Journal of Cell and Molecular Biology 8(2): 125-130, 2010 Research ArticleHaliç University, Printed in Turkey. http://jcmb.halic.edu.tr

Eukaryotic elongation factor eEF1A1 promotes and Ser300 mutantsof eEF1A1 inhibit transition through the S and G2/M phases of thecell cycleKah Wai LIN*and Serhiy SOUCHELNYTSKYI

Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institute, Karolinska UniversityHospital, Stockholm, Sweden (*author for correspondence; kahwai.lin@ki.se)

Received: 17 September 2010; Accepted: 02 December 2010

AbstractThe eukaryotic elongation factor 1 alpha (eEF1A) plays a crucial role in normal physiological processes and carcinogenic transformation. Our recent study showed that the phosphorylation of eEF1A1 at Ser300 by type I transforming growth factor-β (TGF-β) receptor inhibited the protein synthesis and cell proliferation. Inhibition of cell proliferation may occur by accumulation of cells in a specific phase of the cell cycle. We report here an exploration of the role of eEF1A1 and an impact of Ser300 in eEF1A1 on the transition through the phases of the cell cycle by human breast epithelial cells. Our results suggest that the overexpression of eEF1A1 increases proliferation of cells by promoting transition of cells through the S- and G2/M-phases in the cell cycle. Ser300 mutants of eEF1A1 with impaired ability to support protein synthesis inhibited the accumulation of cells in G0/G1 phase. Thus, we showed that eEF1A1 modulates transition of the S and G2/M phases by cells.

Keywords: Elongation factor, phosphorylation, cell cycle, proliferation, breast cancerAbbreviations: eEF1A: eukaryotic elongation factor 1 alpha, aa-tRNA: aminoacyl-tRNA, PTM: Post-translational modification, TGF-β: transforming growth factor-β, WT: wild-type, CDK: cyclin-dependent kinase

Ökaryotik uzama faktörü eEF1A1 G2/M geçişini destekler ve eEF1A1S’nin Ser300 mutantı engeller

ÖzetÖkaryotik uzama faktörü 1 alpha (eEF1A) normal fizyolojik işlemler ve karsinojenik dönüşümde kritik bir rol oynar. Bu çalışmamız eEF1A1’in Ser300’de tip I transforme edici büyüme faktörü- β (TGF- β) ile fosforlanmasının, protein sentezini ve hücre proliferasyonunu engellediğini gösterdi. Hücre proliferasyonunu engellenmesi hücre dönüsünün spesifik bir fazında hücrelerin birikimi ile gerçekleşebilir. Burada, insan meme epitel hücrelerinde hücre döngüsü fazları geçişinde eEF1A1’in rolünün ve eEF1A1’e Ser300’ün etkisinin bulgusunu rapor ediyoruz. Sonuçlarımız eEF1A1’in fazla ifadesinin hücre döngüsünde hücrelerin S- ve G2/M- faz geçişlerini destekleyerek hücre proliferasyonunu artırdığı fikrini vermektedir. eEF1A1’in Ser300 mutantları protein sentezini desteklemek için zayıflamış yeteneği ile G0/G1 fazında hücre birikimini engelledi. Bu sebeple hücrelerin S ve G2/M fazlarından geçişini eEF1A1’in düzenlediğini gösterdik.

Anahtar sözcükler: Uzama faktörü, fosforilasyon, hücre döngüsü, proliferasyon, meme kanseri

Kah Wai LIN and Serhiy SOUCHELNYTSKYI126

Introduction

Inhibition of cell proliferation may occur by accumulation of cells in a specific phase of the cell cycle. The most often observed is an accumulation of cells in the G0 phase, although accumulation of cells in the G2 phase has also been observed (Fukuda and Ohashi, 1983; Pavey, Russell and Gabrielli, 2001). Changes in the rates of transition through the phases of the cell cycle may impact on the cell proliferation, but this issue is less studied. Transition through the cell cycle is dependent on a synthesis and degradation of a number of proteins.

