From the
Insulin-stimulated protamine kinase (cPK) and protein kinase C
(PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E
(eIF-4E) on serine and threonine residues located on an identical
tryptic fragment as judged by two-dimensional phosphopeptide mapping.
With cPK and PKC, the apparent K
Evidence has accumulated that the capped-mRNA binding protein,
eukaryotic protein synthesis initiation factor 4E (eIF-4E),
We have purified
to apparent homogeneity an insulin-stimulated cytosolic Protamine
Kinase (cPK) which was differentiated from other protein kinases
including known PKC isoforms based on its catalytic, regulatory,
chromatographic, and immunological
properties(20, 21, 22) . In addition, in a
preliminary report we showed that up to 1 mol of phosphoryl groups was
incorporated per mol of purified preparations of eIF-4E following
incubation with cPK (23). By contrast, all the other insulin-stimulated
protein kinases examined displayed little or no activity with
eIF-4E(23, 24, 25) , raising the possibility
that cPK may contribute to the insulin-stimulated phosphorylation of
eIF-4E(23) . However, neither the functional significance nor
location of the cPK-catalyzed phosphorylation of eIF-4E were examined.
In this paper, we present evidence that cPK and PKC phosphorylate
eIF-4E at an identical tryptic peptide. By mutational analysis, we show
that cPK phosphorylates eIF-4E at Ser
Purification of eIF-4E and eIF-4 to near
homogeneity from bovine kidney extracts was as
described(23, 33) , except that gel permeation
chromatography on Superose 12 was performed instead of ion-exchange
chromatography on Mono Q. Following SDS-PAGE, fractions containing
eIF-4E were identified by Coomassie Blue staining, pooled, and
concentrated and washed with 50 mM Tris-chloride, pH 7.0,
containing 10% glycerol, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 14
mM
By
contrast to cPK, PKC displayed an apparent K
The results presented in Fig. 3A show that cPK phosphorylated eIF-4E
Interestingly,
relative to wild type eIF-4E, threonine phosphorylation of
eIF-4E
In
contrast to cPK, PKC phosphorylated eIF-4E
Addition of bovine kidney eIF-4E to the
m
Using a two-dimensional phosphopeptide mapping procedure
identical to the one employed in this study, evidence was obtained
earlier that PKC modifies eIF-4E at the serine residue phosphorylated
in insulin-treated 3T3-L1 cells(1) . The results presented
herein indicate that cPK phosphorylates eIF-4E at the serine residue
modified by PKC ( Fig. 1and Fig. 3). In addition, the
mutational analyses described in this paper indicate that this residue
is Ser
The results presented also indicate that phosphorylation of
Ser
Earlier studies
suggest that phosphorylation may alter the affinity of eIF-4E for
capped mRNA(44, 45) . Alternatively, phosphorylation may
trigger and/or stabilize the association of eIF-4E with the other
subunits of eIF-4(4, 40) . The phosphorylation site
mutant cDNAs described in this paper should facilitate studies on the
functional significance of the insulin-stimulated phosphorylation of
eIF-4E and provide further information on the role of cPK in this
regulation.
We are most grateful to Dr. Rosemary Jagus for the
human eIF-4E cDNA. We also thank Dr. Leonard S. Jefferson for helpful
comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for
eIF-4E was about 1.2 and 50 µM, respectively. Relative to
recombinant human eIF-4E, cPK exhibited about 100% and
5% activity
with eIF-4E
and eIF-4E
, respectively,
and eIF-4E
was phosphorylated exclusively on
threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin
synthesis in m
GTP-Sepharose-treated reticulocyte lysates.
In contrast, following incubation with cPK, these eIF-4E preparations
stimulated globin synthesis up to 6-fold. Compared to the
dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte
lysates and highly purified protein phosphatase 2A exhibited marked
preference for the cPK-modified threonine. The results indicate that
cPK phosphorylates eIF-4E on Ser
and Thr
,
that the hydroxyl group or phosphorylation of Thr
is
necessary for cPK to act on Ser
, and that Ser
phosphorylation activates reticulocyte globin synthesis. The
results suggest that cPK could contribute to the insulin-stimulated
phosphorylation of eIF-4E, but that protein phosphatase 2A may confer
the site specificity of this response.
