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Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization
1Division of Cell Biology, Max-Planck Institute for Developmental Biology, 72076 Tübingen, Germany.
Abstract:
Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.
Insights
Axin protein regulates embryonic axis formation by inhibiting Wnt signaling. Key domains for its function involve binding to GSK3beta and APC, crucial for beta-catenin regulation.
Area of Science:
- Developmental Biology
- Molecular Biology
- Cell Signaling
Background:
- Axin is a key regulator of embryonic axis induction in vertebrates.
- It functions within the Wnt signal transduction pathway, inhibiting its activity.
- Axin forms a complex with glycogen synthase kinase 3beta (GSK3beta), beta-catenin, and adenomatous polyposis coli (APC).
Purpose of the Study:
- To investigate the specific roles of different Axin protein domains in embryonic axis formation.
- To determine how Axin domains influence beta-catenin levels and Wnt signaling.
- To understand the localization patterns of Axin and its interactions with other signaling proteins.
Main Methods:
- Epistasis experiments in frog embryos.
- Analysis of Axin domain function through protein interaction studies.
- Coexpression of tagged Axin and Dishevelled (Dsh) proteins to study localization and interaction.
Main Results:
- The regulators of G-protein signaling (RGS) domain and GSK3beta-binding site of Axin are essential for its function.
- COOH-terminal sequences, including the protein phosphatase 2A (PP2A) binding site and DIX domain, are not critical.
- Axin lacking a direct beta-catenin binding site can still regulate beta-catenin levels and axis formation indirectly.
- NH2-terminal sequences are required for Axin localization, while COOH-terminal sequences enhance spot localization.
- Dishevelled (Dsh) colocalizes with Axin, suggesting interaction with the Axin/APC/GSK3/beta-catenin complex.
Conclusions:
- Interaction with APC and GSK3beta is critical for Axin's regulation of beta-catenin signaling in embryonic cells.
- Specific Axin domains dictate its role in axis formation and protein complex formation.
- Axin localization is dependent on its N-terminal sequences, with C-terminal sequences influencing specific localization patterns.
- Dishevelled may modulate the activity of the Axin/APC/GSK3/beta-catenin complex through direct interaction.