Bioscience Reports, Vol. 18, No. 3, 1998
Hypothesis
Multiple Phosphorylation Sites and Quaternary
Organization of Guanine-Nucleotide Exchange
Complex of Elongation Factor-1 (EF-1bgd/ValRS)
Control the Various Functions of EF-1a
Odile Minella,1 Odile Mulner-Lorillon,1 Guillaume Bec,2 Patrick Cormier,1
and Robert Belle1,3
Received March 12, 1998; accepted May 15, 1998
The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1
bgd (EF-1bgd), comprises four different subunits including valyl-tRNA synthetase
(EF-lbgd/ValRS). The factor is multiply-phosphorylated by three different protein kinases,
protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDK1). EF-lbgd/ValRS
is organized as a macromolecular complex for which we propose a new structural model.
Evidence that EF-lbgd/ValRS is a sophisticated supramolecular complex containing many
phosphorylation sites, makes it a potential regulator of any of the functions of its partner
EF-1a, not only involved in protein synthesis elongation, but also in many other cellular
functions.
KEY WORDS: Elongation factor-1; guanine-nucleotide exchange; protein synthesis;
protein phosphorylation; Cyclin dependent kinase 1 (CDK1).
Note: The common nomenclature for EF-1 and its subunits is used throughout the article
for harmonization with reference articles and data bank's key words. Correspondence
with another nomenclature suggested recently is available (Merrick and Hershey, 1996).
INTRODUCTION
A guanine-nucleotide exchange complex is part of the Elongation factor-1 (EF-1)
required for peptide chain elongation during translation (Hershey, 1991). EF-1 is
composed of the guanine-nucleotide exchange complex EF-1bgd/valyl t-RNA synthetase (EF-1bgd/ValRS) and EF-1a, a G-protein, responsible for the binding of
aminoacyl t-RNA to the ribosome (Riis et al., 1990; Nygard and Nilsson, 1990;
Merrick and Hershey, 1996).
1
Biologie Cellulaire de l'Ovocyte. Station Biologique de Roscoff, Centre National de la Recherche
Scientique (CNRS UPR 9042), Universite Pierre et Marie Curie (UPMC), BP 74, 29682 Roscoff cedex.—
France.
2
Centre National de la Recherche Scientifique (CNRS UPR 9002), 15 rue Rene Descartes, 67084
Strasbourg cedex.—France.
3
To whom correspondence should be addressed.
119
0144-8463/98/0600-0119$15.00/0 © 1998 Plenum Publishing Corporation
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Minella, Mulner-Lorillon, Bec, Cormier, and Belle
Recently, a number of reports have revealed the complexity of the structure of
the guanine-nucleotide exchange factor and demonstrated that several of its subunits
are phosphorylated by protein kinases involved in the control of cell division (Belle
et al., 1995). Here we review the structure and phosphorylation of EF-1bgd/ValRS
and we propose a model for its quaternary, supramolecular organization. The
increasing number of functions ascribed to its partner EF-1a, (Merrick and Hershey,
1996), leads to the hypothesis that EF-1bgd/ValRS could play multiple regulatory
roles in cells.
SUBUNITS OF THE GUANINE-NUCLEOTIDE EXCHANGE COMPLEX
AND THEIR PHOSPHORYLATION
The nucleotide exchange factor EF-1bgd/ValRS is composed of four different
subunits: EF-1b, EF-1y, EF-1g and Valyl-tRNA synthetase as judged from the
native complexes purified from at least three sources: Mammals (Bec et al., 1989),
Xenopus (Mulner-Lorillon et al., 1989), and Artemia (Brandsma et al., 1995). Figure
1 summarizes the subunit composition of each native complex, and takes into
account the interactions between each of them reported elsewhere (Janssen et al.,
1991; Belle et al., 1995).
EF-1y is highly hydrophobic with a lysine-rich hydrophilic cluster and has
potential structural, anchoring and regulatory roles. EF-1y contains a glutathione
S-transferase domain which may be involved in the multimerization of EF-1
(Koonin et al., 1994). EF-1y stimulates the guanine-nucleotide exchange activity of
Fig. 1. Subunit composition and phosphorylation sites of
higher eukaryotic EF-1bgd. Phosphorylation sites (in one
lettering amino acids coding) and position in the sequences
refer to the Xenopus sequences.
