF - Developmental Biology
IS - 1
ER -