A phosphoinositide cascade regulates a receptor recycling pathway.

Phosphatidylinositol polyphosphates (PIPs) are essential cellular regulators. Each PIP transiently appears at specific membranes to regulate the spatial and temporal recruitment of unique downstream effectors. Importantly, most PIPs serve as both signaling lipids, and as precursors for other PIPs. These precursor-product relationships likely form cascades of pathways, which would provide a way for PIPs to coordinate events both spatially and temporally. Here we uncover a pathway on endosomes, which is likely governed by a cascade of three phosphoinositide lipids. The pathway is specific for the recycling of some receptors to the plasma membrane and is mediated by sorting-nexin SNX17, and the Retriever, WASH and CCC complexes. We discover that PIKfyve and an upstream PI3-kinase, VPS34 positively regulates this pathway. VPS34 produces PI3P, which provides the substrate for PIKfyve to generate PI3,5P , which is then further converted to PI5P by myotubularin 3-phosphatases. We show that PIKfyve regulates 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. 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. More broadly, these findings provide more general insights into mechanisms for how cells regulate the levels of some receptors at the cell surface.