From the
Initiation factor 4E (eIF-4E) binds to the
m
The initiation of protein synthesis in eukaryotic cells is
divided into discrete stages, each catalyzed by a different initia-tion
factor (eIF-)
Many of the initiation factor
polypeptides exist as phosphoproteins, and several lines of evidence
support the view that the overall rate of translation is regulated by
phosphorylation and dephosphorylation of initiation and elongation
factors(5, 6) . In the case of eIF-4E, there is a
correlation between the rate of protein synthesis and phosphorylation
of the protein. Dephosphorylation of eIF-4E occurs under conditions
where translation of normal cellular mRNAs is inhibited, such as heat
shock, mitosis, and adenovirus infection. Conversely, phosphorylation
of eIF-4E increases under conditions that stimulate protein synthesis
such as exposure of cells to insulin, serum, epidermal growth factor,
platelet-derived growth factor, nerve growth factor, phorbol esters,
lipopolysaccharide, tumor necrosis factor-
Although there are at least five forms of mammalian eIF-4E separable
by IEF, two forms predominate, having pI values of 5.9 and
6.3(9, 10, 11) . Several lines of evidence
suggest that these represent nonphosphorylated and monophosphorylated
forms of the protein. When eIF-4E is labeled in vivo with
[
A
previous study presented evidence that the major phosphorylation site
in eIF-4E was Ser-53(12) . This was based on labeling eIF-4E in vivo with [
The kinase(s)
responsible for in vivo phosphorylation of eIF-4E has not been
identified, although rat brain protein kinase C (27) and bovine kidney
protamine kinase (28) can phosphorylate eIF-4E in vitro at the physiological site, based on generation of a tryptic
phosphopeptide that co-migrates with the in vivo phosphopeptide. Another study indicated that protein kinase C
phosphorylates both Ser and Thr in eIF-4E in
vitro(29) , whereas casein kinase I phosphorylates
predominantly Thr residues(30) .
In order to aid in isolation
of the physiological kinase, we undertook the chemical synthesis of
P-Ser-Lys, the peptide expected from complete tryptic digestion of the
region surrounding Ser-53 in eIF-4E. Such a peptide would be useful as
a standard in studies to determine whether a given kinase
phosphorylates eIF-4E at the physiological site. Surprisingly, the
synthetic peptide did not co-migrate with the natural tryptic
phosphopeptide. Consequently, we have redetermined the phosphorylation
site of eIF-4E.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Z-Ser-Lys(
Based on the assignment of Ser-53 as the major
phosphorylation site of eIF-4E, we predicted a tryptic phosphopeptide
of P-Ser-Lys. To provide an electrophoretic standard for investigating
the in vivo phosphorylation of eIF-4E, we synthesized this
dipeptide. Initial attempts to synthesize 3 by coupling
of either Z-P-Ser or N-t-butyloxycarbonyl-P-Ser to
Lys(
Since the chymotryptic peptide isolated in Fig. 3contained numerous hydroxyamino acid residues, it was
necessary to determine which of them was phosphorylated. Four
independent methods were used. In the first, the release of
radioactivity during Edman degradation was followed. P-Ser and P-Thr
are unstable to Edman degradation and break down during cleavage to
release free phosphate. The acidic phosphate cannot be extracted from
the ionic polybrene matrix used for a sequencing support by butyl
chloride, the usual extraction solvent, and more hydrophilic washes
would extract the peptide. Covalent attachment prior to sequencing
eliminates the necessity for using a polybrene-coated glass fiber disc,
so the free phosphate may be easily extracted from the peptide without
fear of peptide loss. Fig. 4shows that there was an increase in
released radioactivity at cycle 12, which corresponds to Ser-209.
However, released radioactivity was also above background in cycles 1,
2, 5, 13, and 14. It is likely that this represented extraction of
noncovalently bound peptide in the case of the earlier cycles, or
incomplete Edman chemistry in cycle 12, resulting in carryover into
cycles 13 and 14. Cycles 13 and 14 contained Thr, which could
potentially be phosphorylated in eIF-4E. We therefore analyzed the
phosphoamino acid content of the chymotryptic peptide (Fig. 2, lane5) as well as that of intact eIF-4E (lane4). Only P-Ser was detected, confirming earlier results (9, 15) and indicating that the radioactivity in cycles
13 and 14 does not represent P-Thr but rather carryover from cycle 12.
This analysis, however, could not exclude the possibility that Ser-199
(cycle 2) also contained radioactivity.
