Cyclin D1 and c-Myc Internal Ribosome Entry Site (IRES)-Dependent Translation is Regulated by AKT Activity and Enhanced by Rapamycin Through a p38 MAPK and
ERK-Dependent Pathway
YiJiang Shi, Anushree Sharma, Hong Wu*, Alan Lichtenstein and Joseph Gera‡
Departments of Research & Development, Molecular and Medical Pharmacology* and
Medicine, VA Greater Los Angeles Healthcare System, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Howard Hughes Medical Institute*,
University of California, Los Angeles, California, 91343
‡To whom correspondence should be addressed: Tel.: 818-895-9416; Fax: 818-895-
9554; E-Mail: [email protected] Keywords: translation; IRES; AKT/PKB; rapamycin; cyclin D1; c-myc Running Title: AKT-dependent cyclin D1 and c-myc IRES activity 1 The abbreviations used are: IRES, internal ribosome entry site; AKT, AKT/protein kinase B; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; rRNA, ribosomal RNA; MEF, mouse embryonic fibroblast; UTR, untranslated region; ANOVA, analysis of variance.
JBC Papers in Press. Published on January 4, 2005 as Manuscript M407874200 by guest on A
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Abstract The macrolide antibiotic rapamycin inhibits the mammalian target of rapamycin
protein (mTOR) kinase resulting in the global inhibition of cap-dependent protein
synthesis, a blockade in ribosome component biosynthesis and G1 cell cycle arrest. G1
arrest may occur by inhibiting the protein synthesis of critical factors required for cell
cycle progression. Hypersensitivity to mTOR inhibitors has been demonstrated in cells
having elevated levels of AKT kinase activity, whereas cells containing quiescent AKT
activity are relatively resistant. Our previous data suggest that low AKT activity induces
resistance by allowing continued cap-independent protein synthesis of cyclin D1 and c-
myc proteins. In support of this notion, the current study demonstrates that the human
cyclin D1 mRNA 5’ untranslated region contains an internal ribosome entry site (IRES)
and that both this IRES and the c-myc IRES are negatively regulated by AKT activity.
Furthermore, we show that cyclin D1 and c-myc IRES function is enhanced following
exposure to rapamycin and requires both p38 MAPK and RAF/MEK/ERK signaling, as
specific inhibitors of these pathways reduce IRES-mediated translation and protein levels
under conditions of quiescent AKT activity. Thus, continued IRES-mediated translation
initiation may permit cell cycle progression upon mTOR inactivation in cells whose AKT
kinase activity is relatively low.
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Introduction
The global regulation of cap-dependent translation is mediated via the mTOR
signaling cascade (1-2). Activation of mTOR results in phosphorylation of the p70 S6
kinase and the translation repressor 4E-BP1, allowing the formation of functional eIF-4F
complexes resulting in cap-dependent mRNA translation initiation and ribosomal
component biogenesis (3-4). The efficiency with which an mRNA can initiate cap-
dependent translation is a function of the length and degree of secondary structure present
within the 5’ UTR, as well as, the sequence context of the initiation codon (11). Most
eukaryotic mRNAs contain 5’ UTRs with relatively short and unstructured 5’ UTRs
(< 100 nucleotides) which allows efficient cap-dependent ribosomal scanning (11).
However, some key regulators of cell proliferation and apoptosis have leaders which are
quite long, highly structured and contain many upstream AUG or CUG codons, and as a
result, are inhibitory to scanning ribosomes (12). Translation initiation in a number of
these mRNAs is achieved via IRES-mediated mechanisms (13). Protein synthesis via
this alternative form of initiation is typically favored under conditions when the default
cap-dependent pathway is inhibited (14).
The ability of AKT to regulate cap-dependent initiation is mediated via its
inhibitory effects on the mTOR inhibitor complex TSC1/TSC2 (7-9). A direct linkage
between AKT and the mTOR kinase has also been described. AKT can phosphorylate
mTOR and studies in Drosophila have demonstrated that dTOR is downstream and
epistatic to the PI3-kinase/AKT pathway (4-6). However, AKT has recently been shown
to negatively regulate the translation of the Elk-1 and Sap1a mRNAs independent of
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mTOR (52) and our prior work (10) also suggests a role for AKT in the regulation of cap-
independent translation.
Previously, we identified many mRNAs whose translation was unaffected or
induced under conditions of mTOR inhibition following rapamycin treatment (10). Many
of these transcripts remained on actively translated polysomes or shifted from
monosomal to polysomal translational states following the global inhibition of cap-
dependent translation. Interestingly, two of these mRNAs demonstrated remarkable
differential translational states depending on the AKT activity status of the cell. In cells
with relatively active AKT, the cyclin D1 and c-myc mRNAs were translationally
repressed by mTOR inhibition, while in cells containing quiescent AKT, these transcripts
were well-translated and found in polysome structures following treatment with the drug.
In this report we demonstrate that the human cyclin D1 mRNA can mediate internal
translation initiation. Furthermore, we demonstrate that both cyclin D1 and c-myc IRES
function is stimulated following rapamycin exposure in cells with quiescent AKT activity
but not in cells with activated AKT. Lastly, we show that differential AKT-dependent
cyclin D1 and c-myc IRES activity is dependent on p38 MAPK and RAF/MEK/ERK
signaling.
Experimental Procedures
Cell lines and plasmids- The U87, U87PTEN, LAPC-4puro and LAPC-4AKT cell lines have
been described previously (kind gifts of I. Mellinghoff & C. Sawyers, UCLA) (15). The
murine embryonic fibroblasts (MEFs) in which PTEN was deficient, as well as the
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parental control have also been previously described (16). The cell lines were maintained
in media supplemented with 10% fetal calf serum. We validated the expression of wild-
type cyclin D1 and c-myc mRNAs in all of these cell lines by Northern analysis and
sequencing of the 5’ UTRs of these transcripts which were identical to previously
published sequences (17). The parental construct utilized in these studies was pRF (kind
gift of A. Willis, University of Leicester, UK) (18). The 5’ UTR of the human cyclin D1
mRNA (accession no. NM053056) was amplified from total RNA. Subsequently it was
inserted into the intercistronic region of pRF to generate pRCND1F. The 396 nucleotide
c-myc (18) and 365 nucleotide p27Kip1 IRESes (19) were amplified from I.M.A.G.E.
clones 4667496 and 4298338 respectively, and also cloned into the intercistronic region
of pRF to generate pRmycF and pRp27F. The inserts were also cloned into a
promoterless version of pRF, pRF(-P) (kindly provided by J-T. Zhang, Indiana
University). All constructs were confirmed by sequencing.
Sequence Analysis and Secondary Structure Predictions- BESTFIT (GCG® Wisconsin
Package™, Accelrys) was used to compare the cyclin D1 leader to the 18S rRNA
sequences (accession no. X03205). We used the MFOLD web server to predict
secondary structures for the human cyclin D1 leader using the default settings and the
temperature fixed at 37oC (20).
Transient transfection analysis of dicistronic mRNA reporters- The reporter constructs
were transfected into cells using Lipofectamine Plus (Invitrogen) and normalized for
transfection efficiency by co-transfection with pSVβGal (Promega). Cells were
harvested 24 hours following transfection and Renilla, Firefly luciferase and β-
galactosidase activities determined (Dual-Glo Luciferase & β-Galactosidase assay
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systems, Promega). Luminescence of extracts was determined using a microplate
luminometer (Turner BioSystems, Sunnyvale, CA). Northern analysis was performed as
previously described (10), utilizing a PCR probe specific for sequences within the Firefly
luciferase open reading frame. In experiments where rapamycin (Calbiochem) was used,
cells were transfected and subsequently exposed to 10nM rapamycin for 18 hours after
which, extracts were prepared and luciferase and β-galactosidase activities were
determined.
In vitro translation of dicistronic mRNA reporters- The dicistronic plasmids were
linearized using BamHI and capped RNA transcribed in vitro (mMessage T7
Transcription kit, Ambion). Capped RNA transcripts were used to program extracts of
the indicated cell lines as previously described (21). The translation reactions were
performed with either the cap analog m7GpppG (Ambion) or GTP as indicated.
Western blotting and in vitro kinase assays- Western blotting was performed as
previously described (10). For in vitro kinase assays, MEFs treated with SB203580
(25µM) or PD98059 (25µM) for predetermined time points, harvested and lysed in
buffer contain 20mM HEPES [pH7.5]; 130mM NaCl; 25mM β-glycerolphosphate; 2mM
NaPPi; 2mM EDTA; 10% glycerol; 1mM PMSF; 2.5 µg pepstatin; 2.5µg leupeptin;
2.5µg antipain; 0.5mM dithiothreitol and 1% Triton X-100. Cleared supernatants were
subsequently incubated with either anti-p38 or anti-ERK antibody and protein A-
Sepharose overnight. Pellets were washed three times in lysis buffer and once in kinase
buffer containing 25mM HEPES [pH 7.4]; 25mM β-glycerolphosphate; 25mM MgCl2;
0.5mM EDTA and 0.5mM dithiotheitol. P38 kinase reactions were performed in the
presence of 100 µM ATP and 2 µg ATF-2 as a substrate. Phosphorylation of ATF-2 at
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Thr71 was measured by Western blot using a phospho-specific ATF-2 antibody. ERK
kinase reactions were performed using ELK-1 as a substrate and phosphorylation of
ELK-1 was detected by immunoblotting with a phospho-ELK-1 (Ser383) antibody. P38
MAPK, ERK, phosphor-ATF-2 and phosphor-ELK-1 antibodies were from Cell
Signaling. Statistical analysis was performed by using Student’s t test and ANOVA
models using Sigma Stat 3.0 (Jandel Scientific).
Results
The cyclin D1 and c-myc Leaders Function as IRESes in Transfected Cells in an AKT-
Dependent Manner.
The glioblastoma U87-MG cell line has a PTEN-null mutation with resulting
heightened AKT activity. It was stably transfected with a wild-type PTEN construct
which markedly downregulated AKT activity (15). Similarly, the LAPC-4puro prostate
cancer cell line, with relatively quiescent AKT activity was stably transfected with a
myristylated AKT allele (or empty vector control). The differential AKT/mTOR cascade
activation of these paired isogenic lines has already been described (10, 15). In addition,
for this study, we also utilized murine embryonic fibroblasts (MEFs) in which the PTEN
gene had been disrupted (16). The AKT activity in these MEFs is markedly higher as
compared to PTEN +/+ MEFs (16, 22).
To determine whether the human cyclin D1 leader could internally initiate translation,
it was cloned into the intercistronic region of a dicistronic reporter mRNA and tested in
transiently transfected cells. The c-myc leader, containing a known IRES (23), was also
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cloned into this region. The dicistronic mRNAs used in these studies contain the Renilla
and Firefly luciferases as the first and second cistrons, respectively (pRF). Since we had
previously observed a differential pattern of cyclin D1 and c-myc polysome association
in quiescent as compared to activated AKT-containing cells (10), we investigated the
ability of cyclin D1 and c-myc leaders to initiate translation internally in transiently
transfected cell lines containing differential AKT activities. We transiently transfected
these cell lines with the indicated dicistronic constructs shown in figure 1A. Expression
of the downstream Firefly luciferease in the constructs containing the cyclin D1 5’ UTR
or the c-myc IRES as compared to pRF (empty vector) was enhanced dramatically in
those cell lines containing quiescent AKT activity (U87PTEN, LAPC-4puro, PTEN +/+ MEF
in figures 1B, C and D). For example, in the U87MGPTEN, LAPC-4puro and PTEN +/+
MEF cell lines the presence of the 5’ UTR of cyclin D1 or the c-myc IRES resulted in an
~ 3 to 4 fold increase in Firefly luciferase activity. In contrast, Firefly luciferase activity
was minimally affected in the relatively “active AKT” member cell lines of these
isogenic pairs, indicating that AKT activity prevented cyclin D1 and c-myc IRES
function. Only a modest ~ 1.5 to 2 fold increase was seen in myc-IRES activity and no
increase in CCND1 IRES activity. The presence of the human p27Kip1 IRES-containing
sequences (19, 25), in the dicistronic reporter construct resulted in an ~ 10 fold increase
in Firefly luciferase activity in all of the cell lines tested irrespective of AKT activity.
This was consistent with our earlier observations that the p27Kip1 mRNA was well
translated in the face of mTOR inhibition regardless of the AKT status of the cell (10)
and indicates that p27Kip1 IRES function is independent of AKT activity. It also provides
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a control confirming that reporter expression will occur in the “high-AKT” cell lines if an
IRES is functional.
To evaluate whether the AKT-dependent enhanced translation of the downstream
cistron in the cell lines tested was the result of initiation from shorter monocistronic
transcripts or possibly from cryptic promoter activity, we analyzed the dicistronic
mRNAs via Northern blot and luciferase activities in constructs where the SV40
promoter was absent (pRF(-p), pRCD1F(-p) and pRmycF(-p)) (52). Figure 2A shows a
schematic diagram of the promoterless dicistronic constructs transfected into PTEN -/- and
PTEN +/+ MEFs. As shown in figure 2B, introduction of the promoterless constructs
resulted in minimal luciferase activities in both the PTEN -/- and PTEN +/+ MEFs,
indicating that the AKT-dependent Firefly luciferase expression from these constructs
was not the result of internal promoter activities. Furthermore, Northern analysis of
mRNAs from transfected PTEN -/- or PTEN +/+ MEFs detected only the presence of the
full-length dicistronic transcripts when probed for sequences within the downstream
Firefly luciferase open reading frame (Fig. 2C). The 5’ UTR of cyclin D1 is 208
nucleotides in length, while the c-myc IRES sequences were 396 nucleotides in length.
These data further supported the notion that the cyclin D1 5’ UTR was capable of internal
initiation and that both the IRES activities of the 5’ UTR of cyclin and the c-myc IRES
were regulated by AKT activity.
The Cyclin D1 5’ UTR and c-myc IRES Mediate Internal Initiation in an AKT-
dependent Fashion in Cell-free Extracts
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To rule out that Firefly luciferase reporter expression could be due to unusual
cryptic splicing events, we analyzed the AKT-dependent IRES activities of the cyclin D1
and c-myc mRNAs in cell-free extracts. These extracts were prepared according to
Carroll et al., and demonstrated to have high efficiencies in initiating protein synthesis
(21). Capped dicistronic mRNAs that either lacked (pRF) or contained the cyclin D1
(pRCND1F) 5’ UTR, the c-myc IRES (pRmycF), or the p27Kip1 IRES (pRp27F) were in
vitro transcribed and subsequently used to program translation in lysates from U87MG,
U87MGPTEN, LAPC-4puro, LAPC-4myrAKT, PTEN +/+ MEFs or PTEN -/- MEFs.
Translation of the parent pRF mRNA yielded Firefly luciferase activities which were
indistinguishable from the background obtained from control reaction mixtures that
lacked Firefly luciferase reporter mRNAs. In contrast, as shown in figure 3, an
equivalent amount of either pRCND1F or pRmycF mRNAs generated Firefly luciferase
activities which were ~ 2 to 4 fold higher in extracts prepared from cell lines with
relatively quiescent AKT levels. The Firefly luciferase activities generated from the
pRp27F mRNA were consistently ~ 6 fold higher in all cell extracts tested as compared
to pRF mRNA. This again was consistent with the results from the dicistronic in vivo
experiments demonstrating that the IRES-dependent translation of p27Kip1 was
independent of AKT activity.
To evaluate whether the translation of the cyclin 5’ UTR and the c-myc IRES in
these reporter mRNAs was indeed cap-independent within these cell extracts, in vitro
transcribed and capped mRNAs from pRCND1F and pRmycF were translated in the
presence of increasing concentrations of the cap analog m7GTP. This analog blocks cap-
dependent translation by binding to the initiation factor eIF-4E (24). Using mRNA in
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vitro transcribed from pRF and capped, translation of the Renilla luciferase cistron was
blocked by ~ 90-95% at 150µM of m7GpppG, but was not effected by comparable
concentrations of the non-methylated form of the analog GTP and the translation of the
Firefly cistron was unaffected by m7GpppG (data not shown). As shown in figure 4, with
both the in vitro transcribed and capped mRNAs from pRCND1F and pRmycF, the
translation of the Renilla cistron was inhibited by ~ 80-90% at concentrations of 100µM
m7GpppG or higher in all cell extracts tested. However, the translation of the Firefly
cistron remained consistent in the extracts with relatively high levels of active AKT, and
increased ~ 1.5 to 2.5 fold in extracts from the cell lines with relatively quiescent AKT
levels. This again supported the notion that sequences present within the 5’ UTR of
cyclin D1 mRNA could confer cap-independent initiation and that both this sequence and
the c-myc IRES activities were enhanced under conditions of low AKT activity.
AKT-dependent Cyclin D1 5’ UTR and c-myc IRES activity is enhanced by rapamycin
Since our previous studies (10) demonstrated a stimulation of cyclin D1 and c-myc
polysome association and protein levels by rapamycin under conditions of quiescent
AKT activity, we assessed whether cyclin D1 or c-myc IRES activity would also be
enhanced following rapamycin exposure in cells with quiescent AKT activity. To
address the AKT-dependent affects of rapamycin on cyclin D1 or c-myc IRES function,
we transfected our dicistronic constructs into the cell lines shown in figure 5 and
determined Renilla and Firefly luciferase activities prior to and following rapamycin
exposure. Renilla luciferase activity was reduced by rapamycin ~ 70 to 90 % as
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compared to values obtained in the absence of the drug for each construct tested (data not
shown). However, the results show that in the cell lines with relatively quiescent AKT
(U87PTEN, LAPC-4puro, PTEN +/+ MEFs), rapamycin treatment resulted in an ~ 3 to 6 fold
stimulation of Firefly luciferase activity as compared to values obtained for lines treated
identically containing active AKT. Interestingly, rapamycin exposure also stimulated
p27Kip1 IRES activity ~ 4 to 6 fold in all cell lines irrespective of AKT activity again,
consistent with our previous data and results from others demonstrating resistance of p27
IRESKip1 activity following exposure to the PI3-kinase inhibitor LY294002 (19).
