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 were C(16:0) C1P and C(18:0) 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 A(2)alpha. 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.
Lamour
Nadia F
NF
Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Stahelin
Robert V
RV
Wijesinghe
Dayanjan S
DS
Maceyka
Michael
M
Wang
Elaine
E
Allegood
Jeremy C
JC
Merrill
Alfred H
AH
Cho
Wonhwa
W
Chalfant
Charles E
CE
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D004537:Eicosanoic Acids / Q000737:chemistry / Q000378:metabolism; D015777:Eicosanoids / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008954:Models, Biological; D015388:Organelles / Q000378:metabolism; D010169:Palmitic Acids / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D013229:Stearic Acids / Q000737:chemistry / Q000378:metabolism
D002518:Ceramides; D004537:Eicosanoic Acids; D015777:Eicosanoids; D010169:Palmitic Acids; D013229:Stearic Acids; C065576:ceramide 1-phosphate; C119090:CERT1 protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D017346:Protein Serine-Threonine Kinases