Fig. 4

SphK1 plays a role of acetyltransferase, acetylating S565 of COX2 in neurons. a Acetyl-CoA binding activity of SphK1 was analyzed by filter binding assay with 10 μM sphingosine. The binding velocity (V_binding) of [³H] acetyl-CoA to SphK1 was plotted to the acetyl-CoA concentration and the nonlinear regression analysis of the saturated plot yielded the kinetic parameters such as K_cat (catalytic constant) and K_M (Michaelis−Menten constant) for acetyl-CoA and SphK1 binding activity (n = 3 per group). b Dissociation of acetyl group from SphK1 was analyzed by equilibrium dialysis in the presence of 0, 5, 25, and 100 μM free acetyl-CoA. The dissociation rate (V_dissociation) of [³H] acetyl group from [³H] acetyl-CoA and SphK1 complex was plotted against inhibitor-free acetyl-CoA concentration and dissociation constant (K_D) was calculated from the nonlinear regression analysis (n = 3 per group). c Acetyl-CoA binding activity of SphK1 was analyzed by filter binding assay in the presence of 0, 10, and 100 μM sphingosine (n = 3 per group). d Acetylation assay of purified COX2 protein treated with SphK1 and [¹⁴C] acetyl-CoA in the presence of 100 μM sphingosine or not. BSA-treated COX2 protein was negative control (n = 4–6 per group). e LC-MS spectra of peptide 560-GCPFTSFSVPDPELIK-575 (m/z = 918.44) of COX2 acetylated by SphK1, acetyl-CoA and sphingosine. f LC-MS/MS spectra of ac-S565 in 560-GCPFTSFSVPDPELIK-575 of COX2. g Acetylation assay of COX2 WT and COX2 S565A recombinants treated with SphK1 and [¹⁴C] acetyl-CoA in the presence of 100 μM sphingosine (n = 4–6 per group). d One-way analysis of variance, Tukey’s post hoc test. g Student’s t test. ***P < 0.001. All error bars indicate s.e.m.