Ser960 on RapGEF2 is selectively phosphorylated by PKC-θ. (A) Recombinant RapGEF2 wt and neutral exchange SS959/960AA, S959A, and S960A mutant proteins were 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 immunoblotting (middle panel). The enzymatic activity of PKC-θ was controlled in an MBP substrate phosphorylation reaction (bottom panel). Experiments were repeated at least 3 times, with similar results. (B) To analyze the effects of RapGEF2 phosphorylation at Ser960 on Rap1 GTP loading, Jurkat T cells were transiently transfected with RapGEF2 wt or SS959/960EE, S959E, or S960E mutant cDNA expression vectors or with GFP control, as indicated. CD3-induced Rap1 activation, as determined by GTP loading, was enhanced in the SS959/960EE as well as in the S960E mutant RapGEF2-expressing cells. Equal expression levels of the transfected wt and mutant RapGEF2 were confirmed by immunoblotting (not shown). Results shown are representative results of 3 experiments with similar outcomes.