Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases

Kinase-templated abiotic reaction

Protein kinases are quintessential regulators of cellular function. Numerous pathologies are intimately linked to the dysregulated activity of a particular protein kinase. Herein we report a technology based on a proximity-induced chemical transformation that enables the detection and imaging of specific kinases. Using two probes that target the nucleotide-binding site and substrate binding site of a target kinase respectively, the reagents appended on the probes are brought within reactive distance thereby enabling the chemical transformation. The reaction used for sensing is a ruthenium-photocatalyzed reduction of a pyridinium immolative linker, which uncages a fluorophore (rhodamine). We demonstrate that this technology can be used to discriminate between closely related kinases with a high signal to noise ratio. We further demonstrate that the technology operates within the complexity of a cellular context with a good correlation between the level of kinase activity and fluorescence output

PNA-Encoded Synthesis (PES) and DNA Display of Small Molecule Libraries

DNA-encoded library technologies have emerged as a powerful platform to rapidly screen for binders to a protein of interest. These technologies are underpinned by the ability to encode a rich diversity of small molecules. While large libraries are accessible by cycles of mix and split synthesis, libraries based on single chemistries tend to be redundant. Furthermore, the quality of libraries generally decreases with the number of synthetic transformations performed in its synthesis. An alternative approach is to use hybridization to program the combinatorial assembly of fragment pairs onto a library of DNA templates. A broad molecular diversity is more easily sampled since it arises from the pairing of diverse fragments. Upon identification of productive fragment pairs, a focused library covalently linking the fragments is prepared. This focused library includes linker of different length and geometry and offers the opportunity to enrich the selected fragment set with close neighbors. Herein we describe detailed protocols to covalently link diverse fragments and screen fragment-based libraries using commercially available microarray platform