(61b) Endoplasmic Reticulum Sequestration Empowers Phosphorylation Profiling on the Yeast Surface

Cells sense cues in their surroundings using a variety of receptor proteins, which turn an extracellular recognition event into an intracellular response. In many cases, the currency of intracellular response is transient post-translational modification. Chief among these modifications is phosphorylation, the addition of a phosphate to a serine, threonine, or tyrosine residue. There is great interest in understanding phosphorylation/dephosphorylation events that yield responses in cell signaling networks, but to date, methods to characterize the phosphoproteome and interactome rely on challenging low-throughput methods. In this talk, we will discuss our recent progress in developing and applying a yeast surface display system for high-throughput screening of kinase/substrate reactions using an endoplasmic reticulum co-localization strategy. We will demonstrate the ability to 1) display full receptor intracellular domains on the yeast surface, 2) robustly enrich phosphorylated intracellular domains from dilute pools using magnetic selections, 3) profile substrate specificities for a tyrosine kinase, and 4) demonstrate the ability to co-localize multiple units, leading to cascade-dependent phosphorylation of an intracellular substrate. Further progress in generalizing this system will also be discussed.