Endoplasmic reticulum sequestration empowers phosphorylation profiling on the yeast surface

Abstract

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.

Leveraging yeast sequestration to study and engineer posttranslational modification enzymes

“…Importantly, it was shown that ponatinib remained effective against most single mutants of ABL1 kinase, with drug resistance starting only with rare compound mutations. Ezagui et al (2022) recently harnessed ER sequestration to measure tyrosine phosphorylation cascades using full‐length protein substrates. Using a modified version of the YESS plasmid containing a ribosomal skipping sequence (T2A), the protein‐tyrosine kinase (LCK) is expressed and activates ZAP‐70, an intermediate enzyme, through phosphorylation of its catalytic loop.…”

Leveraging yeast sequestration to study and engineer post-translational modification enzymes.

“…Importantly, Taft showed that ponatinib remained effective against most single mutants of ABL1 kinase, with drug resistance starting only with rare compound mutations. Stern and colleagues recently harnessed ER sequestration to measure tyrosine phosphorylation cascades using full-length protein substrates (Ezagui et al, 2022). Using a modified version of the YESS plasmid containing a ribosomal skipping sequence (T2A), the protein-tyrosine kinase (LCK) is expressed and activates ZAP-70, an intermediate enzyme, through phosphorylation of its catalytic loop.…”

Modular and integrative activity reporters enhance biochemical studies in the yeast ER

“…Furthermore, YESS allows the user to measure enzyme activities directly on the cell surface, rather than transcriptional signal amplification (Sanchez and Ting 2020). YESS is an all-in-one platform that allows one to engineer and profile the substrate specificity of PTM-enzymes while also enabling highthroughput drug screening deep mutational scanning assays (Yi, Gebhard et al 2013, Yi, Taft et al 2015, Ezagui, Russell et al 2022.…”

“…Additionally, it has been used in conjunction with machine learning to predict the substrate preference and binding energy of hepatitis C virus (HCV) protease (Pethe, Rubenstein et al 2019). More recently, YESS was used to assay histone acetyltransferase activities (Waldman, Rao et al 2021) and crosstalk and to recapitulate mammalian phosphorylation cascades (Ezagui, Russell et al 2022).…”