Guanine-Nucleotide Exchange Complex
121
EF-1b (Janssen and Moller, 1988a). Specific affinity of EF-1y for both membranes
and tubulin suggests an anchoring function for this subunit (Janssen and Moller,
1988b). Two lines of evidence imply a regulatory role for EF-1y in cell division:
increased expression associated with cancer (Lew et al., 1992; Chi et al., 1992;
Mimori et al., 1995; Mimori et al., 1996) and phosphorylation by cyclin dependent
kinase 1 (CDK1) during meiotic cell division of Xenopus oocytes (Mulner-Lorillon
et al., 1992). Evidence that EF-1y phosphorylation changes under physiological conditions comes from results obtained in Xenopus. The site phosphorylated by CDK1
in the Xenopus protein has been identified (Fig. 1). Since EF-1y proteins from lower
eukaryotes or from the native Artemia complex are not CDK1 substrates (Janssen
et al., 1991), it was of interest to determine whether the mammalian protein could
be a substrate for the kinase. The data shown in Fig. 2, indicates that this is the
case. Therefore, phosphorylation of EF-1y by CDK1 may be a feature acquired
during the evolution of higher eukaryotes.
EF-1b possesses GTP/GDP exchange activity (Merrick and Hershey, 1996).
EF-1b is a substrate for Casein kinase II and is phosphorylated in vivo (Belle et al.,
1989; Venema et al., 1991a; Venema et al., 1991b). The site phosphorylated by Casein kinase II is indicated in Fig. 1. Such phosphorylation inhibits guanine-nucleotide
Fig. 2. EF-1y and EF-1d from rabbit are substrates for
CDK1 in vitro. Rabbit EF-1 was purified and further dissociated into sub-fractions (Bec et al., 1994). Fractions were
phosphorylated in the standard conditions described for the
phosphorylation of the Xenopus complex using the same
CDK1 preparation (Mulner-Lorillon et al., 1992). Left:
Coomassie blue stained bands after SDS-PAGE. Right: corresponding autoradiography. C: control fraction containing
whole EF-1; b/y and d: further purified fractions containing
EF-1byand EF-1d. Right arrow points on the mobility shift
of the phosphorylated form of EF-1d. ValRS is Valyl-tRNA
synthetase.
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Minella, Mulner-Lorillon, Bec, Cormier, and Belle
exchange activity (Janssen et al., 1988). Phosphorylation of EF-1b by Protein kinase
C increases its exchange activity (Venema et al., 1991a; Venema et al., 1991b; Peters
et al., 1995). The existence of both, up- and down-regulation of the guanine-nucleotide exchange activity by phosphorylation suggests that the elongation step of protein synthesis is regulated.
Valyl-tRNA synthetase responsible for aminoacylation of tRNAvaline, is associated with EF-1 in all native purified complexes: mammalian (Bec et al., 1989); Motorin et al., 1988), Xenopus (Belle et al., 1995) and Artemia (Brandsma et al., 1995).
Indeed, it is the only amino-acyl t-RNA synthetase associated with EF-1. The two
forms of Valyl-tRNA synthetase found in Artemia, one free and one associated with
EF-1, may reflect an evolutionary transition from the exclusively free enzyme in
yeast (Bec et al., 1989) to the complexed form found in higher eukaryotes. ValyltRNA synthetase is associated with the complex via EF-1d (Bec et al., 1994). The
mammalian enzyme is phosphorylated by protein kinase C which increases its
activity (Venema et al., 199la). The role of the synthetase and its phosphorylation
should be investigated further, and coordinately with the other components of EF-1
to which it is linked in the higher eukaryotes.
The protein EF-1d is a separate component of the guanine-nucleotide exchange
factor, distinct from the protein b, although both share an identical guanine-nucleotide exchange domain (Morales et al., 1992). After characterization in Xenopus,
EF-1d was identified in other organisms. EF-1d possesses a leucine zipper motif
(Morales et al., 1992) characteristic of a protein-protein interaction motif (O'Shea
et al., 1991). The leucine zipper motif could be involved either in the quaternary
structure of the whole complex (see below), or in the interaction with yet unidentified
ligands. In plant cells, from rice and wheat, two proteins b and b' possess the
guanine-nucleotide exchange activity (Matsumoto et al., 1994) but neither contains
the leucine zipper motif and, therefore, they do not correspond to EF-1d (Belle et
al., 1995).
The presence in the same complex of two distinct proteins EF-1b and EF-1d
sharing an identical activity (Cormier et al., 1993), strongly suggests specific roles.
Since plants, and lower eukaryotes do not contain EF-1d, the protein might have
originated from gene duplication. During subsequent evolution, EF-1d acquired the
leucine-zipper motif, not necessary for protein synthesis as such, but involved in a
specific function in the higher eukaryotic animal cells that remains to be elucidated.
Identification in Xenopus oocytes of not one, but two different EF-1d proteins, each
encoded by a distinct mRNA and simultaneously present in cells in the ratio 1:10
(Minella et al., 1996a), strengthens the idea of specialized function and regulation
for each of them.
