Figure 6

IR induced 4EBP1 dephoshorylation independent of Akt/mTOR. Jurkat cells were treated with 50 μM or 100 μM LY294002 (LY) (A) or irradiated with 10 Gy (B). Lysates were made 6 h after treatment with LY294002 or 4 h, 8 h, 12 h, and 24 h after IR. (A) LY294002 induced dephosphorylation of Akt on threonine 308 (T308) and serine 473 (S473) and on mTOR on serine 2448 and 2481 (S2448, S2481) indicating an inhibition of the Akt/mTOR pathway. Dephosphorylation of p70S6K on threonine 389 (T389) of p85S6K on threonine 412 (T412), and of 4EBP1 on threonine 37/46 (T37/46) and threonine 70 (T70) was also observed after treatment with LY294002. (B) IR did not affect phospho-Akt, phospho-mTOR, and phospho-S6K levels, but reduced phospho-4EBP1 levels as indicated by anti-phospho-4EBP1 antibodies and a shift of 4EBP1 from p20 to p17, suggesting that IR induced 4EBP1 dephoshphorylation independent of Akt/mTOR pathway. Reduction of Mcl-1 levels in response to LY294002 and IR correlated with 4EBP1 dephosphorylation. (C, D) Jurkat cells were treated with 50 μM or 100 μM LY294002. 6 h and 24 h later, mitochondrial dissipation (ΔΨm low, C) and DNA fragmentation (sub G1, D) were analyzed by flow cytometry. LY294002 induced mitochondrial dissipation and DNA fragmentation in a time- and concentration-dependent manner. (E) Jurkat cells were irradiated with 10 Gy. 4–24 h after irradiation, cells were lysed. Phospho-ERK1/2 and ERK levels were analyzed by western blot. No changes of phospho-ERK1/2 and ERK1/2 levels were observed in response to IR. Flow cytometric data shows mean values ± S.D. (n = 3), ** indicates p < 0.01, *** indicates p < 0.001, n.s. indicates no significance (p > 0.05).