title: Oxysterol binding protein-dependent activation of sphingomyelin synthesis in the golgi apparatus requires phosphatidylinositol 4-kinase IIα.
abstract: Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KIIα. PI4KIIα, but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KIIα nor PI4KIIIβ expression was required for 25-hydroxycholesterol-dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KIIα in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KIIα activity, leading to CERT recruitment to the TGN and increased SM synthesis.
authors.affiliation: Departments of Pediatrics and Biochemistry and Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Banerji
authors.forename: Mike
authors.initials: M
authors.lastname: Ngo
authors.forename: Ciaran F
authors.initials: CF
authors.lastname: Lane
authors.forename: Carolyn-Ann
authors.initials: CA
authors.lastname: Robinson
authors.forename: Shane
authors.initials: S
authors.lastname: Minogue
authors.forename: Neale D
authors.initials: ND
authors.lastname: Ridgway
mesh_terms: D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D006224:Cricetinae; D003412:Cricetulus / Q000378:metabolism; D020868:Gene Silencing; D006056:Golgi Apparatus / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D034741:RNA, Small Interfering; D011987:Receptors, Steroid / Q000378:metabolism*; D013109:Sphingomyelins / Q000096:biosynthesis*; D021601:trans-Golgi Network / Q000737:chemistry; D000098825:Oxysterol Binding Proteins
chemical_list: D006888:Hydroxycholesterols; D015778:Minor Histocompatibility Antigens; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D013109:Sphingomyelins; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase