4E-BP1, a repressor of mRNA translation,
is phosphorylated and inactivated
by the Akt(PKB) signaling pathway
Anne-Claude Gingras,1,4Scott G. Kennedy,2,3,4Maura A. O’Leary,3Nahum Sonenberg,1and
1Department of Biochemistry, McGill University Montreal, Quebec, Canada H3G 1Y6; and2Department of Pharmacological
and Physiological Sciences and3T he Ben May Institute for Cancer Research, T he University of Chicago,
Chicago, Illinois 60637 USA
Growth factors and hormones activate protein translation by phosphorylation and inactivation of the
translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive
signaling pathway. T he mechanism by which signals emanating from extracellular signals lead to
phosphorylation of 4E-BPs is not well understood. Here we demonstrate that the activity of the
serine/threonine kinase Akt/PKB is required in a signaling cascade that leads to phosphorylation and
inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and
rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin
resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated
phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1.
Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated
upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation
of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1
by Akt requires the activity of FRAP/mT OR. FRAP/mT OR may lie downstream of Akt in this signaling
cascade. T hese results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mT OR,
induces the phosphorylation of 4E-BP1.
[Key Words: Protein synthesis; phosphorylation; PI 3-kinase; protein kinase B; eIF4E; FRAP/mT OR]
Received November 13, 1997; revised version accepted December 19, 1997.
Numerous cellular processes are controlled by extracel-
lular stimuli that activate signaling cascades. Many
stimuli activate common pathways, such as the well-
described Ras and phosphoinositide 3-kinase pathways.
Phosphoinositide 3-kinase (PI 3-kinase) is activated by
growth factor receptors after growth factor stimulation
and induces cell proliferation and cell survival (for re-
view, see Franke et al. 1997a; Vanhaesebroeck et al.
1997). Several downstream targets of PI 3-kinase have
been identified, including p70 ribosomal protein S6 ki-
nase (p70S6k) (Chou and Blenis 1995; Proud 1996), the
GT Pases Rac (Hawkins et al. 1995), certain protein ki-
nase C isoforms (Nakanishi et al. 1993; Akimoto et al.
1996), and the serine/threonine kinase Akt (also known
as protein kinase B-PKB) (Burgering and Coffer 1995;
Frankeet al. 1995). Upon activation by growth factors, PI
3-kinase phosphorylates the D3 position of phosphati-
dylinositols. T hese phospholipids act as second messen-
gers that mediate the diverse cellular functions of PI 3-
kinase, including activation of Akt (for review, see
Franke et al. 1997a; Hemmings 1997). Wortmannin, a PI
3-kinase inhibitor, blocks activation of Akt after stimu-
lation with growth factors, indicating that the activity of
PI 3-kinase is an obligatory step in Akt activation by
growth factors (Burgering and Coffer 1995; Franke et al.
1997b; Kennedy et al. 1997). T ranslocation of Akt to the
plasma membrane through its pleckstrin homology (PH)
domain is likely required for its activity (Hemmings
1997). Indeed, constitutivetargeting of Akt to theplasma
membrane is sufficient to promote its activation (Bur-
gering and Coffer 1995; Franke et al. 1997b; Kennedy et
al. 1997). Full activation of Akt by growth factors re-
quires the phosphorylation of threonine and serine resi-
dues by upstream kinases (Alessi et al. 1997; Stokoeet al.
1997). Both the upstream activating kinases and the re-
cruitment of Akt to theplasmamembranearethought to
be dependent on the products of PI 3-kinase (Cohen et al.
1997). Akt mediates some of the PI 3-kinase cellular re-
sponses, including protection from apoptosis induced by
4T hese authors made an equal contribution to the work.
5Corresponding author. Present address: Department of Molecular Ge-
netics, University of Illinois at Chicago, Chicago, Illinois 60607.
E-MAIL firstname.lastname@example.org; FAX (773) 702-6260.
502GENES & DEV ELOPMENT 12:502–513 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org
serum and growth factor deprivation (Kennedy et al.
T he immunosuppressive drug rapamycin, an inhibitor
of G1cell cycle progression, blocks the activation of
p70S6k by growth factors and Akt (Chung et al. 1992;
Price et al. 1992; Burgering and Coffer 1995). Rapamycin
forms a complex with the immunophilin FKBP12 to gen-
erate a potent inhibitor of FRAP/mT OR (also termed
RAFT 1 or RAPT 1) (Brown and Schreiber 1996). FRAP/
mT OR activates p70S6k in vivo (Brown et al. 1995); it is
unclear, however, whether FRAP/mT OR is a down-
stream effector in thePI 3-kinaseandAkt signaling path-
way. FRAP, which is the mammalian homolog of the
Saccharomyces cerevisiae targets of rapamycin T OR1
and T OR2 (Hall 1996), is a member, together with AT M
and DNA–PK, of a recently characterized family of phos-
phatidylinositol kinases-related (PIK-related) kinases.
PIK-related kinases activation and mechanisms of action
remain unclear (Hoekstra 1997). FKBP rapamycin-
associated protein /mammalian target of rapamycin
(FRAP/mT OR) could provide a link between cell cycle
progression and the control of mRNA translation, as
rapamycin, which blocks thecell cyclein G1, also causes
a decrease in mRNA translation (Beretta et al. 1996;
Brown and Schreiber 1996). Consistent with this finding,
the yeast T OR has been demonstrated to regulate G1
progression through a translational mechanism (Barbet
et al. 1996).
