Exp Mol Med.  2001 Dec;33(4):198-204.

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

• Kang KR
• Lee SY

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, Ca2+/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 Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ 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 state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.

Keyword

Ca2+; CaM; eEF-2; eEF-2 kinase

MeSH Terms

Animal
CHO Cells
Ca(2+)-Calmodulin Dependent Protein Kinase/*metabolism
Cell Division
Cells, Cultured
Comparative Study
Cytosol/enzymology
Egtazic Acid/pharmacology
Hamsters
Human
Hydrogen Peroxide/*pharmacology
Mice
Okadaic Acid/pharmacology
Oxidants/*pharmacology
Peptide Elongation Factors/metabolism
Phosphoprotein Phosphatase/metabolism
Phosphorylation
Polyethylene Glycols/pharmacology
Trifluoperazine/pharmacology