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Molecular and Cellular Biology, March 1999, p. 1871-1880, Vol. 19, No. 3
Fred Hutchinson Cancer Research Center,
Seattle, Washington 981091; Medical
Scientist Training Program, University of Washington, Seattle,
Washington 981952; and Department of
Cellular and Molecular Physiology, The Pennsylvania State University
College of Medicine, Hershey Medical Center, Hershey, Pennsylvania
170333
Received 13 August 1998/Returned for modification 15 September
1998/Accepted 25 November 1998
Eukaryotic translation initiation factor 4E (eIF4E) binds to the
mRNA 5' cap and brings the mRNA into a complex with other protein
synthesis initiation factors and ribosomes. The activity of mammalian
eIF4E is important for the translation of capped mRNAs and is thought
to be regulated by two mechanisms. First, eIF4E is sequestered by
binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce
the release of eIF4E by stimulating the phosphorylation of 4EBP1.
Second, mitogens and stresses induce the phosphorylation of eIF4E at
Ser 209, increasing the affinity of eIF4E for capped mRNA and for an
associated scaffolding protein, eIF4G. We previously showed that a
mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in
vitro at the physiological site. Here we show that Mnk1 regulates eIF4E
phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies
with eIF4G and eIF4E. We identified activating phosphorylation sites in
Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E
phosphorylation, while expression of activated Mnk1 increases basal
eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive
phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests
that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar
kinase in cells and is independent of other mitogenic signals that
release eIF4E from 4EBP1.
Mitogens stimulate protein and RNA
synthesis (56, 65). The increase in protein synthesis is
partly due to increased initiation on preexisting mRNAs, with the
result that those mRNAs are recruited into larger polysomes. In
addition to an increase in basal translation, specific mRNAs are
preferentially upregulated, suggesting that mitogenic signal
transduction pathways impinge on the components of the translation
machinery that interact with the mRNA.
mRNAs are brought to the ribosome by eukaryotic initiation factor
(eIF4F) (for reviews, see references 58, 60, and
61). eIF4F is a multiprotein complex formed from
25-, 46- and 220-kDa subunits, called eIF4E, eIF4A, and eIF4G,
respectively. eIF4E, also known as cap-binding protein, is responsible
for binding the 5'-terminal 7-methyl-GTP (m7GTP) cap found
on all eukaryotic mRNAs. eIF4A is a subunit of an RNA helicase that
seems to unwind secondary structure in the mRNA. eIF4G is the
scaffolding subunit, to which the other subunits bind. It also has a
binding site for eIF3, which links the eIF4F-mRNA complex to the 40S
ribosomal subunit. In yeast, eIF4G has an additional functional region,
to which the poly(A)-binding protein and the 3' end of the mRNA bind
(64). Besides serving as a passive scaffold, eIF4G plays a
regulatory role, stimulating the binding of capped mRNA to eIF4E
(24). Thus, the eIF4F complex promotes interactions between
the 5' end of the mRNA, the ribosome, and an RNA helicase.
As the main mRNA-binding component of the translation machinery, the
eIF4F complex has the potential to distinguish between mRNAs for
differential translation in mitogen-treated cells. The eIF4E subunit is
thought to be the main regulatory component since it is present in
limiting molar amounts (11, 26, 53), and eIF4E availability
in quiescent cells is further restricted by eIF4E-binding proteins,
including 4EBP1 or PHAS-I (61). 4EBPs prevent eIF4E binding
to eIF4G without altering mRNA binding to eIF4E (23, 44,
52). Mitogenic stimuli induce the phosphorylation of 4EBPs and
the release of eIF4E, allowing eIF4E to associate with eIF4G and
participate in translation. In addition, mitogens stimulate the
phosphorylation of ribosomal protein S6 and several translation
initiation factors, including eIF4E. Phosphorylation of 40S ribosomal
protein S6 specifically stimulates the translation of a class of mRNAs
that have pyrimidine-rich 5' ends (29). However, cells
lacking the S6 protein kinase still exhibit mitogen-induced increases
in basal translation (32), indicating that other mechanisms, such as 4EBP1 and eIF4E phosphorylation, also mediate mitogenic stimulation of translation.
