Kinase Regulation of Microtubule Dynamics

Abstract

Microtubules are dynamic polymers that facilitate important cellular functions, including intracellular transport and chromosome segregation. They are remodeled by plus-end polymerization dynamics, which are commonly targeted to treat cancer. The regulation of microtubule polymerization dynamics by kinases has been extensively studied, but the mechanisms driving this regulation are not well understood. This dissertation covers two orthogonal approaches used to explore kinase regulation of interphase microtubule dynamics. Our first approach systematically explored the effects of kinase inhibition on microtubule growth in live cells. We conducted a high-content screen of preclinical, clinical, and approved kinase inhibitors using an automated quantitative pipeline to score EB3-positive growing plus-ends. Our findings suggest that kinase regulation of microtubule growth varies across cell types and is dependent on the basal activation of signaling pathways. In the case of human retinal pigmented epithelial cells, kinases weakly regulate microtubule growth under baseline conditions, and that regulation is stronger under sensitized conditions when growth is partly inhibited by a microtubule depolymerizing drug. Our second approach tested the hypothesis that kinase-phosphatase signaling functions to maintain plus-end dynamics in response to microtubule depolymerization. This hypothesis was informed by phosphoproteomics data showing the rapid dephosphorylation of dozens of microtubule-binding proteins in response to a microtubule depolymerizing drug, including several that are known to stabilize microtubules. We validated one such protein, CLIP1, as a biomarker of microtubule stability. Our data suggest that CLIP1 phosphorylation may be a viable tool to discover a kinase and/or phosphatase that responds to microtubule depolymerization. In addition to our contributions to fundamental microtubule biology, our work also has implications in translational drug discovery. Our high-content screening approach is useful for quantifying off-target inhibition of tubulin by kinase inhibitors, which is a common challenge in drug development.