Investigating NEK10 as a myosin light chain kinase - Mutations in the motor protein Myosin-7A (Myo7A) result in debilitating sensory disorders in humans including the most common form of autosomal recessive deaf-blindness known as Usher Syndrome. Despite its importance for sensory function, there is a fundamental gap in knowledge about how Myo7A activity is regulated in a cellular setting. There is an urgent need to fill this knowledge gap since Myo7A gene augmentation therapy is currently being explored in both pre-clinical and clinical trials to treat Usher Syndrome patients. The long- term goals of this project are to (i) elucidate how Myo7A is being regulated inside cells, and to (ii) determine how perturbations to this regulation result in Myo7A dysfunction that ends in sensory disease. To achieve these long- term goals, the current objectives of this proposal are to (i) characterize the regulation of Myo7A by the myosin light chains that are associated with its lever arm, and (ii) elucidate how sensory disorder mutations within the lever arm disrupt this normal light chain-mediated regulation. A major roadblock in studying Myo7A is the lack of a malleable cell culture model in which the myosin exhibits observable motor-based activity. Indeed, reconstituting robust motor-dependent activity of Myo7A in conventional cell culture models has been a controversial issue in the field. This roadblock has now been circumvented in this proposal with the discovery of a discrete regulatory element in the lever arm of Myo7A that tightly controls its motor-dependent targeting. Excitingly, robust motor-dependent targeting of Myo7A can now be reconstituted in polarized kidney epithelial cells by augmenting how myosin light chains associate with this regulatory element that was discovered. Based on the light chains that bind to this regulatory element, NIMA-related kinase 10 (NEK10) was further identified as a kinase that has the ability to activate Myo7A targeting in polarized epithelial cells. The central hypothesis of this study is that NEK10 acts as a myosin light chain kinase to control Myo7A targeting in cells by modulating the behavior of light chains associated with its lever arm regulatory element, and that sensory disorder mutations found in this regulatory element disrupt this normal regulation. This hypothesis will be tested through two specific aims: Aim 1 will focus on characterizing NEK10 as a myosin light chain kinase for Myo7A in polarized epithelial cells. Aim 2 will explore how Usher Syndrome mutations found within the Myo7A regulatory element disrupt its ability to act as a molecular switch that is normally controlled by NEK10 in polarized epithelial cells. This study is innovative, since it identifies a completely novel regulatory element within the lever arm of a myosin that has never been described before, and further ascribes a new function to the poorly characterized kinase NEK10. The proposed research is significant since it will be the first to define how Myo7A is regulated via its light chains in a cellular setting, and will also provide insight into how specific Usher Syndrome mutations with the Myo7A lever arm disrupt this normal regulation to result in myosin dysfunction.
