Figure 3.

Activation of PI3K by GyrB.PKR. (A) Serum-starved GyrB.PKR or GyrB.PKRK296H-expressing cells were left untreated or treated with coumermycin (100 ng/ml) for 6 h. Protein extracts (500 μg) were subjected to immunoprecipitation with an anti-PI3K p85 antibody followed by an in vitro lipid kinase assay in the presence of [³²P-γ]ATP and phosphatidylinositol (PI) as a substrate. Radioactive PIP was visualized by TLC and autoradiography (a). P85 levels in the immunoprecipitates were detected by immunoblotting (b). The data represent one of two reproducible experiments. (B) GyrB.PKR-expressing cells were transiently transfected with scrambled control siRNA (SCR) or siRNA targeting the p85 subunit of PI3K for 72 h. (C) GyrB.PKR cells were transiently transfected with pCMV plasmid lacking or containing FLAG-tagged PTEN for 24 h. (D) GyrB.PKR cells were infected with adenovirus expressing a dominant negative mutant of the p85 subunit (dnp85) of PI3K or a control adenovirus for 24 h. (B–D) Transfected or infected cells were left untreated or treated with coumermycin (100 ng/ml) for 6 h, and protein extracts (30 μg) were subjected to immunoblotting for the indicated proteins. The data represent one of three reproducible experiments.