Mechanism of radiation-induced arrest of global protein synthesis in C3H macrophages

Previously we reported radiation-induced apoptosis in macrophages of C3H mice but not other strains of mice. The depletion of Mcl-1, an anti-apoptotic Bcl-2 family protein known to have a very short turnover time, through radiation-induced arrest of global protein synthesis was identified as a major factor for the apoptosis. In the present study, the mechanism by which global protein synthesis was suppressed by irradiation was elucidated. Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) is a well documented mechanism of downregulating protein synthesis under a variety of stress conditions. Western blot analysis using phospho-eIF2alpha antibody to detect phosphorylation at serine 51 showed the increased phosphorylation of eIF2alpha in a dose dependent manner in irradiated macrophages of C3H mice, but not B6 mice. Four kinases have been identified as eIF2alpha kinases. PKR, PERK, GCN2 and HRI are activated by viral infection, endoplasmic reticulum stress, amino acid deprivation and hemin deficiency, respectively. The antibodies to detect the activation-specific phosphorylation site for PKR, PERK and GCN2 were available and used in Western blot analysis. Phospho-PKR and phospho-PERK antibody showed no change of phosphrylation at the activation-specific site by irradiation. On the other hand, phosphorylation of GCN2 at Threonine 898 was markedly increased by irradiation in macrophages of C3H mice, but not B6 mice. GCN2 is known to be activated through the binding of non-aminoacylated tRNA to Histidyl-tRNA Synthetase-related domain located in C-terminal region of GCN2 molecule. Whether radiation induces amino acid deprivation and/or increment of uncharged tRNA in C3H mouse macrophages is under investigation.14th International Congress of Radiation Researc