In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3beta or beta-catenin.
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 (AxinDeltaSgg) or Armadillo (AxinDeltaArm) 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 AxinDeltaSgg 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.
Department of Cell and Developmental Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
Susan A
SA
Kremer
Naz
N
Erdeniz
Wynne
W
Peterson-Nedry
Elizabeth A
EA
Swanson
Marcel
M
Wehrli
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D051186:Armadillo Domain Proteins / Q000378:metabolism / Q000502:physiology*; D060466:Axin Protein; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004331:Drosophila melanogaster / Q000196:embryology*; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D008969:Molecular Sequence Data; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D051155:Wnt1 Protein / Q000502:physiology
C495305:ARM protein, Drosophila; D048868:Adaptor Proteins, Signal Transducing; D051186:Armadillo Domain Proteins; D060466:Axin Protein; C118000:Axn protein, Drosophila; D029721:Drosophila Proteins; D014157:Transcription Factors; D051155:Wnt1 Protein; C074622:wg protein, Drosophila; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3