(Received for publication, April 12, 1995)
From the
Ser-53 has previously been considered the major phosphorylation
site in eukaryotic initiation factor (eIF)-4E, and this appeared to be
supported by studies using a S53A mutant. Recently, however, several
lines of evidence have indicated that Ser-53 might not be the true
phosphorylation site. This prompted us to re-examine the
phosphorylation site in eIF-4E using factor purified from P-labeled, serum-treated Chinese hamster ovary cells.
Isoelectric focusing and phosphoamino acid analysis indicated the
existence of a single phosphorylated serine. Edman degradation of the
major radiolabeled tryptic product from
P-labeled eIF-4E
showed that the phosphorylated site was positioned three residues from
the N terminus of this peptide. There are three serines in the sequence
of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine).
P-Labeled eIF-4E was
digested with trypsin, Lys-C, or trypsin followed by Glu-C and
subjected to two-dimensional mapping; the data obtained eliminated two
of these potential sites, leaving Ser-209. Comigration of the synthetic
peptide SGS(P)
TTK with the radiolabeled tryptic product
on (i) reverse-phase chromatography and (ii) two-dimensional mapping at
different pH values confirmed that Ser-209 is the major phosphorylation
site in eIF-4E in serum-stimulated Chinese hamster ovary cells.
Phosphorylation of eukaryotic initiation factor
(eIF)()-4E (also known as eIF-4
), which binds to the
5`-cap structure on eukaryotic mRNAs, has been shown to correlate
positively with changes in the rate of translation under a wide range
of conditions (for reviews, see (1, 2, 3) ).
eIF-4E is the least abundant of the components of the eIF-4F
complex(4) , which also contains the RNA helicase eIF-4A and
eIF-4
, and translation of messages with a high degree of secondary
structure is enhanced by overexpression of eIF-4E(5) .
Phosphorylation of eIF-4E could therefore provide an important
mechanism for regulating cellular translation and particularly that of
certain mRNAs. Although it remains unclear how phosphorylation of
eIF-4E affects its activity, it has been reported to lead to increased
association of eIF-4E with high molecular mass complexes, perhaps
including the other subunits of eIF-4F(6, 7) . Recent
work also suggests that phosphorylation of eIF-4E may increase its
affinity for the cap(8) , although phosphorylation is certainly
not a prerequisite for it to bind.
Several years ago, Rychlik et al.(9) presented evidence that the major site of phosphorylation of eIF-4E in rabbit reticulocytes was Ser-53. This apparent identification of Ser-53 as the site of phosphorylation was backed up by several studies employing a site-directed mutant of eIF-4E in which Ser-53 was altered to alanine. Such a mutant showed a number of differences from the wild-type protein when overexpressed in intact cells or when studied in vitro in that it did not induce cell transformation or promote abnormal morphology(10, 11) , and it was not incorporated into 48 S initiation complexes(12) .
Evidence has, however, been
presented that other phosphorylation sites exist in eIF-4E based on the
fact that the S53A mutant still underwent
phosphorylation(11, 12, 13, 14) . In
addition, Kaufman et al.(13) found no discernible
differences in the ability of wild-type or S53A mutant eIF-4E to
support translation of selected mRNAs or to become incorporated into
the eIF-4F complex. Furthermore, we recently observed that the major
insulin-stimulated eIF-4E kinase in Chinese hamster ovary (CHO) cells
was able to phosphorylate recombinant eIF-4E (S53A) to a similar extent
compared with the wild-type recombinant protein. ()Taken
together, these findings prompted us to reinvestigate the site of
phosphorylation in eIF-4E. (
)Our data show that the major
site of phosphorylation in eIF-4E in serum-stimulated CHO cells is
Ser-209, close to the C terminus of the protein.
Glu-C digestion of the major radiolabeled
tryptic product of eIF-4E was performed by scraping radioactive
material from a thin-layer cellulose plate following two-dimensional
mapping (see below) and washing extensively with water. The pooled
supernatants were dried under vacuum, resuspended in water, and
incubated with 1 µg of endoproteinase Glu-C in 50 mM ammonium bicarbonate (pH 7.8) for 16 h before processing as
described above. Both the Glu-C and Lys-C preparations used digested
completely 1 µg of rabbit reticulocyte eIF-2 (data not shown)
under the same conditions.