The
third method involved analysis of the labeled chymotryptic peptide by
further proteolytic digestion and separation of peptides by TLE (Fig. 2). The chymotryptic phosphopeptide (
The fourth method was
to alter the codons for positions 207 and 209 in human eIF-4E from Ser
to Ala using site-directed mutagenesis. These mutations represent
single or double base changes in the cDNA coding region that convert
codon 207 from AGC (Ser) to GCC (Ala) and codon 209 from TCC (Ser) to
GCC (Ala). The mutations, and that previously generated at codon 53
(AGC to GCC; Ref. 18) were confirmed by dideoxynucleotide sequencing (Fig. 5, A and B). RNA transcribed from these
plasmid constructs was translated in rabbit reticulocyte lysate in the
presence of [
The effect
of changing Ser-209 to Ala on the association of eIF-4E with the 48 S
initiation complex was tested. Two different protocols were used, one
in which
The evidence presented in this study supports the existence
of a single phosphorylation site in eIF-4E at Ser-209. The release of
radioactivity during Edman sequencing (Fig. 4), the increase in
the PTH-DHA-DTT/PTH-Ser ratio (Fig. 4), the susceptibility to
site-specific proteases (Fig. 2), and the results of
site-directed mutagenesis (Fig. 5) all support the assignment of
Ser-209 as the major phosphorylation site of eIF-4E. The previous
evidence favoring Ser-53 consisted of (i) amino acid composition
data(12) , (ii) the fact that eIF-4E
Reassignment of the major phosphorylation
site does not negate the evidence that Ser-53 is important for the
activity of eIF-4E. This has been demonstrated by the activity of
eIF-4E
Similarly, the evidence that
phosphorylated eIF-4E binds to cap analogs and mRNA caps 3-4-fold
more strongly than nonphosphorylated eIF-4E (7) is not
contradicted by the change in the phosphorylation site. In that study, in vivo phosphorylated eIF-4E was chromatographically resolved
from the nonphosphorylated form, and the binding affinity of each was
measured fluorimetrically. Knowledge that phosphorylation increases the
affinity for caps combined with the localization of the phosphorylation
site at the C terminus suggests that phosphorylation may induce a
conformational change in the protein that makes the cap-binding site
more accessible. C-terminal phosphorylation on Ser/Thr is correlated
with modulation of activity of a number of proteins, such as glycogen
synthase, cAMP-dependent protein kinase, and the
Although the physiological eIF-4E kinase(s)
has not been isolated, the context surrounding Ser-209 indicates that
it would be an optimal phosphorylation acceptor for protein kinase C
based on the consensus sequence
(R/K
The chymotryptic phosphopeptide also contains sequence contexts that
are predicted to be phosphate acceptor sites for casein kinase I and II
(49). One of the five casein kinase I consensus sites
(D/E)XX(S/T) in eIF-4E lies within the chymotryptic
phosphopeptide in the context DTAT, where Thr-205 would be the
phosphate acceptor. If this region of the C terminus of eIF-4E were to
be made accessible for phosphorylation upon phosphorylation of Ser-209,
then the Thr phosphorylation by casein kinase I observed in vitro(30) may be at Thr-205. Ser-199, in the context SHAD, is
predicted to be an acceptor for casein kinase II. Attempts to
phosphorylate eIF-4E with purified casein kinase II, however, were
unsuccessful(45) . No phosphorylation of any secondary sites was
observed in the present study. However, the presence of numerous Ser
and Thr residues in the C terminus of eIF-4E may explain the multiple
minor phosphorylations of eIF-4E that have been previously
observed(9, 16) .
We thank Dr. Robert Smith, Debra Waters, and Wei-Hua
Wu, Department of Biochemistry and Molecular Biology, Lousiana State
University Medical Center, for technical advice and assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
GTP-containing cap of eukaryotic mRNA and facilitates the
entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is
a phosphoprotein, and the phosphorylated form binds to mRNA caps
3-4-fold more tightly than the nonphosphorylated form. A previous
study indicated that the major phosphorylation site was Ser-53
(Rychlik, W., Russ, M. A., and Rhoads, R. E.(1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the
phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic
phosphopeptides did not co-migrate electrophoretically. Accordingly, we
redetermined the phosphorylation site by isolating a chymotryptic
phosphopeptide on reverse phase high performance liquid chromatography.
The peptide was sequenced by Edman degradation and corresponded to
QSHADTATKSGSTTKNRF
. The site of
phosphorylation was determined to be Ser-209 by four methods: the
increase in the ratio of dehydroalanine to serine derivatives during
Edman degradation, the release of
P, the further digestion
of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and
site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not
phosphorylated in a rabbit reticulocyte lysate system, whereas the
wild-type, S53A, and S207A variants were. This site falls within the
consensus sequence for phosphorylation by protein kinase C.
(
)or group of initiation factor
polypeptides(1, 2, 3) . Binding of mRNA to the
43 S initiation complex to form the 48 S complex is catalyzed by the
eIF-4 factors. These factors consist of eIF-4A, a 46-kDa ATP-dependent
RNA helicase, eIF-4B, a 70-kDa RNA-binding protein that stimulates the
activity of eIF-4A, eIF-4E, a 25-kDa cap-binding protein, and
eIF-4
, a 154-kDa protein that forms complexes with the other eIF-4
polypeptides as well as with eIF-3. By promoting formation of these
complexes, eIF-4
may serve to bring the 43 S initiation complex,
the cap-binding function of eIF-4E, and the RNA helicase activity of
eIF-4A and eIF-4B into immediate proximity so as to begin the dual
processes of unwinding and scanning of the mRNA from the cap in the
direction of the first AUG (4).
