Physiological concentrations of PGE₂-induced HPK1 kinase activity.

HA-HPK1 was immunoprecipitated from lysates derived from Jurkat T cells transfected with HA-HPK1. Cells were exposed to varying concentrations of PGE₂ for 2 minutes at 37°C prior to lysis and immunoprecipitation. In vitro immune complex HPK1 kinase assays (IVK) were performed in the presence of exogenous substrate, 5 μg histone H2A. (A) ³²P-incorporated histone H2A. (B)³²P-incorporated auto-transphosphorylated HA-HPK1. (C) Western blot of immunoprecipitated HPK1 from panel B, using the anti-HPK1 no. 7 antibody. (D) In vitro immune complex HPK1 kinase assays were performed on lysates of PGE₂-stimulated (10 nM) transfectants expressing wild-type or a catalytically inactive mutant (K46E) of HPK1. The autoradiographic bands depict³²P-incorporated histone H2A. (E)³²P-incorporated autophosphorylated/transphosphorylated wild-type and (K46E) mutant HPK1. (F) Western blot of immunoprecipitated HPK1 from panel E, using the anti-HPK1 no. 7 antibody. (G) In vitro immune complex kinase assays performed with endogenous HPK1 immunoprecipitated from nontreated cells or from cells treated with 10 nM PGE₂. (H) Western blot of immunoprecipitated HPK1 from panel G, using the anti-HPK1 no. 7 antibody. Lane numbers are indicated at the bottom of all lanes. Data in all figures are representative of at least 3 independent experiments.