Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling F. D. Testai, M. A. Landek, R. Goswami, M. Ahmed and G. Dawson Departments of Pediatrics, Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA Abstract There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methyl- umbelliferyl-phosphorylcholine, and a [ 14 C]choline-labeled sphingomyelin substrate to screen large numbers of phospho- compounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendro- cytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of AS- Mase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P 3 ] was a much more potent inhibitor of ASMase than lysophos- phatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P 2 ]. When PtdIns(3,4,5)P 3 was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P 3 to PtdIns(4,5)P 2 , we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P 3 levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P 3 and is most likely involved in receptor clustering and capping. Keywords: phosphate, phosphatidylinositol-3-kinase, phos- phatidyl-myo-inositol 3,4,5-triphosphate, PTEN, sphingom- yelinase. J. Neurochem. (2004) 89, 636–644. The hydrolysis of sphingomyelin to ceramide is an important event in all cells, and a deficiency of the lysosomal acid sphingomyelinase (ASMase) leads to rapid neurodegenera- tion and death from the massive accumulation of sphingo- myelin (Niemann–Pick disease) (Horinouchi et al. 1995; Lin et al. 2000; Nix and Stoffel 2001). Most ASMase is lysosomal, but an alternately spliced Zn-dependent secretory form exists (Tabas 1999), and a third form localized to detergent-resistant membrane fractions (Rafts) has been proposed (Liu and Anderson 1995; Lin et al. 2000; Grassme et al. 2003; Gulbins 2003). The most recent evidence for a Raft localization for ASMase has come from imaging studies which show it to be concentrated in ‘caps’ or aggregated Rafts (Kirschnek et al. 2000; Yu et al. 2000; Grassme et al. 2001, 2002, 2003). Thus CD95 ligand [acting through a CD95(Fas) receptor of the tumor necrosis factor (TNF) family] appears to cause ASMase to translocate on to the outer leaflet of immortalized lymphoblast cell membranes and co-localize with clustered CD95 and ceramide in cholesterol- and sphingolipid-rich Rafts (Grullich et al. 2000; Kirschnek et al. 2000; Lin et al. 2000; Grazide et al. 2002; Grassme et al. 2003). A small portion of the ASMase Received December 10, 2003; revised manuscript received January 8, 2004; accepted January 9, 2004. Address correspondence and reprint requests to Glyn Dawson, Department of Pediatrics MC4068, University of Chicago School of Medicine, 5841 S. Maryland Avenue, Chicago, IL 60637, USA. E-mail: dawg@midway.uchicago.edu Abbreviations used: ASMase, acid sphingomyelinase; DISC, death- inducing signaling complex; HMU-P-Chol, 6-hexadecanoylamino-4- methylumbelliferyl-phosphorylcholine; HOG, human oligodendrogli- oma cells; Ins(1,4,5)P 3 , myo-inositol 1,4,5-triphosphate; lyso-PA, lyso- phosphatidic acid; NGF, nerve growth factor; NSMase, neutral sphingomyelinase; NT-3, neurotropin-3; NTR, neurotropin receptor; OPC, oligodendrocyte primary cultures; PI3 kinase, phosphatidylinosi- tol-3-kinase; PtdIns(4,5)P 2 , phosphatidyl-myo-inositol 4,5-diphosphate; PtdIns(3,4,5)P 3 , phosphatidyl-myo-inositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog protein; Raft, Triton X-100-insoluble, sphingolipid-rich membrane fraction from sucrose density gradient; TNF, tumor necrosis factor. Journal of Neurochemistry , 2004, 89, 636–644 doi:10.1046/j.1471-4159.2004.02374.x 636 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 636–644