Multi-Site Phosphorylation of Oxysterol Binding Protein (OSBP) Regulates Sterol Binding and Activation of Sphingomyelin Synthesis

Multi-Site Phosphorylation of Oxysterol Binding Protein (OSBP) Regulates Sterol Binding and Activation of Sphingomyelin Synthesis Asako Goto, Xinwei Liu, Carolyn-Ann Robinson and Neale D. Ridgway The Atlantic Research Centre, Depts. of Pediatrics, and Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 *Running Head: Phosphorylation regulation of OSBP Address correspondence to: Neale D. Ridgway ([email protected]). Abbreviations: CERT, ceramide transport protein; CK1, casein kinase 1; ER, endoplasmic reticulum; FFAT, two phenylalanines in an acidic tract; 25OH, 25hydroxycholesterol; OSBP, oxysterol binding protein; ORP, OSBP-related protein; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PH, pleckstrin homology; PKD, protein kinase D; SM, sphingomyelin; TGN, trans-Golgi network; TEM, transmission electron microscopy; VAP-A-A, vesicle-associated-membrane protein-associated protein-A ABSTRACT The endoplasmic reticulum (ER)-Golgi sterol transfer activity of OSBP regulates sphingomyelin (SM) synthesis as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated suggesting that this modification regulates the directionality and/or specificity of transfer activity. Here we report that phosphorylation on two serine-rich motifs, S381-S391 (Site 1) and S192, S195, S200 (Site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation Site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by Site 1 phosphorylation. Individual Site 1 and Site 2 phosphomutants supported oxysterol-activation of SM synthesis in OSBP-deficient CHO