Northern blot analysis further demonstrated that rapamycin had no affect on pRF steady-
state mRNA levels prior to and following rapamycin treatment (data not shown).
Differential AKT-dependent cyclin D1 and c-myc IRES activity is dependent on both p38
MAP Kinase and RAF/MEK/ERK signaling
Since it has been previously demonstrated that c-myc IRES function is dependent
on p38 MAPK activity during apoptosis (23) and both p38 MAPK and ERK signaling
following genotoxic stress (45) we investigated whether these cascades also contributed
to the differential AKT-dependent cyclin D1 and c-myc IRES activity we had observed.
An additional rationale was the known ability of AKT to downregulate p38 (46) and ERK
(53) activity. To determine if differential p38 or ERK signaling could be correlated with
AKT-dependent cyclin D1 or c-myc IRES activity we initially examined the activities of
these kinases in the PTEN -/- and PTEN +/+ MEFs prior to and following rapamycin
exposure. As shown in figure 6A, the basal p38 and ERK kinase activities, as
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determined by in vitro kinase assays, was approximately 3-4 fold higher in the PTEN +/+
MEFs as compared to PTEN -/- MEFs. Additionally, treatment with rapamycin resulted
in a 8 fold induction of p38 and 10 fold induction of ERK activity in PTEN +/+ MEFs
while only modestly increasing basal p38 and ERK activities (~ 1-2 fold) in the PTEN -/-
MEFs. Total p38 and ERK content in the samples was similar demonstrating that
equivalent amounts of material was immunoprecipitated (Fig. 6A). These data suggest
that p38 and ERK signaling is activated by rapamycin exposure in an AKT-dependent
manner and is consistent with the known negative regulatory effects of AKT on p38 and
on ERK (46, 53).
To investigate whether AKT-dependent cyclin D1 and c-myc IRES activity was
regulated by p38 or ERK signaling we planned to transfected PTEN +/+ and PTEN -/-
MEFs with the indicated dicistronic constructs in figure 6B and subsequently treat these
cells with the p38 inhibitor, SB203580, or the ERK inhibitor PD98058. Our preliminary
experiments demonstrated almost complete inhibition of kinase activity using these
inhibitors (data not shown). Treatment of either PTEN +/+ or PTEN -/- MEFs with
SB203580 inhibited basal p38 kinase activity by greater then 95% within 2 hours of
treatment at a concentration of 25 µM. Similarly, treatment of the MEFs with PD98058
inhibited ERK activity by greater then 92% within two hours of treatment at 25µM.
These inhibiting concentrations of SB203580 or PD98059 were found to
significantly affect AKT-dependent IRES function. The experiments in figure 6B and C
are assays performed in the absence or presence of rapamycin, respectively. As shown in
figure 6B, PTEN -/- MEFs expressed relatively little Firefly luciferase activity when
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transfected with the dicistronic constructs containing the cyclin D1 5’ UTR or the c-myc
IRES within the intercistronic regions. However, as previously observed Firefly
luciferase activity was markedly increased (~ 3-4 fold) in the PTEN +/+ MEFs transfected
with pRCND1F or pRmycF. Treatment of these cells with either SB203580 or PD98059
resulted in greater then 75% inhibition of Firefly luciferase activity. The p38 inhibitor
resulted in a modest decrease in Renilla luciferase activity (~5-10% inhibition), while the
ERK inhibitor reduced Renilla luciferase expression by 60-65% in all the constructs
tested at the concentrations used in these experiments (data not shown). Interestingly, the
p27Kip1 IRES was unaffected by treatment with either of the inhibitors and did not
demonstrate differential AKT-dependent activity as shown before.
To address whether these signaling cascades contributed to the rapamycin induced
differential cyclin D1 and c-myc IRES activity we had previously observed, we
performed the same assays in MEFs pre-treated with SB203580 or PD98058 which had
been transiently transfected with the indicated dicistronic constructs in figure 6C. As
shown in the relatively quiescent AKT containing PTEN +/+ MEFs, rapamycin induced
Firefly luciferase expression by approximately four fold relative to control experiments
without the drug. This enhancement of IRES activity was markedly inhibited by pre-
treatment with either SB203580 or PD98059. One hour pre-incubation with either of
these inhibitors resulted in greater than 80% inhibition of the rapamycin induced firefly
luciferase expression in PTEN +/+ MEFs. Renilla luciferase expression was reduced by
rapamycin (~ 65 %inhibition) in all the constructs tested and pre-treatment with
SB203580 or PD98059 in combination with rapamycin did not significantly further
reduce Renilla luciferase expression (data not shown). As before, p27Kip1 IRES activity
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was enhanced by rapamycin, irrespective of AKT activity, but was not affected by
inhibition of p38 or ERK signaling.
