Ca²⁺-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2α on Neurosecretory Vesicles

Abstract

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2␣ and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2␣ knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2␣ knockdown and expression of PI3K-C2␣ catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca 2؉ . We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2␣ is indeed regulated by Ca 2؉ . We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2␣ occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca 2؉ in vitro and in living neurosecretory cells.

A phosphoinositide conversion mechanism for exit from endosomes

“…PI(3)P, synthesized by PI3KC2α on chromaffin granules, positively regulated secretion (Meunier, Osborne et al 2005;Wen, Osborne et al 2008). However, PI(3)P turnover and/ or PI conversion was not addressed by this study.…”

The Clavesin Family, Neuron-specific Lipid- and Clathrin-binding Sec14 Proteins Regulating Lysosomal Morphology

“…Unfortunately, the EEA1 antibody did not work for immunofluorescence in neurons. Thus, we co-transfected hippocampal cultures with GFP-clavesin 1 and a construct encoding tandem copies of the PtdIns 3-phosphatebinding FYVE domain of Hrs fused to mCherry, a marker of early endosomes (27). To allow for increased transfection efficiency, cells were transfected at 6 DIV and imaged at 7 DIV.…”

“…Mutations of clavesin 1 resulting in a defective lipid binding were introduced into its cDNA using a QuikChange XL site-directed mutagenesis kit (Stratagene). Tandem FYVE domain (2ϫFYVE), derived from mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (27) was a gift of Dr. Fred Meunier. The enhanced green fluorescent protein from the pEGFP plasmid was replaced with mCherry.…”

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…In animal cells, PtdIns(3)P also localizes to early endosomes, where it is required for protein trafficking. More recently, it has been reported that PtdIns(3)P associates with the plasma cell membrane during responses to insulin (123) and that plasma membrane-associated PtdIns(3)P is required for both glucose transporter 4 protein translocation to the plasma membrane (123) and neurosecretory granule release (124). PtdIns(3)P is generally found on the cytoplasmic leaflet of internal membranes.…”