As we have previously reported, ()isoelectric
focusing analysis reveals the existence of only two distinct species of
eIF-4E in CHO cells as detected by immunoblotting, and treatment of
isolated eIF-4E with alkaline phosphatase causes conversion of all the
factor to the more basic (i.e. unphosphorylated) form. Fig. 1A shows an immunoblot of eIF-4E from control and
serum-treated CHO.K1 cells: in the former case, only 20% of the eIF-4E
is phosphorylated, while in serum-treated cells, almost all the factor
(
80%) is in the more acidic (phosphorylated) form. These findings
are consistent with the data from a range of other cell types
indicating that eIF-4E contains one major phosphorylation
site(20) .
Figure 1:
Analysis of eIF-4E in
CHO cells. A, isoelectric focusing was carried out using
eIF-4E isolated from serum-starved CHO.K1 cells that had no further
addition (lane1) or that were re-challenged with
serum (10%, v/v) for 10 min (lane2). Shown is a
Western blot; the position of phosphorylated eIF-4E (P) is
indicated. B, phosphoamino acid analysis of P-labeled eIF-4E from serum-treated CHO.K1 cells was
performed as described under ``Experimental Procedures.'' The
positions of migration of phosphoserine (S) and
phosphothreonine (T) markers (
1 µg each) are
indicated. P
,
[
P]orthophosphate; o,
origin.
To determine which type of amino acid was phosphorylated in eIF-4E in CHO.K1 cells, serum-starved cells were incubated with radioactive inorganic phosphate and briefly treated with serum, and eIF-4E was then isolated and subjected to phosphoamino acid analysis. Only phosphoserine was observed (Fig. 1B), which is consistent with several other reports using a range of different cell types (see (20) , for a review), although phosphothreonine has been reported in eIF-4E under some conditions (see, for example, (7) ).
It thus appeared likely that
eIF-4E was phosphorylated at a single serine residue in serum-treated
CHO.K1 cells. To investigate further the location of the phosphoserine
residue in eIF-4E, radiolabeled factor was isolated from serum-treated
cells and subjected to tryptic digestion followed by separation of the
resulting peptides by reverse-phase chromatography (Fig. 2). Two
separate experiments were performed, and in both cases, one major peak
of radioactive material was observed. This peak eluted at 8%
acetonitrile, indicating that the material was very hydrophilic. Some
additional radioactive material eluted later, at 20-30%
acetonitrile, and may have represented products of incomplete
trypsinolysis of radiolabeled eIF-4E. In both experiments, the first
peak contained
75% of the total radioactive material, and this was
contained within one fraction, whereas the later material emerged as a
broad smear.
Figure 2:
HPLC
analysis of the tryptic phosphopeptides from P-labeled
eIF-4E. Reverse-phase chromatography on a C
column was
performed for the products of trypsinolysis of
P-labeled
eIF-4E from serum-treated CHO.K1 cells (A) or for a
nonradiolabeled synthetic phosphopeptide, SGS(P)TTK (B), as
described under ``Experimental Procedures.'' The flow rate
was 0.1 ml/min; 100-µl fractions were
collected.
Two-dimensional phosphopeptide mapping was performed to characterize radiolabeled material contained in the main peak from reverse-phase chromatography. Fig. 3A (panel i) shows that one positively charged phosphopeptide was obtained when electrophoresis was performed at pH 1.9; this phosphopeptide migrated slightly faster than cyanol (which has a net charge of +1 at this pH). The same result was obtained at pH 3.5, except that the peptide and cyanol did not migrate as quickly toward the cathode (data not shown). Consistent with the findings of other groups(14, 19, 21) , the radiolabeled tryptic product from eIF-4E did not move from the origin on chromatography, again testifying to its extremely hydrophilic nature.
Figure 3:
Analysis of the location of the phosphate
label in eIF-4E. A, two-dimensional peptide mapping and
autoradiography were performed for the products of digestion of P-labeled eIF-4E with trypsin (paneli),
Lys-C (panelii), or trypsin followed by Glu-C (paneliii). Paneliv shows a
schematic representation of the map obtained for the nonradiolabeled
synthetic phosphopeptide SGS(P)TTK and serves as a key for the position
of 2,4-dinitrophenyllysine (DNP-lysine) and cyanol in panelsi-iii. o, origin.
