title: High-Throughput Analysis of Mammalian Receptor Tyrosine Kinase Activation in Yeast Cells.
abstract: Tyrosine phosphorylation is an essential posttranslational modification in intracellular signaling molecules. Since tyrosine phosphorylation occurs in less than 0.1 % of all phosphorylated amino acids in mammalian cells, it is difficult to detect the nascent phosphotyrosine at a high signal-to-noise ratio due to high intracellular backgrounds (i.e., unexpected crosstalks among endogenous signaling molecules). In order to address this issue, we reconstituted the mammalian signaling pathway involving an extracellular ligand and a receptor tyrosine kinase (RTK) in Saccharomyces cerevisiae, a lower eukaryote that lacks endogenous tyrosine kinases. In this chapter, we describe a method for high-throughput analysis of ligand-receptor interaction by combining the yeast cell-surface display technique with an automated single-cell analysis and isolation system. Yeast cells coexpressing the cell-wall-anchored form of the human epidermal growth factor (EGF) and the human EGF receptor (EGFR) fused with a signal peptide at the N terminus facilitated the interaction of EGF with EGFR in an autocrine manner, followed by EGFR oligomerization and subsequent autophosphorylation. Furthermore, yeast cells expressing cell-wall-anchored forms of a conformationally constrained random peptide library instead of EGF are treated with a fluorophore-labeled anti-phosphorylated EGFR antibody and then subjected to the automated single-cell analysis and isolation system. The yeast cells with the highest level of fluorescence were shown to display novel and efficient EGFR agonistic peptides. Thus, our yeast display technique serves as a quantitative measurement for RTK activation, which is applicable to high-throughput de novo screening of RTK agonistic peptides.
authors.affiliation: Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan. n-yosi44@sanken.osaka-u.ac.jp.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Yoshimoto
authors.affiliation: Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D015870:Gene Expression; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008024:Ligands; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D020794:Receptor Protein-Tyrosine Kinases / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases
keywords: Automated single-cell analysis and isolation system; Cell-surface display; Conformationally constrained random peptide library; EGFR; RTK; Tyrosine phosphorylation; Yeast