Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate Retriever-mediated recycling on endosomes

Abstract Cell-surface receptors control how cells respond to their environment. Many cell-surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH and CCC complexes. Here we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces PI3P, which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition causes a loss of Retriever and CCC from endosomes, and mutation of the lipid binding site on a CCC subunit impairs its endosomal localization and delays integrin recycling. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.