Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta 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 Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, 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 PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta 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 co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.
Zhao
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X
Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, 49 Convent Drive, Bethesda, MD 20892, USA.
Várnai
P
P
Tuymetova
G
G
Balla
A
A
Tóth
Z E
ZE
Oker-Blom
C
C
Roder
J
J
Jeromin
A
A
Balla
T
T
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002522:Chlorocebus aethiops; D008074:Lipoproteins / Q000378:metabolism; D009227:Myristic Acids; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats
D002135:Calcium-Binding Proteins; D008074:Lipoproteins; D009227:Myristic Acids; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; C101464:frequenin calcium sensor proteins; C037178:phosphatidylinositol 4-phosphate; D019870:1-Phosphatidylinositol 4-Kinase