, and interleukin-1 or
overexpression of oncoproteins such as p21
or
pp60
(for review, see Ref. 6). The
phosphorylated and nonphosphorylated forms of eIF-4E are resolved by
chromatography on RNA-Sepharose, and the phosphorylated form binds to
cap analogs and mRNA caps 3-4-fold more tightly than the
nonphosphorylated form(7) . Since eIF-4E appears to be the least
abundant of the initiation factors and acts at the rate-limiting step
of initiation (for review, see Ref. 8), the phosphorylation of eIF-4E
may directly regulate the rate of protein synthesis initiation.
P]P
, the pI of the major
radioactive form is 5.9, whereas that of the major nonradioactive form
is 6.3, a difference that is consistent with the presence of a single
phosphate residue(9, 10) . Phosphatase treatment
converts the pI 5.9 form to pI 6.3 but has no effect on the pI 6.3
form, suggesting that there are no phosphate residues that are
metabolically inactive and hence not labeled in
vivo(10, 7) . Tryptic digestion produces a single
major phosphopeptide(12, 13, 14) . Finally, the
only phosphoamino acid detected in the in vivo labeled protein
is P-Ser(9, 15) , although treatment of cells with
okadaic acid produces eIF-4E containing P-Thr as well(16) .
P]P
,
isolating tryptic phosphopeptides either directly or after alkylation
of the protein with citraconic anhydride, and determining their amino
acid compositions. Comparison with the cDNA-derived amino acid sequence
of eIF-4E suggested that the phosphorylated residue lay in the peptide
WALWFFKNDKSKTWQANLR
, which contains only a
single Ser residue, Ser-53. Several studies have subsequently
demonstrated the importance of Ser-53 to the activity of eIF-4E, and
these have been interpreted to mean that phosphorylation enhances its
activity. The wild-type protein (eIF-4E
) is
incorporated into the 48 S initiation complex along with
mRNA(17) , whereas the eIF-4E
(18) and
eIF-4E
(8) variants are not. Overexpression or
microinjection of eIF-4E
, but not
eIF-4E
, in cultured mammalian cells causes accelerated
protein synthesis and cell growth, leading to oncogenesis in some cases (19, 20) and to aberrant cellular morphology such as
multiple nuclei in others(21, 8) . Overexpression of
eIF-4E
but not eIF-4E
causes
increased expression of cyclin D1 (22) and chloramphenicol
acetyl transferase encoded by an mRNA with high 5` secondary
structure(23) . The ability to relieve inhibition of protein
synthesis in early sea urchin embryo extracts is a property of
eIF-4E
but not eIF-4E
(24) .
Overexpression of eIF-4E
in Xenopus embryos
induces the differentiation of mesodermal tissues in 90% of the cases,
as opposed to 53% for eIF-4E
(25) . In contrast
to the above, however, overexpressed eIF-4E
and
eIF-4E
are equally phosphorylated and competent to
form eIF-4 complexes in COS cells(26) .
Materials
Z-Ser, Lys(Z)OBn hydrochloride, L-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin
and 1-chloro-3-tosylamido-7-amino-2-heptone-treated
-chymotrypsin
were purchased from Sigma. Protease Glu-C and endoproteinase Asp-N were
purchased from Boehringer Mannheim.
NMR and Mass Spectroscopy
C NMR
spectra were obtained with a Jeol FX90Q spectrometer.
H NMR
spectra were recorded at 400 MHz on a Jeol GX-400 and at 500 MHz on a
Bruker AM 500 spectrometer in
H
O. Chemical
shifts were determined relative to sodium
3-trimethylsilyl-[2,2,3,3-
H
]propionate
as internal standard. Liquid matrix secondary ions mass spectrometry
was performed on a AMD-604 Intectra GmbH spectrometer.
Synthesis and Characterization of P-Ser-Lys
The
scheme of synthesis was as follows. Z)OBn (1) was prepared from Z-Ser
and Lys(
Z)OBn hydrochloride(31) .
C NMR
spectra and other physiochemical data were in accordance with the
predicted structure. This compound (1, 101 mg, 0.17
mmol) was treated with trimethyl phosphate (1 ml), and then phosphorous
oxychloride (32 µl, 0.34 mmol) was added. The mixture was stirred
at 0 °C overnight, poured into water (20 ml), and neutralized with
1 N NH
OH. The precipitate obtained was purified by
semipreparative HPLC on a Supelco LC-18-DB reverse phase column
monitored at 260 nm over 15 min with a linear 40-100% gradient of
buffer A (0.03 M NH
OAc, pH 5.0) and buffer B (33%
buffer A, 67% acetonitrile (v/v)). The material eluting at 6.6 min was
collected and lyophilized (yield, 35 mg; 29% as calculated for the
diammonium salt). The
C NMR spectrum of product 2 and other physiochemical data confirmed the presence
of a phosphate group on the serine moiety. Next, 2 (27
mg) was dissolved in ethanol (8 ml), palladium on charcoal (100 mg) was
added, and hydrogen was passed through the mixture at room temperature
for 8 h. The catalyst and product were collected on a sintered glass
funnel, the ethanol filtrate being discarded, and the product was
eluted by washing twice with water. The water solution was evaporated
to dryness to obtain 3 (10 mg, 78.8%). Analytical HPLC
was performed on a Supelco LC-18-T reversed phase column (250
5
mm) using 0.01 M KH
PO
, pH 3.0, at 2.0
ml/min with monitoring at 210 nm. This produced a single peak with
retention time of 1.4 min. Mass spectrometry revealed peaks at 314
(M+H), 627 (2M+H), and 940, (3M+H); mass calculated for
C
H
N
O
P + 1 was
314.
