Effect of cAMP-elevating agents, the PKA catalytic subunit, and wortmannin on PKB activity in 293 cells. (A) Effect of cAMP-elevating agents on ΔPH-PKB. Serum-starved 293 EBNA cells overexpressing ΔPH-PKB were incubated for 15 min with 1 μM insulin (I), 10 μM forskolin (F), 1 mM CPT-cAMP (CPT), 2.5 μM prostaglandin E1 (PGE1), or 1 mM 8-bromo-cAMP (8-Br). After the incubation, cells were lysed and ΔPH-PKB was immunoprecipitated and its kinase activity was determined with Crosstide as described in Materials and Methods. ΔPH-PKB activity is expressed as counts per minute of ³²P incorporated into Crosstide, and numbers above the bars indicate how many times above the basal level of stimulation PKB was stimulated. (B) Effect of the transfected PKA catalytic subunit. 293 EBNA cells were transfected with PKB and were either stimulated by insulin or cotransfected with the catalytic subunit of PKA. PKB was immunoprecipitated, and its activity was determined as described previously. (C) Effect of H-89 incubation on PKB activity. 293 EBNA cells were transfected with PKB and incubated or not incubated with H-89 (5 μM) and/or forskolin (10 μM; F), CPT-cAMP (1 mM; CPT), and insulin (1 μM; I). PKB was immunoprecipitated, and its activity was determined as described in Materials and Methods. (D) Effect of preincubation with wortmannin on PKA-induced activation of PKB. 293 EBNA cells were transfected with PKB in the presence or absence of PKA without preincubation or prior to preincubation for 30 min with 100 nM wortmannin (W). Subsequently, the cells were not treated or were treated with 1 μM insulin (I) for 5 min. The cells were lysed, PKB was immunoprecipitated, and kinase assays were performed as described in Materials and Methods. Results are expressed as counts per minute of ³²P incorporated into Crosstide. The levels of the expressed enzymes measured by immunoblotting are shown below the graph. The results shown in panels A to D are means ± standard errors of results from a representative of two separate experiments performed in triplicate. Values significantly different from basal values (P < 0.05, Student’s t test) are denoted by ∗. NS, not stimulated.

Effect of the PKB S422A mutation on PKB’s activation by PKA. (A) The PKA phosphorylation consensus site was mutated by changing S422 to alanine (S422A). Then wild-type (WT) or mutant PKB was used to transfect 293 cells and the resulting kinase activity was determined as described in Materials and Methods. Results are expressed as a percentages of the value for the control and are the mean values ± standard errors of three experiments, each of which was performed in triplicate. (B) A PKA kinase assay was performed with Kemptide as a substrate in the presence or absence of PKI as described in Materials and Methods. Statistical significance, according to Student’s t test, is indicated as follows: ∗ indicates a P of <0.01 and ∗∗ indicates a P of <0.005 versus the value for the appropriate control.

Effect of forskolin on PKB S473 phosphorylation. (A) Western blot showing endogenous expression of PKB in COS cells either after immunoprecipitation with anti-PKB (IP) or directly in the lysate (L). (B) COS cells were stimulated for the indicated times with insulin or forskolin prior to lysis. Cell extracts were subjected to SDS-PAGE (10% polyacrylamide) followed by Western blot analysis with antibody to PKB or to phospho-S473 PKB. Results shown are representative of at least two experiments.

Time courses of the effects of forskolin and insulin on PKB and GSK-3 activity in COS cells (A) Western blot showing endogenous expression of GSK-3 in COS cells either after immunoprecipitation with anti-GSK3 (IP) or directly in the lysate (L). (B to E) Time courses of PKB and GSK-3 activities. COS cells were treated or not treated for the times indicated with insulin (1 μM) or forskolin (10 μM) prior to extraction. Cell lysates were immunoprecipitated with antibodies either to PKB or to GSK-3; thereafter, activities in the immune complexes were determined. The results are expressed as percentages of the control value at time zero and are mean values ± standard errors of results from two experiments. ▴ and ▵ represent the GSK-3 activities in the presence of overexpressed dominant negative PKB.

