(Received for publication, July 27, 1995; and in revised form, August 16, 1995)
From the
Eukaryotic translation is believed to be regulated via the
phosphorylation of specific eukaryotic initiation factors (eIFs),
including one of the cap-binding complex proteins, eIF-4E. We show that
in the yeast Saccharomyces cerevisiae, both eIF-4E and another
cap-binding complex protein, p20, are phosphoproteins. The major sites
of phosphorylation of yeast eIF-4E are found to be located in the
N-terminal region of its sequence (Ser and
Ser
) and are thus in a different part of the protein from
the main phosphorylation sites (Ser
and Ser
)
proposed previously for mammalian eIF-4E. The most likely sites of p20
phosphorylation are at Ser
and/or Ser
. All
of the major sites in the two yeast proteins are phosphorylated by
casein kinase II in vitro. Casein kinase II phosphorylation of
cap-complex proteins should therefore be considered as potentially
involved in the control of yeast protein synthesis. Mutagenesis
experiments revealed that yeast eIF-4E activity is not dependent on the
presence of Ser
or Ser
. On the other hand, we
observed variations in the amount of (phosphorylated) p20 associated
with the cap-binding complex as a function of cell growth conditions.
Our results suggest that interactions of yeast eIF-4E with other
phosphorylatable proteins, such as p20, could play a pivotal role in
translational control.
The eukaryotic initiation factor (eIF)()-4E
(eIF-4F
) is an essential component of the eukaryotic translation
apparatus. eIF-4E constitutes part of the so-called cap-binding complex
eIF-4F which, in higher eukaryotes, also contains eIF-4A and
p220(1) . This complex, together with eIF-4B, promotes binding
of the 43S preinitiation complex to mRNA(2, 3) . The
yeast Saccharomyces cerevisiae does not possess a cap-binding
complex directly equivalent to mammalian eIF-4F. Instead, yeast eIF-4E
has been shown to form a complex with two other proteins, p150
(believed to be the homologue of p220) and p20, whose functions are as
yet unknown(4, 5, 6, 7) . However,
up to now it has been assumed that yeast eIF-4F fulfils the same
function(s) as its mammalian counterpart.
The sequencing of several eIF-4E genes has revealed strong homology within the group of known mammalian polypeptide sequences and less extensive, but clearly evident, homology between the yeast eIF-4E sequence and the sequences of the counterpart proteins of mammals and wheat. The mammalian and yeast proteins are immunologically distinct (8) . However, mouse eIF-4E (9) can substitute for the yeast protein in vivo. Thus, there is also at least partial functional homology between the various eIF-4E proteins. At the same time, the functional role(s) of eIF-4E is(are) incompletely defined. Certain reports have already indicated that this protein might be directly or indirectly involved in a number of processes other than translation. For example, the yeast eIF-4E gene has been identified as the locus of a cell-cycle mutation (cdc 33) that arrests the mitotic cycle at the ``start'' stage(10) . Moreover, a fraction of the cellular population of eIF-4E in COS-1 cells (11) and in yeast (12) localizes to the nucleus.
One striking property of mammalian eIF-4E is that its overproduction can lead to the transformation of higher cells(13, 14) . Moreover, increased levels of this initiation factor allow enhanced translation of mRNAs whose leaders bear strongly inhibitory secondary structure (15) . On the other hand, the overproduction of yeast eIF-4E has little effect on growth or on translation limited by mRNA structure in S. cerevisiae(12) .
Various lines of evidence
indicate that the phosphorylation state of eukaryotic initiation
factors can influence their activities in translation(2) . The
most convincing case so far is that of eIF-2. Phosphorylation of the
subunit of this factor by specific kinases at serine 51 in
mammals (2) and yeast (16) inhibits the exchange of
GTP/GDP and effectively reduces the activity of eIF-2. Phosphorylation
of other sites in the C-terminal region of eIF-2
by casein kinase
II also influences eIF-2 activity, albeit in a more subtle
manner(17, 18) . Increased phosphorylation of eIF-4E,
in contrast, seems to correlate with enhanced translational
rates(19, 20, 21, 22, 23, 24, 25) .