Preparation of
Rabbit reticulocytes were incubated with
[P-Labeled
eIF-4E
P]P
, and the eIF-4E purified by
m
GTP-Sepharose chromatography as described previously (12) with the exception that the cells were resuspended in 2
volumes of the labeling buffer and incubated in the presence of 0.4
mCi/ml of radiolabel (method 1) or phosphate-free Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and at
0.2 mCi/ml of radiolabel (method 2). Similar results were obtained with
both methods. The
P- eIF-4E was then mixed with 100 µg
of unlabeled rabbit reticulocyte eIF-4E and further purified by
reverse-phase HPLC as described previously(12) , except that a
C
column (4.6
150 mm, 5 µm particle size,
Vydac, Hesperia, CA) was used.
Proteolytic and Acid
Hydrolysis
P-labeled eIF-4E was dried by vacuum
centrifugation and resuspended at 0.5 mg/ml in 50 mM NH
HCO
. Proteolysis was carried out for 20
h at 37 °C with either trypsin (25 µg/ml) or chymotrypsin (50
µg/ml). Peptides were digested with trypsin (80 µg/ml), Glu-C
(16 µg/ml), or Asp-N (8 µg/ml) for 12 h under the same
conditions except in the case of Glu-C, where 50 mM potassium
phosphate buffer, pH 8.0, was used. Phosphoamino acid analysis was
performed essentially as described previously(9) .
Thin-layer Electrophoresis
TLE was performed as
described previously(9) . Following electrophoresis, peptides or
amino acids were visualized by staining with 0.5% ninhydrin in
1-butanol. Radiolabeled phosphopeptides were visualized by
autoradiography and, when necessary, were recovered from the plates by
scraping and extracting the cellulose with 8% acetic acid, 2% formic
acid.
Isolation of a Chymotryptic Phosphopeptide from
eIF-4E
P-labeled eIF-4E was digested with
chymotrypsin, and the resulting peptides were diluted to 1 ml with 0.1%
trifluoroacetic acid and resolved by reverse phase HPLC using a Vydac
C
column (4.6
150 mm, 5 µm particle size).
Buffer C was 0.1% aqueous trifluoroacetic acid, and buffer D was 0.1%
trifluoroacetic acid in 95% acetonitrile. Buffer D increased linearly
from 0 to 15% over 30 min and then to 85% over 19 min.
P-Labeled peptides were identified by Cerenkov counting.
For rechromatography of isolated peaks, fractions were pooled,
concentrated to approximately 200 µl by vacuum centrifugation, and
diluted to 500 µl with buffer C prior to injection.
Edman Degradation
All peptide sequencing was
performed on an Applied Biosystems 477A peptide sequencer (Foster City,
CA). A polybrene-coated glass fiber disc was the sequencing support for
samples sequenced by the standard noncovalent method. The ratios of
PTH-DHA-DTT to PTH-Ser were calculated at Ser cycles from measurements
of peak heights for the two species. When covalent sequencing was
performed, the peptides were linked through their carboxyl groups to an
arylamine-derivitized polyvinylidine difluoride disc according to the
protocol provided by the manufacturer (Sequelon AA reagent kit,
Millipore, Bedford, MA). The disc was washed after coupling with first
water and then methanol until radioactivity in the wash solutions
remained at background levels. Then the disc was minced and loaded into
a standard sequencer reaction cartridge above an Immobilon-P disc
(Millipore). Ninety percent methanol was substituted for butyl chloride
at the S3 and X2 positions. Approximately one-third of the PTH sample
was analyzed by HPLC. After sample injection, the conversion flask was
washed with 90% methanol from the X2 bottle, and this wash was used to
push the remaining two-thirds of the sample to a fraction collector for
quantitation of radioactivity by Cerenkov detection.
