Decoding protein kinase signaling during mitosis: Exploiting molecular scaffolds and developing drug-targeting tools for studying local kinase biology

Abstract

Fundamental cellular processes such as cell division, migration, differentiation, and growth require environmental signals to be converted into chemical responses that generate biological outputs. Accordingly, these events rely on the precise and timely regulation of signaling proteins inside the cell. Organization of enzymes in space and time becomes particularly important in a process such as mitosis where regulation of mitotic protein kinases ensures that DNA is properly segregated between the two daughter cells. During mitosis, two key protein kinases, Aurora A and polo-like kinase 1 (Plk1) carry out many functions to ensure the fidelity of cell division. Current approaches in which small-molecule kinase inhibitor drugs are used to ascribe function to Aurora A and Plk1 provide important insight into the global roles of these proteins inside the cell. However, since these enzymes share similar localization patterns and many overlapping roles, knowing their individual contributions at specific locations is not possible with existing pharmacological approaches. This is because traditional inhibitor drugs distribute throughout the cell, inhibiting enzymes at many locations, and thus limit the ability to resolve how a protein functions at a distinct organelle. Therefore, decoding the roles of Aurora A and Plk1 at distinct subcellular locations is critical for understanding how cell division is regulated. More importantly, elucidating local kinase action can uncover how signaling becomes dysregulated in disease, thus paving the way for development of more effective therapeutics. Using a combination of chemical genetics, gene-editing, super-resolution microscopy, live-cell imaging, and biochemistry the studies in this thesis uncover how Aurora A and Plk1 coordinate signaling events at distinct locations in the cell during mitosis. In this work, I exploit the local scaffolding ability of Gravin, an A-kinase anchoring protein (AKAP), to study how anchored pools of Aurora A and Plk1 facilitate mitotic events. Additionally, I develop a new drug-targeting system called LoKI (Local Kinase Inhibition) to deliver Aurora A and Plk1 inhibitors to specific subcellular locations to further probe local kinase action. My studies uncover that Gravin localizes active pools of Aurora A and Plk1 at mitotic centrosomes and show that loss of local kinase activity at this location attenuates substrate phosphorylation, produces mitotic spindle defects, and prolongs mitosis. I also discover a new role for this anchoring protein in coordinating the recruitment of γ-tubulin during mitosis and demonstrate that loss of Gravin disrupts protein-protein interactions that facilitate the proper targeting of this key microtubule nucleating component. Together, these studies elucidate how local kinase action drives mitosis and provide evidence that subcellular targeting of protein kinases is a molecular mechanism that underlies precise execution of critical protein phosphorylation events inside cells.