Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P₂ by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P₂-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P₂ hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P₂ are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.