Preparation of cDNA Encoding wild-type, S53A, S207A, and
S209A eIF-4E
The plasmids pTCEEC (17) and
pTCALA(18) , which contain most of the human eIF-4E cDNA, were
modified to form pT4EA+ and pT4ES53A, respectively, by excision of
the NsiI to HindIII region of the 3`-untranslated
region (nucleotides 1085-1353) and replacement with the
3`-untranslated and poly(A) sequence (nucleotides 456-651)
derived from the Xenopus ribosomal protein S22 (32) using an EcoRI, HindIII, BamHI adaptor (Oligos Etc.,
Wilsonville, OR). pT4EA+ served as the template for site-directed
mutagenesis by polymerase chain reaction (33) to introduce a
single-base change (T625G) converting Ser-209 to Ala or a double-base
change (A619G/G620C) converting Ser-207 to Ala. Briefly, 0.1 µg of
linear plasmid was amplified in two separate reactions (10 cycles of 45
s at 94 °C, 45 s at 50 °C, and 90 s at 72 °C; 50 pmol of
each oligonucleotide) with the upstream primer, CBP440
(5`-ATGATGTATGTGGCGCTG-3`), and mut624
(5`-AGTGGTGGCGCCGCTCTTAG-3`) or a downstream primer, S22-3`
(5`-CAATCCACATTACTTTCTTT-3`) and mut624s (5`-CTAAGGCCGGCTCCACCACT-3`)
using PfuI thermostable DNA polymerase (Stratagene, La Jolla,
CA). mut624s and mut624
are complementary with the exception of
three mismatches. The gel-purified products of the initial reactions
were mixed, heated 5 min at 94 °C, and then annealed to one another
by slow cooling to 45 °C. Annealed fragments were subjected to a
second round of amplification (20 cycles of 45 s at 94 °C, 45 s at
45 °C, and 90 s at 72 °C) with PfuI using only the
external primers, CBP440 and S22-3`
. The product was
digested with AccI and NsiI and used to replace the
corresponding 539 base pairs of wild-type eIF-4E sequence in
pT4EA+. The resulting plasmids, pT4ES207A and pT4ES209A, were
sequenced in the amplified region from the AccI site of the
insertion through the termination codon using the antisense
oligonucleotide, CBP 695
(5`-TCTCGATTGCTTGACGCAGT-3`) and a
Sequenase
T7 DNA polymerase dITP kit (U. S. Biochemical Corp.)
as described by the manufacturer. Each of the induced mutations creates
a new restriction endonuclease site (NaeI in pT4ES207A and NarI in pT4ES209A), which was used for independent
verification of the correct mutation. pT4ES53A was also sequenced to
verify the site of mutation with the antisense oligonucleotide, CBP
258
(5`-GTGAGTAGTCACAGCCAGGC-3`).
Cell-free Transcription
Plasmids were linearized
with EcoRI and transcribed with T7 RNA polymerase in vitro as described previously(4) . The resulting mRNA
transcripts, which contain a poly(A) tail, were approximately 5-fold
more efficient in directing eIF-4E synthesis in vitro than
transcripts derived from pTCEEC, which do not contain poly(A).
Cell-free Translation and IEF
Cell-free
translation was performed as described previously(4) . Following
synthesis, 3-µl samples from the reactions were diluted with 70
µl of buffer containing 9.2 M urea, 0.5% Nonidet P-40, 80
mM DTT, 10% glycerol, 1.75% Pharmalytes, pH 5-8, and
0.75% Pharmalytes, pH 4-6.5 (Pharmacia Biotech, Inc.). IEF (10
watts, 16 h, 20 °C) was performed in polyacrylamide gels (5%
acrylamide, 0.15% N,N`-bisacrylamide) prepared in the
same buffer, and using 10 mM NaOH and 20 mM phosphoric acid at the cathode and anode, respectively.
Z)OBn using N, N`-dicyclohexylcarbodiimide
and 1-hydroxybenzotriazole were unsuccessful. Yields were extremely low
due to side reactions in which the phosphate instead of the carboxyl
reacted and also to difficulties with isolation of the coupling product
because of its mixed hydrophilic-hydrophobic properties. The successful
synthesis of 3 was achieved by phosphorylation of
Z-Ser-Lys(
Z)OBn (1) with POCl
followed
by deprotection using a palladium catalyst, the structure being
established by
H NMR spectroscopy (Fig. 1). We also
synthesized Ser-Lys to permit comparison of
H NMR spectra.
Ser-Lys has a simple NMR pattern, which enables one to make a
straightforward assignment of the resonances. In particular, the Ser
methine proton appears as a triplet coupled to the Ser
methylene protons, the two
protons having almost identical
chemical shifts (Fig. 1a). Phosphorylation shifts the
signals so that the Ser
proton overlaps with one of the Ser
protons, whereas the other Ser
proton (now with a different
chemical shift) overlaps with the Lys
proton (Fig. 1b). Other regions of the P-Ser-Lys spectrum did
not differ significantly from that of Ser-Lys (not shown). Several
selective decouplings and an additional spectrum at pH 2 were used to
simplify both multiplets so that the couplings with the phosphorus were
observable. For example, Fig. 1c shows a portion of the
spectrum with decoupling of Ser H-
and H-
. The signal of the
second
proton appeared in this case as a doublet at 4.12 ppm with
a 5.5-Hz coupling constant due to coupling with phosphorus through
three chemical bonds.
