Cells sense cues in their surrounding environment, turning extracellular stimuli into intracellular responses. Transient posttranslational modifications, such as phosphorylation, often guide these responses through tightly regulated signaling networks.There is great interest in understanding phosphorylation events that yield responses in cell signaling, but to date, methods to characterize the phosphoproteome rely on low-throughput methods. To overcome this limitation, this study optimizes a yeast surface display system for high-throughput screening of kinase-substrate interaction using endoplasmic reticulum (ER) sequestration. We demonstrate the ability of yeast to display phosphorylated full-length receptor cytoplasmic domains and additionally optimized magnetic bead selections to efficiently recover yeast displaying phosphorylated domains. These lessons were applied to profiling the substrate specificity of the tyrosine kinase lymphocyte cell-specific protein-tyrosine kinase by mutating a phosphoacceptor motif within the CD28 cytoplasmic domain and to building multienzyme phosphorylation cascades in the yeast ER. Collectively, this study provides a facile approach for profiling kinase-substrate reactions in high throughput.

No abstract