Figure 5. Examining potential essential translation‐related targets and the functional importance of phosphorylation on rates of protein synthesis upon TOR kinase inhibition.

Phosphorylation kinetics of common phosphosites identified for 3 essential translation‐related proteins, and the effects of the tif471_18A phosphosite mutant on rates of protein synthesis upon Torin1 treatment.

• A, B
  Phosphosites identified on (A) Tif212 and (B) Erf1, respectively, showing 2‐fold phosphorylation change within 20 min of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T = 0 min). Grey dashed lines indicate 2‐fold threshold.
• C, D
  Relative phosphorylation levels of the 10 common phosphosites of Tif471 plotted against time after Torin1 treatment. Phosphorylation profiles are grouped based on phosphorylation kinetics, where 6 sites showed more than 1.5‐fold increase by 10 min (C), and the remaining 4 exceeded 2‐fold by 30 min (D). Grey dashed lines indicate 2‐fold threshold.
• E, F
  Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471 ⁺) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 min of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471 ⁺ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log₂ transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from three independent experiments.