Figure 4.

Y87-phosphorylation of PKD2 is mediated by active Src. (A) HeLa cells (2 × 10⁶ cells/10 cm plate) were transfected with vector or constitutively-active Src (Src.CA) as indicated. PKD2 was immunoprecipitated and analyzed using the pY87-PKD2 antibody. Samples were counterstained for total PKD2 and lysates were assessed by Western blot for Src expression (input control). (C) Src in vitro kinase assay using purified PKD2 as a substrate. After the kinase reaction samples were analyzed for Y87 phosphorylation using the pY87-PKD2 antibody. Samples were counterstained for total PKD2 and for Src (input controls). (B,D,E) HeLa cells (0.75 × 10⁶ cells/6-cm plate) were co-transfected with vector or constitutively-Src (Src.CA) and GFP-tagged versions of PKD2 or PKD2.Y87F as indicated. PKD2 was immunoprecipitated (anti-GFP) and assessed for Src-mediated phosphorylation using the pY87-PKD2 antibody (B), assessed for PKD2 activation loop phosphorylation using an anti-pS706/710 antibody (D), or for PKD2 autophosphorylation using an anti-pS876 antibody (E). Immunoprecipitates were re-probed with anti-GFP for expression control of PKD2 and PKD2.Y87F. The lysates were evaluated by Western blot for Src expression (input control). Uncropped blots for Fig. 4 are shown in Supplemental Figures S7, S8 and S9. All experiments have been performed at least three times with similar results.