title: Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis.
abstract: The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, 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 cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.
authors.affiliation: Department of Pediatrics, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada.
authors.forename: Asako
authors.initials: A
authors.lastname: Goto
authors.forename: Xinwei
authors.initials: X
authors.lastname: Liu
authors.forename: Carolyn-Ann
authors.initials: CA
authors.lastname: Robinson
authors.forename: Neale D
authors.initials: ND
authors.lastname: Ridgway
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D013109:Sphingomyelins / Q000096:biosynthesis*; D013261:Sterols / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D000098825:Oxysterol Binding Proteins
chemical_list: D006888:Hydroxycholesterols; D011987:Receptors, Steroid; D013109:Sphingomyelins; D013261:Sterols; C114051:VAPA protein, human; D033921:Vesicular Transport Proteins; D000098825:Oxysterol Binding Proteins; D012694:Serine; C007997:25-hydroxycholesterol; D002784:Cholesterol