Page 1 of 37Articles in PresS. Am J Physiol Cell Physiol (May 9, 2007). doi:10.1152/ajpcell.00499.2006
S6 KINASE INACTIVATION IMPAIRS GROWTH AND TRANSLATIONAL
TARGET PHOSPHORYLATION IN MUSCLE CELLS MAINTAINING
PROPER REGULATION OF PROTEIN TURNOVER
Virginie Mieulet 1,2, Mila Roceri 1,2, Catherine Espeillac 1,2, Athanassia Sotiropoulos 1,2,
Mickael Ohanna 1,2, Viola Oorschot 4, Judith Klumperman 4, Marco Sandri 3, Mario
Pende 1,2
1
INSERM, U845, Paris, F-75015, France
2
Université Paris Descartes, UMRS-845, Paris, F-75015, France
3
Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine, Via Orus
2, 35129 Padova, Italy.
4
Cell Microscopy Center, Department of Cell Biology, University Medical Center
Utrecht and Institute of Biomembranes, 3584CX Utrecht, The Netherlands.
Running title: S6 kinases and muscle protein turnover
Address correspondence to: Mario Pende, Tel: 0033 1 40 61 53 15, Fax: 0033 1 43 06 04 43, E-mail:
[email protected]
Copyright © 2007 by the American Physiological Society.
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A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)
deficient cells are small and display a blunted response to nutrient and growth factor
availability, we have hypothesized that mutant cell atrophy may be triggered by a change in
global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the
mTOR/S6K pathway, here we evaluate the control of translational target phosphorylation and
protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The
phosphorylation of ribosomal protein S6 (rpS6), eIF4B and eEF2 are predominantly regulated
by mTOR in muscle cells. Conversely in liver the MAPK and PI3K pathways also play an
important role, suggesting a tissue specific control. S6K deletion in muscle mimics the effect of
the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2
phosphorylation. To gain insights on the functional consequences of these modifications,
methionine incorporation and polysomal distribution are assessed in muscle cells. Rates and
rapamycin sensitivity of global translation initiation are not altered in S6K deficient muscle
cells. In addition two major pathways of protein degradation, autophagy and the expression of
the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our
results do not support a role of global translation control in the growth defect due to S6K
deletion, suggesting specific modes of growth control and translational target regulation
downstream of mTOR.
Keywords: signal transduction; atrophy; autophagy.
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The phosphorylation of transcription and translation factors is a common mechanism to ensure
rapid and long-term changes in gene expression depending on the environmental cues. In the 40S
ribosomal subunits, the phosphorylation of ribosomal protein S6 (rpS6) was demonstrated more than
twenty years ago in eukaryotic cells after exposure to growth factors, mitogens and nutrients (12; 48;
49). RpS6 is phosphorylated in sequential order on five serine residues (Ser-236, Ser-235, Ser-240,
Ser-244, Ser-247) in the carboxy-terminal tail of the protein (24). The kinases catalyzing this reaction
have been initially identified as two isoforms, p70 and p85 S6 Kinases, encoded by a single gene,
S6K1 (22). However the genetic inactivation of S6K1 in mice does not impair rpS6 phosphorylation
due to the existence in the mammalian genome of a homologous gene coding for S6K2, that also has
S6 kinase activity (46). The combined deletion of S6K1 and S6K2 (S6K1;S6K2-/-) strongly reduces,
but not abrogates, rpS6 phosphorylation, demonstrating that both kinases are the main regulators of
this event in vivo (34).
In resting cells S6K1 is tethered to a translation preinitiation complex comprising the
eukaryotic Initiation Factor 3 (eIF3) (16). When anabolic signals are received, S6K1 is phosphorylated
by the mammalian Target Of Rapamycin (mTOR) and PDK1 protein kinases (23). These
modifications switch on the catalytic activity of S6K1 that dissociates from the eIF3 subunits and in
turn phosphorylates multiple translational targets (16). The S6K1 substrates include not only rpS6, but
also the eukaryotic Initiation Factor 4B (eIF4B) at Ser-422 and the eukaryotic Elongation Factor 2
kinase (eEF2K) at Ser-366 that are also involved in the translational machinery (35; 52). These
substrates share a consensus sequence with two Arginine residues at the -3 and -5 position from the
phospho-serine. Of note, the MAPK-dependent p90 ribosomal protein S6 kinases (Rsk) recognize a
similar substrate consensus sequence and can partly compensate for the loss of S6K activity (34; 44;
52). Therefore, the combination of the mTOR inhibitor rapamycin and MAPK inhibitors is required to
abrogate phosphorylation of rpS6, eIF4B and eEF2K, indicating a certain degree of redundancy in this
regulation by the mTOR/S6K and MAPK/Rsk pathways.
