FIG. 1.

Activation of PKB mutants. (A) Schematic presentation of the PKBα mutants employed. Membrane-targeted forms of PKBα contain either the lck myristylation and palmitylation signal (m/p) at the N terminus or the CaaX box from Ki-Ras at the C terminus. PH, pleckstrin homology domain. All the PKBα constructs are HA epitope tagged at the N terminus, except for PKBα-CaaX. (B) Ectopically expressed PKB variants from extracts of human 293 cells were assayed as described in Materials and Methods. The extracts are derived from the cotransfection experiment with S6K1 described in the legend to Fig. 2A. The cells were serum starved for 24 h and either extracted immediately, stimulated for 30 min with 1 μM insulin, or pretreated with 250 nM wortmannin for 1 h prior to stimulation with insulin, as indicated. Error bars indicate standard deviations.