The fitness cost of spurious phosphorylation

Abstract

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known, but quantifying this is required to understand the constraints faced by cell systems as they evolve. Here, we use the model organismS. cerevisiaeto inducibly express tyrosine kinases. Because yeast lacksbona fidetyrosine kinases, most of the resulting tyrosine phosphorylation is spurious. This provides a suitable system to measure the impact of artificial protein interactions on fitness. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ∼30,000 phosphosites mapping to ∼3,500 proteins. Examination of the fitness costs in each strain revealed a strong correlation between the number of spurious pY sites and decreased growth. Moreover, the analysis of pY effects on protein structure and on protein function revealed over 1000 pY events that we predict to be deleterious. However, we also find that a large number of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species withbona fidetyrosine kinases. Taken together, our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

The allosteric landscape of the Src kinase

“…Src was the first discovered tyrosine kinase, and the first identified, cloned, and sequenced oncogene 27,41 . The enzymatic activity of variants of Src is easy to measure using a highly-validated cellular toxicity assay where the inhibition of cellular growth is directly proportional to the amount of Src-induced protein phosphorylation 42–44 . The solubility of Src can also be quantified in the same cells using a highly-validated protein abundance selection assay, abundancePCA (aPCA), that uses protein fragment complementation to quantify soluble protein concentration over at least three orders of magnitude 12,30,40 .…”

“…Suitable 13 selections exist for many different enzymatic activities, including microfluidic 32 and growth-based complementation 34 and toxicity assays 33 . Indeed, the specific assay employed in this study can likely be used to chart allosteric maps for many different protein kinases 43,67 , including tens of different human cancer genes. This will allow fundamental questions about the conservation and evolution of allosteric sites in protein families to be addressed.…”

“…We quantified the kinase activity and abundance of each genotype in 30 separate pooled selection assays. First, we quantified kinase activity in triplicate using kinase-dependent impairment of cell growth [42][43][44][45] (Figure 1a,b). Activity scores were highly reproducible for all five library tiles (median Pearson's r = 0.90) and strongly correlated with Src-dependent tyrosine phosphorylation 42,43 ( r = -0.89, Figure 1c, Figure S1a,b).…”