Class II PI3-kinase C2[alpha] is essential for ATP-dependent priming of neurosecretory granule exocytosis

Phosphoinositide-3 kinase (PI3K) activity is important for several aspects of neuronal differentiation and plasticity. However, its direct involvement in regulated exocytosis is unclear, despite clear evidence for a requirement for phosphoinositides in this process. Neurotransmitter release from synaptosomes and hormonal secretion from chromaffin cells is only sensitive to high concentrations of the PI3K inhibitors wortmannin and LY294002 pointing to a possible role for the less sensitive PI3K-C2[alpha]. In neurosecretory cells PI3K-C2[alpha] was detected on a subpopulation of mature secretory granules, abutting the plasma membrane. Both PI3K-C2[alpha] antibodies and PI3K inhibitors selectively prevented ATP-dependent priming in permeabilized chromaffin cells. Transient expression of PI3K-C2[alpha] potentiated secretion, whereas its catalytically inactive mutant abolished exocytosis, suggesting a possible role of the main catalytic product of this enzyme, PtdIns-3-phosphate (PtdIns3P), in this process. Treatment of PC12 cells expressing the PtdIns3P-binding FYVE domain with a low concentration of wortmannin selectively abolished early endosomal staining and revealed a full colocalisation with PI3K-C2[alpha] on PC12 granules. Finally, sequestration of PtdIns3P by the overexpression of FYVE domain abolished secretion from PC12 cells. Together these data demonstrate that production of PtdIns3P along with PtdIns4,5[P.sub.2] is required in the acquisition of fusion competence that secretory vesicles undergo during or following docking to the plasma membrane.