Development of Full-Length Ligand-Activated Split-Kinases and Split-Phosphatases for Interrogation of Signal Transduction Pathways
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PublisherThe University of Arizona.
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EmbargoRelease after 12-Sep-2019
AbstractReversible phosphorylation controls both temporal and spatial activity of proteins within almost all signaling cascades. The human complement of >500 protein kinases and 147 protein phosphatases regulate various cellular events from cell division to cell death. The aberrant function of these proteins is implicated in diseases such as cancer, metabolic disorder and neurodegeneration. Thus, understanding the function of a specific kinase or phosphatase, by turning them on or off, is important for understanding their biology and for designing new therapies. Toward this goal, a few emerging methods have made significant advances towards turning-on or turning-off the activity of a specific kinase. However, current methods do not necessarily allow for orthogonal control over two or more kinases or phosphatases in living cells. The major focus of this dissertation is the development of a new method for turning-on a specific kinase or phosphatase temporally, which can be used for orthogonal control of multiple kinases or phosphatases simultaneously. We hypothesized that if we could design fragmented split-kinases that can be turned on by different ligands then two or more kinases or phosphatases could be temporally controlled. We first developed a sequence dissimilarity-based approach to identify sites in the catalytic domain of kinases tolerant to a 25-residue loop insertion. The successful loop insertion sites, guided the fragmentation of the kinases at these sites into two inactive fragments, which were subsequently attached to two proteins, FKBP and FRB that dimerize in the presence of the small molecule, rapamycin. Previously we have demonstrated that the addition of rapamycin to the designed split-kinases could selectively turn-on enzymatic activity. Moreover we demonstrated that the split-enzyme approach could be extended to protein phosphatases. In this work we have successfully tested and extended the in vitro approach and designed several new split kinases and split-phosphatases. Initial studies were performed in an in vitro setting and focused on the catalytic domain of split-kinases and split-phosphatases. Importantly, we extend our design to full-length proteins and demonstrate that full-length split-kinases and split-phosphatases can be successfully controlled by addition of a small molecule ligand, rapamycin, in live cells. Moreover, the known downstream activity of full-length rapamycin-gated split-kinases, Src and Lyn, and the split-phosphatase, HePTP, were validated within signaling pathways. Thus, this work lays the foundation for future temporal interrogation of kinases and phosphatases implicated in a range of cellular pathways. Another section of the dissertation focuses on an interesting discovery that several of these split-kinases can be mixed and matched, where half of the new chimeric enzyme is derived from one parent kinase and the other half comes from a different parent kinase. This chimeric phenomenon has interesting implications for the evolution and design of protein kinases and the approach can potentially be used to design new chimeric-protein kinases. In summary, this work describes the design of ligand-activated split-kinases and split-phosphatases that can be used to study phosphorylation in living cells and potentially be used to rewire biological circuitry.
Degree ProgramGraduate College