Localization of GFP-PKB in response to insulin or forskolin in transfected HeLa cells. HeLa cells were transfected with GFP-PKB as described in Materials and Methods. Forty-eight hours later, HeLa cells were preincubated (C1 to C3) or not preincubated (B1 to B3) for 20 min with wortmannin (100 nM) prior to stimulation for 10 min with insulin (1 μM) (B1 to B3) or for 30 min with forskolin (10 μM) (D1 to D3). Cells were washed and fixed with paraformaldehyde (4%). Slides were mounted and analyzed by confocal microscopy. Images represent the center section of the X-Y plane. (A1 to A3) Nonstimulated cells.

Effect of cAMP-increasing agents on PI3-kinase activity immunoprecipitated with antibody to p85. 293 cells were preincubated for 20 min with 100 nM wortmannin prior to stimulation for 5 min with 1 μM insulin or for 30 min with 10 μM forskolin. Total cellular protein lysates were immunoprecipitated with anti-p85 antibody, and associated phospholipid kinase activities were estimated in vitro with PtdIns or PtdIns-4-P plus PtdIns-4,5-P₂ as the substrate. The lower panel shows a representative thin-layer chromatogram of products of PI3-kinase in the presence or absence of wortmannin. The position of ³²P-labeled PI (PIP) is indicated. The graph shown in the upper panel corresponds to the radioactivity associated with each spot as quantified by phosphorimaging. B represents the nontreated cells. IP, immunoprecipitates.

Effect of phosphatase treatment on PKA-induced PKB activation in 293 cells. PKB and CA-PKB corresponding to mutations of both T308 and S473 to aspartate were expressed in 293 cells in the presence or absence of the catalytic subunit of PKA. Cell lysates were prepared and subjected to immunoprecipitation with an antibody to PKB. Immunoprecipitates were treated with CIP (20 U) for 30 min prior to the kinase assay. The results are the mean values ± standard errors of results from two experiments, each of which was performed in triplicate.

Effect of T308A and S473A on PKB activation by PKA in 293 cells. Single or double mutations of T308 and S472 to alanine were produced in PKB, and the constructs obtained were transfected in 293 cells. Immunoprecipitation was performed following stimulation with forskolin (10 μM), and the PKB activity was determined as described in Materials and Methods. A Western blot with antibody to PKB illustrates expression of PKB below the graph. Values shown are expressed as percentages of the maximal values and are the mean values ± standard errors of results from three separate experiments performed in triplicate. Values significantly different from basal values are denoted by ∗ and ∗∗ (∗ = P < 0.05 and ∗∗ = P < 0.005, Student’s t test).

Phosphopeptide maps of endogenous PKB obtained from COS cells. Results of a two-dimensional peptide map analysis of ³²P-labeled PKB-HA isolated from metabolically labeled COS cells are shown. COS cells were grown in medium containing 1 mCi of [³²P]orthophosphate per ml for 4 h. Untreated, insulin (1 μM)-treated, or forskolin (10 μM)-treated cells were lysed, and equal amounts of lysate protein were immunoprecipitated with antibodies to HA. The immunoprecipitates were resolved by SDS-PAGE. The ³²P-labeled band corresponding to PKB was excised from the gel, eluted, and digested with trypsin (20 μg). Peptides were separated on cellulose thin-layer plates by electrophoresis from left to right (anode on the right), followed by thin-layer chromatography from bottom to top. Autoradiograms of the thin-layer plates are shown.

In vitro phosphorylation of GSK-3. COS cells were treated with either insulin (1 μM) or forskolin (10 μM) for the indicated times, and PKB was immunoprecipitated. At the same time, GSK-3 was immunoprecipitated from nonstimulated cells. PKB and GSK-3 immunoprecipitates were mixed in the presence of [γ-³²P]ATP, and the samples were subjected to SDS-PAGE under reducing conditions followed by autoradiography.

Effect of increasing concentrations of wortmannin on GFP-PKB localization in response to forskolin. HeLa cells were transfected with GFP-PKB as described in Materials and Methods. Forty-eight hours later, HeLa cells were preincubated for 20 min with wortmannin (100 nM [B1 to B3] or 300 nM [C1 to C3]) or buffer (A1 to A3) prior to stimulation for 30 min with forskolin (10 μM). Cells were mounted and analyzed by confocal microscopy. Images represent the center section of the X-Y plane.