PI(3,5)P2 controls the signaling activity of class I PI3K.

3-Phosphoinositides are essential cellular lipids regulating health and disease. Among them, phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] remains the least understood. Using a newly developed ratiometric PI(3,5)P2 sensor that enables spatiotemporally resolved PI(3,5)P2 quantification, we demonstrate that growth factor stimulation generates a distinct PI(3,5)P2 pool on lysosomes and late endosomes. Formed sequentially by class II PI3KC2β and PIKfyve, this PI(3,5)P2 pool terminates growth factor-stimulated class I phosphatidylinositol 3-kinase (PI3K) activity through a specific interaction with its regulatory p85 subunit. Disrupting this p85-PI(3,5)P2 interaction via a small-molecule inhibitor or cancer-causing mutations blocks feedback inhibition, driving sustained class I PI3K activation and promoting neurite growth. Our findings uncover a spatiotemporally specific regulatory function of PI(3,5)P2 that links class I and II PI3Ks to tune growth factor signaling. This mechanism offers therapeutic strategies for treating p85-mutant cancers and advancing tissue regeneration.