FIG. 5.
Characterization of Rat-1:iRas cells expressing a dominant negative Akt protein. (A) Characterization of the Rat-1:iRas–dominant negative Akt cells. Rat-1:iRas cells etopically expressing a plasmid bearing the gene encoding the dominant negative Akt(K179A) protein (DN Akt) or the vector control were generated, as described in the Materials and Methods. Total protein (50 μg) was isolated from Rat-1:iRasV cells (vector control cells), three Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (Pool) in the absence or presence of IPTG (5 mM). Protein samples were resolved on an SDS–10% polyacrylamide gel, transferred to membrane, and analyzed for the presence of Akt, Ras, and α-tubulin. Akt(K179A) protein was detected using a hemagglutinin-specific antibody (BABCO). IPTG-induced p21Ras expression was detected using a pan-Ras monoclonal antibody (Calbiochem, San Diego, Calif.). To ensure equal levels of protein loading, blots were reanalyzed with an α-tubulin-specific antibody (Sigma). Primary antibodies were detected using an HRP-labeled secondary antibody and by performing ECL. (B) Expression of the dominant negative Akt protein blocks H-Ras(V12)-induced endogenous Akt activity. Subconfluent Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were grown overnight in medium containing 2% FBS. Eighteen hours later cells were washed and cultured for 4 h without serum and with or without IPTG (5 mM). Some groups received LY 294002 (10 μM) 3 h after serum deprivation. Immunocomplex kinase assays for Akt were performed as described in Materials and Methods. The fold Akt activity was determined by obtaining values for Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (grown in the absence of IPTG) and normalizing these numbers to 1. Data presented are representative of results of at least three different assays, which generated similar results. (Gel) Immunoblot analysis demonstrating that relatively equal amounts of total Akt protein were immunoprecipitated during the course of the experiment. Both endogenous Akt and hemagglutinin-tagged dominant negative Akt(K179A) proteins are shown.