Diacylglycerol kinase ζ interacts with sphingomyelin synthase 1 and sphingomyelin synthase-related protein via different regions

Abstract

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, 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 N-terminal half catalytic domain of DGKζ. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Keywords: diacylglycerol kinase; sphingomyelin synthase; sphingomyelin synthase-related protein; sterile α motif domain.

Fig. 1

Identification of DGK isozymes interacting with SMSr, SMS1, and SMS2. (A) Immunoprecipitation (IP)‐western blot (WB) analysis of the interaction of vector alone (V5 vector), SMSr‐V5, SMS1‐V5, and SMS2‐V5 with 3×FLAG‐DGK isozymes (α, β, γ, δ1, δ2, η1, η2, κ, ε, ζ, ι, and θ). V5‐tagged protein was immunoprecipitated with an anti‐V5 antibody. SDS/PAGE (10% acrylamide) was performed, and separated proteins were detected by western blotting with anti‐FLAG and anti‐V5 antibodies. A representative of three repeated experiments is shown. Left panel, cell lysate (Input); right panel, IP. (B) Densitometric quantification of 3×FLAG‐tagged DGKs using imagej fiji software (US National Institutes of Health, Bethesda, MD, USA). Binding activity was calculated as the percentage of the band intensity in the IP sample compared with the input band intensity. The values are presented as the means ± SD of three independent experiments. *P < 0.05, ***P < 0.005 versus vector alone.

Fig. 2

DGKζ‐binding activities of SMSr‐NT, SMS1r‐CT, SMSr‐SAMD, SMS1‐NT, and SMS1‐CT. (A) Schematic representation of the structures of AcGFP‐tagged SMSr‐NT, SMS1r‐CT, SMSr‐SAMD, SMS1‐NT, and SMS1‐CT. (B, D) Immunoprecipitation (IP)‐western blot (WB) analysis of the interaction of 3×FLAG‐tagged DGKζ‐FL with AcGFP‐tagged SMSr‐NT, SMSr‐CT, SMS1‐NT and SMS1‐CT (B) and SMSr‐SAMD (D). AcGFP‐tagged proteins were immunoprecipitated with an anti‐GFP antibody. SDS/PAGE (10% acrylamide) was performed, and separated proteins were detected by Western blotting with anti‐GFP and anti‐FLAG antibodies. (C, E) Quantitative analysis of western blotting by densitometry was performed using imagej fiji software. The values are presented as the means ± SD of three independent experiments. (C) **P<0.01, among AcGFP, AcGFP‐SMS1‐NT and AcGFP‐SMS1‐CT, #P<0.05, among AcGFP, AcGFP‐SMSr‐NT and AcGFP‐SMSr‐CT; (E)   **P < 0.01.

Fig. 3

Binding activities of various DGKζ deletion mutants to SMSr‐SAMD and SMS1‐CT. (A) Schematic representation of the structures of 3×FLAG‐tagged DGKζ deletion mutants. (B, C, E, G, J) Immunoprecipitation (IP)‐western blot (WB) analysis of the interaction of AcGFP alone with 3×FLAG‐tagged DGKζ‐NT, DGKζ‐CD, and DGKζ‐CT (B); the interaction of AcGFP‐tagged SMSr‐SAMD with 3×FLAG‐tagged DGKζ‐NT, DGKζ‐CD and DGKζ‐CT (C); the interaction of AcGFP‐tagged SMS1‐CT with 3×FLAG‐tagged DGKζ‐NT, DGKζ‐CD and DGKζ‐CT (E); the interaction of AcGFP‐tagged SMSr‐NT and SMS1‐CT with 3×FLAG‐tagged DGKζ‐CD‐a (G); and the interaction of AcGFP‐tagged SMSr‐NT and SMS1‐CT with 3×FLAG‐tagged DGKζ‐CD‐b (I). AcGFP‐tagged SMSr‐SAMD, SMSr‐NT or SMS1‐CT was immunoprecipitated with an anti‐GFP antibody. SDS/PAGE (12% acrylamide) was performed, and separated proteins were detected by western blotting with anti‐GFP and anti‐FLAG antibodies. (D, F, H, J) Quantitative analysis of western blotting by densitometry was performed using imagej fiji software [51]. Binding activity was calculated as the percentage of the band intensity in the IP sample compared with the input band intensity. The values are presented as the means ± SD of three independent experiments. **P < 0.01, ***P < 0.005 versus AcGFP alone.

Fig. 4

Binding activities of purified DGKζ with SMS1‐CT and SMSr‐NT. (A) Schematic representation of the structures of Twin‐Strep (TS)‐tagged DGKζ (TS‐DGKζ), GST‐tagged SMS1‐CT, and GST‐tagged SMSr‐NT. (B) The interaction of TS‐DGKζ with GST‐SMS1‐CT and GST‐SMSr‐NT. GST‐SMS1‐CT or GST‐SMSr‐NT was pulled down with glutathione‐Sepharose beads. SDS/PAGE (12% acrylamide) was performed and separated proteins were detected by western blotting with anti‐GST and anti‐TS antibodies. (C) Quantitative analysis of western blotting by densitometry was performed using imagej fiji software. Binding activity was calculated as the percentage of the band intensity (TS‐DGKζ) in the pull‐down sample compared with that of GST‐SMSr‐NT‐pull down (set to 100). The values are presented as the means ± SD of three independent experiments. ***P < 0.005, ****P < 0.001 versus GST alone.

Fig. 5

Effects of SMSr and SMS1 on DGKζ activity in vitro. (A) Purification of TS‐DGKζ, SMS1‐TS, and SMSr‐TS. TS‐DGKζ, SMS1‐TS, and SMSr‐TS were expressed in Sf9 insect cells and purified using Strep‐Tactin XT beads. Purified TS‐DGKζ, SMS1‐TS, and SMSr‐TS were detected by immunoblot with anti‐TS antibody. (B) Effects of SMS1 and SMSr on DGKζ activity in vitro in the presence of 34:1 (16:0/18:1)‐DG. The activities of DGKζ (34:1 (16:0/18:1)‐PA production) were measured using LC–MS/MS. The activity of TS‐DGKζ alone was set to 100. The values are presented as the means ± SD of four independent experiments. *P < 0.05 versus TS‐DGKζ alone. (C) Effects of SMSr‐NT on DGKζ activity in vitro in the presence of 34:1 (16:0/18:1)‐DG. The activities of DGKζ (34:1 (16:0/18:1)‐PA production) were measured using LC–MS/MS. The activity of TS‐DGKζ alone was set to 100. The values are presented as the means ± SD of four independent experiments. **P < 0.01 versus TS‐DGKζ/GST alone.

Fig. 6

Schematic representation of the interaction of DGKζ with SMS1 and SMSr. SMSr‐SAMD binds to DGKζ‐CD‐b. SMS1‐CT interacts with DGKζ‐CD‐a and DGKζ‐CD‐b. We previously demonstrated that DGKδ‐SAMD associates with SMSr‐SAMD and that SMSr activates DGKδ [42]. See Results and Discussion for the full description. MG‐PLC, multiglycerophospholipid PLC hydrolase [43].