Several lines of evidence suggest that phosphorylation of eIF4E
stimulates translation initiation. Mitogen-enhanced eIF4E phosphorylation usually correlates with increased protein synthesis (36), and phosphorylation increases the binding of eIF4E to capped mRNA and to eIF4G in vitro (5, 39, 48). The location of Ser 209 adjacent to the cap-binding pocket is consistent with an
effect of phosphorylation on mRNA binding (45, 46).
Significantly, overexpression of wild-type eIF4E, but not of a
substitution mutant that is not phosphorylated, leads to malignant
transformation and high rates of protein synthesis (10, 41).
Studies of cells transformed by eIF4E overexpression show an increased
ability to translate mRNAs with increased cap-proximal secondary
structure (37, 55, 59), consistent with increased binding of
the eIF4F complex and its associated helicase activity. While some
other reports indicate that changes in eIF4E phosphorylation do not invariably correlate with increased translation (34, 49, 51, 54,
57, 70), it seems likely that eIF4E phosphorylation modulates
translation initiation in cells.
Different signal transduction pathways control the phosphorylation of
4EBP1, S6, and eIF4E. In fibroblasts, the phosphorylation of 4EBP1 and
S6 occurs by an extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase-independent signaling pathway that is sensitive to a specific inhibitor, rapamycin (4, 6, 18, 25, 68, 69). In contrast, mitogen-stimulated eIF4E
phosphorylation occurs via a rapamycin-insensitive, Ras- and ERK/MAP
kinase-dependent pathway (14-16). This means that
phosphorylation of eIF4E is independent of phosphorylation of 4EBPs.
eIF4E is also phosphorylated in response to stresses, including
anisomycin and hypertonicity, dependent on the p38 MAP kinase (49,
51, 70). This suggests that the MAP kinases ERK and p38, or a
protein kinase activated by ERK or p38, may phosphorylate eIF4E in
cells. However, neither ERK nor p38 is a candidate to phosphorylate
eIF4E directly, because the residue phosphorylated, Ser 209 (13,
31), lacks proline residues required for MAP kinase recognition
(1, 7). Therefore, eIF4E is more likely to be phosphorylated
by one or more MAP kinase-dependent protein kinases.
A number of MAP kinase-dependent kinases are now known, and of these,
Mnk1, MAPKAPK-3 (3pK), and Msk1 are activated by both ERK and p38
(9, 17, 43, 71). Direct in vitro phosphorylation assays
suggest that MAPKAPK-3 and Mnk1 can both phosphorylate eIF4E directly,
while certain other MAP kinase-dependent protein kinases, including Rsk
and MAPKAPK-2, cannot (51, 52a, 71). Under a variety of
stimulation and inhibitor conditions, the in vivo phosphorylation of
eIF4E correlates with the activity of Mnk1 (70). This
correlative evidence suggests that Mnk1, or a similarly activated
protein kinase, phosphorylates eIF4E in cells. In view of the potential
importance of eIF4E phosphorylation in regulating translation
initiation, we have investigated whether Mnk1 phosphorylates eIF4E in
vivo. In this report, we demonstrate that Mnk1 is a member of the eIF4F
complex, binding directly to eIF4G. We have identified activating
phosphorylation sites in Mnk1 and created activated and dominant
negative mutants. Coexpression of a dominant negative mutant Mnk1
inhibits mitogen-induced and basal phosphorylation of eIF4E, while
expression of an activated mutant Mnk1 results in constitutive
phosphorylation of eIF4E. Phosphorylation of eIF4E by Mnk1 also occurs
in the presence of excess 4EBP1. We suggest that Mnk1 or a protein
kinase with similar binding properties and enzymatic specificity
phosphorylates eIF4E in mitogen- and stress-stimulated cells.
Plasmids and mutagenesis.
The plasmids pLexA-Mnk1,
pEBG-Mnk1, and pEBG-T2A2 (Mnk1 containing Thr197Ala/Thr202Ala
mutations) have been described previously (71). Untagged
Mnk1 was expressed from pCS2+ (66), and myc-tagged (MT-) Mnk1 from pCS3+MT (66a) (this vector encodes six myc
tags at the N terminus of the fusion protein). The mutations Thr197Ser (T197S), Thr202Ser (T202S), Thr332Ser (T332S), Thr197Asp/Thr202Asp (T2D2), Thr332Asp (T332D), Thr197Asp/Thr202Asp/Thr332Asp (T3D3), and
Thr197Ala/Thr202Ala/Thr332Ala (T3A3) were created by
Pfu-mediated mutagenesis (Quickchange, Stratagene) and
confirmed by nucleotide sequencing. Two-hybrid screens.
