FIGURE 7.

Mutations in phosphorylation sites decrease enzymatic activity of CERS isozymes. A, HEK 293T cells were transfected with a vector or a plasmid encoding WT HA-CERS1–6 or MT HA-CERS2–6, as indicated. B and C, HEK 293T cells were transfected with a vector or a plasmid encoding mouse WT HA-Cers2 or MT HA-Cers2, as indicated. A and B, total membrane fractions (10 μg of protein) prepared from the transfected cells were incubated with 5 μm deuterium-labeled sphingosine and 25 μm of the indicated acyl-CoA at 37 °C for 30 min. Lipids were extracted, and deuterium-labeled ceramides were analyzed by a Xevo TQ-S LC/MS system and quantified by MassLynx software. The values represent the activity of each CERS relative to that of the vector sample, and are the mean ± S.D. from three independent assays. Statistically significant differences in the activities between WT and MT are indicated; **, p < 0.01; t test. C, total membrane fractions (10 μg of protein) prepared from the transfected cells were separated by SDS-PAGE and subjected to immunoblotting with anti-HA antibodies.