Fig. 6.

A. In vitro kinase activity of the mutants. The kinase activity of the in vitro translated proteins was examined in the presence of dsRNA or heparin. In vitro translated proteins (3 μL) were immunoprecipitated by anti-PKR mAb and protein A-Sepharose. The kinase activity present in the immune complexes was assayed. The positions of PKR and eIF2α bands are indicated by arrows. The different mutants are as indicated under the panels. (B) In vivo PKR activity assayed by translation inhibition assay. The transfections were performed using HT-1080 cells grown in six-well plates. The reporter used was pGL2C. An 800 ng aliquot of pGL2C was cotransfected using Lipofectamine reagent with 200 ng of the expression constructs for the proteins indicated. At 24 h after transfection, luciferase activity was measured in the cell extracts and normalized to the amount of total protein in the extract. All expression constructs were in pCDNA3; Control indicates the empty-vector (pCDNA3) control. Each sample was assayed in triplicate and the data represent means of six samples from two separate experiments. Error bars indicate SD. The expression of all proteins was ascertained to be at the same level by western blot analysis. (C) Western blot analysis was performed on the HT1080 cell extracts. All the expression constructs were in pCDNA3 and encoded flag-tagged PKR proteins. The western blot analysis was performed with anti-flag mAb and the same blot was stripped and reprobed with anti-β-actin mAb to ensure equal loading in all lanes. (D) Yeast growth phenotype of the PKR mutants. Growth of transformed INVSc1 yeast strain containing wtPKR/pYES2 (wt), K296R/pYES2 (K296R), K299A/pYES2 (K299A), DM/pYES2 (DM), hep2/pYES2 (hep2), and DM,hep2/pYES2 (DM,hep2). Cells were grown for 2 days at 30 °C on synthetic medium lacking uracil with 2% glucose (bottom panel) or 10% galactose (top panel) as sole carbon source.