Strain L40 yeast cells (67)
expressing pLexA-Mnk1 were transformed with a human HeLa cDNA library
(Clontech). From an estimated 5 × 106 primary
transformants, 51 HIS3-positive colonies were selected. Of
25 which possessed strong In vitro binding to eIF4G.
Recombinant eIF4GI was made in
Sf9 cells by using the baculovirus expression system and purified as
described for eIF2 (33). Glutathione
S-transferase (GST)-Mnk1 or GST-T2A2 proteins were made in
Escherichia coli and purified on glutathione-Sepharose. Beads were mixed with eIF4G in a buffer containing 100 mM
CH3COOK, 2 mM (CH3COO)2Mg, 1 mM
phenylmethylsulfonyl fluoride, 7 mM 2-mercaptoethanol, and 50 mM HEPES
(pH 7.4). Samples were mixed at 4°C for 1 h and washed three
times by centrifugation with the same buffer containing 0.1% Triton
X-100. Bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with rabbit antiserum to eIF4G, raised as described previously (35).
Copurification assays.
293 cells in 50-mm dishes were
transiently transfected (Lipofectamine; Gibco-BRL) with pEBG-Mnk1 or
pCS3+MT-Mnk1 DNA (4 µg) and grown for 48 h after the addition of
DNA. The cells were then serum starved overnight with 0.1 to 0.5%
fetal bovine serum. They were lysed in buffer containing 1% Triton
X-100 and immunoprecipitated (anti-myc monoclonal antibody 9E10 for
MT-Mnk1) or purified with glutathione-Sepharose (GST-Mnk1)
(71). The glutathione-Sepharose was washed three times in
the same buffer before SDS-PAGE and Western blotting were performed.
Anti-HA monoclonal antibody 12CA5 was from D. Kremer and L. Breeden.
Anti-Flag monoclonal antibody M2 was from Kodak. Rabbit antiserum to
the 9E10 epitope, used for Western blotting, was from Babco. Rabbit
antiserum to GST was raised by standard procedures.
Immunolocalization.
NIH 3T3 cells were transiently
transfected with pSR Phosphopeptide analysis.
For labeling, 50-mm dishes of 293 cells were transfected with pEBG-Mnk1 and starved as described above;
at 4.5 h before lysis, the cells were washed once and incubated
for 30 min in phosphate-free Dulbecco modified Eagle medium containing
1 mM pyruvate, 1 mg of fatty acid-free bovine serum albumin per ml, and
20 mM HEPES (pH 7.4). The fluids were replaced with the same mixture
containing 0.5 mCi of [32P]orthophosphate (NEN) per ml,
and incubation was continued for 4 h. The cells were stimulated as
needed, and GST-Mnk1 was isolated by using glutathione-Sepharose and
SDS-PAGE. The protein was eluted from the dried gel, oxidized with
performic acid, and digested with trypsin followed by thermolysin as
described previously (3). Phosphopeptides were resolved by
electrophoresis in cellulose thin layers at pH 1.9 followed by
ascending chromatography in a buffer containing isobutyric acid
(3), with the anode at the left. Individual phosphopeptides
were extracted from the cellulose, partially hydrolyzed in 6 M HCl at
110°C for 2 h, mixed with nonradioactive phosphoserine,
phosphothreonine, and phosphotyrosine, and analyzed by electrophoresis
at pH 3.5 (3).
In vitro kinase assays.
GST-Mnk1 was purified from starved
or tetradecanoyl phorbol acetate (TPA)-stimulated 293 cells as
described above, except that a wash in 0.5 M LiCl was used to remove
associated MAP kinases, as described previously (71).
Protein kinase assays were performed (71) with 5 µg of
recombinant eIF4E substrate and 10 µCi of [ Analysis of the eIF4E phosphorylation state.
293 cells were
transiently transfected with the following plasmids as needed (the
quantity of DNA per 50-mm dish is given in parentheses): wild-type or
mutant Mnk1 in vector pEBG (4 µg), HA-eIF4E in vector pSR Mnk1 binds to eIF4G and other components of the eIF4F initiation
complex.
