The

International
Journal of
Biochemistry
& Cell Biology
PERGAMON The International Journal of Biochemistry & Cell Biology 31 (1999) 2529

Molecules in focus

Eukaryotic initiation factor eIF2

Scot R. Kimball *
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, P.O. Box 850, Hershey,
PA 17033, USA

Abstract

eIF2 plays a central role in the maintenance of what is generally considered a rate-limiting step in mRNA
translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S ribosomal subunit.
At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and the eIF2
GDP complex is
released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi
and is mediated by a second initiation factor, eIF2B. In what is probably the best-characterized mechanism for the
regulation of mRNA translation, phosphorylation of eIF2 on its smallest, or a-, subunit converts eIF2 from a
substrate of eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2a eectively prevents formation of
the eIF2
GTP
Met-tRNAi complex and inhibits global protein synthesis. Phosphorylation of eIF2a occurs under a
variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain
stresses. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: EIF2B; EIF5; Protein synthesis; Phosphorylation

1. Introduction 2. Structure of eIF2

Eukaryotic initiation factor eIF2 originally was
identied over 25 years ago as a protein that
bound GTP and initiator methionyl-tRNAi (Met-
tRNAi) and mediated the association of Met-
tRNAi to the 40S ribosomal subunit (reviewed
in [1]). Subsequent studies showed that a variety
of stimuli modulate eIF2 activity which in turn
regulates mRNA translation. This article will
briey describe the structure and function of
eIF2.
* Tel.: +1-717-531-8970; fax: +1-717-531-7667; e-mail:
skimball@psu.edu.
eIF2 is a multimeric protein consisting of three
dissimilar subunits termed a, b, and g, in order
of increasing molecular mass (Fig. 1). cDNAs for
each of the subunits have been cloned and
sequenced from a variety of species, and the pre-
dicted amino acid sequences of the individual
subunits show an exceptional level of conserva-
tion among species. For example, the human
eIF2a, b, and g sequences are respectively, 58%,
47%, and 72%, identical to the corresponding
subunits from S. cerevisiae (cf. [2] and refs.
therein). The conservation among species
underscores the important role eIF2 plays in cell
viability.

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26 S.R. Kimball / The International Journal of Biochemistry & Cell Biology 31 (1999) 2529

Fig. 1. Domain structure of human eIF2 a-, b-, and g-subunits. The number of amino acids in each eIF2 subunit is shown to the
right and just below the open boxes representing the polypeptide chains. Known phosphorylation sites are represented by a `P'
above the boxes with the residue number shown below it. In eIF2b, CK-II phosphorylates Ser2 and Ser67, PKC phosphorylates
Ser13, and PKA phosphorylates Ser218 [4]. Polylysine domains are shown as gray boxes, guanine nucleotide binding domains are
shown as black boxes, and the putative `zinc nger' motif in eIF2b is shown as a box with cross hatching. The eIF2B and eIF5
binding domains in eIF2b are denoted by black lines beneath the eIF2b polypeptide.