Differential cyclin D1 and c-myc protein levels were also assessed in the PTEN
+/+ and PTEN -/- MEFs treated with rapamycin alone and in combination with either the
p38 or ERK inhibitors. As shown in figure 7, treatment of PTEN -/- MEFs with
rapamycin resulted in downregulation of cyclin D1 and c-myc expression, however
PTEN +/+ MEFs maintain or modestly increase expression in response to the drug. This
differential response in cyclin D1 and c-myc expression is ablated by pre-treatment of the
cells with either the p38 or ERK inhibitors prior to exposure to rapamycin. As
determined by densitometry, pre-treatment with SB203580 inhibited cyclin D1 protein
levels in rapamycin treated PTEN +/+ MEFs by 7.5 fold (figure 7, compare lanes 7 and
8), while pre-treatment with the ERK inhibitor PD98059 reduced cyclin D1 expression
by 9 fold in these cells (lanes 11 and 12). Pre-treatment with either SB203580 or
PD98059 reduced c-myc protein expression to below detectable levels in quiescent AKT
containing PTEN +/+ MEFs upon rapamycin exposure (lanes 7 and 8, lanes 11 and 12).
Sequence analysis and secondary structure prediction of the Cyclin D1 5’ UTR
The 5’ UTRs of the major human cyclin D1 and c-myc mRNAs are 209 and 400
nucleotides, respectively. Both leader sequences contain the hallmarks of 5’ UTRs from
other mRNA demonstrated to exhibit IRES activity. Both leaders are relatively long,
highly structured and contain upstream AUG or CUG initiation codons (17, 26). While a
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model of the c-myc IRES structure has been described (27), the leader of the human
cyclin D1 mRNA has not been characterized in this regard.
Some mRNAs capable of internal translation initiation have been shown to contain
sequence complementarity to 18S ribosomal RNAs (28, 29). It has been proposed that
these regions of complementarity may serve as cis-acting elements involved in the direct
recruitment of ribosomal 40S subunits to mRNAs and possibly regulate cap-independent
translation (30). To address whether the cyclin D1 leader contained regions of sequence
complementarity to 18S ribosomal RNAs, we performed sequence comparisons.
Comparisons of 18S ribosomal RNAs and the cyclin D1 5’ UTR identified several
complementary sequence matches (see supplemental data). Seven of these regions
ranged from 80% to 94% similarity to 18S rRNA over 11-22 nucleotides for the cyclin
D1 leader.
A secondary structural model of the cyclin D1 5’ UTR was derived by free energy
calculations using the MFOLD algorithm (31). Thirteen structures were calculated which
ranged in initial free energies (dG) from –71.5 to –88.9 kcal/mole. The most stable
predicted structure is shown as supplemental data. The structure is highly complex with
several long stems, junctions and higher order bifurcations. Taken together, these data
support the notion that the cyclin D1 5’ UTR contains a bona fide IRES element.
Discussion
Our previous studies (10) suggested that the cyclin D1 and c-myc mRNAs are
transcripts which could be effectively translated under conditions of reduced cap-
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dependent initiation. In this report we have demonstrated that under specific signaling
conditions, the leader of the human cyclin D1 mRNA can initiate protein synthesis via an
IRES. While the cyclin encoded by the Kaposi’s sarcoma-associated herpes virus has
also been reported to contain an IRES (37), to our knowledge, this is the first report of
this mRNAs ability to initiate translation internally. We have also shown that AKT
activity regulates cyclin D1 and c-myc IRES function and demonstrated that rapamycin
increases cyclin D1 and c-myc IRES activity in an AKT-dependent manner. These
results are consistent with our previously observed AKT-dependent effects on cyclin D1
and c-myc protein synthesis following rapamycin exposure. Furthermore, we have
extended our studies by implicating the p38 MAPK and RAF/MEK/ERK signaling
cascades in the regulation of AKT-dependent cyclin D1 and c-myc IRES activity. Our
results support a working model in which the AKT-dependent control of cyclin D1 and c-
myc IRES function in response to rapamycin may regulate the expression of these critical
determinants resulting in either G1 arrest or tumor cell survival. When AKT activity is
relatively low, rapamycin treatment results in the inhibition of cap-dependent translation,
but stimulates the selective translation of cyclin D1 and c-myc via their IRESes mediated
via p38 MAPK and RAF/MEK/ERK signaling, thus maintaining expression. However,
when AKT is elevated, the rapamycin-induced inhibition of cap-dependent translation is
not associated with enhanced IRES function, most likely due to the negative regulatory
affects of AKT activity on the p38MAPK and RAF/MEK/ERK pathways, thus, cap-
independent translation is prevented and protein levels fall. This differential regulation
of cap-independent translation and overall cyclin D1/c-myc expression accounts for the
differential sensitivity of “high versus low” AKT-activity cell targets.