Regulation of protein translation is an important as-
pect of the control of cell growth. A rate-limiting step in
this process is the binding of the mRNA to the small
ribosomal subunit, a step mediated by the eIF4 group of
initiation factors (for review, see Sonenberg 1996). eIF4F,
through its smaller subunit eIF4E, recognizes the cap
structure (m7GpppX, where X is any nucleotide) that is
present at the 5? end of all cellular, except organellar,
mRNAs. eIF4F, in conjunction with eIF4B, is thought to
unwind the secondary structure in the mRNA 5?-UT R to
facilitate ribosome binding (Sonenberg 1996). Overex-
pression of eIF4E in rodent cells leads to cellular trans-
formation and eIF4E has been implicated in cell cycle
control (Lazaris-Karatzas et al. 1990; Sonenberg 1996). In
addition, a role for eIF4E in cell survival has been pro-
posed, as NIH 3T 3 cells that express eIF4E ectopically
are refractory to apoptosis induced by serum deprivation
(Polunovsky et al. 1996). eIF4E is the target of a family of
translational repressors termed the 4E-BPs (for eIF4E-
Binding Proteins; also known as PHAS). T heserepressors
bind to eIF4E and prevent its association with eIF4G and
incorporation into the eIF4F complex, which leads to
inhibition of cap-dependent, but not cap-independent,
translation (Sonenberg 1996). Overexpression of 4E-BP1
or 4E-BP2 in cells transformedby eIF4E, Ha-v-ras or v-src
partially reverts their transformed phenotypes (Rousseau
et al. 1996).
Inhibition of translation by 4E-BPs is reversible. After
treatment of cells with serum, growth factors, or hor-
mones, 4E-BP1 (the prototype member of the family), is
hyperphosphorylated in a wortmannin- and rapamycin-
sensitive manner, and dissociates from eIF4E (Beretta et
al. 1996; von Manteuffel et al. 1996, 1997). T he rapid
increase in 4E-BP1 phosphorylation after serum or
growth factor stimulation provides a very attractive
mechanism for explaining the increase in translation
rates of several mRNAs after stimulation.
Because phosphorylation of 4E-BP1 is wortmannin
sensitive, and mutants in the PDGF receptor that fail to
activate PI 3-kinase also fail to phosphorylate 4E-BP1
after PDGF treatment (Beretta et al. 1996; von Man-
teuffel et al. 1996), it was suggested that phosphorylation
of 4E-BP1 by serum and growth factors is mediated by PI
3-kinase. However, it is not clear whether PI 3-kinase
lies directly upstream of 4E-BP1 in a phosphorylation
cascade. T his is an important question, particularly in
light of a recent report demonstrating that wortmannin
can inhibit FRAP/mT OR activity directly (Brunn et al.
1996). Here we provide direct evidence that PI 3-kinase
and its downstream effector Akt lie in a pathway leading
to the in vivo phosphorylation of 4E-BPs. T his phos-
phorylation is sensitive to rapamycin. T he rapamycin
sensitivity can be overridden by coexpression of a rapa-
mycin-resistant mutant of FRAP/mT OR. T hus, FRAP/
mT OR may lie downstream of Akt in the 4E-BP1 phos-
P110?, the catalytic subunit of PI 3-kinase,
and its downstream effector Akt/PKB mediate
the phosphorylation of 4E-BP1
T o study the role of Akt in the phosphorylation of 4E-
BP1, a hemagglutinin-tagged 4E-BP1 (HA–4E-BP1) was
generated. Wefirst examined whether thetransiently ex-
pressed HA–4E-BP1 exhibits a change in electrophoretic
mobility after phosphorylation, as was observed for the
endogenous 4E-BP1. Human embryonic kidney (HEK)
293 cells were transfected transiently with a HA–4E-BP1
expression vector. After transfection, the cells were de-
prived of serum for 36 hr and then stimulated with in-
sulin for 30 min. Immunoblot analysis demonstrated a
clear shift in mobility of HA–4E-BP1 with insulin stimu-
lation (Fig. 1A, lanes 1,2). T he mobility shift was not
observed when cells were preincubated with either wort-
mannin or rapamycin (lanes 3,4), consistent with what
has been observed previously for endogenous 4E-BP1
(von Manteuffel et al. 1996).
Previous studies have indicated a role for PI-3-kinase
in the phosphorylation of 4E-BP1 by serum and growth
factors (Beretta et al. 1996; von Manteuffel et al. 1996).
However, it was also suggested that the effects of extra-
cellular stimuli on 4E-BP1 phosphorylation could be ex-
plained by direct activation of FRAP, as the in vitro au-
tokinase activity of FRAP is also inhibited by wortman-
nin (Brunn et al. 1996). T o examine whether PI 3-kinase
can affect the phosphorylation state of 4E-BP1, we tran-
siently cotransfected HA-4E-BP1 and PI 3-kinase expres-
sion vectors into serum-deprived 293 cells. Cotransfec-
tion of HA-4E-BP1 with the catalytic subunit of PI 3-
kinase p110? induced phosphorylation of 4E-BP1, as
manifestedby ashift in its mobility (Fig. 1B, lane2). T his
Akt regulates 4E-BP1 phosphorylation
GENES & DEV ELOPMENT503
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