The Mnk1 primary sequence predicts a protein kinase
domain flanked by an N-terminal region of unknown function and a
C-terminal region that binds the MAP kinases ERK1, ERK2, and p38
(17, 71). To identify potential substrates for Mnk1, we used
the yeast two-hybrid system (67, 71). From 5 × 106 transformants, we isolated 2 cDNAs that encoded
proteins that interacted with full-length Mnk1 but not with control
proteins. The cDNAs encoded portions of an
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Phosphorylation of the Cap-Binding Protein
Eukaryotic Translation Initiation Factor 4E by Protein Kinase Mnk1
In Vivo
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
N Mnk1 was created by
Taq-mediated PCR (Perkin-Elmer), resulting in a 23-residue
deletion from the N terminus, and C Mnk1 was made similarly to
delete 85 residues from the C terminus. The human eIF4E open reading
frame was PCR amplified from a bacterial expression clone
(62) and inserted into the BglII site of
pcDL-SR
456 (63) to encode eIF4E with a single HA tag at
the N terminus. eIF4E-209Ala was subcloned into the same vector by
using a mutant C-terminal oligonucleotide and Taq-mediated PCR. Its
sequence was confirmed. The eIF4E open reading frame was also cloned
into pCS3+MT for expression of MT-eIF4E. Vectors expressing HA-tagged and Flag-tagged 4EBP1 were the kind gifts of Anne-Claude Gingras and
Nahum Sonenberg (18).
-galactosidase activity, 12 were specific
for the original bait plasmid and did not interact with N Mnk1.
Three of these clones encoded p220-2 or eIF4GII (22) (human
EST Z34918), and nine of them encoded human hRch1, a form of
-importin (19, 21).
-eIF4E, fixed with 4% paraformaldehyde in
phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and
stained with 12CA5 anti-HA antibody or chicken anti-Mnk1 antiserum.
Antiserum was raised to GST-Mnk1, made in E. coli
(71), by immunizing chickens. Immunoglobulin was purified
from eggs (Aves Inc., Tigard, Oregon) and affinity purified with
GST-Mnk1, coupled to CNBr-Sepharose, and eluted with urea. Secondary
antibodies were fluorescein-conjugated anti-chicken antibody and Texas
red-conjugated anti-mouse antibodies (Jackson Immunoresearch
Laboratories). Images were obtained with a Deltavision image-deconvoluting microscope and represent optical sections.
-32P]ATP.
Recombinant eIF4E was prepared as described previously (62)
and was the kind gift of C. G. Proud (University of Dundee).
(1 µg), and Flag-4EBP1 in vector pcDNA3 (4 µg). Vector DNA was added
as needed. Transfected cells were serum starved between 48 and 60 h after DNA addition), labeled with [32P]orthophosphate
between 60 and 64 h as described above, and treated with 0 or 100 nM TPA for the last 15 min before lysis. [32P]HA-eIF4E
was recovered from cells lysed in 1% Triton buffer by using
immunoprecipitation with 12CA5 anti-HA antibody and analyzed by
SDS-PAGE and autoradiography. Samples for isoelectric focusing were
prepared by lysing nonradioactive cells (in 100-mm dishes) by Dounce
homogenization in 4E buffer (50 mM -glycerophosphate, 1 mM
phenylmethylsulfonyl fluoride, 2 mM Na3 VO4, 1 µM microcystine [Calbiochem]). Protein concentrations were
equalized, and samples were removed for immunoprecipitation and for
eIF4E purification as needed. Immunoprecipitations were performed as
above, and the immunoprecipitates were washed with 1% Triton buffer.
eIF4E was purified with m7GTP-Sepharose (Pharmacia).
Samples were washed in four times in 4E buffer, eluted in buffer
containing urea and ampholytes, and separated on a one-dimensional
denaturing isoelectric focusing gel containing equal parts of pH 4 to 6 and pH 6 to 8 ampholytes, as described previously (13).
RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References
-importin relative
(20, 21) and of eIF4GII, one of two known forms of eIF4G
(22). Both fragments bind to full-length Mnk1 and to a
mutant which lacks the C-terminal 85 amino acids but do not bind to a
mutant in which the N-terminal 23 amino acids are deleted (Fig.
