Identification and characterization of diacylglycerol kinase ζ as a novel enzyme producing ceramide-1-phosphate

Abstract

Ceramide-1-phosphate (C1P) is a sphingolipid formed by the phosphorylation of ceramide; it regulates various physiological functions, including cell survival, proliferation, and inflammatory responses. In mammals, ceramide kinase (CerK) is the only C1P-producing enzyme currently known. However, it has been suggested that C1P is also produced by a CerK-independent pathway, although the identity of this CerK-independent C1P was unknown. Here, we identified human diacylglycerol kinase (DGK) ζ as a novel C1P-producing enzyme and demonstrated that DGKζ catalyzes the phosphorylation of ceramide to produce C1P. Analysis using fluorescently labeled ceramide (NBD-ceramide) demonstrated that only DGKζ among ten kinds of DGK isoforms increased C1P production by transient overexpression of the DGK isoforms. Furthermore, an enzyme activity assay using purified DGKζ revealed that DGKζ could directly phosphorylate ceramide to produce C1P. Furthermore, genetic deletion of DGKζ decreased the formation of NBD-C1P and the levels of endogenous C_18:1/24:1- and C_18:1/26:0-C1P. Interestingly, the levels of endogenous C_18:1/26:0-C1P were not decreased by the knockout of CerK in the cells. These results suggest that DGKζ is also involved in the formation of C1P under physiological conditions.

Keywords: Ceramide kinase (CerK); Ceramide-1-phosphate (C1P); Diacylglycerol kinase (DGK); Sphingolipids.