Figure 2
Abl is an upstream kinase for Tyr717 in PKD2. (a) Effect of the Abl inhibitor STI-571 and activator DPH on the phosphorylation of Tyr-717 following oxidative stress. HEK293 cells were transfected with wild type (WT) PKD2 or an Y717F mutant. 48 h after transfection, cells were treated with the indicated compounds and 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. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S8 (b) Co-transfection of c-Abl with PKD2 results in Tyr-717 phosphorylation without additional stimulus. HEK293 cells were transfected with GST-tagged wild type (WT) PKD2 or an Y717F mutant and FLAG-tagged c-Abl. 48 h after transfection, PKD2 was precipitated from the cells using Glutathione-sepharose beads. 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. S9 (c) siRNA mediated downregulation of Abl expression results in decreased Tyr-717 phosphorylation of PKD2. Cells were transfected with siRNA targeted against c-Abl and subsequently transfected with FLAG-PKD2. PKD2 was precipitated from the cells using FLAG antibody and phosphorylation on Tyr-717 was followed with the PSSA. Quantification of three independent experiments is shown. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S10 (d) in vitro kinase assay showing direct phosphorylation of PKD2 by Abl. PKD2 (kinase dead, K580A mutant) was incubated with or without 50 ng Abl and incubated for 30 min at 30 °C. 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. S11.
