Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells

Abstract

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca 2+ /CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca 2+ and CaM in the presence of eEF-2 and [γ-32 P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca 2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation statespecific antibody, we show that oxidant such as H 2 O 2 , which triggers a large influx of Ca 2+

Sustained Hydrogen Peroxide Induces Iron Uptake by Transferrin Receptor-1 Independent of the Iron Regulatory Protein/Iron-responsive Element Network

“…Thus, H 2 O 2 was shown to inhibit translation (52,53) by mechanisms such as the inhibition of the 70-kDa ribosomal protein S6 kinase (52), dephosphorylation of the eukaryotic initiation factor 4E-binding protein 1, or increased binding of this repressor protein to eukaryotic initiation factor 4E and concomitant loss of eukaryotic initiation factor 4F complexes (52,57). Translation can also be inhibited by H 2 O 2 via phosphorylation of the elongation factor eukaryotic elongation factor 2 (eEF2) (52), which was either mediated by activation of the eEF-2 specific, Ca 2ϩ /calmodulin-dependent protein kinase III (55) or reversible inhibition of the protein phosphatase 1 (56). On the other side, H 2 O 2 has been shown to stimulate translation in cell-free systems (58), plants (59), bacteria (60), and mammalian cells (54).…”

Translational reprogramming in cellular stress response

“…Like some initiation factors, one common regulatory mechanism of elongation factors is phosphorylation. For instance, elongation factor eEF2 undergoes phosphorylation at Thr56 within the GTP‐binding domain in response to oxidative stress and this modification interferes with its ability to bind to the ribosome . mTORC1 negatively regulates its cognate kinase eEF2K and thereby activates eEF2 .…”

The role of protein synthesis in cell cycling and cancer

“…Such a reduction could hypothetically benefit a cancer cell since the presence of free radicals can result in the phosphorylation of EF2 (Figure 1). For example, exposure to hydrogen peroxide results in an increase in intracellular Ca +2 levels, thereby activating EF2 kinase and subsequently inhibiting protein synthesis via phosphorylation of EF2 (Kang and Lee, 2001). Because the Warburg effect may limit the generation of free radicals, it may also limit one mechanism of EF2 phosphorylation.…”