Wnt signalosome assembly is governed by conformational flexibility of Axin and by the AP2 clathrin adaptor

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• Wnt signal transduction is crucial for cellular processes and relies on the inhibition of GSK3 by phosphorylated motifs on the LRP6 co-receptor.
• The precise mechanism of GSK3 recruitment to LRP6 has remained an unresolved question in the field.

Purpose of the Study:

• To elucidate the molecular mechanisms governing GSK3 recruitment to LRP6.
• To identify the binding sites and interactions involved in Wnt signal transduction complex formation.

Main Methods:

• Nuclear magnetic resonance (NMR) spectroscopy to identify binding sites.
• Co-immunoprecipitation assays and CRISPR-engineered mutations to study protein interactions.
• Analysis of intrinsically disordered regions and specific motifs (PPPSPxS, LIR, PRTxR).

Main Results:

• NMR identified the membrane-proximal PPPSPxS motif and flanking sequences as the primary binding site for Axin and GSK3.
• An intrinsically disordered segment of Axin functions as the LRP6-interacting region (LIR).
• Axin's LIR exhibits phospho-dependent foldback, and a PRTxR motif facilitates a multi-pronged Axin-GSK3 interaction, antagonizing LRP6 docking.

Conclusions:

• The study reveals a detailed mechanism for GSK3 recruitment to LRP6 via Axin scaffolding.
• Wnt-driven clustering of LRP6 within clathrin-coated pits facilitates Axin-GSK3 complex docking and kinase activity modulation.
• This provides critical insights into the regulation of Wnt signal transduction pathways.