The long range goal of the proposed studies is to determine how phosphatidylinositol (3, 5)-bis phosphate (PI3, 5P2) levels are dynamically regulated. PI3, 5P2, a very low abundance, yet essential cellular regulator, is transiently synthesized as specific membranes at specific times. Emerging studies from us and others indicate that PI3, 5P2, is critical for normal human physiology. A mutation predicted to lower PI3, 5P2 causes a severe congenital disorder that results in infant mortality and has severe pathological effects on multiple tissues. Minor mutations predicted to result in a modest defect in the ability to dynamically regulate PI3, 5P2 levels underlie a severe form of CMT4J, a neuropathy, as well as some cases of amyotrophic lateral sclerosis (ALS). Another minor mutation has been linked to epilepsy, abnormalities in brain development and severe psychiatric problems. Notably, we discovered that proteins required for the regulation of PI3, 5P2 are critical for modulation of synaptic activity. These findings underscore the importance of determining how PI3, 5P2 levels are dynamically regulated. Our previous studies revealed that the dynamic regulation of PI3, 5P2 occurs through a large protein complex which includes a conserved lipid kinase called Fab1 in yeast, and PIKfyve in mammals, as well as two conserved regulatory proteins, Vac14 and Fig4. Based on its sequence, and in vitro studies, it was assumed that Fig4 provides the major route for turnover of PI3, 5P2. However, our studies suggest that a major function of Fig4 is in the activation of Fab1/PIKfyve; the mechanism of this activation is unknown. In addition the upstream signaling pathways that activate the PIKfyve/Fab1-Vac14-Fig4 complex are unknown, and this presents another critical gap in determining how PI3, 5P2 levels are dynamically regulated. We will address these gaps with the following specific aims: 1). Determine roles of Fig4 in the regulation of Fab1/PIKfyve activity; 2) Determine whether Fab1 kinase activity is regulated in part via an intramolecular inhibitory domain; 3). Determine whether phosphorylation of Fab1 and Vac7 is the major pathway for Pho85- Pho80 regulation of PI3, 5P2 levels, and whether Cdk5-p35 is an upstream regulator of PIKfyve. Results of these studies will provide significant insights into the mechanisms that regulate PI35P2, and may provide new avenues to develop therapeutic strategies to treat selected neurological and psychiatric disorders.
Mutations in Fig4 are linked to several neurological diseases. Evaluation of these mutations in yeast suggests a specific defect in the dynamic regulation of PI3, 5P2. Using yeast and mammalian cells, we will use multiple approaches to determine mechanisms that dynamically regulate PI3, 5P2.
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