Abstract
Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3β (GSK3β) activity. We reveal the GSK3β substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3β site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3β-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.