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Introduction Mammalian sphingomyelin (SM) synthase (SMS) consists of two isoform (SMS1, SMS2). SMS produces diacylglycerol (DG) by the transfer of phosphocholine from phosphatidylcholine (PC) to ceramide. SMS‐related protein (SMSr) also generates DG via synthesis of ceramide phosphoethanolamine, a SM analog, using phosphatidylethanolamine. Mammalian DG kinase (DGK) consists of ten isozymes (α, β, γ, δ, η, ϵ, κ, ι, ζ, θ) and governs a wide range of physiological and pathological events such as type 2 diabetes. Among them, SMS1, SMSr, DGKδ and DGKη contain a steraile a motif domain (SAMD), a protein‐protein interaction module. We previously reported that DGKδ interacts with SMSr, but not SMS1, via SAMDs [Murakami et al. J. Biol. Chem. 295, 2932–47, 2020]. Moreover, SMSr provided DG to DGKδ. However, it is still unclear whether other DGK isozymes interact with SMSs. In the present study, we analyzed interaction between DGKs and SMSs by co‐immunoprecipitation analysis. Results and Conclusion First, we determined whether SAMD‐containing DGKs (DGKδ and DGKη) interact with SAMD‐containing SMSs (SMS1 and SMSr). Although DGKη‐SAMD co‐sedimented with DGKη‐SAMD itself, SMS1‐ and SMSr‐SAMD failed to interact with DGKη‐SAMD. Furthermore, only the combination of full‐length DGKδ and full‐length SMSr could form a heteromeric complex. These results suggest that the interaction via heteromerization of SAMDs is specific to DGKδ and SMSr. Next, we analyzed whether other DGKs, which contain no SAMD, interact with SMSs. We found that DGKζ was strongly co‐immunoprecipitated with SMSr and SMS1, but not SMS2. These results suggest that DGKζ can interact with SMS1 and SMSr without heteromerization between SAMDs. Taken together, these results suggest that, in addition to DGKδ, DGKζ is a new candidate that acts downstream of SMSr and SMS1. SMSr produces palmitic acid (16:0)‐containing DG molecular species and SMS1 generates PC‐derived DG species including 16:0‐containing DGs. Moreover, DGKζ preferably phosphorylates 16:0‐containing DG species during neuronal cell differentiation. Therefore, it is possible that these enzymes compose a new pathway independent of the phosphatidylinositol turnover, which primary utilizes arachidonic acid (20:4)‐containing DG species.

We previously reported that diacylglycerol (DG) kinase (DGK) d interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile a motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKf, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N-terminal cytosolic region of SMSr binds to the Nterminal half catalytic domain of DGKf. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKf. Taken together, these results indicate that DGKf associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to convert it to phosphatidic acid (PA) [1][2][3][4][5]. Mammalian DGK consists of 10 isoforms, which can be divided into five groups: type I (a, b and c), type II (d, g and j), type III (e), type IV (f and ι), and type V (h) [1-5]. In addition, there are several splice variants, such as DGKd1 and 2 [6] and DGKg1 and 2 [7]. DG binds to and regulates C1 domain-equipped proteins, such as conventional protein kinase C (PKC) and novel PKC and Ras guanine nucleotide releasing protein (GRP) [8][9][10][11]. Moreover, various PA-binding proteins (more than 70) have been reported, including C-Raf, cAMP phosphodiesterase 4A1, atypical PKC (PKCf), novel PKC (PKCd and e), sporulation-specific