Figure 3.

The mechanism underlying tyrosine phosphorylation of PKD2 in oxidative stress is different from that of PKD1. (a) Graphical representation of the different phosphorylation sites covered in this study in PKD1/2 (b) Activation model of PKD1 in oxidative stress (for details see text). (c) Tyrosine phosphorylation of PKD2 is dependent on activation loop Ser-706/710 phosphorylation. HEK293 cells were transfected with wild type (WT) PKD2 or the indicated mutants (S2E = S706/710E, S2A = S706/710 A). 48 h after transfection, cells were stimulated with H₂O₂ (10 mM, 10 min) and PKD2 was precipitated from the cells using FLAG antibody. Immunoprecipitates were subjected to western blot and probed with the indicated antibodies. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S12. (d) Effect of PKC inhibitors and PDB on tyrosine phosphorylation of PKD2. HEK293 cells were transfected with FLAG-PKD2. 48 h after transfection, cells were treated with inhibitors (3 µM of BimI, 10 µM of Gö6983 or 5 µM STI571) or activator (500 nM PDB) and subsequently stimulated with H₂O₂ (10 mM, 10 min). Immunoprecipitates were subjected to western blot and probed with the indicated antibodies. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S13. (e) The activation mechanism of PKD1 in oxidative stress differs from that of PKD2. HEK293 cells were transfected with wild type (WT) PKD1 or the indicated mutants (S2E = S738/742E, S2A = S738/742A). 48 h after transfection, cells were stimulated with H₂O₂ (10 mM, 10 min) and PKD1 was precipitated from the cells using HA antibody. Immunoprecipitates were subjected to western blot and probed with the indicated antibodies. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S14.