Protein Engineering Methods to Understand Kinase Signaling and Protein-Protein Interactions

Abstract

Tyrosine phosphorylation is one of the key covalent post-translational modifications through which multicellular organisms communicate. Phosphorylation of tyrosine residues on proteins can modulate enzymatic activity and can create binding sites for the recruitment of downstream signaling proteins. Conservation of enzyme structure across the human kinome makes it difficult to design small molecules to selectively modulate kinase activity, which can help elucidate their roles in signaling pathways and in diseases. To circumvent this limitation, we have developed a split-protein method to control the activity of individual kinases by utilizing chemical inducer of dimerization (CID) proteins. The split-protein approach relies on the identification of viable fragmentation sites in a protein that can be used to generate ligand-gated control of protein activity. The major focus of this dissertation is the identification of new kinase and firefly luciferase fragmentation sites for temporal control of a specific kinase and for the study of protein-protein interactions, respectively. With a sequence dissimilarity-based approach and structure-guided analysis, we successfully identified new split-Src sites, the first split-Syk site, the first split-Abl-1aFL, and new firefly luciferase fragmentation sites utilizing the CIDs of rapamycin and/or abscisic acid. Temporal control of split-Abl-1a coupled with quantitative phosphoproteomics analysis aided in the understanding of Abl-1 cellular signaling. Both known and novel substrates were identified, and validation of novel phosphotyrosine targets like AFDN, AMOT, DDX3X, NCAPH present opportunities for studying and understanding unanticipated functions of Abl-1. In summary, this work describes the split-protein approach to selectively control and dissect the complex signaling processes of specific protein tyrosine kinases (PTKs) and for monitoring PPIs. The new tools developed could potentially be used to rewire signaling pathways and aid in the development of novel therapeutic targets.