EF-1d is a substrate for three different protein kinases. Casein kinase II
phosphorylates a consensus site present in all sequences and also phosphorylates
Xenopus protein on another phospho-acceptor site involving a threonine residue
(Belle et al., 1995). EF-1d, at least in mammals, is phosphorylated by protein kinase
C (Venema et al., 1991a; Venema et al., 1991b).
Phosphorylation of EF-18 by CDK-1 (Mulner-Lorillon et al., 1994) appears
interesting in the light of the importance of cyclin dependent kinases (CDKs) in the
cell cycle control. During meiotic cell division of Xenopus oocytes, phosphorylation
Guanine-Nucleotide Exchange Complex
123
in vivo of the two d subunits by CDK1 increases on two acceptor sites (MulnerLorillon et al., 1994; Minella et al., 1996a). The protein resolves as a doublet on
SDS-PAGE, the upper band corresponding to a bi-phosphorylated form, involving
phospho-serine and phospho-threonine residues, the lower band to a monophosphorylated form on a phospho-threonine residue (Belle et al., 1995; Minella et al., 1996a;
see Fig. 1). This feature was used to show that the phosphorylation of EF-1d by
CDK1 constitutes a memory signal in early development (Minella et al., 1994). It
was of interest to analyze if mammalian protein could also be a substrate for CDK1.
Figure 2 shows that this is the case. Interestingly, the mammalian protein shows an
electrophoretic shift phosphorylation (Fig. 2, right arrow) as does the Xenopus protein (Belle et al., 1995). Since in the Xenopus protein, the shift was ascribed to
the Serine phosphorylation, and not to the threonine site (Minella et al., 1996a),
phosphorylation of mammalian EF-15 involves the Serine site. CDK1 and Casein
kinase II phosphorylation sites on EF-15 are indicated in Fig. 1. The phosphoacceptor sites are compatible with the latest results obtained with the Xenopus
complex (Minella et al., 1996a).
A MODEL FOR QUATERNARY ORGANIZATION OF
GUANINE-NUCLEOTIDE EXCHANGE COMPLEX
Experimental observations and results from different sources do not suggest a
simple model for the quaternary structure of EF-1bgd/ValRS. The model proposed
for the Artemia complex (Janssen et al., 1994) does not fit with the results in
Xenopus (Belle et al., 1995) and does not take into account the presence of ValyltRNA synthetase in the complex (Brandsma et al., 1995). Reconstitution experiments, from dissociated individual subunits did not resolve the quaternary structure,
because various multimerisations occur in vitro particularly with EF-1d (Janssen et
al., 1994; Bec et al, 1994). Native purified complex from Xenopus, resolved by SDSPAGE, has complex stoichiometry (Belle et al., 1995; Fig. 3, left). Western blotting
of the purified complex with polyclonal antibodies against the whole complex are
comparable with western blots performed on cytosolic fractions, indicating that purification does not eliminate constituents (Belle et al., 1995; Minella et al., 1996b).
EF-lyand EF-1b are found in all eukaryotic native complexes in a ratio 1:1. On
the other hand, EF-15 and valyl-tRNA synthetase are also in a ratio 1:1 both in
Xenopus and mammalian complexes, consistent with a role of EF-1d in binding
valyl-tRNA synthetase to the whole complex (Bec et al., 1994; Belle et al., 1995).
Interpretation becomes difficult, however, when it appears that the ratio of EF-1b
to EF-1d is close to 1:3 which implies an excess of b/g over d/ValRS. Chromatography on Superose 6, resolves the Xenopus complex into two peaks (I to II) with
molecular masses of 750 and 550 kDa (Minella et al., 1996a); peak I contains 70%
and peak II 30% of total protein.
Our model (Fig. 3) proposes that the isolated Xenopus complex is a mixture of
two supramolecular forms in ratio 70:30; the 760 kDa peak contains b/g/d/ValRS,
and the 550 kDA peak contains b/y. We suggest that b/y forms a stable hexamer
with a 3:3 stoichiometry; it is called the B unit in Fig. 3. This hypothesis is compatible with the molecular weights of the b/y complex from rabbit, reconstituted in
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Minella, Mulner-Lorillon, Bec, Cormier, and Belle
Fig. 3. A model for quaternary organization of EF-1. Left: Xenopus EF-1 constituents
after resolution by SDS-PAGE and Coomassie blue staining of the gel. Right: quaternary
structure (see text).
