Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase β stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca2+-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III P14K beta with functional consequences affecting the kinase. Recombinant P14K beta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4K beta in PI4K beta -immunoprecipitates. When expressed in COS-7 cells, PI4K beta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed colocalization with endogenous PI4K beta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4K beta inhibited the development of this vesicular phenotype. Transfection of PI4K beta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [P-32]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca2+-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4K beta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.