Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling.
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-methylumbelliferyl-phosphorylcholine, and a [(14)C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase 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 lysophosphatidic 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.
Testai
F D
FD
Department of Pediatrics, University of Chicago, Chicago, Illinois, USA.
Landek
M A
MA
Goswami
R
R
Ahmed
M
M
Dawson
G
G
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051059:PTEN Phosphohydrolase; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D010710:Phosphates; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D025521:Tumor Suppressor Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D000249:Adenosine Monophosphate; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D013108:Sphingomyelin Phosphodiesterase; C032881:lysophosphatidic acid