Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism.
Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.
Marchesini
Norma
N
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
Luberto
Chiara
C
Hannun
Yusuf A
YA
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division; D002518:Ceramides / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007477:Ions; D007700:Kinetics; D051379:Mice; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013107:Sphingolipids / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000502:physiology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014343:Trypan Blue / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D000814:Aniline Compounds; D001597:Benzylidene Compounds; D002518:Ceramides; D004396:Coloring Agents; C468773:GW 4869; D007477:Ions; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; D010740:Phospholipases; C419391:ISC1 protein, S cerevisiae; D010738:Type C Phospholipases; C491484:SMPD3 protein, human; C491483:Smpd3 protein, mouse; D013108:Sphingomyelin Phosphodiesterase; C022616:thiazolyl blue; D014343:Trypan Blue