The eIF2a amino acid sequence is unremark-
able with the exception of Ser51, which is a phos-
phate acceptor for three protein kinases: heme-
regulated inhibitor (HRI), double-stranded
RNA-activated protein kinase (PKR), and the
nutrient-regulated protein kinase (GCN2). Yeast
eIF2a additionally contains three casein kinase II
(CK-II) sites in the C-terminal region which are
not conserved in the mammalian protein [3].
In contrast to eIF2a, the eIF2b amino acid
sequence contains a number of interesting fea-
tures including phosphorylation sites for CK-II,
protein kinase C (PKC), and cAMP-dependent
protein kinase (PKA) [4]. The N-terminal half of
the protein contains three domains, each consist-
ing of 68 lysine residues. The binding site for a
second initiation factor, eIF5, overlaps the sec-
ond poly-lysine domain [5]. The N-terminal half
of eIF2b also contains two of three consensus
guanine nucleotide binding domains. However,
the distance between the two domains is larger
than usual and mutations in the second have lit-
tle eect on GDP binding [6], suggesting that
eIF2b is not important in guanine nucleotide
binding. The C-terminal portion of eIF2b con-
tains the binding domain for the guanine nucleo-
tide exchange factor, eIF2B [7], as well as a
predicted zinc nger motif. However, zinc has no
eect on the activity of the protein and attempts
to demonstrate binding of zinc to eIF2 have been
unsuccessful. Finally, cross-linking of Met-tRNAi
to eIF2b results in the labeling of four peptides
distributed throughout the protein [8]. Whether
or not all of these sites are important in the bind-
ing of Met-tRNAi to eIF2 is unknown.
The primary amino acid sequence of eIF2g
contains all three consensus guanine nucleotide-
binding domains. The binding of GDP to eIF2g
is greatly decreased by mutations in the guanine
nucleotide binding domains of either the
human [6] or yeast [2] protein, suggesting that
eIF2g, rather than eIF2b, contains the primary
guanine nucleotide binding site. Cross-linking
analysis has also implicated the N-terminus of
eIF2g in the binding of Met-tRNAi [8].
3. Biological function
The primary role of eIF2 in translation in-
itiation, as depicted in Fig. 2, is to transfer Met-
tRNAi to the 40S ribosomal subunit (reviewed
 S.R. Kimball / The International Journal of Biochemistry & Cell Biology 31 (1999) 2529
Fig. 2. Scheme depicting the role of eIF2 in the initiation of mRNA translation.
in [1]). Following the association of mRNA with
the 40S subunit and location of the subunit at
the AUG start codon, eIF5 binds to eIF2 and
stimulates the hydrolysis of eIF2-bound GTP. It
has been proposed that either eIF5 or the b- or
g-subunits of eIF2 contains the GTPase activity
responsible for GTP hydrolysis. However, eIF5
stimulates GTP hydrolysis only when eIF2 is
bound to the 40S subunit as a ternary complex
with GTP and Met-tRNAi [9], suggesting that
either the GTPase is intrinsic to the 40S subunit
or that a component of the 43S preinitiation
complex is required for hydrolysis by eIF2 or
eIF5. Interestingly, one of the two CK-II phos-
phorylation sites on eIF2b, Ser67, is adjacent to
the eIF5 binding site, suggesting that phosphoryl-
ation by CK-II might regulate the binding of
eIF5 to eIF2. Following GTP hydrolysis, the
eIF2
GDP complex is released from the ribo-
some. Although the release mechanism has not
been delineated, it is noteworthy that eIF2 has
been found associated with the 60S ribosomal
subunit and it has been suggested that transfer to
27
the 60S subunit is important in eIF2
recycling [10].
Prior to binding Met-tRNAi, the GDP bound
to eIF2 must be exchanged for GTP, a reaction
mediated by eIF2B [11]. The best characterized
mechanism for regulating eIF2B activity involves
phosphorylation of the eIF2a (reviewed in [1]).
Phosphorylation of eIF2a on Ser51 converts eIF2
from a substrate into a competitive inhibitor of
eIF2B. Therefore, it has always been assumed
that one, or more, subunits of eIF2B bind to
eIF2a. However, in a recent study, the only
detected interaction between eIF2 and eIF2B was
the binding of eIF2b to the d- and e-subunits of
eIF2B [7], suggesting that eIF2a phosphorylation
might cause a structural alteration in eIF2b that
results in a change in the anity of eIF2B for
eIF2. In addition, in yeast, point mutations in
the a-, b-, and d-subunits of eIF2B result in an
apparent change in the anity of the protein for
phosphorylated eIF2. This result suggests that in
vivo the a- and b-subunits of eIF2B may also be
28 S.R. Kimball / The International Journal of Biochemistry & Cell Biology 31 (1999) 2529
important in regulating interactions between eIF2
and eIF2B.
In yeast, phosphorylation of eIF2a causes an
increase, rather than a decrease, in synthesis of
the transcription factor GCN4. Translation of
GCN4 mRNA is regulated by four open reading
frames in the 5 0 -untranslated region of the mess-
age that suppress GCN4 synthesis when eIF2B
activity is high, but play a permissive role under
conditions where eIF2B activity is reduced. For a
more complete discussion of this complicated
process, the reader is referred to a review
article [1] and references therein.
Phosphorylation of eIF2b by PKA also in-
directly modulates the activity of eIF2B, at least
in vitro [7]. Interestingly, the binding site for
eIF2B on eIF2b is close to the PKA phosphoryl-
ation site. This observation leads to the specu-
lation that phosphorylation of eIF2b by PKA
might alter the anity of eIF2B for eIF2.
Studies in yeast strongly suggest that eIF2 has
a role in selecting the AUG codon used to in-
itiate mRNA translation. In fact, the a- and b-
subunits of eIF2 were initially identied in yeast
as suppressors of initiation codon mutants and
were given the names sui2 and sui3,
respectively [12]. The mechanism through which
eIF2 aids in selecting the correct AUG start
codon is unknown. However, Huang et al.
suggest that hydrolysis of GTP bound to eIF2 is
important in start site selection [13].
A recent study by Ting et al. [14] presents the
intriguing possibility that, in addition to its role
in translation initiation, eIF2 might be involved
in events that occur in the nucleus. In that study,
a fraction consisting of ve polypeptides puried
from placenta interacted with DNA-dependent
protein kinase (DNA-PK) and stabilized for-
mation of a complex containing DNA, DNA-
PK, and a DNA binding protein termed Ku.
Three of the ve polypeptides were the a-, b-,
and g-subunits of eIF2. In addition, eIF2b was
phosphorylated by DNA-PK. Finally, the nding
that a portion of eIF2 localizes to the nucleus,
suggests that eIF2 might play a physiological role
in DNA repair through its interaction with
DNA-PK.
4. Possible medical applications
eIF2a phosphorylation is a critical control
point in the regulation of gene expression under
conditions such as viral infection, apoptosis, and
cell transformation (reviewed in [15]).
Furthermore, interferon treatment has been used
to induce PKR, and thus stimulate eIF2a phos-
phorylation, during viral infection. However,
interferon is not a specic regulator of PKR and
interferon administration can cause a variety of
complications in vivo. Therefore, if more specic
modulators of eIF2a kinase activity can be devel-
oped in the future, they may prove benecial in
the treatment of pathologies as diverse as viral
infection and cancer.
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