vitro in which the reconstituted particle elutes as a trimer from a molecular size
chromatography (Bec et al., 1994). In the native Xenopus complex the B unit
dimerizes due to the highly hydrophobic nature of EF-1y, thereby neutralizing a
hydrophobic interface. The B/B dimer has a theoretical Mr of 460 kDa which
approximates the size of the 550 kDa material resolved by Superose 6 chromatography (Minella et al., 1996a, Fig. 3). Secondly we note that there are striking
sequence homologies in ValRS and EF-1d. Previous work shows that ValRS
interacts through its N-terminal domain (homologous with EF-ly) with EF-1d(homologous with EF-lb). Thus it is tempting to postulate that the association of ValRS
with EF-1d mimics the b/y interaction and that ValRS and EF-1d form a hexamer
with a 3:3 stoichiometry; it is called the A unit in Fig. 3. The A unit could dimerize
with the B unit, forming an A/B complex. Such a complex perfectly fits the stoichiometry and the molecular weight found in the Xenopus and Mammalian cell
lines. Furthermore, the EF-1b and y subunits are more abundant in the cytoplasm
than ValRS and EF-1d; thus the B unit should participate in two sub-complexes:
A/B and B/B. This explains the apparent heterogeneous repartition of the elongation factor found in higher eukaryotes (Nagata et al., 1976).
The leucine zipper motif present in EF-1d may be involved in the interaction
between three 8 molecules. The specific length of the leucine zipper motif in EF-1d,
twice that of other leucine zipper proteins (Belle et al., 1995) is compatible with the
trimeric hypothesis. The glutathione S-transferase of EF-ly domain may be involved
in the interaction of B/B and A/B units.
CONCLUSIONS AND PERSPECTIVES
We propose a model for the quaternary organization of EF-1bgd/ValRS complex. Our model is based on results from the analysis of native complexes obtained
Guanine-Nucleotide Exchange Complex
125
from purification procedures and not from reconstitution experiments with individually expressed proteins. The model we propose could be tested by different
approaches. First, identical purification procedures should lead to comparable
structural complexes between species. Second, cross-linking experiments should
provide evidence for the binding of subunits between each other. Probably the
most difficult would be to ascertain the trimeric structures of both A and B units
(see text) which would necessitate other methods for mass measurements. This
could only be done after improvement of the chromatographic separation of A/B
and B/B units.
EF-1bgd/ValRS is a highly complex molecular factor: it contains various combinations of individual proteins; it can be phosphorylated to a variable extent on
multiple acceptor sites. It is generally admitted that reversible protein phosphorylation in the most universal mechanism for regulation of protein activity and that
almost every aspect of cell life is controlled by the reversible phosphorylation of
proteins. Single cells must, therefore, contain different forms of the EF-1bgd/ValRS
complex which may have specific roles. EF-1bgd/ValRS associates (Janssen et al.,
1994) and co-localizes (Minella et al., 1996b; Sanders et al, 1996) with EF-la in
cells. Therefore, it is of interest to investigate roles for EF-1bgd/ValRS regarding
functions of EF-1a.
EF-1 a, initially characterized as a factor involved in the protein synthesis elongation, now appears to be implicated in multiple cellular functions, including protein
metabolism, other than the elongation step of protein synthesis, and also including
cell proliferation, cytoskeletal organization and nucleic acid metabolism (Riis et al.,
1990; Hershey, 1991; Belle et al., 1995; Merrick and Hershey, 1996).
The role of EF-1a in the elongation step of protein synthesis, as a G-protein
(Bourne et al., 1992), is firmly established (Moldave, 1985; Merrick and Hershey,
1996). The exchange factor EF-1bgd/ValRS stimulates EF-la; a typical function of
such a guanine-nucleotide exchange factor. The role of each individual form of the
factor should now be investigated. Little is known on how EF-1a is regulated as a
multiple functional protein (Merrick and Hershey, 1996). Three lines of evidence
favor that EF-1bgd/ValRS is of importance in some EF-1a functions. The first is
the multiple phosphorylation of EF-1bgd/ValRS during meiotic cell division correlated with protein synthesis changes (Belle et al., 1995). The second involves EF-la
function in translation accuracy and frameshifting (Thompson, 1988; Dinman and
Kinzy, 1997). It was reported on the basis of genetic experiments in yeast the EF1b influences the function of EF-1a in frameshifting (Kinzy and Woolford, 1995).
Third, recent findings on Herpes Simplex virus 1 a regulatory protein ICPO (Infected
Cell protein 0) shows that ICPO acts through its interaction with EF-1d to mediate
effects in the human infected cells with changes in the phosphorylation state of
EF-1d by CDKs (Kawaguchi et al., 1997).
Altogether, we can hypothesize that regulations, in protein synthesis and
other cellular functions, involving the multifunctional protein EF-1a, will be
fruitfully associated to regulation through EF-1bgd/ValRS in the next years. This
hypothesis can be tested for any activity of EF-1a in vitro by addition of purified
EF-1bgd/Va1RS complex, previously phosphorylated by the different protein kinases involved.
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Minella, Mulner-Lorillon, Bec, Cormier, and Belle
ACKNOWLEDGMENTS
This work was supported by Association de la Recherche contre le Cancer
(ARC) and Conseil Regional de Bretagne.
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