(
)undergoes phosphorylation on serines coincident with
the stimulation of protein synthesis in response to insulin (1, 2, 3) and numerous other extracellular
stimuli (4-14). Conversely, dephosphorylation of eIF-4E
correlates with decreased rates of protein synthesis (e.g. 15,
16). However, to date, a direct effect of eIF-4E phosphorylation on
protein synthesis has not been established. In addition, the protein
kinases and phosphatases responsible for controlling this
phosphorylation have not been identified. Nonetheless, two-dimensional
phosphopeptide mapping and phosphoamino acid analysis indicated that
PKC phosphorylates eIF-4E at the site(s) modified in response to
insulin(1) . The exact location of the modified site(s) was not
determined. However, based on an earlier report(17) , which
appears to be in error,
(
)it was suggested that
eIF-4E may be phosphorylated on Ser
in response to
insulin(1) . In addition, because insulin-stimulated
phosphorylation of eIF-4E was abolished following prolonged incubation
with phorbol esters(1) , a treatment which inactivated
PKC(1) , it was suggested that this kinase may contribute to the
insulin-stimulated phosphorylation of eIF-4E(1) . However, in
cells, eIF-4E is present largely in the soluble fraction (18), and PKC
appeared to be a reasonable eIF-4E kinase only when eIF-4E was purified
from ribosomes(19) . From this fraction, eIF-4E was isolated as
part of a complex termed eIF-4 which also contains eIF-4
of
apparent M
46,000 and eIF-4
of apparent M
220,000(18) .
and
Thr
, that phosphorylation at Ser
activates
eIF-4E, and that PP2A, a major cytoplasmic protein serine threonine
phosphatase(26) , preferentially dephosphorylates
Thr
. The results are consistent with the idea that cPK
contributes to the insulin-stimulated phosphorylation of eIF-4E and
concomitant activation of protein synthesis, but indicate an important
role for PP2A in this insulin response.
Materials
N-Tosyl-L-phenylalanine
chloromethyl ketone-treated trypsin and
Na-p-Tosyl-L-lysine chloromethyl ketone-treated
-chymotrypsin were from Sigma. DH10B Escherichia coli containing cDNA encoding human eIF-4E in expression vector pMW19 (27) was provided by Dr. Rosemary Jagus, Center Marine
Biotechnology, University of Maryland. TAQuence DNA sequencing kit was
from United States Biochemicals. Rat brain PKC and
m
GTP-Sepharose were from Calbiochem and Pharmacia/LKB,
respectively. Globin mRNA was from Life Technologies, Inc.
Haemin-containing nuclease-treated rabbit reticulocyte lysates were
from Promega. [
S]Methionine (1100 Ci/mmol) was
from Amersham Corp. Sources of other materials are given
elsewhere(28, 29, 30) .
Enzyme Preparations
Myelin basic
protein(29) , PP2A(28) , and cPK (20) were purified to apparent homogeneity, and the activities
of cPK(20) , PKC(20) , and PP2A
(29) were determined as described. Protein was determined
as described(31) . Homogeneity of enzyme preparations was
determined by SDS-PAGE(32) . Protein bands were detected with
Coomassie Brilliant Blue.
-mercaptoethanol, using Centricon-10
microconcentrators (Amicon). The preparations were aliquoted and stored
at -70 °C.
Phosphorylation of eIF-4E
The incubations (0.05
ml) were performed at 30 °C in 50 mM Tris-chloride, pH
7.0, containing 10% glycerol, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, 14 mM -mercaptoethanol, 0.01 units of cPK, 2 mM MgCl
, 0.2 mM [
-
P]ATP (1000 cpm/pmol), and 4 µg
of eIF-4E. Incubations with PKC also contained 20 µg/ml
phosphatidylserine and 0.25 mM CaCl
. Reactions
were initiated with ATP and MgCl
. Control incubations were
performed in the absence of cPK, PKC, or eIF-4E. Reactions were
terminated with sample buffer(32) , and the mixtures were heated
at 100 °C for 5 min. Following SDS-PAGE, proteins were
electrophoretically transferred onto Immobilon
-P transfer
membranes. Membrane strips were stained with Ponceau S, washed
extensively with H
O, dried, and exposed to Kodak X-Omat
AR-5 film. In some experiments, radiolabeled bands identified by
autoradiography were cut out from membrane strips, immersed in
scintillant, and counted.
