A protein kinase network regulates the function of aminophospholipid flippases

Limited exposure of aminophospholipids on the outer leaflet of the plasma membrane is a fundamental feature of eukaryotic cells and is maintained by the action of inward-directed P-type ATPases ("flippases"). Yeast S. cerevisiae has five flippases (Dnf1, Dnf2, Dnf3, Drs2, and Neo1), but their regulation is poorly understood. Two paralogous plasma membrane-associated protein kinases, Pkh1 and Pkh2 (orthologs of mammalian PDK1), are required for viability of S. cerevisiae cells because they activate several essential downstream protein kinases by phosphorylating a critical Thr in their activation loops. Two such targets are related protein kinases Ypk1 and Ypk2 (orthologs of mammalian SGK1), which have been implicated in multiple processes, including endocytosis and coupling of membrane expansion to cell wall remodeling, but the downstream effector(s) of these kinases have been elusive. Here we show that a physiologically relevant substrate of Ypk1 is another protein kinase, Fpk1, a known flippase activator. We show that Ypk1 phosphorylates and thereby down-regulates Fpk1, and further that a complex sphingolipid counteracts the down-regulation of Fpk1 by Ypk1. Our findings delineate a unique regulatory mechanism for imposing a balance between sphingolipid content and aminophospholipid asymmetry in eukaryotic plasma membranes.