Abstract 1868 Fig4 Influences Cellular Homeostasis Through MAP Kinase Related Pathways

Fig4 is a highly conserved phosphoinositide (PI) phosphatase that plays crucial roles in human development and long-term health. Fig4 contributes to cellular homeostasis through dephosphorylating PI3,5P2 at the 5-position of the inositol head group and recent evidence suggests that it may also play cellular roles through protein phosphatase activity. Through participation in a large protein complex with its opposing lipid kinase, Fab1, and the scaffold protein Vac14, Fig4 contributes to both the production and turnover of PI3,5P2 through both catalytic and non-catalytic mechanisms. The Fab1-Vac14-Fig4 complex dynamically regulates levels of PI3,5P2 in response to intracellular signals and environmental stresses to modulate cellular processes including actin dynamics, endosomal membrane trafficking, ion channel gating, and autophagy. In humans, point mutations in Fig4 have been implicated in neurological diseases including Charcot-Marie-Tooth type 4J and leukoencephalopathy, pathologies which involve axonal loss and demyelination. Disease states resulting from mutation of Fig4 have been attributed primarily to dysregulation of PI3, 5P2. Here we present evidence that expression of disease-related mutants of Fig4 that bind poorly to Fab1 and Vac14 confer resistance to rapamycin at 37C in S. cerevisiae. Our data indicate that this effect is conferred in a dominant manner independent of Fig4 catalytic function or association with the Fab1-Vac14-Fig4 complex. The growth advantage conferred by Fig4 disease-related mutants requires the kinase activity of the p21 activated kinase Ste20 (sterile 20 protein), a MAP kinase kinase kinase kinase involved in cellular responses to extracellular conditions including amino acid starvation, exposure to mating pheromone, and osmotic stress. Our results demonstrate that MAP kinase pathway components operating both upstream and downstream of Ste20 are also required for this Fig4-dependent growth advantage. We are investigating whether activation of Ste20 by Cdc42 occurs upstream or downstream of the Fig4-dependent role by testing for the ability of constitutively active Ste20 mutants to confer a growth advantage in the absence of Fig4 to narrow down where Fig4 interacts with this MAP kinase pathway to mediate this phenotype.