Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells w...

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Bibliographic Details
Published in:Journal of lipid research 2007-06, Vol.48 (6), p.1293-1304
Main Authors: Lamour, Nadia F, Stahelin, Robert V, Wijesinghe, Dayanjan S, Maceyka, Michael, Wang, Elaine, Allegood, Jeremy C, Merrill, Alfred H. Jr, Cho, Wonhwa, Chalfant, Charles E
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Cell Membrane - metabolism
Ceramides - chemistry
Ceramides - metabolism
Eicosanoic Acids - chemistry
Eicosanoic Acids - metabolism
Eicosanoids - metabolism
Golgi Apparatus - metabolism
HeLa Cells
Humans
Immunoblotting
Mass Spectrometry
Microscopy, Confocal
Models, Biological
Organelles - metabolism
Palmitic Acids - chemistry
Phosphotransferases (Alcohol Group Acceptor) - genetics
Phosphotransferases (Alcohol Group Acceptor) - metabolism
Protein-Serine-Threonine Kinases - metabolism
RNA Interference
Stearic Acids - chemistry
Stearic Acids - metabolism
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Summary:Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C₁₆:₀ C1P and C₁₈:₀ C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A₂α. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide "pool" transported in an active manner via CERT.
ISSN:0022-2275
1539-7262
DOI:10.1194/jlr.M700083-JLR200