Journal of Cell and Molecular Biology 8(2): 125-130, 2010
Haliç University, Printed in Turkey.
http://jcmb.halic.edu.tr
Research Article
Eukaryotic elongation factor eEF1A1 promotes and Ser300 mutants
of eEF1A1 inhibit transition through the S and G2/M phases of the
cell cycle
Kah Wai LIN*and Serhiy SOUCHELNYTSKYI
Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institute, Karolinska University
Hospital, Stockholm, Sweden
(*author for correspondence; [email protected])
Received: 17 September 2010; Accepted: 02 December 2010
Abstract
The 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 cancer
Abbreviations: 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
Özet
Ökaryotik 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/Mfaz 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
126 Kah Wai LIN and Serhiy SOUCHELNYTSKYI
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 127
with either vehicle (4 mM HCl, 0.1% BSA) or TGF
β1 (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
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 and
S300E mutants did not show TGF-β1 responsiveness. (*p<0.05)
128 Kah Wai LIN and Serhiy SOUCHELNYTSKYI
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.
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
Figure 2. eEF1A1-WT promotes transition of cells through the S- and G2/M-phases, and accumulation in
G0/G1 phase. S300A and S300E mutants slow down S-phase transition, and inhibit TGF-β-dependent
accumulation of cells in G0/G1 phase.
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 Sand 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,
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.
eEF1A1 in regulation of cell cycle 129
Figure 3. eEF1A1 promotes transition of cells through the S- and G2/M-phases, and TGF-β-dependent
accumulation in G0/G1 phase. S300A and S300E mutants of eEF1A1 slow down S-phase transition,
and inhibit TGFb-dependent accumulation of cells in G0/G1 phase.
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