FIGURE 7.

Gcn2 phosphorylates Kog1 directly. A, schematic presentation of Kog1 fragments used for the kinase assay. RNC, raptor N-terminal conserved domain; HEAT, HEAT domains; WD, WD40 domains. B, purified recombinant GST-Kog1 fragments were incubated with lysates from cells expressing GCN2-FLAG. Gcn2-FLAG co-purified with GST-Kog1 fragments was assayed by Western blotting. C, immunopurified FLAG-tagged Gcn2(E803V) (active), wild type, and Gcn2(K628R) (kinase-dead) versions of Gcn2 were assayed for kinase activity against recombinant GST-fused Kog1 fragments. The experiments were repeated three times, and representative data are shown.