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An interesting question arises; under what circumstances might there be a
requirement for IRES-mediated translation initiation of cyclin D1 mRNA, particularly,
when cyclin D1 expression has been shown to be dependent on eIF-4E (38). Recent data
suggest that cyclin D1 also normally accumulates during the G2 phase of the cell cycle
and synthesis during this phase may contribute to rapidly achieving levels of cyclin D1
required for the ensuing G1 transit in actively proliferating cells (39). It has also been
recently appreciated that there is a reduction in cap-dependent protein synthesis during
the G2/M cell cycle transition (40, 41) and interestingly, it is known that both AKT
activity and protein levels transiently drop during the G2/M transition (42). While it has
been demonstrated that post-translational mechanisms contribute to the accumulation of
cyclin D1 during G2 (43, 44), it is also possible that the IRES-mediated synthesis of
cyclin D1 normally occurs during this phase of the cell cycle and supplements
expression.
Our data imply that the factor(s) responsible for AKT-dependent cyclin D1 and c-
myc IRES function are downstream of p38 and ERK. This is consistent with the results
of others who have demonstrated roles for these effectors in regulating the IRES-
mediated synthesis of c-myc during apoptosis or in response to genotoxic agents (23, 45).
It is possible that these effectors regulate cyclin D1 and c-myc IRES activity, direct or
indirectly, via phosphomodulation of an IRES trans-acting factor(s) (ITAF). Recently,
three members of the poly(rC)-RNA binding family, PCBP1, PCBP2 and hnRNPK have
been shown to be required for c-myc IRES activity and stimulate IRES-mediated
translation when overexpressed and bound to the mRNA (47). Moreover, it is known that
the activity of these proteins is regulated by phosphorylation (48-50). Alternatively, p38
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or ERK activity may lead to changes in ITAF expression thereby affecting IRES
function. Along these lines it has been demonstrated that the expression of PCBP1 under
hypoxic conditions is dependent on p38 activity in cortical neurons (54). Experiments
designed to address these questions are currently in progress.
The observation that p27Kip1 IRES activity was not AKT-dependent following
rapamycin exposure is interesting and suggests that the regulation of this IRES is similar
to but distinct from the cyclin D1 and c-myc IRESes in this setting. P27Kip1 IRES
function may be regulated by a specific ITAF(s) which enhances its function following
rapamycin exposure, but is nonresponsive to changes in p38, ERK or AKT activities.
The p27kip1 IRES has been demonstrated to be active under conditions of elevated cyclic
AMP (25) and repressed by the neuronal ELAV HuD (19), while enhancement of c-myc
IRES activity has been shown to be dependent on PCBP1, PCBP2 and HnRNPK (47). It
is certainly possible that the factors mediating cap-independent translational control of
p27kip1 are distinct from those regulating other cellular IRESes.
Our data also suggest that the ability of tumor cells to respond to mTOR inhibitors
by stimulating cap-independent mechanisms of initiation of critical cell cycle proteins
may constitute a mechanism of cellular resistance to these drugs. In particular, tumors
which have relatively little dependence on the PI3-K/AKT/mTOR signaling cascade (i.e.
low AKT activity) appear to markedly increase the cap-independent synthesis of cyclin
D1/c-myc following mTOR inhibitor exposure. Further understanding the mechanisms
regulating the expression of these determinants may assist in the development of
compounds which function in a synthetically lethal manner with mTOR inhibitors.
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Acknowledgements- We thank I. Mellinghoff, C. Sawyers, A. Willis and J-T. Zhang for
providing cell lines and plasmids. We also thank M. Rettig for helpful discussions and
critical reading of the manuscript. This work was supported by research funds of the
Veteran’s Administration including the Research Enhancement Awards Program (REAP)
entitled “Cancer Gene Medicine” and NIH CA096920.