1A and data not shown). Since the C
terminus is needed for ERK or p38 MAP kinase binding (71), this suggests that the binding of -importin and eIF4GII is not mediated by MAP kinase. The -importin clone encodes residues 43 to
239, which contain the region hypothesized to interact with nuclear
localization signal peptides. Since the N terminus of Mnk1 is rich in
basic residues, it may be recognized by -importin as a nuclear
localization signal. However, we have not detected a significant
portion of Mnk1 within the nucleus (Fig.
2), suggesting that Mnk1 may not bind
functionally to -importin in mammalian cells. The two-hybrid isolate
of eIF4GII encodes the C-terminal 192 residues, implying that this
region of eIF4GII is sufficient to confer Mnk1 binding. The amino acid
sequences of eIF4GI and eIF4GII are 68% identical in this region,
suggesting that Mnk1 may bind to both forms of eIF4G.
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FIG. 1.
(A) Growth of yeast strain L40 expressing LexA-Mnk1 and
VP16-eIF4GII (top), LexA-Mnk1- N and VP16-eIF4GII (middle), and
LexA-Mnk1-C and VP16-eIF4GII (bottom) on medium lacking histidine.
Growth indicates transactivation of the HIS3 reporter gene.
(B) GST-Mnk1 and GST-Mnk1-T2A2 (T197A/T202A) were purified from
E. coli and incubated with purified mammalian eIF4G. GST
fusion proteins were isolated, and bound eIF4G was eluted and analyzed
by SDS-PAGE and Western blotting. Input lanes represent one-fifth of
the sample used for binding. (C to E) 293 cells were transiently
transfected with pEBG, pEBG-Mnk1, or pEBG-Mnk1N, together with
pCS3+eIF4E. GST-Mnk1 was purified with glutathione-Sepharose, and bound
proteins were visualized with GST antibody (C), eIF4G antibody (D), or
9E10 anti-MT antibody (E). Lysate lanes represent 1/10 of the sample
used for purification. (F) Diagram of characterized eIF4G binding
sites. See the text for details. aa, amino acids.
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FIG. 2.
Subcellular localization of Mnk1 and eIF4E. NIH 3T3
cells were transfected with pCS2-Mnk1 and pSR -eIF4E. Untagged Mnk1
and HA-eIF4E were visualized with an affinity-purified
chicken-anti-Mnk1 antibody and 12CA5 (anti-HA) ascites fluid, followed
by fluorescein-conjugated anti-chicken antibody and Texas
red-conjugated anti-mouse antibody. (A and C) Mnk1 immunofluorescence;
(B and D) HA-eIF4E. Cells were either serum starved (A and B) or
treated with phorbol ester for 60 min (C and D).
Subcellular localization of Mnk1. The initiation factors eIF4E and eIF4G are localized predominantly in the cytoplasm, with 12 to 25% of eIF4E being found in the nucleus (40, 42). To determine the subcellular distribution of Mnk1 within cells, we expressed untagged Mnk1 and HA-tagged eIF4E in NIH 3T3 cells and examined their localization by indirect immunofluorescence. Anti-Mnk1 antiserum did not stain untransfected cells significantly. When Mnk1 was overexpressed, the bulk of Mnk1 localized in the cytoplasm in serum-starved cells and a small population was found within the nucleus (Fig. 2A). Upon stimulation with TPA, Mnk1 shifted to a perinuclear ring (Fig. 2C). HA-eIF4E was also mostly cytoplasmic and shifted to a perinuclear ring in a similar way after TPA stimulation (Fig. 2B and D). The shift in localization of both Mnk1 and eIF4E after TPA treatment could reflect a general reorganization of the cytoplasm or a change in the localization of a subset of proteins. Under both unstimulated and stimulated conditions, the majority of Mnk1 and eIF4E colocalize, consistent with the binding of Mnk1 to eIF4G and eIF4E. To confirm these data, we fractionated extracts of cells expressing Mnk1 or HA-eIF4E. Both Mnk1 and eIF4E were present predominantly within the cytosolic compartment, consistent with our immunofluorescence data (data not shown).
Phosphorylation site substitution mutants of Mnk1.
