Regulation of alkaline ceramidase activity by the c-Src-mediated pathway.
Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca(2+) suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca(2+)-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca(2+)-independent manner.
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Hirotsune
H
Sasaki
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Kaori
K
Toyomura
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Wataru
W
Matsuzaki
Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Aya
A
Okamoto
Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Naoto
N
Yamaguchi
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Hiroyuki
H
Nakamura
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan. Electronic address: murayama-toshi@faculty.chiba-u.jp.
Toshihiko
T
Murayama
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D055574:Alkaline Ceramidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000378:metabolism; D002208:Caproates / Q000378:metabolism; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D008246:Lysophospholipids / Q000378:metabolism; D049990:Membrane Transport Modulators / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013194:Staining and Labeling; D013449:Sulfonamides / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D002208:Caproates; D002518:Ceramides; D004791:Enzyme Inhibitors; D007211:Indoles; D008246:Lysophospholipids; D049990:Membrane Transport Modulators; D034741:RNA, Small Interfering; C416927:SU 6656; D013449:Sulfonamides; C060506:sphingosine 1-phosphate; D014638:Vanadates; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D000081247:CSK Tyrosine-Protein Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C526755:ACER2 protein, human; D055574:Alkaline Ceramidase; D009327:4-Chloro-7-nitrobenzofurazan; D013110:Sphingosine; D002118:Calcium
Ceramidase; Lyn; Sphingolipid metabolism; Vanadate; c-Src