Peptide Mapping and Phosphoamino Acid
Analysis
Two-dimensional phosphopeptide mapping was performed
essentially as described(1) . Briefly, P-labeled
eIF-4E (4 µg) prepared by incubation with cPK or PKC was subjected
to SDS-PAGE and then electrophoretically transferred onto
Immobilon
-P membranes. Membrane strips were stained with
Ponceau S, washed extensively with H
O, and dried. Bands
corresponding to eIF-4E were excised and incubated at 37 °C for 16
h with trypsin (50:1 w/w) in 0.05 ml of 0.2 M ammonium
bicarbonate, pH 8.4. An additional aliquot of trypsin was added, and
the mixture was incubated for a further 6 h. About 80-90% of the
P-label was released following incubation with trypsin.
The solution was then dried under vacuum, redissolved in a solution
containing 10% pyridine and 0.4% acetic acid, spotted onto a thin layer
chromatography plate, and subjected to electrophoresis in the same
solution at 600 V for 1.5 h, and then to ascending chromatography in a
solution containing 60% n-butanol and 20% acetic acid. After
drying, the plates were exposed to x-ray film. Phosphoamino acid
analysis was performed at pH 3.5 as described(33) .
Phosphoserine, phosphothreonine, and phosphotyrosine standards were
identified by staining with ninhydrin.
Construction and Expression of eIF-4E
Expression vector pMW19 containing cDNA
encoding human eIF-4E (27) was used as template to change to Ala
the codons for Ser and
eIF-4E
and Thr
by inverse
polymerase chain reaction site-directed mutagenesis(35) .
Mutagenic primers used to independently introduce these changes in the
sense strand of eIF-4E cDNA were pGCGGCGCTACCACTAAAAATAGG
(Ala
) and pGCGGCTCCGCTACTAAAAATAGG (Ala
)
(changed nucleotides are underlined.) A single primer,
pTCTTAGTAGCTGTGTCTGCGTGG, was used for antisense strand amplification.
The reaction mixtures (0.1 ml) contained 100 pmol each of sense and
antisense primer, 10 fmol of pMW19 DNA in 20 mM Tris-chloride,
pH 8.7, 10 mM KCl, 10 mM ammonium sulfate, 2 mM MgCl
, 0.1% Triton X-100, 0.1 mg/ml bovine serum
albumin, 200 µM of each dNTP, and 2.5 units of Pfu DNA polymerase (Strategene). The mixtures were subjected to an
initial 3 min denaturation at 94 °C, followed by 30 cycles of
denaturation at 94 °C (1 min), annealing at 50 °C (1 min), and
extension at 72 °C (12 min). Reaction products with relative
electrophoretic motility of about 6.2 kilobase pairs were excised from
1.8% low melting agarose gels and recovered using glass milk (GeneClean
II, BIO 101). Approximately 200 ng of this DNA was incubated overnight
at 12 °C in 0.01 ml of 20 mM Tris-chloride, pH 7.6,
containing, 1 mM ATP, 5 mM MgCl
, 5
mM dithiothreitol, 50 µg/ml bovine serum albumin, and 4
units of T4 DNA ligase (New England Biolabs) as
recommended(36) . The mixture was then used to transform One
Shot
E. coli (Invitrogen), and plasmids were
isolated and analyzed by restriction mapping and by sequencing both
cDNA strands to verify for mutations and the integrity of the remaining
coding region. Plasmids containing wild type and mutant cDNA were then
separately used to transform BL21(DE3)pLysS E. coli (Novagen),
and expression was induced as described(27) . Purification of
recombinant wild type and mutant proteins was also as
described(27) , except that, chromatography on
m
GTP-Sepharose was omitted because >90% of the eIF-4E
preparations did not bind to this resin and are therefore considered
inactive in capped mRNA binding.
