Figure 1

Identification of PKD2 Tyr-717 phosphorylation during oxidative stress. (a) Upper panel: alignment of the PKD1/2/3 activation segment. Activation loop serine residues are denoted in purple, the identified pTyr-residue in green. The borders of the activation segment are indicated in bold. Lower panel: position of the activation loop Ser residues and Tyr residue in a PKD2 homology model (generated by the Phyre2 server). (b) Mass spectrometry based identification of the p-Tyr717 containing peptide following oxidative stress. (c) Tyr-717 phosphorylation after oxidative stress is detected by a PSSA. 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. Phosphorylation of Tyr-717 was assessed using the home-made PSSA. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S4. (d) Phosphorylation of endogenous PKD2 following oxidative stress. HEK293 cells were stimulated with H₂O₂ (10 mM, 10 min) and PKD2 was precipitated from the cells using a PKD2 antibody. Phosphorylation of Tyr-717 was assessed using the home-made PSSA. N.S.: non-stimulated cells. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S5. (e) Sub-millimolar doses of H₂O₂ elicit Tyr-717 phosphorylation. HEK293 cells were transfected with wild type (WT) PKD2. 48 h after transfection, cells were stimulated with H₂O₂ at the indicated concentrations for 10 min and PKD2 was precipitated from the cells using FLAG antibody. Phosphorylation of Tyr-717 was assessed using the home-made PSSA. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S6. (f) Tyr-717 phosphorylation is not observed in classical PKD activation by GPCRs or phorbol-12,13-dibutyrate (PDB). HEK293 cells were transfected with wild type (WT) PKD2. 48 h after transfection, cells were serum starved for 6 h prior to stimulation with bradykinin (BK; 1 µM, 5 min), neurotensin (NT; 1 µM, 10 min), Lysophosphatidic acid (LPA; 10 µM, 5 min), PDB (500 nM, 15 min) or H₂O₂ (10 mM, 10 min). FLAG immunoprecipitates were subjected to western blot and probed with the indicated antibodies. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S7.