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

Protein kinases are subject to multiple layers of regulation that modulate their activity, localization and substrate specificity. However, the details of kinase regulation are incompletely understood, even for some of the most well-characterized kinases. Here, we measure the functional effect of thousands of single amino acid mutants of Src kinase's catalytic domain to identify residues involved in novel regulatory interactions. A barcoded plasmid library of Src variants was expressed in S. cerevisiae and timepoints were sampled throughout growth. Plasmids were extracted and barcodes amplified and counted with Illumina sequencing. Barcodes were related back to their cognate variant using barcode-variant map.