MOLECULAR AND CELLULAR BIOLOGY, Nov. 2010, p. 5160–5167
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 21
Regulation of Eukaryotic Initiation Factor 4E (eIF4E)
Phosphorylation by Mitogen-Activated Protein
Kinase Occurs through Modulation
of Mnk1-eIF4G Interaction?
Mayya Shveygert,1Constanze Kaiser,1† Shelton S. Bradrick,2and Matthias Gromeier1*
Division of Neurosurgery, Department of Surgery,1and Department of Molecular Genetics and
Microbiology,2Duke University Medical Center, Durham, North Carolina 27710
Received 15 April 2010/Returned for modification 13 May 2010/Accepted 23 August 2010
The m7G cap binding protein eukaryotic initiation factor 4E (eIF4E) is a rate-limiting determinant of
protein synthesis. Elevated eIF4E levels, commonly associated with neoplasia, promote oncogenesis, and
phosphorylation of eIF4E at Ser209 is critical for its tumorigenic potential. eIF4E phosphorylation is catalyzed
by mitogen-activated protein kinase (MAPK)-interacting serine/threonine kinase (Mnk), a substrate of Erk1/2
and p38 MAPKs. Interaction with the scaffolding protein eIF4G, which also binds eIF4E, brings Mnk and its
substrate into physical proximity. Thus, Mnk-eIF4G interaction is important for eIF4E phosphorylation.
Through coimmunoprecipitation assays, we showed that MAPK-mediated phosphorylation of the Mnk1 active
site controls eIF4G binding. Utilizing a naturally occurring splice variant, we demonstrated that the C-
terminal domain of Mnk1 restricts its interaction with eIF4G, preventing eIF4E phosphorylation in the
absence of MAPK signaling. Furthermore, using a small-molecule Mnk1 inhibitor and kinase-dead mutant, we
established that Mnk1 autoregulates its interaction with eIF4G, releasing itself from the scaffold after phos-
phorylation of its substrate. Our findings indicate tight control of eIF4E phosphorylation through modulation
of Mnk1-eIF4G interaction.
In eukaryotes, initiation of protein synthesis is facilitated by
eukaryotic initiation factor 4F (eIF4F), a complex consisting of
the scaffolding factor eIF4G and its interaction partners, the
m7G cap binding protein eIF4E and the helicase eIF4A. In the
preinitiation complex, eIF4G serves as a central ribosome
adaptor module, attracting 40S ribosomal subunits to the 5?
end of mRNAs via direct association with eIF3 (4). Interac-
tions of eIF4E with the m7G cap and eIF4G are recognized as
rate-limiting steps in translation. They are tightly controlled by
key mitogenic signals, such as the phosphoinositide-3-kinase/
mammalian target of rapamycin (PI3K/mTOR) and Ras/
mitogen-activated protein kinase (MAPK) signal transduc-
tion pathways. A myriad of effects of PI3K/mTOR and MAPK
mitogenic signals on translation of discrete mRNA subsets and
the global transcriptome have been reported (15).
Dysregulated translational control is a significant factor in
tumorigenesis and constitutes a prominent target for therapy.
Thus, it is of central interest to mechanistically unravel the
effects of mitogenic signals on the translation apparatus. A
signature oncogenic signaling effect is eIF4E phosphorylation
at Ser209 upon activation of Erk1/2 or p38 MAPKs (2, 22).
Erk1/2 and p38 MAPK signals converge on Mnk, which is
uniquely capable of catalyzing eIF4E Ser209 phosphorylation
(21). eIF4E has been implicated in tumorigenesis (9, 10), and
Ser209 phosphorylation has been shown to be required for
eIF4E’s oncogenic potential (3, 20, 23). Unraveling mecha-
nisms of protein synthesis modulation due to eIF4E phosphor-
ylation has been pursued intensely, but the consequences of
eIF4E phosphorylation for the regulation of translation initi-
ation remain a matter of debate (19).
The Mnk proteins are serine/threonine kinases encoded by
two distinct genes, Mnk1 and Mnk2 (17). Both Mnk1 and
Mnk2 transcripts are subject to alternative splicing, giving rise
to full-length versions (Mnk1a/2a) as well as truncated versions
(Mnk1b/2b) lacking the MAPK binding domain (11). Mnk1a
and Mnk2a are activated by p38 and/or Erk1/2 MAPKs. How-
ever, Mnk2 has a high basal level of catalytic activity toward
eIF4E phosphorylation and can be active even in unstimulated
cells (17). Interestingly, Mnks do not form a stable binary
complex with eIF4E to achieve Ser209 phosphorylation. In-
stead, they interact with the scaffolding protein eIF4G, bring-
ing the kinase and its substrate into physical proximity (14).
Consequently, association of Mnk with eIF4G is essential for
eIF4E Ser209 phosphorylation. We report here that phosphor-
ylation of Mnk1 by p38 or Erk1/2 MAPKs not only activates its
kinase activity but also modulates Mnk1 interaction with
eIF4G, thereby facilitating eIF4E phosphorylation. MAPK-
mediated control over Mnk1-eIF4G binding constitutes an
added level of regulation over eIF4E phosphorylation.
MATERIALS AND METHODS
Cloning of expression plasmids. pcDNA5/FRT/TO myc-eIF4GI-flag was gen-
erated by modifying pcDNA5/FRT/TO myc-eIF4GI-b (7) with a C-terminal Flag
tag. Briefly, a C-terminal eIF4G fragment fused to Flag protein was generated by
PCR using primers 1 and 2 (Table 1), digested with NheI-NotI, and inserted into
* Corresponding author. Mailing address: Department of Surgery,
Duke University Medical Center, Durham, NC 27710. Phone: (919)
668-6205. Fax: (919) 681-4991. E-mail: firstname.lastname@example.org.
† Present address: European Molecular Biology Laboratory, Hei-
?Published ahead of print on 7 September 2010.
would enable Mnk1 to bind successive eIF4F complexes, thus
allowing rapid turnaround of the substrate.
Our findings suggest that after MAPK activation, Mnk1 not
only becomes catalytically active but is also actively recruited to
the vicinity of eIF4E through increased interaction with the
scaffolding protein eIF4G. The latter is biologically signifi-
cant, because the ?4G Mnk1 mutant, which is a fully active
kinase but cannot bind eIF4G, is unable to reconstitute an
adequate eIF4E phosphorylation response to TPA stimula-
tion in Mnk1/2 DKO MEFs.
The data presented here uncover an additional level of con-
trol over Mnk1 activity and consequently eIF4E phosphoryla-
tion. This complex regulation is likely influenced by other as-
pects not addressed in this study. For example, it is possible
that phosphorylation of eIF4G by signal transduction events
elicited by TPA may also play a role in regulation of eIF4E
phosphorylation, e.g., by modulating Mnk1 association. Con-
sidering that phosphorylation of eIF4E by Mnk proteins ap-
pears to be critical for malignant transformation (3, 20, 23), it
will be of interest to fully define both the events that govern
this posttranslational modification and the consequences it has
for control of mRNA translation.
We thank E. Y. Dobrikova for technical assistance, C. Goetz for
providing recombinant eIF4E, and R. Fukunaga for generously sharing
Mnk1/2 DKO MEFs.
This work was supported by PHS grant CA140510 (to M.G.) and a
grant from the Susan G. Komen Foundation.
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VOL. 30, 2010 MAPKs CONTROL Mnk1 BINDING TO eIF4G5167