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.

We demonstrate that recycling through the endocytic recycling compartment (ERC) is an essential step in FcRI-induced activation of extracellular signal-regulated kinase (ERK)1/2. We show that ERK1/2 acquires perinuclear localization and colocalizes with Rab 11 and internalized transferrin in FcRI-activated cells. Moreover, a close correlation exists between the amount of ERC-localized ERK1/2 and the amount of phospho-ERK1/2 that resides in the nucleus. We further show that by activating phosphatidylinositol 4-kinase ␤ (PI4K␤) and increasing the cellular level of phosphatidylinositol(4) phosphate, neuronal calcium sensor-1 (NCS-1), a calmodulin-related protein, stimulates recycling and thereby enhances FcRI-triggered activation and nuclear translocation of ERK1/2. Conversely, NCS-1 short hairpin RNA, a kinase dead (KD) mutant of PI4K␤ (KD-PI4K␤), the pleckstrin homology (PH) domain of FAPP1 as well as RNA interference of synaptotagmin IX or monensin, which inhibit export from the ERC, abrogate FcRI-induced activation of ERK1/2. Consistently, NCS-1 also enhances, whereas both KD-PI4K␤ and FAPP1-PH domain inhibit, FcRI-induced release of arachidonic acid/metabolites, a downstream target of ERK1/2 in mast cells. Together, our results demonstrate a novel role for NCS-1 and PI4K␤ in regulating ERK1/2 signaling and inflammatory reactions in mast cells. Our results further identify the ERC as a crucial determinant in controlling ERK1/2 signaling. INTRODUCTIONNeuronal calcium sensor-1 (NCS-1), a 22-kDa calmodulinrelated protein, belongs to the superfamily of EF-hand Ca 2ϩ -binding proteins (Burgoyne and Weiss, 2001;Chen et al., 2002). NCS-1 is conserved through evolution with orthologues identified in yeast, Xenopus, Caenorhabditis elegans, Drosophila (first identified and termed frequenin), and avian and mammalian cells (Braunewell and Gundelfinger, 1999;Hendricks et al., 1999;Jeromin et al., 1999;Burgoyne and Weiss, 2001;Chen et al., 2002). In mammalians, NCS-1 is mainly, but not solely, expressed in neurons and neuroendocrine cells where it functions to modulate synaptic transmission as well as synaptic plasticity (McFerran et al., 1998;Chen et al., 2002;Sippy et al., 2003). 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. These lipids have emerged as key players in the control of a variety of cellular functions. By binding proteins that ...