Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase βinteract in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis

Abstract

Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms. The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase β (PI4Kβ). However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kβ. Here we have compared the subcellular distribution of NCS-1 and PI4Kβ and investigated whether they are capable of forming complexes. In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals. A consistent portion of NCS-1 and PIK4β is cytosolic,whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex. Very small amounts of NCS-1 and PI4Kβ are present in synaptic vesicles. Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4βinteract to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions. Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles. Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kβ associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions. Together,these findings demonstrate that mammalian NCS-1 and PI4Kβ interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kβ to target membranes.

Calneurons provide a calcium threshold for trans -Golgi network to plasma membrane trafficking

“…Yeast null mutant strains of Frequenin, the Drosophila (13)/yeast orthologue (10) of NCS-1, and those of the yeast PI-4K␤ orthologue Pik1 are not viable, pointing to the essential role of both proteins in Golgi-toplasma membrane trafficking (10,(14)(15)(16). This finding is, however, at variance with the situation in mammalia where NCS-1 seems to be more diffusely distributed in neurons with considerable amounts of the protein localized outside of the Golgi (17,18). Moreover, its binding to PI-4K␤ seems to be of lower affinity as compared with the yeast proteins (11).…”

Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

“…It remains possible that type III PI 4-kinases may have an important role in brain but require appropriate stimulation. For example, there is evidence that PI 4-kinase type III␤, the brain homologue of yeast Pik1, is stimulated by frequenin (34)(35)(36)(37).…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…NCS-1 can also interact with the mammalian Golgi enzyme PI4KIIIb and enhances its activity three-to 10-fold (Taverna et al 2002;Haynes et al 2005;de Barry et al 2006). The interaction with Golgi-associated PI4KIIIb suggests that it may regulate secretion through the modulation of phosphatidylinositol-dependent trafficking steps (Hendricks et al 1999;Zhao et al 2001;Haynes et al 2005).…”