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PI-3 kinase type II C2α is essential for ATP-dependent priming of neurosecretory granules prior to exocytosis

F.A. Meunier1, G. Hammond1, P.J. Parker1, G. Schiavo1, F.T. Cooke2 and J. Domin3, 1Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK,2Biochemistry and Molecular Biology, University of College, London, Darwin Building, Gower Street, W1E 6BT, London, UK and 3Renal section, Faculty of Medicine, Imperial College School of Medicine, London W12 0NN, UK. (Introduced by D.J. Adams)

Phosphatidylinositol 3-kinases (PI3K) are implicated in a variety of synaptic functions including axonal guidance and long-term depression and potentiation (reviewed in Osborne et al., 2001). However, a direct involvement of this class of enzymes and their lipid products in neuroexocytosis has been questioned (Chasserot-Golaz et al., 1998), based on the low sensitivity of exocytosis to PI3K inhibitors wortmannin and LY294002 (Martin et al., 1997; Wiedemann et al., 1996).

Neurotransmitter release from synaptosomes and hormonal secretion from chromaffin cells are only sensitive to high concentrations of the PI3K inhibitors wortmannin and LY294002, pointing to a possible role for the less sensitive PI3K-C2α. In support of this, PI3K-C2α was detected on a subpopulation of mature secretory granules abutting the plasma membrane in neurosecretory cells. Furthermore, both PI3K inhibitors and sequestration of PI3K-C2α with specific antibodies selectively prevented ATP-dependent priming in permeabilised chromaffin cells.

Transient over-expression of PI3K-C2α in PC12 cells potentiated evoked secretion, whereas its dominant negative mutant abolished exocytosis, suggesting PtdIns3P, the main catalytic product of this enzyme plays a role in neuroexocytosis. Consistent with this, treatment of PC12 cells transiently expressing PtdIns3P-sequestering FYVE domain with low concentrations of wortmannin selectively abolished early endosomal staining and revealed a full co-localisation of the FYVE domain with PI3K-C2α on PC12 granules. Finally sequestration of PtdIns3P by the FYVE domain also abolished secretion from PC12 cells demonstrating that PtdIns3P production is needed in the process of acquisition of fusion competence secretory vesicles undergo, during or following docking to the plasma membrane.

Chasserot-Golaz, S., Hubert, P., Thierse, D., Dirrig, S., Vlahos, C. J., Aunis, D., & Bader, M.F. (1998) Journal of Neurochemistry, 70, 2347-2356.

Martin, T.F., Loyet, K.M., Barry, V.A., & Kowalchyk, J.A. (1997) Biochemical Society Transactions, 25, 1137-1141.

Osborne, S.L., Meunier, F.A., & Schiavo, G. (2001) Neuron, 32, 9-12.

Wiedemann, C., Schafer, T., & Burger, M.M. (1996) Embo Journal, 15, 2094-2101.