RasGRP3 is positively regulated by PKC-θ and directly phosphorylated by PKC-θ and PKC-β2. (A) HEK-293 cells were cotransfected with 10 μg RasGRP3 and 10 μg constitutively active mutants of PKC-α (A25E) or PKC-θ (A148E). At 24 hours after transfection, cells were stimulated with either 10 nM PMA or DMSO (vehicle control) for 10 minutes. Cell lysates were assayed for PKC-θ, PKC-α, RasGRP3, Ras-GTP, phospho-Erk1/2, and total Ras. (B) Sf9 cells were mock infected or infected with PKC-θ or PKC-β2 expressing baculovirus. Lysates were either immunoprecipitated with anti–PKC-θ, anti–PKC-β, or primary antibody was omitted, as a control. An immunocomplex kinase assay was conducted using either a 43-kDa MBP or a 127-kDa MBP-RasGRP3 fusion protein followed by resolution by SDS-PAGE and autoradiography. The positions of MBP-RasGRP3, autophosphorylated PKC-θ, PKC-β2, and MBP are shown. Autoradiographic exposure was for 20 minutes.