Mammalian cell signaling relies on the rapid assembly and disassembly of protein complexes containing enzymes, scaffolding proteins and downstream effectors. The co-localization of proteins into complexes provides increased signaling fidelity and diversifies cellular signaling behaviors. Despite their importance, our understanding of the intra-cellular composition of signaling complexes during specific signaling events remains incomplete. To address this need, we have developed a suite of affinity reagents that allow selective enrichment of activated signaling complexes from cell lysates. These reagents selectively enrich signaling complexes that contain both a protein kinase and a specified post-translational modification (PTM). Use of these reagents with SILAC-based quantitative proteomics allows multiple signaling complexes to be studied in parallel. Our progress in using these reagents to interrogate growth factor-stimulated signaling cascades and the intracellular wiring of different cancer cell lines will be presented.

Affinity reagents for studying active signaling complexes

251st National Meeting (2016)

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