Despite the discovery that molecular motors are phosphorylated 25 years ago, fundamental questions on the identity of the protein kinase(s) or the particular phosphorylation sites, and how they function to control motors remain unanswered. Since kinase cascades display considerable crosstalk and play multiple roles in cell home- ostasis, deciphering which kinase is involved in a particular process has been difficult. Further, there is some debate as to the extent to which phosphorylation inhibits or stimulates intracellular transport, the extent regulatory mechanisms are conserved between species, and how in vitro mechanisms translate to in vivo systems. Thus, what is lacking is a cohesive strategy to successfully unravel how phosphorylation contributes to the spatial and temporal regulatory mechanisms that control intracellular transport in vivo, without which targeting effective treat- ments to a pathway that is likely disrupted early in disease such as cancer or neurodegeneration is unattainable. The long-term goal is to identify the cellular/molecular mechanisms involved in the regulation of intracellular transport in vivo. The overall objective is to develop an in vivo platform to tease out how a specific kinase controls motor function by identifying the precise functional sites involved, and by isolating the regulatory steps from a complex network of mechanisms. The central hypothesis is that the kinase glycogen synthase kinase-3beta (GSK3b) differentially phosphorylates particular sites on kinesin-1 to control intracellular transport in vivo. The rationale for the proposed research is that once the in vivo mechanisms of how GSK3b is involved in kinesin- mediated transport are known, the field will be a step closer to identifying the complex mechanisms that govern the motility of numerous cellular cargoes on MT tracks for their delivery to distal sites. Guided by strong prelimi- nary data, this hypothesis will be tested by pursuing the specific aim; identify that GSK3b-regulates kinesin-1 function during intracellular transport in vivo. Two objectives will be pursued; generate heritable GSK3b phos- phorylation defective/active KHC/KLC fly lines using the CRISPR/Cas system (Objective 1), and identify the in vivo mechanisms of how GSK3b-mediated phosphorylation controls kinesin-1 function during intracellular transport (Objective 2). The experimental strategy used employs an already proven in vivo approach, coupled with Drosophila genetics, integrated with biochemical analysis and biophysical paradigms. This methodology is innovative in the applicant?s opinion, because it departs from the status quo by enabling the analysis of particular GSK3b-phosphorylation events on kinesin-1 subunits in vivo, which will lead to a better understanding of the mechanistic details of how kinesin-1 functions; which appear to be considerably different from what is currently known from in vitro studies. The proposed research is significant, because it is expected to vertically advance and transform what is currently known, under physiological conditions, in a whole organism setting. The knowledge acquired will dramatically propel the development of precise pharmacological/genetic modifiers against defects in this pathway which will benefit the treatment of cancer and neurodegeneration.
Currently there is an urgent need to understand the cause of neurodegenerative disease and cancer as these are devastating, often hereditary, degenerative disorders for which there are still no cures and only a handful of FDA-approved treatments exist mostly for symptomatic relief. The proposed research is relevant to public health because the discovery of the physiological mechanistic details of how a specific kinase is involved in regulating kinesin-1 motor function during intracellular transport will transform current investigations on targeting this pathway for cure. Thus, the proposed research is highly relevant to the part of NIH?s mission that pertains to developing fundamental knowledge that will enhance health, lengthen life, and reduce the burdens of illness and disability.