Conversely, dephosphorylation has been found to accompany the
inhibition of protein synthesis under various
conditions(26, 27, 28) . However, these are
indirect correlations which are insufficient to prove the existence of
a causal link between phosphorylation and the activity of eIF-4E. A
more direct demonstration of a functional change associated with
phosphorylation was reported by Minich et al.(29) ,
who found that the phosphorylated form of mammalian eIF-4E has a 3-fold
enhanced affinity for the cap.
What is(are) the site(s) of
phosphorylation in eIF-4E? Ser was identified as a residue
whose mutation to alanine nullifies certain properties of the wild-type
factor(30) . Thus, whereas the overproduction of wild-type
eIF-4E induced aberrant growth in rodent and Hela cells, this effect
was not observed when the Ala
mutant was
used(13, 14) . Similarly, overexpression of the
wild-type gene, but not of the Ala
mutant form, seemed to
allow enhanced translation of mRNAs with structured leaders in mouse
cells(15) . It was also reported that the Ala
mutation prevents incorporation of eIF-4E into the 48 S
initiation complex(31) . Certain lines of evidence have
indicated that eIF-4E may be phosphorylated by protein kinase
C(21, 22, 23, 24) , and this enzyme
has therefore been assumed to be involved in the regulation of eIF-4E
activity.
However, conflicting evidence has thrown doubt on the
significance of phosphorylation at position 53 in the mammalian eIF-4E
amino acid sequence. It was observed that mutation of Ser
to Ala did not affect the phosphorylation level of transiently
overexpressed eIF-4E in COS-1 monkey kidney cells(32) . These
authors also detected no effects of the overexpression of either
wild-type or mutant forms of mammalian eIF-4E on translational
initiation in this cell type. This therefore suggests that there may be
an alternative explanation of the results obtained by others using
Ser
mutation(s). For example, the Ser to Ala substitution
at position 53 may result in a conformational mutant form of eIF-4E
with altered properties that are unrelated to the phosphorylation state
of this factor. Indeed, it was more recently reported that the major
site of phosphorylation in mammalian eIF-4E is
Ser
(33) .
Up to now, there has been no
information available about the phosphorylation sites in the yeast
cap-binding complex eIF-4F. Given the widely acknowledged significance
of S. cerevisiae as an experimental organism for studies of
translation, as well as the uncertainty surrounding the results
obtained so far with the mammalian factor, it has become especially
important to investigate eIF-4F phosphorylation in this lower
eukaryote. We have used biochemical and genetic techniques to show that
both eIF-4E and p20 in yeast are phosphoproteins and that they are
phosphorylated by the same kinase. We examine the functional
significance of these phosphorylation events. The results provide a new
perspective on the pathway and role of phosphorylation in the
regulation of the eukaryotic mRNAcap-binding complex eIF-4F.
Figure 1:
In vivo labeling of
eIF-4E and p20 with P
. A and B, SDS-polyacrylamide gel and autoradiograph, respectively, of
eIF-4E and p20 labeled in vivo. LP, log-phase-growing cells
were labeled as described under ``Materials and Methods.'' HS, in vivo labeling during heat shock. The cells
were grown up to the desired OD at 28 °C and then incubated with
200 µCi/ml
P
at 37 °C for 1.5 h. SP, proteins isolated from cells labeled in the stationary
phase. The cells were grown up to stationary phase, then 200 µCi/ml
P
was added to the culture, which was
incubated for another 1.5 h at 30 °C. C and D,
SDS-polyacrylamide gel and autoradiograph, respectively, of proteins
isolated from yeast strains bearing eIF-4E mutant genes labeled in
vivo. S2A, eIF-4E serine 2 alanine mutant; S2+15A,
serine 2 and alanine 15 double mutant; del19, N-terminal
mutant deleted up to amino acid position 19. E,
isoelectrofocusing of wild-type eIF-4E labeled in vivo. 1,
Western blot; 2, autoradiography.
