Athol, H. E.; Thompson, A.; O'Connor, N.; Shirts, M. R.; Kralj, J. M.; Fox, J. M.

Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their ability to discriminate between highly similar substrate sequences (i.e., their sensitivity to minor changes in sequence within different regions of a substrate, and the sequence-dependent nature of this sensitivity) remains poorly understood. Here, we use a genetically encoded biosensor for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could enhance selectivity for one PTK over others, confer sensitivity to substrate length, or improve PTK compatibility beyond the consensus. These effects were not predicted by classical specificity maps or advanced molecular modeling tools. Using a dual-selection screen that incorporates decoy substrates, we found additional sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. Our findings show how context-specific sequence features alter PTK substrate specificity far beyond what might be expected from classical consensus models and establish an experimental framework for defining the limits of substrate overlap between closely related kinases.