Reticulocyte Protein Synthesis
Reticulocyte
lysates (0.1 ml of 90 mg/ml) were subjected to chromatography on
a 1-ml column of m
GTP-Sepharose equilibrated and developed
in 50 mM HEPES-KOH, pH 7.6, containing 0.1 M KCl, 10%
glycerol, 0.1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 14 mM
-mercaptoethanol. Fractions
(0.2 ml) were collected and those exhibiting the highest absorbance at
280 nm employed. Protein synthesis in these fractions was then measured
with globin mRNA and [
S]methionine essentially
as recommended by the manufacturer (Promega). Reactions contained 0.015
ml of the m
GTP-Sepharose-treated lysates, 0.004 ml of
H
O, 0.001 ml of RNasin inhibitor (40 units), 0.001 ml of 1
mM amino acid mixture lacking methionine, 0.001 ml of a 25
µg/ml solution of globin mRNA, 0.002 ml of
[
S]methionine (final concentration
500
µCi/ml), and a 0.005-ml aliquot of highly purified bovine kidney
eIF-4E as indicated. After 5 min at 30 °C, 0.25 ml of a solution
containing 1 N NaOH and 2% H
O
was
added. After 10 min at 37 °C, 1 ml of 25% trichloroacetic acid
(w/v) was added, and the mixture was centrifuged at 12,000
g
for 5 min in a microcentrifuge. The supernatant was discarded, and
pellets were washed five times with 1 ml of 10% trichloroacetic. To the
final pellets was added 1 ml of scintillation fluid, and the
radioactivity was determined. Control incubations were performed in the
absence of globin mRNA. Chromatography on m
GTP-Sepharose
reduced the rate of reticulocyte lysate globin synthesis by about
7.2-fold.
Phosphopeptide Mapping
As a first step to
examine the sites modified on eIF-4E by cPK, and to relate the results
to insulin action, we examined the tryptic phosphopeptides derived from P-labeled eIF-4E prepared by incubation with cPK or PKC.
The results presented in Fig. 1show that a single co-migrating
tryptic phosphopeptide was obtained from recombinant human eIF-4E
incubated with either cPK or PKC. Identical results also were obtained
with bovine kidney eIF-4E, and when eIF-4E was isolated as part of
eIF-4 from this tissue source (not shown). Phosphoamino acid analysis
showed that both cPK (Fig. 2) and PKC (Fig. 3)
phosphorylated eIF-4E on serines and threonines. Assuming that a single
serine and threonine residue was modified, we estimate that cPK
incorporated up to 0.45 and 0.55 mol, and PKC incorporated up to 0.04
and 0.06 mol of phosphoryl groups on serine and threonine residues,
respectively. Little or no difference was detected in the rates of
phosphorylation of these residues by cPK (Fig. 2) or PKC (not
shown).
Figure 1:
Phosphopeptide mapping. Recombinant
human eIF-4E was incubated for 30 min with cPK (1 unit/ml) or PKC (22
units/ml) in the presence of [-
P]ATP, and
the mixtures were electrophoretically transferred onto
Immobilon
-P membranes as described under
``Experimental Procedures.'' Membrane strips were exposed to
x-ray film (10 min with cPK, 3 days with PKC), and bands corresponding
to eIF-4E were cut out, treated with trypsin, and two-dimensional
phosphopeptide mapping was performed as described under
``Experimental Procedures.'' The figure shows the
autoradiograms of the dried thin layer chromatography plates indicating
the pattern derived from the eIF-4E incubations with cPK (panel
A), PKC (panel B), and a mixture from each of the
incubations (panel C). The arrows denote the position
the samples were applied to the plates. The radioactivity applied to
the plates was panel A, 6,000 cpm, panel B, 3,400
cpm, and panel C, 5,200 cpm made up of 2,600 cpm from the
incubation with cPK and 2,600 cpm from the incubation with
PKC.
Figure 2:
Phosphorylation of eIF-4E by cPK.
Recombinant human eIF-4E was incubated with cPK in the presence of
[-
P]ATP, and, at the indicated times,
reactions were terminated, and electrophoretically transferred onto
Immobilon
-P membranes as described under
``Experimental Procedures.'' Bands corresponding to eIF-4E
were cut out, incubated with trypsin, and phosphoamino acid analysis
was performed as described under ``Experimental Procedures.''
After drying, the thin layer plate was exposed to x-ray film. The
figure shows an autoradiogram of the dried plate. The position of
phosphoserine (S), phosphothreonine (T), and
phosphotyrosine (Y) standards is indicated. Control
incubations from which eIF-4E or cPK was omitted displayed little or no
phosphorylation (not shown).
