EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 33, No. 4, 198-204, December 2001 Effect of serum and hydrogen peroxide on the Ca2+/calmodulin- dependent phosphorylation of eukaryotic elongation factor 2(eEF-2) in Chinese hamster ovary cells Kee Ryeon Kang1,2 and So-Young Lee1 Introduction 1 Department of Biochemistry and Gyeongsang Institute of Health The regulation of protein synthesis in eukaryotes is inte- Science, Gyeongsang National University College of Medicine, grated with the translation process and other metabolic Chinju 660-751, Korea pathways of the cell, i.e. altered translation rates occur 2 Corresponding author: Tel, +82-55-751-8730; during mitosis, upon nutrient starvation or oxidant stress, Fax, +82-55-759-8005; E-mail, [email protected] and following treatment with hormones or growth factors. The process of mRNA translation takes place by three Accepted 30 November 2001 distinct phases termed (peptide-chain) initiation, elonga- tion, and termination. The acute regulation of mRNA Abbreviations: eEF-2, eukaryotic elongation factor 2; CaM, calmod- translation depends on changes in the activity of initia- ulin; CHO, Chinese hamster ovary tion factors or elongation factors, which is also common- ly mediated by alteration in their states of phosphory- lation (Hershey et al., 1996). Elongation factor 2 (eEF- 2), one of two elongation factors involved in the elonga- Abstract tion phase of translation in higher eukaryotes, facilitates translocation of peptidyl-tRNA from the ribosomal A site Eukaryotic elongation factor eEF-2 mediates regula- to the P site. Phosphorylation of eEF-2 reduces the tory steps important for the overall regulation of affinity of the factor for the ribosome, thereby decreasing mRNA translation in mammalian cells and is acti- the rate of protein synthesis (Nilsson and Nygard, 1991). vated by variety of cellular conditions and factors. In The translational activity of the phosphorylated eEF-2 2+ this study, eEF-2 specific, Ca /CaM-dependent pro- can be restored by the action of phosphoprotein phos- tein kinase III (CaM PK III), also called eEF-2 kinase, phatase 2A and 2C (PP2A and PP2C, respectively) was examined under oxidative stress and cell prolif- (Redpath and Proud, 1990). eEF-2 is phosphorylated by eration state using CHO cells. The eEF-2 kinase a specific eEF-2 kinase previously also known as Ca2+ activity was determined in the kinase buffer contain- and calmodulin-dependent protein kinase III, on threo- 2+ ing Ca and CaM in the presence of eEF-2 and [γ- nine residues (Calberg et al., 1991). Calmodulin medi- 32 P] ATP. The eEF-2 kinase activity in cell lysates ates many of the intracellular effects of Ca2+ (Park, 2+ was completely dependent upon Ca and CaM. 2001), and the eEF-2 kinase is entirely dependent on Phosphorylation of eEF-2 was clearly identified in Ca2+/CaM for activity in mammalian tissues. Increased proliferating cells, but not detectable in CHO cells phosphorylation of eEF-2 occurs in several types of arrested in their growth by serum deprivation. The cells in response to stimuli, which elevates cellular Ca2+ content of the eEF-2 protein, however, was equiva- levels or stimulates cellular proliferation (Enkemann et lent in both cells. Using a phosphorylation state- al., 1999). Decreased kinase activity is seen in the specific antibody, we show that oxidant such as presence of drugs that increase the concentration of 2+ H2O2, which triggers a large influx of Ca , dramati- cAMP (Hovland et al., 1999). Previous study has demon- cally enhances the phosphorylation of eEF-2. In strated that oxidants such as hydrogen peroxide (H2O2) addition, H2O2-induced eEF-2 phosphorylation is stimulate several early growth response events such as 2+ dependent on Ca and CaM, but independent of the stimulation of protein tyrosine phosphorylation (Rao, protein kinase C. In addition, okadaic acid inhibits 1997). Modification of the redox thiol status of the phosphoprotein phosphatase 2A(PP2A)-mediated cytosol could also contribute to the wide-ranging effects eEF-2 dephosphorylation. These results may pro- of H2O2. In particular, H2O2 and oxidized glutathione has vide a possible link between the elevation of intra- been shown to potentiate the spontaneous release of 2+ cellular Ca and cell division and suggest that Ca2+ from inositol triphosphate-sensitive Ca2+ from phosphorylation of eEF-2 is sensitive cellular reflex inositol triphosphate-sensitive Ca2+ stores (Missiaen et 2+ on stimuli that induces intracellular Ca flux. al., 1991). Increased intracellular Ca2+ is also associated with phosphorylation of eEF-2 and inhibition of protein 2+ Keywords: Ca , CaM , eEF-2, eEF-2 kinase synthesis (Marin et al., 1997). Phosphorylation of eEF-2 by eEF-2 kinase 199 In this report we have studied regulation of the eEF- phenylmethylsulfonyl fluoride (PMSF), vortexed, and then 2 kinase activity in Chinese hamster