Yeast can accommodate phosphotyrosine: v-Src toxicity in yeast arises from a single disrupted pathway

Abstract

Tyrosine phosphorylation is a key biochemical signal that controls growth and differentiation in multicellular organisms. Saccharomyces cerevisiae and nearly all other unicellular eukaryotes lack intact phosphotyrosine signaling pathways. However, many of these organisms have primitive phosphotyrosine-binding proteins and tyrosine phosphatases, leading to the assumption that the major barrier for emergence of phosphotyrosine signaling was the negative consequences of promiscuous tyrosine kinase activity. In this work, we reveal that the classic oncogene v-Src, which phosphorylates many dozens of proteins in yeast, is toxic because it disrupts a specific spore wall remodeling pathway. Using genetic selections, we find that expression of a specific cyclic peptide, or overexpression of SMK1, a MAP kinase that controls spore wall assembly, both lead to robust growth despite a continuous high level of phosphotyrosine in the yeast proteome. Thus, minimal genetic manipulations allow yeast to tolerate high levels of phosphotyrosine. These results indicate that the introduction of tyrosine kinases within single-celled organisms may not have been a major obstacle to the evolution of phosphotyrosine signaling.

Parallel Chemoselective Profiling for Mapping Protein Structure

“…Site saturation mutagenesis was employed to generate single amino acid variants of Src's kinase domain in the context of full-length enzyme (3,506, $70% of possible mutants were made and assayed). This mutant library was adapted to a yeast growth assay (Kritzer et al, 2018). Once transformed into S.…”

A Combined Approach Reveals a Regulatory Mechanism Coupling Src’s Kinase Activity, Localization, and Phosphotransferase-Independent Functions

“…To identify residues important for regulating Src's activity, we used deep mutational scanning (DMS) (Fowler and Fields, 2014;Fowler et al, 2010), adapting an assay based on S. cerevisiae growth (Figure 2A) (Kritzer et al, 2018). Yeast provides a eukaryotic cellular environment to probe intrinsic regulation in the absence of extrinsic regulatory factors, with growth rates correlating to overall levels of cellular phosphotyrosine (Figures 2B and 2C).…”

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to catalytic domain dynamics

“…A yeast-based growth assay for profiling drug resistance. To comprehensively profile mechanisms of inhibitor resistance in Src, we used an assay that relies on the correlation between Src's phosphotransferase activity and its toxicity in S. cerevisiae (Ahler et al, 2019;Brugge et al, 1987;Kritzer et al, 2018). We reasoned that it would be possible to determine the drug sensitivity of individual Src variants by measuring Src-mediated yeast toxicity in the presence of various ATP-competitive inhibitors (Figure 1A).…”