PI3K-C2α associates with secretory granule fraction in chromaffin cells. Equivalent amount of proteins of the indicated fractions (Gasman et al., 1998) were separated by SDS-PAGE and immunoblotted with the indicated antibodies.

PI3K-C2α localizes to mature secretory granules. Fixed chromaffin cells were immunostained with anti-PI3K-C2α and either anti-Syt I (A) or anti-P18 antibodies (B) and imaged by confocal microscopy. Colocalized pixels (>100 a.u.) were highlighted (in white) in the z-stack series of optical sections shown below. Scale bar: 10 μm.

Expression and kinase activity of PI3K-C2α-wt and mutant PI3K-C2α-R1251P. HEK 293 cells were transfected with control vector, PI3K-C2α-wt or mutant PI3K-C2α-R1251P. After 72 h, recombinant enzymes were isolated from cell lysates using an anti-EE mAb and protein A-sepharose beads and blotted with anti-EE antibody (A) or used in a lipid kinase assay using PtdIns as substrate (B).

PI3K-C2α enhances hGH secretion from PC12 cells whereas its catalytically-inactive mutant is inhibitory. (A) PC12 cells transfected with a human growth hormone (hGH)-expressing vector (pXGH5) were treated with indicated concentrations of wortmannin, LY294002 or its inactive analogue LY303511 for 20 min prior stimulation with high K⁺ buffer A containing Ca²⁺ for 20 min in the continuous presence of the inhibitors. (B, C) PC12 cells were cotransfected with pXGH5 and with wild-type PI3K-C2α or PI3KC2α-R1251P. (B) Cells were fixed and coimmunostained for PI3KC2α and hGH. Scale bar: 10 μm. (C) Transfected PC12 cells were stimulated in high K⁺ buffer A containing Ca²⁺ for 20 min. (A, C) Aliquots were removed and assayed for hGH secretion, ** p <0.01).

PtdIns3P is required for neurosecretion in PC12 cells. (A, B) PC12 cells cotransfected with pXGH5 encoding hGH and GFP-2xFYVE or GFP-2xFYVE^C215S for 48 h and then immunostained for hGH (A). Insets show high-magnification details of the hGH-containing structures (white rectangle). Scale bar: 10 μm. (B) Cotransfected PC12 cells were briefly washed and stimulated in high K⁺ buffer A containing Ca²⁺ for 20 min. Aliquots were removed and assayed for hGH secretion, which was expressed as a percentage of the total hGH content. (C) Chromaffin cells were permeabilized with 20 μM digitonin in Ca²⁺- and ATP-free KGEP buffer in the presence or absence of bacterially expressed GST or GST-2xFYVE (17 μM) and incubated for 15 min. The supernatant was then removed and replaced by stimulation KGEP buffers as indicated, in the continuing presence of recombinant proteins for 5 min. Aliquots were removed and assayed fluorimetrically for catecholamine content (n = 4 experiments, ** p <0.01).

Effect of PI3K inhibitors on catecholamine secretion from chromaffin cells. (A) Chromaffin cells were incubated for 20 min with increasing concentration of inhibitors and stimulated with carbachol (100 μM) in the continuing presence of inhibitors for 20 min. (B) EE-tagged PI3K-C2α was transiently expressed in COS cells and immunoprecipitated with anti-EE monoclonal antibodies followed by protein A-Sepharose. PI3K-C2α was eluted with an excess of EE peptide and aliquots treated at 4°C for 5 h with either preimmune serum or anti-PI3K-C2α antibody. This mixture was then incubated with PtdIns in a standard lipid kinase assay as described in the method section. Phosphorylated products were visualized by autoradiography after separation by TLC. The experiment was done in triplicate. (C) Chromaffin cells were permeabilized with 20 μM digitonin in Ca²⁺- and ATP-free KGEP buffer alone or containing affinity purified anti-PI3K-C2α (0.38 μg/ml) or preimmune serum (88 μg/ml) for 10 min. (D) GST-PI3K-C2α (5 μg/ml) or GST alone (5 μg/ml) were preincubated for 30 min with or without 0.38 μg/ml anti-PI3K-C2α before addition to permeabilized chromaffin cells for 10 min. (E) Cells were preincubated with LY294002 (50 μM) for 20 min before permeabilization in the continuing presence of inhibitors. (C, D, E) The supernatant was removed and replaced by stimulation KGEP buffers as indicated in the continuing presence of antibodies/inhibitors for 5 min. (A, C, D, E) Aliquots were removed and assayed fluorimetrically for catecholamine content (n = 4 experiments; ** t test p <0.01).