Activation of sphingomyelin synthesis by oxysterol binding protein involves phosphatidylinositol 4‐phosphate kinase activation at the Golgi

Abstract

Oxysterol binding protein (OSBP) is targeted to the Golgi apparatus after stimulation of cells with 25‐hydroxycholesterol (25OH). OSBP translocation triggers ceramide transfer protein (CERT) translocation leading to increased sphingomyelin (SM) synthesis. The mechanism for CERT translocation is unknown. Our hypothesis is that OSBP translocation leads to increased phosphatidylinositol 4‐phosphate (PI4) synthesis at the Golgi and this promotes translocation of CERT via its PH domain. To test this, CHOK1 cells were treated with 25OH or solvent and isolated Golgi were assayed for PI4 kinase activity. A transient 50% increase in Golgi‐associated PI4 kinase activity was observed after 30 min of stimulation with 25OH that was dependent on OSBP expression. PI4 kinase IIα and IIIβ are localized to the Golgi, and PI4 kinase IIIβ was recently shown to be required for ceramide transport and SM synthesis. However, knockdown of PI4 kinase IIIβ by RNAi reduced PI4 kinase activity of isolated Golgi fractions by only 20%. Moreover, knockdown of PI4 IIIβ did not lead to reduced basal or 25OH‐stimulated SM synthesis in CHOK1 cells, indicating that the kinase was not required for OSBP‐mediated CERT activation. Immunofluorescence experiments further confirmed that OSBP targeting to the Golgi was not affected by PI4 IIIβ knockdown and that the two proteins localized to medial/trans Golgi compartments. In conclusion, SM synthesis is regulated by sterol‐signaling through OSBP leading to PI4 kinase activation at the Golgi. Ongoing experiments suggest that PI4 kinase IIα is required for OSBP‐mediated activation of SM synthesis.