ovary cells treated stood for 10 min on ice. The cell lysates were cleared by o with various stimuli including H2O2 to influence intra- centrifugation at 15,000 g at 4 C for 10 min. Cell ly- cellular Ca2+ concentration. In addition, this study was to sates containing equal amounts of protein from control determine whether induction of cell division by serum and each treatment were used for SDS-polyacrylamide was linked to the activation of eEF-2 kinase. gel electrophoresis (PAGE) and immunoblotting. Purification of eEF-2 protein Materials and Methods Purification of eEF-2 from chicken embryo was carried out according to Kim et al. (1991) with some modifi- Materials cation. The peak fractions of eEF-2 obtained from Dulbecco’s modified Eagle medium (DMEM), antibiotic- DEAE-Sephacel column chromatography were dialyzed antimycotic mixture, and trypsin-EDTA were purchased against 50 mM potassium phosphate buffer, pH 6.7, from Gibco/BRL Life Technologies, Inc (Gaithersburg, MD). containing 10% glycerol, and applied to CM-Sephadex Sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) C-50 column. Finally, gel filtration chromatography was were obtained from Boehringer Mannheim. [γ-32P] ATP done using Ultrogel AcA-44 in 50 mM Tris/HCl, pH 7.5, β (6,000 Ci/mmol) was obtained from NEN Life Science 5 mM MgCl2, 100 mM KCl, 10 mM -mercaptoethanol, Products (Boston, MA). Okadaic acid was from Calbio- and 25% glycerol. eEF-2 activity was assayed by ADP- 32 chem, diacyl ethylene glycol (DAEG) was from Avanti ribosylation with diphtheria toxin in the presence of [ P] Polar Lipids, Inc., fetal bovine serum (FBS) was from NAD(Kang et al., 1994). Hyclone, and ECL Western blotting detection kit was from Amersham Pharmacia Biotech. Hydrogen peroxide Production of antibodies against total or phosphor- ylated eEF-2 (H2O2), phosphoprotein phosphatase 2A (PP2A), trifluo- perazine (TFP), Nutrient mixture F-12 HAM, and all The serum that specifically recognizes the phosphorylat- other chemicals were from Sigma. ed form of eEF-2 was obtained by immunization of rab- bits with a peptide encompassing eEF-2 phosphorylation Cell culture sites [GETRFT(P)DTRK]. This peptide was synthesized, HeLa cells, Vero cells, and NIH3T3 cells were grown in and the threonine at the position corresponding to Thr DMEM containing 25 mM D-glucose, 100 units/ml peni- 56 in the native protein was phosphorylated chemically cillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin (Czernik et al., 1995). The serum recognizing total eEF- 2 was obtained by immunization of rabbits with a B and 10%(v/v) FBS in a humidified 5% CO2 incubator at 37oC. Chinese hamster ovary(CHO)-K1 cells were peptide derived from the same region of the protein grown in Nutrient mixture F-12 supplemented with 10% [ARAGETRFTDTRKD] (Alirezaei, et al., 2001). FBS and same concentrations of antibiotic-antimycotic mixture described above. Serum deprivation was carri- Determination of eEF-2 kinase activity ed out by lowering the concentration of FBS from 10% For determination of the eEF-2 kinase activity, cell to 0.5% for the period of 24 h prior to specific treatment. homogenate was incubated with reaction buffer (50 mM Hepes, pH 7.6, 1.5 mM CaCl2, 1.0 mM DTT, 0.25 mM Prepatation of cell lysates Na3VO4, 1.0 mM MgCl2, and 1.5 mM MnCl2) containing CHO cells were plated onto 60-mm dishes and grown in 1 µmol of purified eEF-2 in the presence of 0.25 mM [γ- 32 Nutrient mixture F-12 containing 10% FBS. At 70-80% P]ATP. The reaction mixtures were incubated at 37°C confluence, cells were growth-arrested by incubating for for 10 min, and terminated by the addition of 10 µl of 5 X 24 h in Nutrient mixture F-12 containing 0.5% FBS. Laemmli sample buffer containing 0.2 M Tris, pH 6.8, Growth-arrested CHO cells were treated with and with- 6% SDS, 30% glycerol, and 15% β-mercaptoethanol. out various concentrations of H2O2, TFP, DAEG, or EGTA Samples were boiled for 5 min and proteins were sepa- for 30 min at 37oC. In the case of time course experi- rated by a 8% SDS-PAGE. Gels were stained with ments, CHO cells were treated with and without 200 µM Coomassie Brilliant Blue R-250 and dried using a o Hoefer Scientific gel drier. X-ray film (Fuji Super RX) H2O2, for the indicated times at 37 C. After treatments, medium was removed, and cells were rinsed with ice- was exposed at -70°C using an intensifying screen. The cold phosphate-buffered saline (PBS) and collected with autoradiograms were analyzed with GS-710 Calibrated a rubber policeman, suspended in 0.5 ml of suspension Imaging Densitometer (Bio-Rad). buffer (10 mM Tris/HCl, pH 7.5, and 2 mM MgCl2) and centrifuged at 2,700 g at 4oC for 5 min. Then they were Analysis of eEF-2 phosphorylation by sequential freeze-thawed, suspended in 20 µl of suspension buffer immunoblotting containing 1% Triton X-100, 5 mM DTT, and 1 mM Phosphorylation of eEF-2 was analyzed by sequential 200 Exp.