Figure 1:
H NMR spectra of Ser-Lys
and P-Ser-Lys. a, spectrum of Ser-Lys, showing the region for
H-
of Ser and Lys and H-
of Ser. b, spectrum of
P-Ser-Lys for the corresponding protons. Note that a different region
of the spectrum is shown. c, spectrum of P-Ser-Lys with
decoupling of Ser H-
and H-
.
The synthetic dipeptide was compared by TLE to
the product of tryptic digestion of eIF-4E labeled in vivo with [P]P
(Fig. 2).
Tryptic digestion resulted in all of the radioactivity in the eIF-4E
migrating as a single species (lane1). However, the
peptide migrated more slowly than P-Ser-Lys (lane3).
This was not due to the presence of salt or other contaminants that
might have retarded mobility of one of the peptides; when the two
peptides were mixed before TLE, the result was the same (not shown).
This suggested that Ser-53 was not the major phosphorylation site in
eIF-4E.
Figure 2:
TLE of phosphopeptides and phosphoamino
acids. The anode in each panel is indicated by +, and the cathode
is indicated by -. For peptides, the running time was 50 min, and
for phosphoamino acids it was 90 min. A, autoradiograms of
tryptic peptides derived from either P-eIF-4E (lane1) or from the chymotryptic phosphopeptide (lane2). B, ninhydrin staining of synthetic
P-Ser-Lys. PanelsA and B are from the same
TLE run. C, autoradiogram of phosphoamino acids obtained by
acid hydrolysis of
P-labeled eIF-4E (lane4) or the chymotryptic phosphopeptide (lane5). The dashedcircles represent the
positions of ninhydrin-stained P-Ser (upper) and P-Thr +
P-Tyr (lower), added as internal standards to the samples
before hydrolysis. D, the
P-labeled chymotryptic
peptide was further digested by Asp-N, and the resultant radioactive
peptide was purified by TLE. A portion was run without further
digestion (lane6) or with tryptic digestion (lane7). An autoradiogram is shown. E, same as D except that the order of Asp-N and trypsin
treatments was reversed. Proof that the enzymes were active in panelsD and E came from inclusion of a
synthetic peptide, CQSHADTATKSGSTTKN, in the reaction mixtures.
Cleavage by trypsin and Asp-N was verified by ninhydrin staining of the
TLE plates prior to autoradiography. The peptide was synthesized by
Fmoc (9-fluorenylmethyloxycarbonyl) chemistry at the University of
Kentucky Macromolecular Structure Analysis
Facility.
The tryptic phosphopeptide of eIF-4E is too hydrophilic to
be retained on C or C
columns(12) .
Attempts to purify and analyze the peptide by other means (hydrophilic
interaction chromatography, ion-exchange chromatography) or to generate
a longer, more hydrophobic peptide with other proteases (Glu-C, Asp-N,
subtilisin, Arg-C) were unsuccessful. However, chymotryptic digestion
generated radioactive peptides that could be retained on C
(Fig. 3). Three major peaks of radioactivity were observed (Fig. 3A, fractions1-3). When
each of these was subjected to a second round of chromatography on
C
, the radioactivity co-eluted with a peak of optical
density (Fig. 3, B-D). The peak fractions (bracketed) were then subjected to Edman degradation. Fraction 1 produced the sequence QSHADTATKSGSTTKN,
which exactly matches residues 198-213 of rabbit eIF-4E, as
determined from the cDNA(35) . Based on the specificity of
chymotrypsin, the peptide is likely to be
QSHADTATKSGSTTKNRF
, but low yields in late
Edman cycles prevented exact identification of the C terminus. The
sequence of Fraction 2 was the same as that of
Fraction 1; the difference in elution time from C
is not
understood but may represent C-terminal heterogeneity. Fraction 3
produced the sequence XPQVQSXX, which does not match
eIF-4E but which is similar to residues 124-131 of the
chain of rabbit hemoglobin (TPQVQSAW; apparently this protein was a
contaminant in the eIF-4E preparation).
Figure 3:
Resolution of chymotryptic
phosphopeptides. eIF-4E was labeled by incubation of rabbit
reticulocytes with [P]P
and purified
by affinity chromatography and HPLC on C
. A, the
protein was digested with chymotrypsin, applied to a C
reverse phase column, and both absorbance and radioactivity were
monitored. B, fractions eluting at 20.7-22.2 min (panelA, bracket1) were reapplied
to the same C
column. Fractions eluting at 21.2-21.7
min (bracketed) were subjected to Edman degradation. C, same as B except fraction eluting at
22.2-23.7 min in panelA were subjected to
rechromatography. D, same as B except that the
fractions eluting from 23.7-24.7 min in panelA were used.
Subsequent characterization
was performed on Fraction 1 from this and other preparations of eIF-4E.
Since the chromatogram of Fig. 3A exhibited several
peaks of radioactivity, it was important to show that Fraction 1
contained the major phosphorylation site. Hence, Fraction 1 was
digested with trypsin and subjected to TLE (Fig. 2, lane2). The resultant peptide migrated identically to the
tryptic peptide derived from the entire eIF-4E molecule (lane1).