Figure 2:
Phosphoamino acid analysis. A, in vivo labeled eIF-4E and p20 proteins were subjected to
partial acid hydrolysis and the phosphoamino acids analyzed by
electrophoresis on cellulose plates using a buffer system at pH 3.5. B, phosphoamino acid analysis of proteins labeled in vitro with casein kinase II. R-WT and R-S2+15A,
recombinant wild-type and double mutant serine 2 and 15 to alanine
eIF-4E, both isolated from E. coli. The lanes labeled
with the prefix Y are analyses of proteins isolated from
yeast: WT and S2+15A eIF-4E wild-type and double
mutant; and p20. The positions of the phosphoamino acids used
as standards (P-Ser (P-S), P-Thr (P-T), and P-Tyr (P-Y)) as well as phosphate (P) and the
origin (OR) are indicated. Partial hydrolysis products are
visible in certain lanes.
Figure 3:
Two-dimensional phosphopeptide mapping of
eIF-4E wild-type and mutants. In vivo labeled eIF-4E protein
was digested with trypsin and analyzed by electrophoresis (1st
dimension) and chromatography (2nd dimension) on cellulose plates. The
positions of -dinitrophenyl-lysine (dnp-K) and
xylene-cyanol (XC) used as standards, and the origin (
),
are indicated. WT, wild-type eIF-4E; Del7, N-terminal
deletion up to amino acid position 7; S2A, serine 2-alanine
mutant; S7A, serine 7-alanine mutant; S15A, serine
15-alanine mutant; S2+15A, double mutant serine 2 and
15-alanine. The phosphopeptides referred to in the text are numbered.
HPLC analysis of the tryptic peptides derived from in vivo phosphorylated eIF-4E also yielded two major radioactive peaks, plus four weaker ones (Fig. 4), consistent with the data obtained from two-dimensional peptide mapping. Thus, the above analytical methods indicated the existence of at least two major (serine) phosphorylation sites in yeast eIF-4E. We attempted to identify the phosphorylated peptides in the two major fractions via peptide sequencing. However, the low level of labeling combined with the incomplete separation of the peptides prevented us from obtaining unequivocal data, and we turned to an alternative approach.
Figure 4:
HPLC analysis of peptides generated by
tryptic digestion of eIF-4E labeled in vivo. A, profile of the
80 min 5-50% acetonitrile gradient (A,
flow-rate 0.5 ml/min). B, quantitation of the radioactivity of
the fractions collected during the run shown in A. The elution
positions of the radioactive peaks identified in B are
indicated in A.
Figure 6: Confirmed and potential phosphorylation sites in the S. cerevisiae cap-complex proteins eIF-4E and p20. Shown are the N-terminal (indicated by the subscript N) amino acid sequence of eIF-4E, and the two regions of the now corrected version of the p20 amino acid sequence containing potential CK-II sites. The serines that were mutated to alanines in the eIF-4E mutants are numbered, and the positions of the start codons of the deletion mutants eIF-4EDel7 and eIF-4EDel19 are indicated by arrowheads. The serines in the revised p20 reading frame suspected to be phosphorylated by CK-II are also numbered.
Figure 5:
The phosphorylation of eIF-4E in vivo. Mutant derivatives in which serine phosphorylation sites have been
eliminated (compare Fig. 3) are compared to wild-type. The
measured values of protein and P incorporation for
wild-type eIF-4E (averages of two independent experiments) were each
normalized to 1, and the equivalent data obtained with the three
mutants were expressed as fractions of the wild-type values. See Fig. 1for an explanation of the strain nomenclature. Light
shaded, protein; dark shaded,
radioactivity.
Identification of the two phosphorylated serines at
positions 2 and 15 via mutational analysis also provides an explanation
of the observed mobilities of the phosphopeptides in two-dimensional
mapping (Fig. 3; compare Fig. 6). These mobilities are
consistent with the expected cleavage sites of trypsin in the eIF-4E
amino acid sequence (compare Ref 48). For example, phosphopeptide 1
could be generated by tryptic cleavage at Lys and at
either Lys
(expected net charge +1 at pH 3.5) or at
Lys
(neutral). Phosphopeptide 2, on the other hand, could
be generated by cleavage at Lys
(expected net charge
-1).
We investigated whether there was a growth phenotype
associated with the double mutation in eIF-4E. Expression of the mutant
gene in strain 4-2 lacking the wild-type eIF-4E gene allowed
growth at a rate reduced by no more than 5% relative to wild-type under
standard growth conditions both in YEPD and minimal medium. We also
investigated the rates of total protein synthesis in whole cells
(measured as [S]L-methionine
incorporation) containing only the S2+15A mutant (data not shown).
A decrease of less than 10% compared to the equivalent strain bearing
wild-type eIF-4E was detectable during log-phase growth at 30 °C or
under heat shock conditions (39 °C).
It might be argued that the described mutations induce changes in the phosphorylation state of eIF-4E via indirect conformational changes. However, this is unlikely for at least three reasons. First, each of the single mutations, S2A and S15A, eliminates the phosphorylation of one specific peptide, and the combination of both mutations results in an additive effect (Fig. 3), whereas S7A has no effect on the phosphorylation pattern. Second, the results obtained with the deletion mutants Del7 and Del19 are fully consistent with the former observations ( Fig. 3and Fig. 5), despite the fact that deletions of this kind would be more likely to induce significant conformational alterations in the overall structure of the protein. Third, the fact that none of the amino acid substitutions have a readily detectable phenotype under log-phase growth conditions indicates that if they do cause conformational changes in the protein these are probably small.
Figure 7: Phosphorylation in vitro using casein kinase II. A, autoradiograph of a time course of the in vitro phosphorylation of recombinant wild-type (WT) and double mutant serine 2 and 15-alanine eIF-4E (S2+15A). B, autoradiograph of a time course of the in vitro phosphorylation of cap-binding proteins isolated from yeast strains bearing either the wild-type or the double mutant eIF-4E gene. The positions of eIF-4E and p20 are indicated. The other larger molecular weight bands are attributable to autophosphorylation of casein kinase II. C, comparison of the results of incubation of wild-type recombinant eIF-4E (4E) with casein kinase II for 20 min at 30 °C and an equivalent control experiment lacking eIF-4E as substrate (C).
In light of the above
discrepancy between the in vitro and in vivo labeling, we isolated cap-binding complexes from yeast cells
containing either the wild-type or the S2+15A mutant and
phosphorylated the resulting preparations in vitro using
casein kinase II. The two major phosphopeptides 1 and 2 observed in the
maps prepared from in vivo labeled wild-type eIF-4E could
again be observed in the wild-type, but not in the S2+15A mutant (Fig. 8, panels WT and S2+15A). In
conclusion, casein kinase II can phosphorylate both Ser and
Ser
in vitro. The difference in substrate
behavior of recombinant eIF-4E from E. coli might be due to an
altered conformation relative to that of the natural yeast protein.
Alternatively, the association of other yeast proteins, such as p20,
with eIF-4E isolated from yeast might influence the accessibility of
site 2. The relative strengths of peptides 2 and 3 are reversed in the in vitro and in vivo labeling of yeast eIF-4E
(compare Fig. 8and Fig. 3, WT). Ser
is evidently poorly phosphorylated in vitro, whereas the
third site (peptide 3) seems to be a better substrate.
Figure 8:
Phosphopeptide mapping of eIF-4E and p20
isolated from yeast and phosphorylated in vitro using casein
kinase II. WT and S2+15A, eIF-4E wild-type and
double mutant, respectively. p20 in vivo, peptide mapping of
p20 labeled in vivo; p20 in vitro, peptide mapping of
p20 phosphorylated in vitro using casein kinase II. The
positions of the eIF-4E phosphopeptides 1-3, the major (A) and minor (B) phosphopeptides of p20, the
standards -dinitrophenyl-lysine (dnp-K) and xylene-cyanol (XC), and of the origin (
) are
indicated.
