Figure 6

Oxidative stress-induced Tyr717 phosphorylation of PKD2 increases kinase activity towards syntide-2. (a) Kinetics of WT PKDs and the indicated mutants after activation by oxidative stress. FLAG-PKDs were purified from HEK293 cells after stimulation with H₂O₂ (10 mM, 10 min). Protein concentration and purity (100%) was analyzed side by side by densitometry of a coomassie stained SDS-polyacrylamide gel using a BSA standard. Michaëlis-Menten kinetics for Syn-2 phosphorylation by each protein was followed in a radiometric kinase assay (see Materials and methods). (b) FLAG-PKDs were purified from HEK293 cells after stimulation with PDB (500 nM, 15 min). The purified proteins were subjected to the same analysis as in 6a. (c) Activation segment Ser and Tyr phosphorylation of stimulated enzymes. The purified enzymes described in 6a and 6b were analyzed for Ser and Tyr phosphorylation in the activation segment via Western Blotting with the indicated antibodies. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S22. (d) PKD2 Y717F displays higher activation loop Ser phosphorylation compared to PKD2 WT. HEK293 cells were transfected with wild type (WT) PKD2 or an Y717F mutant. 48 h after transfection, cells were stimulated with H₂O₂ (10 mM, 10 min) and PKD2 was precipitated from the cells using FLAG antibody and subsequently probed for Ser-706/710 phosphorylation. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S23. (e) Correlations between activation loop Ser phosphorylation and kinase activity towards peptide substrate are indicated by full lines. Pools of PKD1 and PKD2 WT enzymes harbor similar levels of activation loop Ser phosphorylation in oxidative stress, but PKD2 has higher activity towards Syn-2, which is due to Tyr-717 phosphorylation.