(
)
Figure 4:
Radioactivity released during Edman
degradation. A preparation of the chymotryptic phosphopeptide similar
to that shown in Fig. 3B (bracketedfractions) was subjected to Edman degradation, except
that in this case, the peptide was covalently attached to the solid
support as described under ``Experimental Procedures.''
Radioactivity released at each cycle was determined by Cerenkov
counting (bars). Superimposed on each bar is the amino acid
sequence of human, rabbit and mouse eIF-4E, beginning at residue 198
(7). At the top (DHA/SER) is given the ratio of
PTH-DHA-DTT to PTH-Ser for cycles 2, 10 and 12 of a noncovalent Edman
degradation of fraction 1 (Fig.
3B).
The
second method relied on the formation of dehydroalanine. Automated
Edman degradation of Ser and Thr normally produces some breakdown
products, notably the DTT adduct of PTH-dehydroalanine (PTH-DHA-DTT)
for Ser and PTH-dehydroaminobutyric acid for Thr. The ratio of the
amount of breakdown product to the amount of PTH-Ser or PTH-Thr has
been found to be elevated in cycles containing P-Ser or
P-Thr(36, 37) , and this effect has been employed as a
nonradioactive means of identifying phosphorylation
sites(38, 39) . Fig. 4presents the ratios of
PTH-DHA-DTT to PTH-Ser for each of the cycles in which Ser was
detected. The ratio was considerably lower in cycles 2 and 10 than in
cycle 12, again suggesting that only Ser-209 is phosphorylated.
QSHADTATKSGSTTKNRF
) was further digested
with Asp-N. This resulted in a shift in mobility of the radiolabel,
indicating that it contains an Asp-N site (data not shown). From the
specificity of Asp-N, this should produce two peptides,
QSHA
and
DTATKSGSTTKNRF
, in which Ser-199 and
Ser-209 are separated. The results indicated that only one of these
peptides was labeled. To determine which peptide this represented, the
sample was recovered from the TLE plate and further incubated without
and with trypsin (Fig. 2, lanes 6 and 7). The
fact that the phosphopeptide changed mobility indicates that
DTATKSGSTTKNRF
, which contains tryptic
sites, was radioactive. The converse experiment was also performed. The
chymotryptic phosphopeptide was digested first with trypsin, which
should produce
QSHADTATK
,
SGSTTK
,
NR
, and
F
. Only one of these peptides was labeled (lane8). Further digestion with Asp-N did not change its
mobility (lane9), indicating that the radioactivity
was in
SGSTTK
. Similar results were
obtained when Glu-C was used in place of Asp-N (not shown). In all
cases, cleavage activity of the enzymes was verified with a synthetic
peptide of the same sequence as the chymotryptic peptide, which was
used as an internal standard with products detected by ninhydrin (see
the legend to Fig. 2). These results demonstrate that
radioactivity was not associated with Ser-199.
S]Met. Exogenous
S-labeled human eIF-4E has been shown to be phosphorylated
in this system(17, 18) . The
S-labeled
eIF-4E (wild-type) contained nearly equal amounts of the basic (pI 6.3)
and acidic (pI 5.9) forms, as indicated by IEF, indicating efficient
phosphorylation (Fig. 5C, lanes2 and 3). The more acidic form was found to co-migrate with in
vivo
P-labeled rabbit eIF-4E (data not shown).
Likewise, in vitro synthesized eIF-4E
(lanes4 and 5) and eIF-4E
(lanes6 and 7) were resolved into
acidic and basic forms, indicating that each could be phosphorylated.
eIF-4E
could not be modified during incubation,
however, resulting in only the basic form (lanes8 and 9). This further supports the assignment of
Ser-209 as the major site of phosphorylation in eIF-4E. Interestingly,
even though the amount of protein synthesized was similiar, the extent
of phosphorylation of eIF-4E
was lower than that of
either eIF-4E or eIF-E
. Since this residue is just two
amino acids from the phosphorylation site, it is possible that the
change to Ala at position 207 affected the accessibility of the
kinase(s) or phosphatase(s) involved.
Figure 5:
Production and analysis of variant eIF-4E
forms by site-directed mutagenesis, cell free translation, and IEF. A, the plasmids pT4EA+ (WT) and pT4ES53A were
sequenced using the antisense oligonucleotide primer, CBP258 (see
``Experimental Procedures''). The region of the gel
surrounding the mutation site is shown. The bases complementary to the
respective dideoxynucleotide triphosphate included in each lane are
shown at the bottom of each panel. B,
plasmids pT4ES207A, pT4EA+, and pT4ES209A were sequenced using the
antisense oligonucleotide primer CBP695
. The GCC codon of
pT4ES207A lies within a GC-rich region, which causes some termination
of the polymerase in all four lanes. This sequence was independently
verified by digestion with NaeI. C, mRNA-dependent
reticulocyte lysate translation systems containing
[
S]Met (1 mCi/ml, 1100 Ci/mmol, ICN Biomedicals,
Costa Mesa, CA) were programmed with either no mRNA (lane1), or in vitro transcribed mRNA from the
plasmids described above encoding wt eIF-4E (lanes2 and 3), eIF-4E
(lanes4 and 5), eIF-4E
(lanes6 and 7), or eIF-4E
. Following incubation
(30 °C, 1.5 h) in the presence or absence of 5 mM 2-aminopurine as indicated, samples were removed for the
separation of
S-labeled eIF-4E isoforms by IEF. The
positions of nonphosphorylated and phosphorylated eIF-4E are indicated
on the left.
