Effect of serum and hydrogen peroxide on the Ca2+/calmodulin-dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells.
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.
Kang
K R
KR
Department of Biochemistry and Gyeongsang Institute of Health Science, Gyeongsang National University College of Medicine, Chinju, Korea. krkang@gaechuk.gsnu.ac.kr
Lee
S Y
SY
D000818:Animals; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division; D002478:Cells, Cultured; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010445:Peptide Elongation Factors / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D054648:Protein Phosphatase 2; D014268:Trifluoperazine / Q000494:pharmacology
D016877:Oxidants; D010445:Peptide Elongation Factors; D019319:Okadaic Acid; D014268:Trifluoperazine; D011092:Polyethylene Glycols; D004533:Egtazic Acid; D006861:Hydrogen Peroxide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2