Abstract
The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ 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. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ 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 DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.
Highlights
The ␦ isozyme of diacylglycerol kinase (DGK␦) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA)
We previously demonstrated that Diacylglycerol kinase (DGK)␦ 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
The results of pairwise comparisons showed that the SMS-related protein (SMSr)–SAMD was more similar to the DGK␦–SAMD than the SMS1–SAMD (32.8% identity versus 27.5% identity)
Summary
We first compared the amino acid sequences of human DGK␦–SAMD, DGK2–SAMD, SMS1–SAMD, and SMSr– SAMD using the ClustalW alignment tool (Fig. 2A). The identity between the SMSr–SAMD and the DGK␦–SAMD was higher than that between the SMSr–SAMD and the SMS1–SAMD (32.8% versus 30.9%) (Fig. 2) These results raised the possibility that SMSr and DGK␦ formed heterodimeric complexes. We performed co-immunoprecipitation analysis using COS-7 cells co-expressing 3ϫFLAG-tagged DGK␦–SAMD and either AcGFP-tagged DGK␦–SAMD, DGK2–SAMD, SMSr–SAMD, or SMS1– SAMD (Fig. 1, B and D). We found that 3ϫFLAG– DGK␦2 and either V5-tagged full-length SMSr (SMSr–V5) or SAMD-deleted SMSr (SMSr–⌬SAMD–V5) (Fig. 1) were co-expressed in COS-7 cells. To address the functional relationship between SMSr and DGK␦2, we analyzed changes in the amounts of PA in COS-7 cells overexpressing 3ϫFLAG–DGK␦2 and/or SMSr–V5 (Fig. 5). Compared with COS-7 cells transfected with vector alone, total PA levels were not substantially changed in the cells overexpressing DGK␦2 or SMSr alone. Increase), and 34:1-PA (a 21% increase) in COS-7 cells expressing both SMSr and DGK␦2 were significantly increased more than 20% compared with those in control cells (Fig. 5C)
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