Fig. 2: SPHK1 is directly phosphorylated by activated PKR.
From: Cell fate determined by the activation balance between PKR and SPHK1
a The phosphorylation levels of SPHK1 were evaluated during PKR overexpression and PolyI:C administration. HEK293T cells were transfected with pCDNA3.1–PKR or pCDNA3.1 for 24 h and then treated with 10 μg/mL PolyI:C for 3 h. The cells were then harvested and subjected to western blotting analysis. b The levels of SPHK1 phosphorylation were evaluated in PKR knockout cells. HEK293T control and PKR knockout cells at 90% confluence were harvested and subjected to western blotting analysis. c, d The expression levels of phosphorylated SPHK1 were evaluated following treatment with DON or TNF‐α in PKR knockout cells. HEK293T control and PKR knockout cells at 70% confluence were incubated with 400 nM DON or 10 ng/mL TNF-α for 3 h. The cells were then harvested and subjected to western blotting analysis. e Co-IP analysis of the interaction between SPHK1 and PKR. HEK293T cells were co-transfected with SPHK1-Flag and PKR-Myc for 24 h. The cell lysates were subjected to immunoprecipitation with the indicated antibodies, and visualized by western blotting. pCDNA3.1 transfection was used as a negative control. f GST pulldown analysis of the interaction between SPHK1 and PKR. Purified GST and GST–SPHK1 protein bound to agarose beads were added to the lysate of 293T cells overexpressing PKR-HA. GST protein was used as a negative control. g Whole extracts from HEK293T cells transfected with 0, 1, 2, and 3 μg of DNA encoding SPHK1-Flag protein were processed for immunoprecipitation with antibodies against Flag or PKR. h Immunoprecipitation analysis of the endogenous interaction between phosphorylated SPHK1 and PKR. Cell lysates of HEK293T cells were subjected to immunoprecipitation with the indicated antibodies, and visualized by western blotting. i, j The effect of the PKR Thr451 amino acid position on SPHK1 phosphorylation. HEK293T cells at 70% confluence were transfected with wild-type PKR, the PKR451A mutant, or the PKR451D mutant for 24 h. The cells were then harvested and subjected to western blotting analysis. k, l In vitro kinase assay to determine the activity of SPHK1 using γ-32P ATP labeling. Cell lysates from transfected wild-type PKR or its mutants were purified using Flag (M2) magnetic beads, and mixed with equal aliquots of SPHK1. The kinase 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 products were then transferred to a phosphor screen and developed using a PerkinElmer scanner. The reactions of PKR and its substrate eIF2α were shown as positive controls in Supplementary Fig.10.
