Triple SILAC to determine stimulus specific interactions in the Wnt pathway

Area of Science:

• Molecular Biology
• Cell Signaling
• Proteomics

Background:

• Dynamic multiprotein complexes regulate cellular functions and adapt to stimuli.
• Understanding stimulus-dependent protein interactions is crucial for deciphering complex biological processes.
• The Wnt signaling pathway plays vital roles in development, tissue homeostasis, and cancer.

Purpose of the Study:

• To develop and apply a quantitative mass spectrometry approach to identify stimulus-dependent protein interactions.
• To investigate the dynamic interactions of key Wnt signaling components under different pathway activation states.
• To uncover novel protein interactors and regulatory mechanisms within the Wnt pathway.

Main Methods:

• Quantitative affinity purification coupled with mass spectrometry (AP-MS).
• Stable Isotope Labeling by Amino acids in Cell culture (SILAC) in a triple-labeling format for differential quantification.
• Expression of full-length proteins fused to green fluorescent protein (GFP) under endogenous control as baits.
• Application to Wnt signaling pathway components: APC, Axin-1, DVL2, and CtBP2.

Main Results:

• The method successfully distinguished specific interactors from background noise and quantified stimulus-dependent interactions.
• Identified known Wnt signaling complex components and novel candidates, including FAM83B and Girdin.
• Girdin interacts with DVL2 independently of Wnt3a stimulation but binds Axin-1 and APC in a stimulus-dependent manner.
• The core beta-catenin destruction complex showed minimal changes in composition upon Wnt pathway activation.

Conclusions:

• The developed quantitative AP-MS/SILAC approach is effective for characterizing dynamic, stimulus-dependent protein interactions.
• FAM83B and Girdin are identified as novel interactors with multiple Wnt pathway players, suggesting their roles in Wnt signaling regulation.
• While Girdin exhibits stimulus-dependent interactions with key components, the core destruction complex appears relatively stable, highlighting specific regulatory dynamics within the Wnt pathway.