Figure 3:
Phosphorylation site analysis of eIF-4E.
Recombinant human eIF-4E (lane 1), eIF-4E (lane 2), and eIF-4E
(lane 3)
each at 4 µg were incubated with 1 unit/ml cPK (panels A and C) or 25 units/ml PKC (panels B and D) in the presence of [
-
P]ATP for
30 min, and the mixtures were then electrophoretically transferred onto
Immobilon
-P membranes as described under
``Experimental Procedures.'' The membrane strips were exposed
for 10 min (panel A) and 3 days (panel B) to x-ray
film. An autoradiogram of each membrane is shown. The position of
eIF-4E is denoted by the arrow. Control incubations from which
cPK or PKC were omitted displayed little or no phosphorylation (not
shown). Control incubations from which eIF-4E, eIF-4E
,
and eIF-4E
were omitted from the reactions with cPK
showed little or no phosphorylation. In panel B, the band
corresponding to autophosphorylated PKC is denoted by an arrow. Autophosphorylation of PKC was also noted in
incubations from which eIF-4E, eIF-4E
, and
eIF-4E
were omitted were omitted (not shown). In panels C and D, the eIF-4E bands were subjected to
phosphoamino acid analysis after trypsinolysis as described under
``Experimental Procedures.'' The autoradiograms of the dried
thin layer plates are shown. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards is indicated.
Purified preparations of cPK displayed an apparent K of about 1.4 and 1.2 µM
with recombinant human and bovine kidney eIF-4E, respectively.
Similarly, with the eIF-4E component of bovine kidney eIF-4, cPK
exhibited an apparent K
of about 1.0
µM, and up to 1 mol of phosphoryl groups was incorporated
per mol of the eIF-4E component of eIF-4 following incubation with cPK
indicating that eIF-4
and eIF-4
, the other subunits of eIF-4,
may have little or no effect on cPK activity with eIF-4E.
of about 40 and 52 µM with recombinant human
and bovine kidney eIF-4E, respectively. In addition, consistent with
earlier observations(19, 23, 37, 38) ,
PKC incorporated up to 0.1 mol of phosphoryl groups/mol of these eIF-4E
preparations. Highly purified PKC also displayed an apparent K
of about 29 µM with the
eIF-4E component of bovine kidney eIF-4. With eIF-4 as substrate, PKC
incorporated up to 0.3 mol of phosphoryl groups/mol of the eIF-4E
component. These observations appear to be at variance with an earlier
study which indicated that 1 mol of phosphoryl groups could be
incorporated per mol of the eIF-4E component of eIF-4(19) . The
reason for this apparent discrepancy is unclear.
Identification of Target Sites
Because there was
little or no difference in the tryptic phosphopeptide patterns (Fig. 1) and kinetics of phosphorylation, the results indicated
that information on the sites modified by cPK on native eIF-4E could be
derived from the recombinant eIF-4E preparations even though these
preparations appeared to be largely inactive in capped mRNA binding. In
addition, because only one tryptic phosphopeptide was derived from the
eIF-4E preparations incubated with either cPK or PKC, the results
suggested that the modified serines and threonines (Fig. 1) were
located on the same tryptic fragment. Examination of the deduced amino
acid sequence suggested that this tryptic fragment may be SGSTTK
located at the C terminus of eIF-4E, residues 207-212. To test
this idea, eIF-4E cDNA in which Ala replaced Ser or
Thr
(SGSTTK, underlined residues) was prepared, and each
cDNA was expressed separately in BL21(DE3)pLysS E. coli. Mutant proteins were then purified as described(27) , and
the activities of cPK and PKC with these proteins examined and compared
to wild type eIF-4E.
to an extent
similar to wild type eIF-4E, but that, relative to these preparations,
the kinase exhibited
5% activity with eIF-4E
. The
purified preparations of cPK displayed an apparent K
of about 1.2 µM with eIF-4E
,
also similar to that observed with wild type eIF-4E. However, in
contrast to wild type eIF-4E, phosphoamino acid analysis showed that
cPK phosphorylated eIF-4E
exclusively on threonines (Fig. 3C). Together, these results indicate that
Ser
and Thr
are target sites for cPK. They
also indicate that either the hydroxyl group or phosphorylation of
Thr
may be required for cPK to phosphorylate
Ser
. Arguing against the latter possibility is that there
were no discernable differences in the rates that cPK phosphorylated
these residues on wild type eIF-4E (Fig. 2).
was enhanced by about 1.8-fold (Fig. 3C). Because with wild type eIF-4E, cPK
incorporated up to 0.45 mol and 0.55 mol of phosphoryl groups/mol of
the modified serine and threonine residues, respectively, these
observations suggest that phosphorylation of eIF-4E on Ser
may inhibit Thr
phosphorylation and vice
versa. However, the exact location of the threonine(s)
modified by cPK on eIF-4E
remains to be determined.