A striking aspect of the in vitro phosphorylation of the cap-binding complex isolated from yeast is that p20 is very rapidly labeled, apparently becoming fully phosphorylated after 5 min of incubation under the conditions described (Fig. 7B). In the process of analyzing the potential sites of phosphorylation in p20, we discovered a discrepancy in the gene sequence compared with that published previously. Our sequencing analysis revealed the existence of an additional A nucleotide in the series of As(370-374) in the published reading frame(51) . The revised reading frame encodes a protein of 161 amino acids and contains two apparent casein kinase II consensus sequences at serines 91 and 154 (Fig. 6). Phosphoamino acid analysis of p20 (Fig. 2), together with peptide mapping of this protein labeled in vivo and in vitro (Fig. 8), indicates that casein kinase II is the major enzyme responsible for p20 phosphorylation in vivo. The in vivo peptide mapping data reveal the existence of at least one major (peptide A), and one minor (peptide B), phosphorylation site in p20. The same major phosphopeptide (A) was also generated by tryptic digestion of p20 that had been phosphorylated by casein kinase II in vitro (Fig. 8).
Figure 9:
Phosphorylation of eIF-4E and p20 in
vivo during log phase growth (LP), heat shock (HS), and stationary phase (ST). The amounts of
eIF-4E or p20 and of P incorporation into these proteins
are expressed as values relative to the log-phase growth data
(normalized to 1.0).
The results described in this paper show that
eIF-4E and p20 in yeast are at least partially phosphorylated in
vivo. The level of phosphorylation of eIF-4E detected was
particularly low, with most of this factor being present in the
non-phosphorylated form. The pattern of phosphorylation was unexpected.
The major positions of phosphorylation of eIF-4E are not protein kinase
C sites and are not equivalent to the sites reported to be present in
the mammalian counterpart protein. Instead, casein kinase II, a
serine/threonine kinase ubiquitously present in eukaryotic cells, has
been found to phosphorylate both proteins. This kinase has a wide range
of substrates, including proteins linked to the cell-cycle, cell
growth, and cell differentiation(52) . In yeast, casein kinase
II is essential for cell viability, and there is evidence for its
involvement in cell-cycle regulation(53) . For example, casein
kinase II phosphorylation of yeast topoisomerase II is
cell-cycle-dependent(54) . Here, as in yeast
eIF-2(17, 18) , the phosphorylation sites are
close to the C terminus of the protein. The main casein kinase II
phosphorylation sites of yeast eIF-4E are close to the N terminus, and
one of the p20 sites is predicted to be close to the C terminus (Fig. 6). Mutation of the two major phosphorylation sites in
eIF-4E results in only a minimal phenotype in yeast under standard
growth conditions. Indeed, yeast can survive with an eIF-4E protein
that has no detectable phosphorylation (see Fig. 5). In this
context, it is significant that the mutation of Ser
to
Ala in mammalian eIF-4E has no effect on this factor's
association with the 48 S initiation complex(33) . However, as
with eIF-2
(17) , our data do not rule out that there are
conditions under which such sites are functionally important. Of at
least equal importance is the observed variation in binding of
phosphorylated p20 to eIF-4E, which will need to be investigated in
considerable detail.
In conclusion, this study establishes the
necessity to reassess the pathway and regulation of phosphorylation of
yeast (and possibly mammalian) eIF-4E. Indeed, the variations in the
amount of phosphorylated p20 we have described suggest that the
possible involvement of other proteins that interact with eIF-4E may
play important regulatory roles in translational initiation. We see
here a potential parallel to the recently described regulatory system
for mammalian eIF-4E. The binding of PHAS-I to mammalian eIF-4E is
regulated via phosphorylation of Ser in the regulatory
protein by mitogen-activated kinase(50, 55) . Thus,
the mitogen-induced stimulation of protein synthesis is thought to be
mediated via this regulator binding mechanism. On the basis of our new
data on the yeast cap-binding proteins, future work will need to
explore analogous paths for potential regulatory mechanisms in this
lower eukaryote.