FIG. 5.

PKCα and PKCθ mediate IKK activation following CD3/CD28 stimulation with different kinetics. (A) Jurkat T cells were pretreated with different PKC inhibitors for 20 min prior to CD3 and CD28 cross-linking: Gö6976 and Gö6890 were each used at 2 μM, and rottlerin was used at 30 μM. Cells were treated with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 45 min on ice and cross-linked with goat anti-mouse antibodies in solution at 37°C for the indicated periods of time. Cells were harvested, and the IKK complex kinase activity was measured in an IVK assay, as described in the legends to Fig. 1 to 4. (B) Membrane (M) and cytoplasmic (C) fractions from CD3/CD28-stimulated Jurkat T cells were isolated and resolved by SDS-PAGE. The purity of the digitonin-extractable fraction (C) and NP-40-soluble fraction (M) was tested with antibodies specific for the raft-associated protein, LAT, and the cytoplasmic NF-κ B protein, p65. The quantity of PKCα and PKCθ was analyzed by immunoblotting. (C) Jurkat T cells were transfected with the reporter plasmids κB-luc (0.19 μg) and Tk-Renilla (0.01 μg) by the FuGENE6 method. Eighteen hours later, transfected Jurkat T cells were pretreated with Gö6976 at 2 μM and rottlerin at 30 μM for 20 min andwere then cross-linked with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies. Four hours later, luciferase activity was measured, normalized as described above, and expressed as relative luciferase units (RLU). (D) Jurkat T cells (10 × 10⁶ per sample) were electroporated with 40 μg of pFRT-PKCθ or control vector pFRT-H1. Forty-eight hours later, transfected Jurkat T cells were treated with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 45 min on ice and cross-linked with goat anti-mouse antibodies in solution at 37°C for the indicated periods of time. IKK activity was measured in an IVK assay as described above. Suppression of endogenous PKCθ expression, but not PKCα, was confirmed by immunoblotting. (E) Jurkat T cells were electroporated with 40 μg of pFRT-PKCθ (lane 2), pFRT-PKCα (lane 3), or control vector pFRT-H1 (lane 1) together with reporter κB-luc (3.6 μg) and Tk-Renilla (0.4 μg) plasmids. Forty-eight hours later, transfected Jurkat T cells were treated with anti-CD3 (3 μg/ml) and anti-CD28 (3 μg/ml) antibodies for 45 min on ice and cross-linked on plated goat anti-mouse antibodies at 37°C. Four hours later, luciferase activity was measured, normalized as described above, and expressed as relative luciferase units (RLU). Suppression of endogenous PKCθ and PKCα protein levels was confirmed by immunoblotting.