Mutually Inhibitory RAS-PI(3,4)P2 Feedback Loops Mediate Cell Migration

Abstract

Cell migration mediates a large number of key physiological activities during development and in the adult. These processes require coordination of signal transduction and cytoskeletal events which display excitability, but the mechanisms are poorly understood. Phosphoinositides have played a prominent role in the molecular definition of excitable signal transduction networks. Previously our lab identified a novel Pleckstrin Homology domain containing protein, CynA, which localizes to the lagging edge of cells as well as at the base of protrusions at the front. Here, we find CynA preferentially binds to PI(3,4)P2 on lipid strips and on reconstituted lipid vesicles. Using CynA as a PI(3,4)P2 biosensor, we then show that cells maintain complementary spatial and temporal distributions of Ras activity and PI(3,4)P2 during random migration and in response to chemoattractants. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2 and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive feedback loop essential for excitability. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a novel framework for Ras regulation which may play a key role in many physiological processes.