Each reaction was conducted in
the presence or absence of 2-aminopurine, a kinase inhibitor. Although
the amount of protein synthesized was greater in the presence of this
compound, the ratio of phosphorylated versus nonphosphorylated
eIF-4E was decreased about 30%. A similar effect was reported by Huang
and Schneider (40) when human embroynic kidney cells were
treated with 2-aminopurine, but that treatment resulted in an
inhibition of translation. Since the drug has been shown to inhibit the
action of two eIF-2 kinases, heme-controlled repressor and protein
kinase R(41) , which, when active, lead to an inhibition of
translation, there may be a mixed effect of this drug on translation
rates: stimulation due to prevention of eIF-2
phosphorylation and
inhibition due to prevention of eIF-4E phosphorylation.
S-labeled eIF-4E was synthesized by cell-free
translation and the reaction mixture was then added to a second
translation reaction containing globin mRNA(4) , and the other
in which
S-labeled eIF-4E was purified on
m
GTP-Sepharose before being added to the second translation
reaction(18) . With either method, wild-type eIF-4E and
eIF-4E
accumulated to the same degree on the 48 S
initiation complex (data not shown). This result is not entirely
unexpected; our previous study showed that phosphorylation of eIF-4E
increased the affinity of the protein for caps(7) , but the
relationship between the cap-binding and ribosome-binding activities of
eIF-4E has not yet been elucidated.
was not
phosphorylated (24) or was phosphorylated more slowly (18) than eIF-4E
in a rabbit reticulocyte
translation system, and (iii) the fact that protein kinase C
phosphorylated eIF-4E
in vitro but was
inactive with eIF-4E
(42) . In our original
study(12) , the citraconylated phosphopeptide was purified by
C
reverse phase HPLC. To correct for background amino
acids, a second run was performed on the same column but with no
injected peptide. Corresponding fractions were collected, and amino
acid analysis was performed on both peptide and blank fractions. The
difference was used to deduce the peptide's composition and
choose the best candidate among the eIF-4E peptides. Apparently there
was sufficient experimental error in this method that we incorrectly
chose peptide 5 (see of Ref. 12) instead of peptide 4,
which has a similar composition. With regard to the other studies,
however, it is not clear why phosphorylation of eIF-4E
would be impaired in a complete translation system or with
purified protein kinase C, unless perhaps the conformation of eIF-4E is
altered by the S53A modification in a way that affects the
accessibility of Ser-209.
compared with the inactivity of
eIF-4E
in a variety of biological systems: the
stimulation of NIH 3T3 and Rat2 cells to grow in soft agar and form
tumors in nude mice(19) , the acceleration of growth of HeLa (21) or HBL100 (8) cells, leading to multiple nuclei and
cell death, the stimulation of DNA synthesis in 3T3 cells(20) ,
the binding of eIF-4E to the 43 S initiation complex(18) , the
specific stimulation of translation of mRNAs containing high secondary
structure (22, 23), and the relief of inhibition of protein synthesis
in early sea urchin embryo extracts(24) . These studies were
conducted with cDNAs encoding both human (18) and mouse (19) eIF-4E, independently subjected to site-directed
mutagenesis to convert Ser-53 to Ala-53. Also, these effects were seen
with both integrating (19, 8) and episomal (21) vectors. Thus, although Ser-53 is not the major
phosphorylation site, this position appears to be important for the
proper functioning of the protein.
-adrenergic
receptor(43) .
,X
)(S/T)(X
,R/K
)
(44). Furthermore, the pseudosubstrate sites of all known isozymes of
protein kinase C, with the exception of protein kinase C
, contain
Gly just before the phosphorylation site, which would correspond to
Gly-208 in eIF-4E. Indeed, protein kinase C has been found to
phosphorylate eIF-4E in vitro within the same tryptic
phosphopeptide as is isolated from in vivo phosphorylated
eIF-4E(45, 46) . In vivo, stimulation of
reticulocytes (46) and 3T3 L1 (14) cells with phorbol
esters, which activate protein kinase C, leads to increased
phosphorylation of eIF-4E and correlates with an increase in
translational rate. In contrast, long term treatment of B lymphocytes
with phorbol ester failed to demonstrate a reduction in the rate eIF-4E
phosphorylation(47) . This led the authors to question the
involvement of protein kinase C. However, it is now known that long
term phorbol ester treatment does not result in down-regulation of all
protein kinase C isoforms (for review, see Ref. 48). Hence, protein
kinase C is a likely candidate for the physiological kinase of eIF-4E.
column.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.