Rap1 activation is a novel PKC-θ effector function. (A) To analyze the role of PKC-θ in CD3-induced Rap1 activation, wt and PKC-θ–KO CD3⁺ T cells were subjected to a Rap1 GTP pull-down assay. As a result, PKC-θ–deficient CD3⁺ T cells did demonstrate a severe Rap1 activation defect. As reported,³²  CD3-induced MAPK activation, via detection of dually phosphorylated ERK1/2, was intact, validating our activation protocol of PKC-θ-KO T cells. (B) Transient transfection of Jurkat T cells with wt PKC-θ or kinase-dead PKC-θ K409R mutant cDNA. Vertical lines have been inserted to indicate a repositioned gel lane. Equal expression levels of the transfected wt and K409R mutant PKC-θ were confirmed by immunoblotting (inset). To analyze the modulation of LFA-1 avidity (C) and Rap1 activation (D) by RapGEF2 phosphorylation at Ser960, Jurkat T cells were transiently transfected with 10 μg RapGEF2 SS959/960AA or SS959/960EE mutant cDNA expression vector or with GFP control, as indicated. Transfected Jurkat T cells were left unstimulated or were stimulated with anti-CD3 antibodies or PDBu/ionomycin. Equal expression levels of the transfected wt and mutant RapGEF2 were confirmed by immunoblotting (insets). On stimulation with PDBu/ionomycin, the RapGEF2 SS/EE mutant caused significantly stronger adhesion to ICAM-1 than did the RapGEF2 wt or SS/AA mutant (wt RapGEF2, n = 3; RapGEF2 SS/AA, n = 3; RapGEF2 SS/EE, n = 3; P = .003). (E) To induce stronger overexpression, 30 μg RapGEF2 SS959/960AA mutant cDNA expression vector or GFP control was used to transfect Jurkat T cells. Results shown in panels B, D, and E are representative results of 3 experiments with similar outcomes; data in panels A and C are shown as the mean plus or minus SD of 3 independent experiments.