Electrophoresis was performed at pH 1.9. Only the parts of the map
containing marker or peptide are shown; no other radiolabeled material
was observed. B, manual Edman degradation was performed using
a sample of the HPLC-purified tryptic product of
P-labeled
eIF-4E. Samples of starting material (lane0) or
following each round of degradation (lanes 1-3) were
analyzed by one-dimensional electrophoresis at pH 3.5, followed by
autoradiography. P
,
[
P]orthophosphate
marker.
To determine
the position of the phosphoserine residue in the tryptic product,
manual Edman analysis was performed, with samples being retained after
every cycle for further analysis by one-dimensional mapping. The
initial peptide and the products obtained after one or two cycles had a
net positive charge (Fig. 3B). However, after the third
cycle, 75% of the radioactivity no longer migrated with positively
charged material, but instead moved in the opposite direction and
migrated with inorganic phosphate. Furthermore, the fourth round of
degradation yielded [
P]orthophosphate only, with
no counts remaining in the position of the original phosphopeptide
(data not shown). These data indicate that the third residue in the
peptide bears the radiolabel, and given the conclusions drawn from the
phosphoamino acid analysis (Fig. 1B), the tryptic
peptide therefore contains a phosphoserine at position 3.
There are
no sequence data available for eIF-4E from the Chinese hamster.
However, there is a very high degree of identity between rabbit, mouse,
and human eIF-4E(22, 23, 24) ; the only
significant differences are in the extreme N terminus of the factor
(residues 12, 16, and 17), while the remainder of the eIF-4E sequence
is 99.5% identical. We therefore examined the sequences of these
proteins to identify possible candidates for the tryptic peptide
obtained in our studies. In each case, there are three serines that lie
three residues C-terminal to a tryptic cleavage site (Lys or Arg).
Attempts to purify sufficient amounts of the tryptic phosphopeptide
from eIF-4E in CHO.K1 cells were unsuccessful. Therefore, to
distinguish between these three possible phosphorylation sites, further
proteolytic digests were performed. In the case of Ser-64 in the
tryptic peptide LISK, digestion of the intact
P-labeled factor with Lys-C would yield a much larger and
more hydrophobic phosphopeptide (TWQANLRLISK) than that obtained by
tryptic digestion, which would migrate quite differently on
two-dimensional peptide mapping. However, as shown in Fig. 3A (panelii), the migration of
the phosphopeptide produced with Lys-C was identical to that of the
tryptic product at pH 1.9 (and also at pH 3.5; data not shown), ruling
out Ser-64 as the phosphorylation site.
In the case of Ser-24 in the
tryptic peptide TESNQEVANPEHYIK, treatment of the tryptic
phosphopeptide with Glu-C would be expected to generate a smaller
species with altered mobility on electrophoresis and chromatography.
However, Fig. 3A (paneliii) shows
that after Glu-C treatment, the migration of the phosphopeptide was
unchanged on two-dimensional mapping at pH 1.9 (and also at pH 3.5;
data not shown), indicating that Ser-24 is not the radiolabeled
residue. By a process of elimination, then the phosphorylation site in
eIF-4E in CHO cells appears to be Ser-209 in the tryptic peptide
SGS
TTK, consistent with the recent findings of the
Rhoads' laboratory.
As a final confirmation of the
identity of the phosphorylation site in eIF-4E, a synthetic
phosphopeptide (SGS(P)TTK) that corresponds to residues 207-212
of eIF-4E was prepared. This peptide showed identical behavior to that
of the tryptic product from P-labeled eIF-4E on (i)
reverse-phase chromatography (Fig. 2), (ii) electrophoresis at
pH 1.9 (Fig. 3A (paneliv)) or (iii)
at pH 3.5 (data not shown), and (iv) thin-layer chromatography (Fig. 3A (paneliv)). This strongly
supports the identification of the labeled phosphopeptide and confirms
that the phosphorylated residue is indeed Ser-209.
In conclusion,
all these data indicate that the major (and probably the only)
phosphorylation site in CHO cells is Ser-209. Reappraisal of the role
of phosphorylation of eIF-4E is therefore required using mutants based
on Ser-209. This work also highlights the need for caution when using
Ser to Ala mutants of proteins to investigate the role of
phosphorylation, as this approach has given phenotypes not related to
phosphorylation in two translation initiation factors, i.e. eIF-4E (discussed above) and eIF-2(25, 26) .