Consistent with the possibility that cPK phosphorylated
eIF-4E
in the tryptic fragment SGATTK containing
Thr
(underlined residue) is that two-dimensional peptide
mapping of
P-labeled eIF-4E
showed a
single tryptic phosphopeptide which comigrated with the one derived
from
P-labeled wild type eIF-4E (not shown, cf. Fig. 1). However, in this tryptic fragment, cPK could also have
phosphorylated eIF-4E
on Thr
.
and
eIF-4E
to approximately 50% of the level observed with
wild type eIF-4E (Fig. 3B). The highly purified PKC
preparations displayed an apparent K
of
about 70 and 80 µM with eIF-4E
and
eIF-4E
, respectively. These values are about 1.8-fold
higher than those displayed by PKC with wild type eIF-4E. Phosphoamino
acid analysis showed that PKC phosphorylated eIF-4E
on
threonines and eIF-4E
on serines and threonines (Fig. 3D). By contrast to cPK (Fig. 3C),
threonine phosphorylation of eIF-4E
by PKC was
equivalent to that observed with wild type eIF-4E (Fig. 3D). These results indicate that, like cPK, PKC
acts on Ser
, but that, unlike cPK, PKC phosphorylated a
threonine other than Thr
. The location of this threonine,
which does not appear to affect PKC activity with Ser
,
remains to be determined.
Effect of eIF-4E Phosphorylation on Protein
Synthesis
As a first step to assess the functional consequence
of the cPK-catalyzed phosphorylation of eIF-4E, bovine kidney eIF-4E
was incubated with and without cPK for various times. An aliquot of the
incubations was then added to reticulocyte lysates treated with
mGTP-Sepharose to deplete them of endogenous eIF-4E, and
the initial rate of globin mRNA translation was measured by the
incorporation in 5 min of [
S]methionine into
protein as described under ``Experimental Procedures.''
GTP-Sepharose-treated lysates enhanced the rate of globin
synthesis by about 1.8-fold (not shown). In contrast, following
incubation with cPK, these eIF-4E preparations enhanced the rate of
globin synthesis up to 6-fold (Fig. 4A). Control
incubations, from which eIF-4E was omitted, showed that cPK (0.075
units) displayed little effect, if any, on the initial rate of globin
synthesis (not shown). However, at 30-fold higher concentrations of
cPK, globin synthesis was markedly inhibited (not shown). The molecular
basis of this cPK effect remains to be determined.
Figure 4:
Effect of eIF-4E on protein synthesis. In panel A, highly purified bovine kidney eIF-4E was incubated
with 0.2 mM ATP instead of [-
P]ATP
in the absence (
) or presence (
) of cPK (30 units/ml) as
described under ``Experimental Procedures.'' At the indicated
times, EDTA to a final concentration of 4 mM was added. A
0.0025-ml aliquot of the mixtures (5 µg of eIF-4E) was then added
to m
GTP-Sepharose-treated reticulocyte lysates, and globin
synthesis was measured after 5 min as described under
``Experimental Procedures.'' In panel B, parallel
incubations of eIF-4E with cPK were performed in the presence of 0.2
mM [
-
P]ATP. After 30 min at 30
°C, a 0.005-ml aliquot of 30 mM EDTA was added, and a
0.005-ml sample of the mixture was then incubated with the reticulocyte
lysates as described above. At the indicated times, reactions were
terminated, and phosphoamino acid analysis was performed after
electrophoretic transfer onto Immobilon
P membrane strips
and trypsinolysis as described under ``Experimental
Procedures.''
