Pho85/Cdk5 is a Positive Regulator of Phosphatidylinositol 3,5-Bisphosphate via Direct Phosphorylation of Fab1/PIKfyve

Abstract

The signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) plays critical roles in multiple tissues and multiple organisms. PI(3,5)P2 is regulated in part via a protein complex that contains the PI3P-5-kinase Fab1, the PI(3,5)P2 lipid phosphatase, Fig4, and the scaffold Vac14. In yeast, as well as in neurons, PI(3,5)P2 levels are transiently and rapidly elevated in response to selected signals. However to date, the upstream signaling pathways that regulate PI(3,5)P2 have not been identified. Here we report that in the yeast Saccharomyces cerevisiae, the CDK, Pho85 and the cyclin, Pho80 are required for the acute elevation of PI(3,5)P2 upon hyper-osmotic shock. Pho85-Pho80 directly phosphorylates Fab1 and positively regulates the synthesis of PI(3,5)P2. Furthermore, we find that Pho85-Pho80 phosphorylation of Fab1 likely generates a conformation change that activates Fab1 lipid kinase activity. Cdk5- p35, the mammalian homologue of Pho85-Pho80, is particularly critical in neuronal physiology and regulate multiple pathways. Notably, we find that mammalian Cdk5-p35 directly phosphorylates peptides of mammalian Fab1/PIKfyve, which strongly suggests that PIKfve is a direct target of Cdk5-p35 in tissue culture cells. These studies reveal a conserved, new down stream target of Cdk5, and provide insights into how PI(3,5)P2 is regulated.