FIGURE 5.

Gcn2 inhibits the intrinsic kinase activity of TORC1. Wild type (Y1599) and gcn2 deletion (Y1600) cells expressing KOG1-HA (+) or empty vector (−) were subjected to the indicated treatments for 15 min. Cells were collected and lysed. TORC1 was immunopurified from cell extracts with HA antibody and assayed for kinase activity toward GST-4E-BP1. A, TORC1 kinase activity from wild type cells. A TORC1 sample from untreated wild type (ctrl) was included as a control. B, TORC1 kinase activity from gcn2 deletion cells. TORC1 samples from untreated wild type (ctrl 1) and gcn2 (ctrl 2) cells were included as controls. TORC1 samples from untreated wild type and gcn2 deletion cells were assayed in the presence of rapamycin and GST-FKBP12 and shown in A and B, respectively, as rapamycin treatment controls (Rap). Bar graphs in A and B are quantitative presentations of TORC1 kinase activities from treated samples that are expressed as percentages of the untreated control. The relative kinase activity was calculated as the ratio between the level of GST-4E-BP1 phosphorylation and that of immunopurified Kog1-HA protein. Data were from five independent experiments and expressed as means ± S.D. (error bars).