Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase β Regulate IgE Receptor-Triggered Exocytosis in Cultured Mast Cells

Abstract

We examined the possible occurrence and function of neuronal Ca2+ sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase β (PI4Kβ) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kβ, increases IgE receptor (FcεRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and stimulates FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kβ reduces PI4Kβ activity, decreases FcεRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcεRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kβ, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P2 that serves as substrate for the generation of lipid-derived second messengers in FcεRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kβ and positive regulation of its activity.

Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase β Stimulate Extracellular Signal-regulated Kinase 1/2 Signaling by Accelerating Recycling through the Endocytic Recycling Compartment

“…We have described these cell lines and demonstrated their viability and ability to undergo Ca 2ϩ -triggered exocytosis in previous publications (Kapp-Barnea et al, 2003). At time 0, following 1 h of binding at 4°C, all cells displayed comparable amounts of bound Tfn; therefore, indicating that their Tfnbinding capacity was neither affected by NCS-1 nor by the KD mutant of PI4K␤ (Figure 1).…”

“…PI4K␤ is a downstream target for NCS-1 in a number of exocytic processes, including control of regulated secretion in both neuroendocrine (Rajebhosale et al, 2003) and nonneuronal cells (Kapp-Barnea et al, 2003;Gromada et al, 2005) and regulation of biosynthetic transport (Zhao et al, 2001). Increasing the pool of PI(4,5)P 2 available for PLCinduced hydrolysis and the consequent increase in second messengers generation provide the molecular mechanism by which NCS-1 stimulates exocytosis.…”

“…Indeed, we (Kapp-Barnea et al, 2003) have previously shown that NCS-1 colocalizes with PI4K␤, whose Golgi localization is well established (De Matteis et al, 2002), at a perinuclear location. However, such perinuclear location might also include the ERC, which also resides at the microtubule organizing center (Hopkins and Trowbridge, 1983).…”

“…Mock-transfected rat basophilic leukemia (RBL) cells and RBL cells stably transfected with full-length cDNAs encoding NCS-1 or the kinase dead (KD) mutant of PI4K␤(KD-PI4K␤D656A) were described previously (Kapp-Barnea et al, 2003). The cells were maintained as adherent cultures in DMEM supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO 2 at 37°C.…”

“…Additionally, NCS-1 stimulates the activity of phosphatidylinositol 4-kinase ␤ (PI4K␤) (Hendricks et al, 1999;Zhao et al, 2001; KappBarnea et al, 2003;Rajebhosale et al, 2003;Haynes et al, 2005) and thereby modulates phosphatidylinositol-dependent signaling (Kapp-Barnea et al, 2003;Rajebhosale et al, 2003). Previously, we have shown that NCS-1 regulates FcRItriggered exocytosis in mast cells by stimulating PI(4)P production and increasing the pool of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] that is required for lipid-derived second messenger generation by the receptor-activated phospholipase C (PLC) (Kapp-Barnea et al, 2003). However, the role of phosphoinositides is not restricted to second messenger generation.…”