The eukaryotic elongation factor 1 alpha (eEF1A) plays a crucial role in protein synthesis. During the mRNA translation, eEF1A catalyzes the GTP-dependent binding of aminoacyl-tRNA (aa-tRNA) to the A site of a ribosome which contains the growing polypeptide chain (Moldave, 1985). In addition, eEF1A is a key regulator in various physiological processes, such as embryogenesis, aging, proliferation, apoptosis, protein degradation and cytoskeletal rearrangement (Condeelis, 1995; Kato et al., 1997; Lamberti et al., 2004). There are two eEF1A isoforms, eEF1A1 and eEF1A2, that are expressed in tissue-specific manner (Lee et al., 1992; Knudsen et al., 1993). eEF1A is also involved in the carcinogenic transformation of various tumors. The increased expression of eEF1A1 correlates with hepatocellular carcinoma (Grassi et al., 2007; Zhang et al., 2009), prostate carcinoma (Liu et al., 2010), and increase metastatic potential in mammary adenocarcinoma (Edmonds et al., 1996). The overexpression of eEF1A2 was found in over 30% of ovarian tumor (Anand et al., 2002) and 83% of pancreatic cancers (Cao et al., 2009). Targeting eEF1A, as a strategy to combat apoptotic-resistant melanoma, has also been reported (Van Goietsenoven et al. 2010).

Post-translational modification (PTM) of protein plays a key role in the regulation of cellular functions. Several reports implicate that the PTM of eEF1A is associated with regulatory function. Phosphorylation of eEF1A1 is involved in GDP/GTP-exchange activity in rabbit reticulocytes (Peters, Chang and Traugh, 1995) and binding to

F-actin (Izawa et al., 2000). The interaction of F-actin and eEF2 has been studied and it’s inhibition by EF1A has been showed (Betkas et al., 1994). Methylation of eEF1A in mouse 3T3B cells is associated with the SV40-dependent transformation (Coppard, Clark and Cramer, 1983). Our recent study showed that phosphorylation of eEF1A1 at Ser300 by type I transforming growth factor-β (TGF-β) receptor inhibit the protein synthesis and cell proliferation, and that the decreased phosphorylation at Ser300 is associated with human breast carcinomas (Lin et al., 2010). Here we report that the eEF1A1 and Ser300 mutants of eEF1A1 have an impact on transition of cells through the phases of the cell cycle.

Materials and methods

Cells and constructs

The MCF-7 cells were obtained from ATCC (LGC Promochem, Boras, Sweden). Cells were maintained under DMEM, 10% FBS, 1% penicillin/streptomycin. The stable transfection of pMEP4-eEF1A1 into MCF-7 cells has been described earlier (Lin et al., 2010). Cell clones were selected and maintained in a culture medium with hygromycinB (Calbiochem, San Diego, CA). Protein expression was induced by treatment of cells with 5 μM CdCl2 for 3-4 h prior to experiments.

Cell proliferation

Cell proliferation was measured by using [3H]thymidine incorporation assay. The same numbers of MCF-7 parental and stably transfected cells were seeded in plates. The cells were treated with and without human TGF-β1 and incubated with 0.1 μCi/ml of [3H]thymidine for 8 hours, and radioactivity incorporated in DNA was measured, as described earlier (Lin et al., 2010).

FACS analysis

The cells were plated at a density of 3.5 X 104 cells/ml in 6-well plates in DMEM medium supplemented with 5% foetal calf serum (FCS). The cells were synchronized (at G0) by incubation in serum-free DMEM for 24 hrs. Cells were pretreated

eEF1A1 in regulation of cell cycle

with either vehicle (4 mM HCl, 0.1% BSA) or TGFβ1 (10 ng/ml) for 1 hr before release, and then incubated for indicated time in the presence of 5% FCS. Trypsinized cells were centrifuged at 128×g at 4°C, and were then fixed in 4% buffered formaldehyde for 18 hours at room temperature. For DNA histograms, cells were harvested and analysed as described (Castro et al., 1993). Briefly, after fixation formaldehyde was removed by 95% ethanol for 1 hour followed by rehydration in distilled water for 1 hour. After treatment with subtilisin Carlsberg solution (0.1% Sigma protease XXIV, 0.1 M Tris and 0.07 M NaCl (pH 7.5)) and staining with DAPI-Sulforhodamine solution (8 mM DAPI, 50 mM Sulforhodamine 101, 0.1 M Tris and 0.07 M NaCl (pH 7.5)), samples were analyzed by flow cytometry (FASC system, Becton Dickinson, San Jose, CA). The percentage of cells in G1(G0), S and G2/M phases was analyzed by ModFit LT 3.0 software (Verity Software House, Topsham, ME). The S-phase was fitted to a broadened trapezoidal model.