The binding
of Mnk1 to eIF4G and eIF4E and the in vitro phosphorylation of eIF4E by
Mnk1 suggested that Mnk1 may phosphorylate eIF4E in cells. To test
this, we designed dominant negative and activated mutants of Mnk1.
Because Mnk1 is activated by phosphorylation by ERK or p38 MAP kinases,
we reasoned that replacement of regulatory phosphorylation sites by
nonphosphorylated or acidic residues might inhibit or activate Mnk1,
respectively. We examined the Mnk1 sequence for likely MAP kinase
phosphorylation sites. The MAP kinase consensus, 
X[S/T]P, where
is proline or aliphatic (1, 7), occurs three times in
Mnk1, at Thr 197, Thr 202, and Thr 332. Thr 197 and Thr 202 are located
within the T-loop or activation loop (30) of Mnk1, and Thr
332 is close to the COOH terminus. We tested whether these residues are
phosphorylated in vivo by peptide mapping.
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Effects of phosphorylation site mutants on Mnk1 kinase activity. The kinase activities of substitution mutants of Mnk1 were assessed by expressing wild-type or mutant GST-Mnk1 in 293 cells. Cells were unstimulated or stimulated with TPA before purification of GST-Mnk1 and assay with recombinant eIF4E substrate (Fig. 4B). Expression levels were assessed by Western blotting and found to be comparable (Fig. 4A). When incubated with radioactive ATP, all mutants were autophosphorylated or phosphorylated by a contaminating kinase, but the mutants differed in their ability to phosphorylate eIF4E (Fig. 4B). Wild-type GST-Mnk1 exhibits TPA-stimulated eIF4E phosphorylating activity, whereas two mutants lacking the T-loop threonines, T197A/T202A (T2A2) and T197A/T202A/T332A (T3A3), were unable to phosphorylate eIF4E in vitro (Fig. 4B) (71). Consistent with our negative evidence for phosphorylation at Thr 332, replacement of Thr 332 with Ala did not inactivate Mnk1 and actually caused partial activation (T332A) (Fig. 4B).
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Mnk1 regulates eIF4E phosphorylation in cells. To determine the ability of Mnk1 to phosphorylate eIF4E in vivo, we cotransfected 293 cells with HA-tagged eIF4E and wild-type or mutant GST-Mnk1. The phosphorylation status of eIF4E was analyzed either by metabolic labeling with [32P]orthophosphate labeling or by isoelectric focusing and Western blotting (Fig. 5).
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Effects of overexpressed activated mutant Mnk1 in cells overexpressing 4EBP1. The preceding results indicate that increased Mnk1 activity leads to increased phosphorylation of eIF4E in transfected cells. However, these experiments had made use of overexpressed, epitope-tagged eIF4E to assay the phosphorylation state of eIF4E in the cells that were overexpressing Mnk1. Thus, the normal balance between eIF4E and 4EBP1 levels was not maintained in the transfected cells. To determine whether increased Mnk1 activity would also increase eIF4E phosphorylation in cells where eIF4E is associated with 4EBP1 and to test whether eIF4E phosphorylation altered the binding of eIF4E to either eIF4G or 4EBP1, we cotransfected 293 cells with various combinations of plasmids expressing mutant GST-Mnk1, HA-tagged eIF4E, and Flag-tagged 4EBP1 (18) and analyzed HA-eIF4E complexes and phosphorylation state (Fig. 6).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Previous studies have shown that the cap-binding protein eIF4E is phosphorylated at a single site, Ser 209, when cells are stimulated with a variety of mitogens or stresses, but the kinase responsible has not been identified. The ERK and p38 MAP kinase-activated protein kinase, Mnk1, is able to phosphorylate eIF4E in vitro at Ser 209 (71). We have now found that Mnk1 is bound to the eIF4E-associated scaffold eIF4G and regulates the phosphorylation state of eIF4E in transfected cells. This suggests that endogenous Mnk1 may phosphorylate eIF4E in response to mitogen and stress stimuli. We have also found that Mnk1 can lead to extensive eIF4E phosphorylation under conditions where most eIF4E is associated with the inhibitor, 4EBP1. This suggests that Mnk1 can operate independently of 4EBP1 phosphorylation and may explain how eIF4E becomes phosphorylated in stressed cells, where 4EBP1 is not phosphorylated (51, 70).
