Activation of Akt-ER by addition of 4-HT.

(A) Myr Akt-ER–expressing cells were incubated with 4-HT for the indicated times and left unstimulated or stimulated by BCR cross-linking for 5 minutes. The cells were lysed, and the kinase activity of Akt-ER was analyzed in anti-HA immunoprecipitates by phosphorylation of a GSK-3–derived substrate peptide (see “Materials and methods”). The small increase seen with BCR cross-linking after 30 minutes is not a reproducible result. The non-Myr Akt-ER did not show any activity above background before or after 4-HT addition in a similar experiment (data not shown). The data are representative of at least 3 independent experiments. (B) In an experiment similar to A, one set of cells were pretreated with 100 nmol/L wortmannin for 15 minutes before addition of 4-HT for 1 hour. HA-immunoprecipitates were tested for Akt-ER kinase activity as described above. (C) Untreated, 4-HT–treated or 4-HT+wortmannin–treated cells were lysed and phosphorylation of Akt-ER was assessed by immunoblotting using phosphospecific Akt antibodies reactive against Ser⁴⁷³ or Thr³⁰⁸. Akt-ER protein was present in all lanes as assessed by anti-HA immunoblotting (data not shown).