Triple SILAC to determine stimulus specific interactions in the Wnt pathway.
Many important regulatory functions are performed by dynamic multiprotein complexes that adapt their composition and activity in response to different stimuli. Here we employ quantitative affinity purification coupled with mass spectrometry to efficiently separate background from specific interactors but add an additional quantitative dimension to explicitly characterize stimulus-dependent interactions. This is accomplished by SILAC in a triple-labeling format, in which pull-downs with bait, with bait and stimulus, and without bait are quantified against each other. As baits, we use full-length proteins fused to the green fluorescent protein and expressed under endogenous control. We applied this technology to Wnt signaling, which is important in development, tissue homeostasis, and cancer, and investigated interactions of the key components APC, Axin-1, DVL2, and CtBP2 with differential pathway activation. Our screens identify many known Wnt signaling complex components and link novel candidates to Wnt signaling, including FAM83B and Girdin, which we found as interactors to multiple Wnt pathway players. Girdin binds to DVL2 independent of stimulation with the ligand Wnt3a but to Axin-1 and APC in a stimulus-dependent manner. The core destruction complex itself, which regulates beta-catenin stability as the key step in canonical Wnt signaling, remained essentially unchanged.
Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry , Am Klopferspitz 18, D-82152 Martinsried, Germany.
Maximiliane
M
Hilger
Matthias
M
Mann
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D025601:Adenomatous Polyposis Coli Protein / Q000378:metabolism; D060466:Axin Protein / Q000378:metabolism; D016000:Cluster Analysis; D000072261:Dishevelled Proteins; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D051153:Wnt Proteins / Q000378:metabolism; D060449:Wnt Signaling Pathway
C555451:AXIN1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D025601:Adenomatous Polyposis Coli Protein; D060466:Axin Protein; C000606581:DVL2 protein, human; D000072261:Dishevelled Proteins; D010750:Phosphoproteins; D051153:Wnt Proteins