The detection of the C-terminal region of eIF4G as a binding partner for Mnk1 in an unbiased yeast two-hybrid screen suggests that Mnk1 has a high specificity for binding eIF4G in cells. eIF4E could be copurified with Mnk1 and eIF4G from transfected cells. The proximity of Mnk1 to eIF4E bound to eIF4G suggests that Mnk1 is likely to phosphorylate eIF4E even when not overexpressed. Mnk1 binds to eIF4G independently of the C terminus of Mnk1, to which ERK and p38 MAP kinases bind. Thus, it seems likely that a complex of Mnk1 and MAP kinase is associated with eIF4G in cells. Together with the specificity of Mnk1 for the physiological phosphorylation site in eIF4E in vitro (71) and the effects of overexpressing Mnk1 mutants, it is highly likely that Mnk1 phosphorylates eIF4E in vivo when ERK or p38 is activated. In addition, Mnk1 may phosphorylate other components of the eIF4F complex. This possibility remains to be explored.
The second protein found to bind to Mnk1 in the yeast two-hybrid screen
was -importin. -Importin binding requires the N-terminal basic
region of Mnk1, which resembles a nuclear localization signal. However,
our immunofluorescence experiments suggest that the large majority of
Mnk1 is cytosolic in fibroblasts, under unstimulated and stimulated
conditions. Mnk1 redistributed to the perinuclear region in
TPA-stimulated cells but still appeared to be outside the nucleus (Fig.
2). It remains to be determined whether Mnk1 enters the nucleus under
certain conditions or perhaps shuttles rapidly out of the nucleus once
it enters. We also found that the majority of epitope-tagged eIF4E is
in the cytoplasm, either by viewing immunofluorescence or by cell
fractionation. While the cytoplasmic localization of eIF4E is
consistent with an Mnk1-eIF4G-eIF4E complex, it is not fully consistent
with the results of a previous study of eIF4E localization
(42). Those authors found that a monoclonal antibody to
eIF4E stained the nucleus more intensely than it stained the cytoplasm.
In addition, biochemical fractionation showed that about 12 to 25% of
the protein was nuclear and the remainder was split equally between
perinuclear membranes and the cytosol (42). It is possible
that perinuclear eIF4E appears to be inside the nucleus under some
conditions. Newly synthesized capped RNA is bound to a distinct
cap-binding protein inside the nucleus (28) and may be
transferred to perinuclear eIF4E during export.
Biochemical and mutational analysis shows that phosphorylation of Mnk1
occurs in mitogen- and stress-stimulated cells at a common set of
serine and threonine residues. We found that Thr 197 and probably Thr
202, both in the T loop of the Mnk1 kinase domain, are phosphorylated
at increased levels in mitogen- or stress-stimulated cells. These sites
both lie in the known MAP kinase phosphorylation consensus,
X[S/T]P, and thus are likely to be phosphorylated directly by ERK
and p38 MAP kinases. The MAP kinase-activated protein kinases, Rsk and
MAPKAPK-2, are similarly phosphorylated in their T loops by ERK and
p38, respectively (2, 8). T-loop phosphorylation is critical
for activation of a variety of protein kinases, and in some cases,
replacement of T-loop phosphorylation site residues with acidic
residues is sufficient for activation (30). However,
replacement of Thr 197 and Thr 202 with aspartate did not activate
Mnk1. Instead, replacement of a C-terminal residue, Thr 332, which does
not appear to be phosphorylated, is sufficient for Mnk1 activation when
expressed in mammalian cells. This residue may be critical for
maintaining an inhibitory conformation, since small C-terminal
deletions also activate Mnk1 (61a). It is likely that the
activation of Mnk1 mutant T332D involves phosphorylation, since this
mutant is activated further by mitogens and is not activated when made
in bacteria. Detailed analysis of Rsk and MAPKAPK-2 has shown that
phosphorylation at several sites in the C- and N-terminal noncatalytic
regions contributes to activity (2, 8). Additional
phosphorylation sites in Mnk1 may well regulate activity and remain to
be identified.
