Figure 1

IR induced caspase-dependent processing of eIF4G1, eIF4B, as well as eIF3A, and caused disassembly of the cap-dependent initiation complex. Jurkat cells were irradiated with 10 Gy. (A) Lysates were made 4–24 h after IR. Cleavage of eIF4G1 and eIF3A, as well as eIF4B decline, were observed in response to IR. The cleavage coincided with processing of caspase-3 (C3) and the caspase-3 substrate PARP indicating caspase-3 activation. No IR-induced processing of eIF4E, eIF4A, and eIF4H was detected. Densitometric analysis shows relative protein levels normalized to the respective levels in non-irradiated cells (0h after IR). (B) 24 h after irradiation, cells were lysed and a pull-down assay was made using 7-methyl GTP agarose to mimic the cap structure of mRNAs. Whereas the recruitment of eIF4E to the cap structure remained unchanged, recruitment of eIF4A, eIF4G1, and eIF3A into the cap-dependent initiation complex was reduced after IR. (C) Jurkat cells were irradiated with 10 Gy and co-treated with 30 μM of caspase inhbitor zVAD or the respective amount of solvent. 24 h later, cells were lysed. Treatment with zVAD blocked IR-induced cleavage of eIF4G1, eIF3A, PARP, and the decline of eIF4B, as well as the processing of caspase-3 to the active p19 and p17 forms indicating that the processing of the three initiation factor occurred after caspase activation. However, caspase inhibitor zVAD did not block IR-induced 4EBP1 dephsophorylation on Mcl-1 reduction.