Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

The delta isozyme of diacylglycerol kinase (DGKdelta) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKdelta preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKdelta and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile alpha motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKdelta-SAMD. Full-length DGKdelta co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKdelta interacted only weakly with full-length DGKdelta and SMSr, respectively. These results strongly suggested that DGKdelta interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKdelta and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKdelta and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKdelta-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKdelta activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKdelta and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.