Phosphatidylinositol 4-kinase IIα is a glycogen synthase kinase 3-regulated interaction hub for activity-dependent bulk endocytosis

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. [Display omitted] •PI4KIIα is required for activity-dependent bulk endocytosis (ADBE)•PI4KIIα phosphorylation is essential for this role, not its enzyme activity•Phosphorylation at PI4KIIα Ser-47 causes binding of five presynaptic proteins•Two of these interactors, AGAP2 and CAMKV, are essential for ADBE Blumrich et al. reveal that phosphatidylinositol 4-kinase IIα (PI4KIIα) is required for activity-dependent bulk endocytosis (ADBE) at the presynapse. PI4KIIα acts as an interaction hub that liberates essential ADBE molecules, such as AGAP2 and CAMKV, via the activity-dependent control of its phosphorylation at key residues.