Statistics

All experiments were performed in triplicate. The Student’s t-test was used and P < 0.05 was considered as statistical significance.

Results and Discussion

We observed that the enhanced expression of the wild-type (WT) eEF1A1 promoted proliferation of MCF-7 cells (Figure 1). Mutations of the Ser300 residue in eEF1A1 result in inhibition of aa-tRNA loading onto eEF1A1, and subsequently in inhibition of protein synthesis. Expression of the Ser300Ala or Ser300Glu mutants of eEF1A1 decreased proliferation of MCF-7 cells (Figure 1).

TGF-β1 is known to have a direct effect on the cell cycle by regulating the activity of CDKs, CDK inhibitors and cyclins (Miyazono, Suzuki and Imamura, 2003; Feng and Derynck, 2005; Massague, 2008). When the MCF-7 cells were treated with TGF-β1, the [3H]thymidine incorporation was dose-dependently inhibited in the WT eEF1A1 expressing cells and in the empty

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Figure 1. Cell proliferation ([3H]thymidine incorporation, 8 hours). eEF1A1-WT increased proliferation,S300A and S300E mutants decrease proliferation. TGF-β1 inhibited proliferation of eEF1A1-WT, S300A andS300E mutants did not show TGF-β1 responsiveness. (*p<0.05)

Kah Wai LIN and Serhiy SOUCHELNYTSKYI128

vector expressing control cells. No TGF-β1 responsiveness was observed in cells expressing Ser300 mutants of eEF1A1. This is expected, as the mutants cannot be phosphorylated by TGF-β receptor type I (Lin et al., 2010). [3H]thymidine incorporation test measures synthesis of the genomic DNA and provides a readout of how fast the cell cycle is.In order to examine the effect of eEF1A1 on the regulation of the cell cycle, we analysed progression of the cells transfected with WT or various mutants of eEF1A1 through G0/G1, S, and G2/M phases.

Using FACS analysis, we monitored distribution of cells in the various phases of the cell cycle (Fig. 2). FACS results showed that the WT eEF1A1 promoted transition of MCF-7 cells through the S- and G2/M-phases and accumulation in G0/G1 phase (Figure 2). Abrogation of the binding of aa-tRNA, and therefore inhibition of protein synthesis, by mutating Ser300 in eEF1A1, resulted in slower transition of the S-phase, as compared to the WT eEF1A1. Upon treatment with TGF- β1,

eEF1A1-WT decreased the accumulation of cells in G0/G1 phase. Abrogation of Ser300 (S300A) and mimic phosphorylation at Ser300 (S300E) of eEF1A1 inhibited TGFβ-dependent accumulation of cells in G0/G1 phase.

In conclusion, our study suggested that overexpression of eEF1A1contributed to the increased proliferation of cells by promoting transition of cells through the S- and G2/M-phases of the cell cycle (Figure 3). aa-tRNA binding-impaired mutants of eEF1A1, especially

Ser300Glu, showed the opposite effect. This indicates that the main contribution of eEF1A1 to the cell cycle regulation is by the promotion of the transition through the cell cycle. Future study will focus on investigating the detail signaling pathway of eEF1A1 and the Ser300 mutation in the regulation of cell cycle.

Figure 2. eEF1A1-WT promotes transition of cells through the S- and G2/M-phases, and accumulation inG0/G1 phase. S300A and S300E mutants slow down S-phase transition, and inhibit TGF-β-dependentaccumulation of cells in G0/G1 phase.

eEF1A1 in regulation of cell cycle 129

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