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Figure Legends
FIG. 1. AKT-dependent cyclin D1 and c-myc IRES activity in vivo. A. Schematic diagrams of the dicistronic constructs used in this study. Constructs used are pRF, pRCD1F, which contains the 5’ UTR of human cyclin D1, pRmycF, containing the human c-myc IRES, and pRp27F, containing the human p27Kip1 IRES. B. Normalized (to values obtained for pRF alone) Renilla (white bars) and Firefly (black bars) luciferase activities of U87 and U87PTEN cells transfected with the indicated constructs. C. & D. are identical to B except for the cell line used as indicated. Transfection with each construct was performed in quadruplicate with each construct in each indicated cell line. FIG. 2. Transfection experiments with promoterless dicistronic mRNA reporter constructs in vivo. A. Diagram of promoterless constructs. pRF(-p), pRCD1F(-p) and pRmycF(-p) are identical to the constructs in FIG. 1A, except the SV40 promoter sequences have been removed (51). B. Relative luciferase activities of the indicated promoterless construct transfected into PTEN -/- MEF or PTEN +/+ MEF. C. Northern analysis of dicistronic mRNAs in PTEN -/- MEF or PTEN +/+ MEF. The positions of the 28S and 18S ribosomal RNAs are shown. FIG. 3. AKT-dependent cyclin D1 and c-myc IRES activity in cell-free extracts. In vitro transcribed dicistronic RNAs from the indicated constructs were used to program translation in cell-free extracts in the three cell line pairs shown. Renilla and Firefly luciferase activities of U87 vs. U87PTEN A, LAPC-4myrAKT vs. LAPC-4puro B and PTEN -/- MEF vs. PTEN +/+ MEF C translation extracts. Values were normalized to those obtained for pRF alone and the data are representative of three independent experiments for each cell line. FIG. 4. Effects of cap-analog on AKT-dependent cyclin D1 and c-myc IRES activity in vitro. Cell-free extracts of the three paired cell lines were programmed to translate in vitro transcribed RNA from either pRCND1F (boxes) or pRmycF (circles) in the presence of the cap analog m7GpppG. Changes in Renilla (unshaded boxes, unshaded circles) and Firefly (shaded boxes, shaded circles) luciferase activities are shown relative to activities obtained in the absence of m7GpppG. A. U87 vs. U87PTEN, B. LAPC-4myrAKT vs. LAPC-4puro, C. PTEN -/- MEF vs. PTEN +/+ MEF. Experiments were repeated three times with similar results. FIG. 5. Rapamycin stimulation of cyclin D1 and c-myc IRES activities are dependent on AKT status. The indicated cell lines were transfected with pRF, pRCD1F, pRmycF or pRp27F and treated with 10nM rapamycin. Relative fold change in Firefly luciferase activity is shown as compared to activities obtained in the absence of rapamycin and
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normalized to values obtained for pRF in each cell line. Data are representative of three similar individual experiments. FIG. 6. AKT-dependent cyclin D1 and c-myc IRES activity requires p38MAPK and ERK signaling. A. p38MAPK and ERK activities in PTEN -/- and PTEN +/+ MEFs prior to and following rapamycin exposure. In vitro kinase reactions were immunoblotted for the indicated phosphorylated substrate and total p38 or ERK protein levels. B. PTEN -/- and PTEN +/+ MEFs were transfected with the indicated dicistronic constructs and treated with either SB203580 or PD98059. Changes in Firefly luciferase (cap-independent) expression are shown. Values were normalized to pRF without treatment and were performed in triplicate. C. PTEN -/- and PTEN +/+ MEFs were transfected with the indicated dicistronic constructs and pre-treated with either SB203580 or PD98059 for 1 hour and subsequently exposed to rapamycin (rapa) as shown. Relative fold changes in Firefly (cap-independent) luciferase activities are shown as compared to activities obtained in the absence of rapamycin. Data are representative of three independent experiments. FIG. 7. Effects of p38MAPK and ERK inhibitors on AKT-dependent cyclin D1 and c-myc protein expression following exposure to rapamycin. Cyclin D1, c-myc and actin protein levels were determined in PTEN -/- or PTEN +/+ MEFs subsequent to rapamycin treatment alone or in combination with SB203580 or PD98059 as indicated. by guest on A
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p27 IRES
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Figure 1
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28S-
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Figure 2
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Figure 3
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U87PTENU87 LAPC-4AKT LAPC-4puro PTEN+/+PTEN-/-
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Figure 5
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APTEN +/+PTEN -/-
- + - + rapamycin
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Figure 6
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PTEN+/+PTEN-/-
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Figure 7
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YiJiang Shi, Anushree Sharma, Hong Wu, Alan Lichtenstein and Joseph GeraERK-dependent pathway
regulated by AKT activity and enhanced by rapamycin through a p38 MAPK and Cyclin D1 and c-Myc internal ribosome entry site (IRES)-dependent translation is
published online January 4, 2005J. Biol. Chem.
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