Dynamic Regulation of Phosphatidylinositol 3,5-bisphosphate and its Upstream Lipid Kinase Fab1/PIKfyve

Abstract

Eukaryotes maintain homeostatic balance in part via signal transduction cascades, which generate cellular adaptations in response to extracellular cues. Some of these cascades are regulated in part via phosphorylated phosphoinositide lipids (PPIs), which are low-abundance signaling molecules. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is generated from phosphatidylinositol 3-phosphate (PI3P) by the conserved lipid kinase Fab1/PIKfyve, which resides in a multiprotein complex known as the Fab1 complex. PI(3,5)P2 controls multiple pathways including calcium storage, lysosomal pH, and TORC1 signaling. PI(3,5)P2 is essential for multiple organ systems. Moreover, defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases, especially those of the nervous system. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here, we report a new mechanism that regulates Fab1 and dynamically controls cellular PI(3,5)P2 synthesis. Using multiple sequence alignment and secondary structure prediction we report a defined domain architecture of each member of the Fab1 complex. We identify new domains in Fab1/PIKfyve and Vac7, a Fab1 activator. Using a forward genetic screen optimized for the isolation of dominant-active Fab1 alleles, we identify point mutations within each domain of Fab1 that alter the dynamic regulation of PI(3,5)P2 levels. We characterize a subset of these dominant-active alleles and show that they disrupt a newly identified, inhibitory, intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We report preliminary evidence that this mechanism may be conserved in a second lipid kinase—the PI4 5-kinase, Mss4. Point mutations in Mss4 were generated based on Fab1 dominant-active alleles. These Mss4 mutations increase Mss4 activity under basal conditions and in response to stimulus-induced Mss4 activation. These studies identify a mechanism of regulation that may be conserved among lipid kinases to dynamically control PPI levels in response to external stimuli.