PKC-θ physically interacts with and phosphorylates RapGEF2. (A) PKC-θ/RapGEF2 complex formation was first observed in the LexA Y2H library screen with the COOH-terminal CD of PKC-θ as bait. In retransformation analysis, the PKC-θ CD, but not the PKC-θ RD or the PKC-α CD or RD domain, interacted with RapGEF2 in parallel assays. Values are mean plus or minus SE of at least 3 independent experiments. (Inset) Immunoblot of the distinct subfragments of the cloned bait and prey proteins, the latter minimally representing the NH₂-terminal region (amino acids [aa] 1-173) of RapGEF2, obtained from the Y2H system. (B) Coimmunoprecipitation analysis of Jurkat T cells transiently cotransfected with hemagglutinin (HA)–tagged RapGEF2 and PKC-θ or GFP expression control. Twenty-four hours after transfection, RapGEF2 wt and SS959/960AA mutant IPs were immunostained with the PKC-θ–specific antibody. (Bottom panel) The RapGEF2 input control. No PKC-θ coimmunoprecipitated with control serum (MOCK), indicating the specificity of the interaction. Similar results were obtained in 3 independent experiments. (C) Jurkat T cells were stimulated with anti-CD3 antibodies or with PDBu/ionomycin, as indicated. Then, RapGEF2 was immunoprecipitated and immunostained with a broadly reactive (p)Ser (top panel) or RapGEF2 (bottom panel) antibody. *A nonspecific upper protein band, recognized by the pan RapGEF2 antiserum. (D) Similarly, a phosphopeptide antibody to Ser960 reacted with immunoprecipitates of RapGEF2 in an epitope-specific manner on CD3 stimulation of transfected Jurkat cells. (E) RapGEF2 is selectively phosphorylated by PKC-θ but not PKC-α in vitro. Recombinant RapGEF2 wt or neutral exchange SS959/960AA mutant protein was incubated with γ³²P-ATP as indicated. The reaction was stopped, and ³²P_i incorporation was analyzed by SDS-PAGE and autoradiography (top panel). Equal loading of RapGEF2 GST fusion proteins was validated by an anti-GST immunoblot (middle panel). Comparable enzymatic activities of these baculo-derived PKC-α and PKC-θ preparations were verified with an MBP substrate phosphorylation reaction (bottom panel). Experiments were repeated at least 3 times, with similar results.