In Silico Design and in Vitro Characterization of Universal Tyrosine Kinase Peptide Substrates

A majority of the 90 human protein tyrosine kinases (PTKs) are understudied “orphan” enzymes with few or no known substrates. Designing experiments aimed at assaying the catalytic activity of these PTKs has been a long-running problem. In the past, researchers have used polypeptides with a randomized 4:1 molar ratio of glutamic acid to tyrosine as general PTK substrates. However, these substrates are inefficient and perform poorly for many applications. In this work, we apply the KINATEST-ID pipeline for artificial kinase substrate discovery to design a set of candidate “universal” PTK peptide substrate sequences. We identified two unique peptide sequences from this set that had robust activity with a panel of 15 PTKs tested in an initial screen. Kinetic characterization with seven receptor and nonreceptor PTKs confirmed these peptides to be efficient and general PTK substrates. The broad scope of these artificial substrates demonstrates that they should be useful as tools for probing understudied PTK activity.