Ceramide kinase profiling by mass spectrometry reveals a conserved phosphorylation pattern downstream of the catalytic site.
Ceramide kinase (CERK) is essential for production of ceramide-1-phosphate (C1P), a bioactive lipid whose formation critically modulates ceramide levels. To explore how CERK is regulated, we used insect cell-expressed, recombinant hCERK and searched for post-translational modifications, using mass-spectrometry techniques. This led to identification of two phosphorylated serine residues, at positions 340 and 408. Point mutations preventing phosphorylation at either of these sites did not lead to detectable changes in subcellular localization or activity. However, preventing phosphorylation at S340 resulted in CERK instability as revealed by the behavior of the S340A mutant protein under various assay conditions in vitro. Phosphorylation of a cognate serine residue in sphingosine kinases was previously shown to be important. Therefore, phosphorylation within a conserved "regulation loop" downstream of the catalytic domain emerges as a new paradigm for regulation of kinases of the diacylglycerol kinase family. This "regulation loop" is reminiscent of the "activation loop" that controls AGC protein kinases, being a similar distance from the critical ATP binding site determinants in the primary sequence.
Chen
Wei-Qiang
WQ
Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria.
Graf
Christine
C
Zimmel
David
D
Rovina
Philipp
P
Krapfenbauer
Kurt
K
Jaritz
Markus
M
Parker
Peter J
PJ
Lubec
Gert
G
Bornancin
Frédéric
F
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism
D010750:Phosphoproteins; D011994:Recombinant Proteins; D012694:Serine; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase