Nucleic Acids Research, 2001, Vol. 29, No. 7 1453–1457
Evidence for regulation of protein synthesis at the
elongation step by CDK1/cyclin B phosphorylation
Annabelle Monnier, Robert Bellé*, Julia Morales, Patrick Cormier, Sandrine Boulben and
Odile Mulner-Lorillon
Station Biologique de Roscoff, Université Pierre et Marie Curie (UFR 937), Centre National de la Recherche
Scientifique (UPR 9042), Institut National des Sciences de l’Univers, BP 74, 29682 Roscoff Cedex, France
Received December 22, 2000; Revised and Accepted January 29, 2001
ABSTRACT
Eukaryotic elongation factor 1 (eEF-1) contains the
guanine nucleotide exchange factor eEF-1B that
loads the G protein eEF-1A with GTP after each cycle
of elongation during protein synthesis. Two features
of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the
significance of target sites for the cell cycle protein
kinase CDK1/cyclin B. The roles of these two features
were addressed by elongation measurements in vitro
using cell-free extracts. A poly(GUA) template RNA
was generated to support both poly(valine) and
poly(serine) synthesis and poly(phenylalanine)
synthesis was driven by a poly(uridylic acid) template.
Elongation rates were in the order phenylalanine >
valine > serine. Addition of CDK1/cyclin B decreased
the elongation rate for valine whereas the rate for
serine and phenylalanine elongation was increased.
This effect was correlated with phosphorylation of
the eEF-1δ and eEF-1γ subunits of eEF-1B. Our
results demonstrate specific regulation of elongation
by CDK1/cyclin B phosphorylation.
INTRODUCTION
Changes in the protein synthetic machinery are important in
the regulation of gene expression in eukaryotes (1). Compared
to initiation, the elongation phase of protein synthesis requires
only a small number of factors, namely eukaryotic elongation
factors 1 and 2 (eEF-1 and eEF-2) (2). eEF-1 is composed of
two elements, eEF-1A and eEF-1B. The G protein eEF-1A
(formerly eEF-1α) is responsible for binding of aminoacyltRNA to the ribosome; the complex eEF-1B exchanges GDP
for GTP on eEF-1A (2–5). On the basis of its structure and
phosphorylation by several protein kinases, eEF-1B has been
postulated to play a regulatory role(s) (6,7). The structure of
eEF-1B has been analysed (7–12). Higher eukaryotic eEF-1B,
from animal sources, contains two different guanine nucleotide
exchange proteins, eEF-1β and eEF-1δ, a putative anchoring
protein eEF-1γ and the unique valyl-tRNA synthetase responsible for attachment of valine to its cognate tRNA (7–12). The
eEF-1B components are all substrates for different protein
kinases (7,13). The cell cycle protein kinase CDK1/cyclin B
(14) phosphorylates eEF-1B in vivo during meiotic maturation
of Xenopus oocytes (15–17) in a manner correlated with changes
in protein synthesis (18). Furthermore, phosphorylation of
eEF-1B persists after fertilisation (19), along with a further
increase in protein synthesis (20). eEF-1B contains three
different phosphoacceptor sites for CDK1/cyclin B: one in the
eEF-1γ component and two on each of the eEF-1δ isoforms
present in Xenopus oocytes (21).
The functions of components of the protein synthetic
machinery have been successfully characterised using cell-free
extracts (22). However, two of the features of eEF-1B, the
presence of the unique valyl-tRNA synthetase and the presence
of phosphorylation sites for CDK1/cyclin B, have not been
related to any biological function. Experiments performed
using reconstituted systems revealed no difference between
phosphorylated and dephosphorylated forms of eEF-1B from
Xenopus oocytes (7,23). Lysates from rabbit reticulocytes (2)
readily perform translation from exogenous template RNAs
and mammalian eEF-1B was shown to be phosphorylated by
CDK1/cyclin B on both the eEF-1γ and eEF-1δ subunits (7).
Based on the hypothesis that phosphorylation of eEF-1B could
influence valyl-tRNA synthetase associated with the eEF-1B
complex, we designed a template RNA for the analysis of
poly(valine) synthesis compared to poly(serine) synthesis. The
template RNA was used in lysates adapted for elongation
determination in the absence of factor-directed initiation.
Using this cell-free system we demonstrate a new regulation of
protein synthesis elongation by CDK1/cyclin B phosphorylation.
MATERIALS AND METHODS
Production of the poly(GUA) template RNA
The oligonucleotide 5′-CCGGCGGAATTCTAG(GTA)37TAGGGATCCGGCCGC-3′ containing EcoRI and BamHI
restriction sites was synthesised by Eurogentec. The oligonucleotide was amplified by PCR at 48°C using the upstream
and downstream primers 5′-CCGGCGGAATTCTAGGTA-3′
and 5′-GCGGCCGGATCCCTATA-3′. The PCR product was
separated on a 2% agarose gel, excised and purified using a
QiaQuick gel extraction kit (Qiagen). The purified DNA fragment was digested with EcoRI and BamHI (New England
Biolabs) and inserted into the pBluescriptII KS– phagemid
vector (Stratagene). Epicurian Coli XL1-Blue supercompetent
*To whom correspondence should be addressed. Tel: +33 2 98 29 23 46; Fax: +33 2 98 29 23 06; Email: [email protected]
1454 Nucleic Acids Research, 2001, Vol. 29, No. 7
bacteria (Stratagene) were transformed with the plasmid, positive
clones selected and the plasmid purified with a Flexiprep kit
(Pharmacia). A T3 sequencing kit (Pharmacia) was used to
select the clones containing the correct constructs. The plasmid
was linearised with NotI (New England Biolabs) and used as a
template for transcription using a Megascript T3 kit (Ambion).
The product, referred to as poly(GUA) template RNA, was
quantified by absorbance at 260 nm.
Cell-free elongation assay
Rabbit reticulocyte lysate (containing an ATP regenerating
system, yeast and calf liver tRNAs and hemin) (Retic Lysate
IVT™; Ambion) was used according to the manufacturer’s
instructions. The incubation mix supplied in the kit was
replaced by a mix devoid of added cold amino acids and
supplemented with 5 mM MgCl2. The high concentration of
MgCl2 allows non-specific binding of the RNA template to
ribosomes, resulting in elongation of polypeptides in the
absence of specific initiation (24). [3H]phenylalanine (55 Ci/mmol),
[3H]valine (23–27 Ci/mmol) and [3H]serine (26–34 Ci/mmol) were
purchased from Amersham. Assays were performed with
5 µCi labelled amino acid corresponding, respectively, to 7.5,
15.5–18.5 and 12–16 µM for phenylalanine, valine and serine.
Optimum efficiency of translation from poly(uridylic acid)
(Sigma) as RNA template was obtained using poly(uridylic
acid) at a concentration of 2.2 µg/µl incubation. The
poly(GUA) template RNA synthesised as indicated above was
used at 3 µg/incubation. Assays were performed using 8 µl of
lysate extract in a total volume of 12 µl and incubated for 1 h
at 30°C. Incubations were stopped by dilution (1:2) and
duplicate aliquots were subjected to TCA precipitation on
Whatman filters (22). Radioactivity on the filters was determined by liquid scintillation spectroscopy.
Phosphorylation conditions
Purified CDK1/cyclin B was obtained from Dr Laurent Meijer
(Roscoff, France). The specific activity of the enzyme was
5.7 pmol phosphate incorporated in histone H1 per min under
standard conditions (25). One microlitre of enzyme solution
was added to the 12 µl lysate assays just prior to elongation
determination. The inhibitor of CDK1/cyclin B, roscovitine
(26), was a gift from Dr Laurent Meijer (Roscoff, France) and
was added at 3 mM to the lysates alone or together with CDK1/
cyclin B. Denatured CDK1/cyclin B was obtained by boiling
the enzyme for 1 min at 100°C.
The activity of purified CDK1/cyclin B in the lysate was
measured as follows. The 12 µl translation lysate assays were
incubated for 1 h at 30°C in the presence of 20 µCi [γ-32P]ATP,
1 µl of purified enzyme and 12 µg H1 histone HIII-S-S (Sigma).
Control incubations were performed without exogenous kinase
and/or without exogenous substrate. Reactions were stopped
by addition of electrophoresis sample buffer (27). Phosphorylated proteins were analysed by autoradiography on Kodak
X-Omat film after resolution by 12% SDS–PAGE. Quantification
of phosphorylation of histone H1 was performed by excision
and Cerenkov counting of the histone H1 band.
Phosphorylation of eEF-1B by CDK1/cyclin B in the lysates was
performed as follows. The 36 µl translation lysate assays were
incubated for 1 h at 30°C in the presence of 20 µCi [γ-32P]ATP and
2 µl of purified enzyme. The reaction was stopped by dilution
in 1 ml of immunoprecipitation medium [50 mM Tris–HCl
pH 7.4, 1% Nonidet P-40, 1% bovine serum albumin, 50 mM
NaF, 10 mM pyrophosphate, 100 µM sodium orthovanadate,
10 mM β-glycerophosphate, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride]. The eEF-1B complex was
then immunoprecipitated using antibody to anti-human eEF-1β, a
gift from Dr G. Janssen (Leiden, The Netherlands). The
antigen–antibody complex was isolated on protein A–Sepharose
beads, resolved by 12% SDS–PAGE and transferred to nitrocellulose membranes (28).
RESULTS AND DISCUSSION
Elongation from template RNAs
Reticulocyte lysates were used for translation of mRNA
templates. Under our experimental conditions accumulation of
poly(phenylalanine) readily occurred from the poly(uridylic
acid) template (Fig. 1, left). Since the mRNA template does not
contain an AUG initiation codon, incorporation of radioactivity corresponds to elongation in the absence of specific
initiation. Elongation was linear with time for up to 2 h and
resulted in incorporation of 33% of the amino acid amount in
the reaction mixture, which compares favourably with elongation
rates obtained in reconstituted systems (29). Synthesis of
Figure 1. Elongation of amino acids from template RNAs. Poly(phenylalanine) was determined using poly(uridylic acid) as template and poly(valine) and
poly(serine) synthesis were determined using poly(GUA) as template. Reactions were initiated by radioactive amino acid addition in a total volume of 110 µl at
30°C. Aliquots of 10 µl were taken at the indicated times for the determination of elongation in c.p.m.
Nucleic Acids Research, 2001, Vol. 29, No. 7 1455
poly(valine) and poly(serine) was analysed under the same
experimental conditions using poly(GUA) as template RNA.
Elongation rates for both amino acids (Fig. 1, middle and right)
were linear with time in the first hour. The relative efficiency
of elongation was in the order phenylalanine > valine > serine.
Increasing the amount of the poly(GUA) template (9, 27 and
81 µg) did not increase elongation and, moreover, elongation
of both poly(valine) and poly(serine) was inhibited at higher
template concentrations. Elongation of poly(serine) was
always lower than elongation of poly(valine) and had higher
background values in the absence of template RNAs (see
Fig. 1, compare valine and serine elongation).
In different independent 1 h assays 31.2 ± 0.8 pmol phenylalanine (n = 16) was elongated under the standard conditions,
whereas the rate for valine was 3.5 ± 0.1 pmol (n = 21) and for
serine barely detectable over the background at 1.2 ± 0.1 pmol
(n = 17).
Since the assay conditions were optimised to perform
elongation by random attachment of ribosomes to RNAs in the
absence of factor-directed initiation, the results reflect specific
elongation rates for the three amino acids. Such differential
efficiencies of translation from different synthetic mRNA
templates have been observed previously for poly(phenylalanine) synthesis compared to others (30,31). A higher
efficiency of poly(phenylalanine) synthesis could be related to
the ability of a ribosome to enter a correct reading frame by
attachment to any of the three nucleotides of a UUU codon.
Figure 2. Effect of CDK1/cyclin B on elongation. Elongation rates were
determined as indicated in Materials and Methods for a 1 h incubation at 30°C.
Columns represent elongation in the presence of the indicated component,
expressed as percent of the corresponding control. Bars represent the standard
errors of four determinations in the same experiment.
Effect of CDK1/cyclin B on elongation
The effect of phosphorylation by the protein kinase CDK1/
cyclin B was first analysed on poly(phenylalanine) synthesis
from a poly(uridylic acid) template. In independent experiments the translation rate was found to increase to 230 ± 20%
(n = 4) when CDK1/cyclin B was added to the extracts. Using
poly(GUA) as template the synthesis of poly(serine) was also
increased to 150 ± 20% (n = 7) in the presence of CDK1/cyclin
B kinase. Therefore, the protein kinase is able to increase elongation rates of two unrelated codons. In contrast, using the
poly(GUA) template, synthesis of poly(valine) was reduced
significantly, by 25 ± 10% (n = 4), on inclusion of CDK1/
cyclin B in the incubations. Elongation of valine was decreased
in parallel with the increase in serine elongation. Although
there was variability in the amplitude of the effect among the
different experiments, as judged by the large standard error,
the differential effect of CDK1/cyclin B was significant and
always displayed an increase in poly(valine) synthesis and a
decrease in poly(serine) [and poly(phenylalanine)] synthesis.
The effect of CDK1/cyclin B was further characterised. In
the experiment shown in Figure 2, CDK1/cyclin B induced an
increase in poly(serine) and poly(phenylalanine) synthesis to
218 and 191%, respectively, and a decrease in poly(valine)
synthesis to 87%. Roscovitine, a specific inhibitor of the
enzyme, totally prevented the effects of CDK1/cyclin B on
elongation (Fig. 2) and boiled enzyme had no effect on the
elongation rates (Fig. 2). Thus, the effect of CDK1/cyclin B
was related to its kinase activity.
It was important to assess the activity of CDK1/cyclin B
under the incubation conditions required in the translation
assays with the lysates. Under standard conditions CDK1/
cyclin B readily phosphorylated histone H1, as judged by the
observed labelling (Fig. 3). Activity of the kinase was also
Figure 3. CDK1/cyclin B activity under the translation assay conditions.
Autoradiography of 32P-labelled proteins after incubation as indicated in
Materials in Methods and resolution by SDS–PAGE. Each lane corresponds to
the indicated incubation conditions. Left, molecular weight markers are indicated
in kDa.
assayed at a concentration of 1 mM ATP, a condition
favouring stoichiometric phosphorylation of the substrate
(Fig. 3). Under these conditions CDK1/cyclin B activity
towards histone H1 was 104 pmol phosphate transferred in 1 h.
Phosphorylation was then assayed under the conditions of the
lysate incubations. The labelling of endogenous substrates or
added histone H1 substrate by endogenous protein kinases
present in the lysates was poorly detectable on the gels (Fig. 3),
but with addition of CDK1/cyclin B, histone H1 labelling was
clearly observed (Fig. 3). The activity of the kinase was calculated
to be 2.5 pmol/h, assuming an ATP concentration of 1 mM. This
activity was likely underestimated due to the ATP regenerating
system, which lowers the specific activity of the ATP pool, and
1456 Nucleic Acids Research, 2001, Vol. 29, No. 7
Figure 4. Phosphorylation of eEF-1B under the translation assay conditions of
the lysates. Western blot and corresponding autoradiography of the eEF-1B
complex immunoprecipitated from the lysate after incubation with [32P]ATP, as
indicated in Materials and Methods. (Left) Western blot performed with anti-eEF-1β
antibody from incubations performed without (–) or with (+) CDK1/cyclin B. (Right)
Corresponding autoradiogram. Left, molecular weight markers are indicated in kDa.
to the presence of high levels of potential endogenous
competitive substrates. Thus, CDK1/cyclin B is readily active
under the experimental conditions of the lysates.
Two of the mammalian eEF-1B subunits, namely eEF-1γ
and eEF-1δ, were reported to be very efficient substrates for
CDK1/cyclin B under standard assay conditions (7). The
experimental conditions for the determination of translation
activities of the lysates utilised a high concentration of ATP
and the use of an ATP regenerating system. Such conditions
impede the analysis of protein phosphorylation since they lead
to poor labelling of the phosphorylated proteins. To ascertain
that eEF-1B subunits were effectively phosphorylated under
our experimental conditions, immunoprecipitation of eEF-1B
was performed using antibodies directed against human eEF-1B
subunits. The antibody to human eEF-1β was efficient in
immunoprecipitating the rabbit eEF-1B complex as judged
from immunoblots of eEF-1β in the eEF-1B immunoprecipitate
(Fig. 4, left). When immunoprecipitation was performed with
lysates incubated with [32P]ATP, radioactivity was detected in
a 47 kDa protein migrating at the level expected for eEF-1γ.
Radiolabel was also present to a lesser extent in a 35 kDa
protein migrating at the level expected for eEF-1δ (Fig. 4,
right). As a control, the p47 and p35 proteins were not radiolabelled in a lysate that did not contain CDK1/cyclin B (Fig. 4,
right). We therefore conclude that eEF-1B is a substrate for
CDK1/cyclin B under the experimental conditions of the translation assay in the lysates.
The effect of CDK1/cyclin B on elongation was analysed.
Our results show that the rate of elongation of poly(valine)
compared to poly(serine) is changed concomitantly and in an
opposite manner by CDK1/cyclin B. Both determinations were
made under rigorously identical experimental conditions,
including measurements from the same mRNA template.
Among the components of the translation machinery, eEF-1
may discriminate between valine and serine elongation due to
the presence of valyl-tRNA synthetase, and no other
synthetases, in a pool of eEF-1B (7). Furthermore, eEF-1B is a
substrate for CDK1/cyclin B on both its eEF-1δ and eEF-1γ
subunits. The eEF-1B complex is present as two pools, one
containing the eEF-1β and eEF-1γ subunits and the other
containing, in addition, valyl-tRNA synthetase and eEF-1δ (7).
The simplest interpretation of our results involves a phosphorylation-dependent general increase in the activity of eEF-1B,
which in turn activates eEF-1A and increases the general
elongation rate. The increase in eEF-1B would be related to
phosphorylation of eEF-1γ by CDK1/cyclin B. In parallel,
phosphorylation of the eEF-1δ subunit in the pool of eEF-1B
containing valyl-tRNA synthetase would lead to inhibition of
the enzyme and therefore to specific inhibition of poly(valine)
synthesis. Our results provide experimental support for a new
type of translational regulation at the level of elongation,
which affects translation of the valine codon in an opposite
manner to other codons.
Our results suggest potential regulation of translation of
valine-rich proteins compared to other proteins depending on
the activation state of the protein kinase CDK1/cyclin B.
During cell division CDK1 is inactive during the G1 and S
phases in which the cells grow by increasing their mass and
CDK1/cyclin B is activated when the cells reach the G2/M
border and have finished growing (32). Since cells contain abundant amounts of structural proteins, cell growth is associated with
quantitatively more translation of structural than other proteins.
Most structural proteins contain coiled-coil valine-rich motifs
(33); therefore the synthesis of such proteins would be
favoured at the level of elongation when CDK1 is inactive,
during the growing phase of the cell cycle, and translation of
such proteins would be reduced at the G2/M transition, when
CDK1 is activated.
Previously it was reported that CDK1/cyclin B phosphorylates
eEF-1B during meiotic maturation of Xenopus oocytes and that
phosphorylation of at least the eEF-1δ component remains
after fertilisation (19). The present findings suggest that this
phosphorylation could favour the synthesis of regulatory
proteins during the rapid cleavage phase of early development,
in which cells divide and increase in number without growth
and therefore without the necessity of producing structural
proteins.
ACKNOWLEDGEMENTS
We are grateful to Dr Laurent Meijer for the gift of CDK1/
cyclin B and roscovitine. We thank Dr Georges Janssen for the
gift of antibodies directed against human subunits of eEF-1B.
This work was supported by the Association de la Recherche
contre le Cancer (ARC) and Conseil Régional de Bretagne.
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