CD40 triggers AKT activities via PI3K activation.

(A) Serum-starved MM.1S cells were activated by CD40 with 2 μg/mL G28.5 anti-CD40 mAb and collected at indicated time intervals. PI3K kinase assay was performed using equal amounts of lysates immunoprecipitated with antiphosphotyrosine 4G10 mAb, and immunocomplexes were assayed for the ability to phosphorylate PIP₂. Equal amounts of PI and PIP₂ produced in each sample demonstrate a specific induction of PI3K by CD40 activation. Control (c) indicates a PI3K kinase assay performed on protein A alone. (B) Serum-starved MM.1S cells, with or without pretreatment with PI3K inhibitors Wortmannin (0.2 μM) and LY294002 (30 μM) or anti-AKT peptide (16 μg/mL), were incubated with 2 μg/mL G28.5 anti-CD40 mAb for 30 minutes. Cell lysates were prepared from each sample and immunoprecipitated with an anti-AKT Ab with (lane 5) or without (lanes 1-4) competitor peptide. Kinase activity was measured with GSK-3α/β as a substrate and visualized by Western blotting with an antiphospho GSK-3 antibody, according to manufacturer's protocol. Western blotting with an anti-AKT Ab (lower panel) served as a loading control. (C) Serum-starved MM.1S cells, with or without pretreatment with indicated inhibitors, were activated by CD40 as described in “Materials and methods.” Cells were collected 30 minutes following CD40 activation, and cell lysates were prepared and subjected to Western blotting using anti-pAKT and anti-pERK Abs. Total AKT and total ERK were detected using anti-AKT and anti-ERK Abs on the same blots, demonstrating equal loading of each sample. PD indicates PD098959 (30 μM); Wort, Wortmannin (0.2 μM); and LY, LY294002 (30 μM).