Previous evidence has indicated that eIF4E phosphorylation depends on ERK MAP kinase in mitogen-stimulated cells and on p38 MAP kinase in stressed cells (14-16, 49, 51, 70). Mnk1 binds to and is activated by the ERK and p38 MAP kinases in vitro and in transfected cells (17, 71). Of the MAP kinase-activated protein kinases tested, Mnk1, its relative Mnk2, and MAPKAPK-3 can specifically phosphorylate eIF4E at Ser 209 in vitro (51, 70a, 71). MAPKAPK-3 was reported to be activated by the ERK, p38, and Jun N-terminal kinase (JNK) classes of MAP kinases (43). Since JNK is activated by stresses and since blockade of p38 but not JNK inhibits eIF4E phosphorylation in stressed cells (51, 70), it is unlikely that a JNK-stimulated protein kinase such as MAPKAPK-3 phosphorylates eIF4E in vivo. Our new results indicate that Mnk1 can phosphorylate eIF4E in cells when overexpressed, and they implicate Mnk1 in stimulating eIF4E phosphorylation in mitogen-stimulated 293 cells. However, it is also possible that a kinase other than Mnk1 is the physiological eIF4E kinase in other cell types or under other stimulation conditions. By binding to the C terminus of eIF4G, the kinase-defective T197A/T202A (T2A2) mutant Mnk1 may displace the physiological eIF4E kinase and inhibit eIF4E phosphorylation. One obvious candidate for another in vivo eIF4E kinase is Mnk2. However, it is unlikely that Mnk2 is responsible for stress activation of eIF4E phosphorylation, since Mnk2 is not activated by p38 MAP kinase (71).
The relationship between eIF4E phosphorylation and 4EBP1 phosphorylation is complex. As discussed above, inhibitor studies indicate that different signal transduction pathways activate the kinases that phosphorylate eIF4E and 4EBP1. In particular, rapamycin inhibits 4EBP1 phosphorylation (61). However, Mendez et al. (47) showed that insulin-stimulated phosphorylation of eIF4E is inhibited by rapamycin and Morley and McKendrick (51) showed that rapamycin inhibited serum-induced eIF4E phosphorylation. On the other hand, the latter authors also showed that rapamycin did not inhibit stress (anisomycin)-induced eIF4E phosphorylation (51). Wang et al. (70) found that 4EBP1 reduced eIF4E phosphorylation by Mnk1 in vitro, although they used large quantities of 4EBP1. We have now found that excess 4EBP1 did not prevent eIF4E phosphorylation by overexpressed Mnk1. This suggests either that Mnk1 can phosphorylate the eIF4E-4EBP1 complex or that free eIF4E is phosphorylated by Mnk1 and exchanges with unphosphorylated eIF4E in the 4EBP1 complex. Either way, it is clear that eIF4E can be phosphorylated to high stoichiometry when Mnk1 is activated in the absence of 4EBP1 phosphorylation. Our experiments do not address the possibility that eIF4E bound to the eIF4G-Mnk1 complex is phosphorylated faster than the free eIF4E or eIF4E bound to 4EBP1.
In conclusion, we have shown that Mnk1 binds to the C terminus of eIF4G and that overexpressed activated Mnk1 increases the stoichiometry of eIF4E phosphorylation in transfected cells, even in the presence of excess 4EBP1. Mnk1 activation correlates with stress- and mitogen-stimulated eIF4E phosphorylation, and dominant-negative Mnk1 interferes with mitogen-stimulated and basal eIF4E phosphorylation, suggesting that Mnk1 regulates eIF4E phosphorylation independently of 4EBP1 phosphorylation in vivo.
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ACKNOWLEDGMENTS |
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We are very grateful to A.-C. Gingras, A. Vojtek, D. Turner, Y. Gotoh, C. Proud, L. Breeden, I. Scheffler, L. Shantz, E. Foulstone, and A. Geballe for reagents; R. Fukunaga, T. Hunter, N. Sonenberg, and C. Proud for interesting discussions; and B. Howell, S. Morris, and C. Sachsenmaier for their comments on the manuscript.
This work was supported by NIH grants R01-CA-73987 (to J.A.C.) and R01-DK-13499 (to S.R.K.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, A2-025, 1100 Fairview Ave. North, Seattle, WA 98109. Phone: (206) 667-4454. Fax: (206) 667-6522. E-mail: jcooper{at}fhcrc.org.
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REFERENCES |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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