Fig. 4: Phosphorylated SPHK1 affects PKR activation and ER stress signal pathway.
From: Cell fate determined by the activation balance between PKR and SPHK1
a The expression levels of phosphorylated PKR and ER stress-related protein were evaluated following SPHK1 overexpression. HepG2 cells were transfected with pCDNA3.1-SPHK1 or pCDNA3.1 for 24 h. The cells were then harvested and subjected to western blotting analysis. The exposure time for SPHK1 was 0.3 s, and the exposure time for phosphorylated PKR, phosphorylated eIF2α, IRE1α, XBP1(s), and CHOP was 6 s (the exposure time for these endogenous proteins was 2 s). b The expression levels of phosphorylated PKR and ER stress-related protein were evaluated in shSPHK1 cells. HepG2 shLacZ or shSPHK1 cells at 90% confluence were harvested and subjected to western blotting analysis. c–e DON, TNF‐α, and UV-induced phosphorylated PKR and eIF2α activation were evaluated following SPHK1 overexpression. HepG2 cells at 70% confluence were transfected with pCDNA3.1–SPHK1 or pCDNA3.1 for 24 h. The cells were then incubated with 2 μM DON or 10 ng/mL TNF-α for 3 h, or exposed to UVC radiation for 10 min. The cells were then harvested and subjected to western blotting analysis. f–h DON, TNF-α, and UV-induced phosphorylated PKR and eIF2α activation in shSPHK1 cells. HepG2 shLacZ or shSPHK1 cells at 70% confluence were incubated with 2 μM DON or 10 ng/mL TNF-α for 3 h, or exposed to UVC irradiation for 10 min. The cells were then harvested and subjected to western blotting analysis. (i) The expression levels of phosphorylated PKR and ER stress-related protein were evaluated following S1P exposure. HepG2 cells at 70% confluence were incubated with 5 or 10 μM S1P for 3 h. The expression levels of the target proteins were then determined by western blotting analysis. j, k The effect of the position of amino acid Ser225 in SPHK1 on PKR phosphorylation. HEK293T cells at 70% confluence were transfected with wild-type SPHK1, the SPHK1225A mutant, or the SPHK1225D mutant for 24 h. The cells were then harvested and subjected to western blotting analysis. l, m In vitro kinase assay of the activity of PKR using γ-32P ATP labeling. Cell lysates from transfected Flag-tagged SPHK1 or its mutants were purified using Flag (M2) magnetic beads, and were mixed with equal aliquots of PKR. The kinase 32P labeling reactions were conducted in the presence of 0.5 μCi γ-32P ATP, and the products were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The 32P-incorporated proteins were then transferred to a phosphor screen and developed using a PerkinElmer scanner. The results are the means ± SEMs of at least three independent experiments. Statistical significance was defined as *p < 0.05, **p < 0.01, or ***p < 0.001.
