Regulation of IL-2–mediated Akt activation by Rac1.
(A) IL-2–starved NK92 cells, pretreated by LY294002 (25 μM), piceatannol (25 μM), or DMSO control or infected with recombinant vaccinia virus encoding p85(DN), kinase-deficient Syk (SykT), or CD56 irrelevant control gene, were stimulated with IL-2 (100 U/mL) for 5 minutes at 37°C and analyzed for IL-2–triggered Rac1 activation before and after the impairment of Syk function. Rac1 was immunoprecipitated from NK92 cell lysates and examined for activity by affinity precipitation (AP) with PAK1 PBD, which binds only to activated Rac1-guanosine triphosphate (GTP) but not to inactivated Rac1-guanosine diphosphate (GDP). The IL-2–activated Rac1, Rac1-GTP, was precipitated by PAK1 PBD agarose, then resolved by 12.5% SDS-PAGE and examined by anti-Rac monoclonal antibody provided in the kit. (B) IL-2–starved NK92 cells, infected with recombinant vaccinia virus encoding dominant-negative Rac1 (N17Rac1), constitutively active Rac1 (V12Rac1), or CD56 irrelevant control gene, were stimulated with IL-2 (100 U/mL) for 5 minutes at 37°C. Cells were analyzed for Akt activation by Western blot (WB) analysis. (C) IL-2–starved NK92 cells, treated with 25 μM piceatannol or 25 μM LY294002 or DMSO, were infected with recombinant vaccinia virus encoding constitutively active V12Rac1 or CD56 irrelevant control gene, then stimulated with IL-2 (100 U/mL) for 5 minutes at 37°C. Cells were analyzed for Akt activation by Western blot analysis with Ser473-specific antibody. The same membranes were stripped and reprobed with anti–pan-Akt. In parallel, these NK cells were also analyzed by in vitro kinase assays for Akt activation with H2B as the substrate. Results are representative of 1 of 4 independent experiments.
