Inhibition of the protein kinase activity of Wt HRI by co-expression with the HRI mutants.

Wt and mutant HRI (K199R, Δ10, and Δ20) were expressed individually or co-expressed in Sf9 cells as described previously.28Protein kinase assays were performed with the use of cytoplasmic cell extracts and purified eIF-2 as described previously.28 (A) eIF-2α kinase activity of Wt, K199R, Δ10, or Δ20 HRI. (B) eIF-2α kinase activity of co-expressed HRI. Wt HRI alone (lanes 1, 5, and 9); co-expression of Wt HRI with K199R (lanes 2, 6, and 10), with Δ10 (lanes 3, 7, and 11), or with Δ20 (lanes, 4, 8, and 12). The time intervals of protein kinase assays are 2, 5, and 10 minutes as indicated. (C) The extent of eIF-2α phosphorylation was quantified by scintilation counting of the gel slices containing eIF-2α. ■, wt; ●, Δ10/wt; ▴, Δ20/wt; ⧫, K199R/wt.