P-Labeled serine (
) and
P-labeled threonine (
) residues were identified by
autoradiography and comigration with ninhydrin-staining phosphoserine
and phosphothreonine standards, and then scraped from the thin layer
chromatography plate into a microcentrifuge tube. The radioactivity was
determined after addition of 1 ml of scintillant. The figure shows the
percent of
P-label remaining at the indicated time
points.
The
dephosphorylation of eIF-4E by reticulocyte lysates was then examined.
Phosphoamino acid analysis showed that, by contrast to the cPK-modified
serine (t 15 min), the cPK-modified threonine was
dephosphorylated rapidly (t
1 min) by the lysates (Fig. 4B). Because the rate of reticulocyte lysate
globin synthesis was linear during the 5-min period of the measurements
in the absence or presence of added eIF-4E, eIF-4E that had been
incubated with cPK up to 30 min, or 0.075 units of cPK (not shown), the
results indicate that phosphorylation of eIF-4E on Ser
activated globin synthesis, and that Thr
phosphorylation may have had little, or no, effect.
Dephosphorylation by PP2A
Earlier studies
indicated that PP2A could dephosphorylate
eIF-4E(38, 39) . Therefore, we examined whether this
phosphatase exhibited preference for the cPK-modified serine and
threonine residues. The results presented in Fig. 5show that
PP2A preferentially dephosphorylated the cPK-modified threonine. The
rate of this dephosphorylation was about 60-fold higher than the rate
of Ser dephosphorylation. However, whether PP2A is a
physiologically relevant eIF-4E Thr
phosphatase remains
to be determined. In this connection, it is pertinent that only in
cells incubated with okadaic acid has the phosphorylation of eIF-4E on
both serines and threonines been observed(40) . However, okadaic
acid is not only a potent inhibitor of PP2A(26) , but also of
protein phosphatase 1 (26) and protein phosphatase
X(41) . Whether these protein phosphatases, and/or the recently
described eIF-4E-binding protein, PHAS-1(42) , also termed
4E-BP1(43) , regulate the phosphorylation of eIF-4E remains to
be determined. Phosphorylation of PHAS-1 by mitogen-activated protein
kinase in response to insulin releases eIF-4E from the
eIF-4E
PHAS-1 complex present in the soluble fraction, and
increases the levels of eIF-4E available to participate in protein
synthesis(42, 43) .
Figure 5:
Dephosphorylation of eIF-4E by PP2A.
Recombinant human eIF-4E was incubated with cPK (1 unit/ml) in the
presence of [-
P]ATP as described under
``Experimental Procedures.'' After 60 min, a 0.005-ml aliquot
was incubated at 30 °C in a final volume of 0.025 ml in the
presence of PP2A
(50 units/ml) in 50 mM
Tris-chloride, pH 7.0, containing 10% glycerol, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, and
14 mM
-mercaptoethanol. At the indicated times, reactions
were stopped, and then electrophoretically transferred onto
Immobilon
-P membranes as described under
``Experimental Procedures.'' Panel A shows an
autoradiogram of the membrane strips. The arrow denotes the
bands corresponding to eIF-4E. These bands were cut out, incubated with
trypsin, and phosphoamino acid analysis was performed as described
under ``Experimental Procedures.'' Panel B shows an
autoradiogram of the dried plate. The position of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards is indicated. Control incubations from which
PP2A
was omitted showed little or no dephosphorylation of
the eIF-4E preparations (not shown).
(Fig. 3). Thus, eIF-4E appears to be
phosphorylated on Ser
in insulin-treated cells. The
results indicate that cPK, an insulin-stimulated enzyme(21) ,
may contribute directly to this phosphorylation, but that PKC is
unlikely to do so because PKC is a relatively poor eIF-4E kinase.
enhances the activity of eIF-4E in globin synthesis
in reticulocyte lysates depleted of endogenous eIF-4E (Fig. 4A). They also establish that, whereas cPK
phosphorylates serine and threonine residues on eIF-4E at equivalent
rates (Fig. 2), reticulocyte lysates (Fig. 4B) and purified
PP2A (Fig. 5) dephosphorylate the modified threonine
preferentially. These observations suggest that PP2A may play an
important role in controlling the site specificity of the
insulin-stimulated phosphorylation of eIF-4E.
, protein phosphatase 2A
;
PAGE, polyacrylamide gel electrophoresis; cpm, counts/min.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.