Studies defining the functional consequences of these post-translational modifications have
largely relied on mTOR inhibition by rapamycin. Rapamycin has multiple effects on protein synthesis,
as it decreases amino acid uptake, cap-dependent translation initiation and ribosome biogenesis while
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increasing protein degradation by autophagy and ubiquitination (7; 53). The effect on translation
initiation is especially evident for mRNAs carrying a long 5’ untranslated region (5’UTR) or a 5’
terminal oligopyrimidine tract (5’TOP) (1; 19). However mTOR activity affects a variety of molecular
targets in addition to S6K, such as the eIF4E-binding proteins (4EBPs), eIF4G and eIF2 (53). Thus it
remains to be demonstrated whether S6 kinases and their substrates are directly involved in any of
mTOR actions on protein synthesis. Overexpression of a rapamycin-resistant mutant allele of S6K1
protects against the inhibitory effect of rapamycin on cap-dependent translation and 5’TOP mRNA
recrutment onto polysomes (18). Inconsistent with these findings, the combined deletion of S6K1 and
S6K2 in mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells affects neither global
translation initiation, as judged by the polysomal profile, nor 5’TOP mRNA translation (34). In
addition the substitution of the five phosphorylatable rpS6 serine residues to alanine does not alter
5’TOP mRNA translation and surprisingly increases translation initiation rates in MEFs (38). The
overexpression of a phosphomimetic eIF4B mutant stimulates or inhibits cap-dependent translation
depending on the reporter mRNA and experimental conditions (16; 35). The role of the S6K pathway
in long-lived protein degradation by autophagy is also unfirm. RpS6 dephosphorylation correlates with
the induction of autophagy in rat livers during fasting (2; 45). However Drosophila S6K loss-offunction mutants do not display increased autophagy in standard feeding conditions and are actually
protected against starvation-induced autophagy (43). Clearly, further studies are needed to address the
contribution of S6K to protein synthesis and degradation.
S6K deletion in Drosophila fruitflies and mice decreases organismal size due to a specific
defect in cell size, highlighting an essential role for S6K in cell growth control (27; 46). S6K1;S6K2-/mice display an increased frequency of perinatal lethality and a 20% reduction of body weight (34). Of
note, S6K1 deletion is sufficient to cause the small size phenotype, suggesting that S6K2 activity does
not participate in growth control. Although the weight of all organs is reduced, S6K1-/- adipose tissue,
skeletal muscles and pancreatic beta cells are predominantly affected (31; 33; 50). Importantly, S6K1
deletion in skeletal muscle cells mimics the inhibitory action of rapamycin and amino acid starvation
on cell size while blunting the hypertrophic effects of insulin like growth factor 1 (IGF1) and
constitutively active Akt (MyrAkt) (31). Since recent studies have proposed that skeletal muscle mass
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results from a balance between protein synthesis and degradation (9), it is tempting to speculate that
S6K1 may control cell growth through the regulation of one or more mTOR-dependent targets in
protein turnover. To address this question, here we evaluate whether S6K1 and/or S6K2 deletion is
sufficient to affect the phosphorylation of translational targets in muscle and liver cells. Since we
uncover a muscle specific control of translational target phosphorylation by S6K, we measure
molecular markers of protein synthesis and degradation in S6K muscle cells, and perform functional
assays. Our results do not support the current model of cell growth control by S6K1 through global
protein synthesis regulation, and instead indicate distinct atrophy programs upon inactivation of
specific mTOR targets.
Experimental Procedures
Animals and in vivo procedures - Generation of S6K deficient mice has been previously described (31;
34). For in vivo analysis of protein expression, five week old male mice were either sacrificed after 48
h starvation, or after 48 h starvation plus 1 h stimulation with oral administration of 1.4 g/kg leucine
(Sigma) and intraperitoneal injection of 1U/kg insulin (Sigma), or after 48 h starvation plus 1 h normal
chow refeeding. For in vivo analysis of atrophy-related gene expression, five week old male mice were
either sacrificed at the randomly fed state, after 48 h starvation, or after 48 h starvation plus 48 h
normal chow refeeding. Denervation induced-muscle atrophy of lower limb muscles was achieved by
severing the right sciatic nerve in the mid-thigh region in anaesthetized two month old male mice. Left
sciatic nerves were exposed but not severed to serve as sham-operated controls. Mice were sacrificed
after 3, 7 or 12 days and gastrocnemia muscles were weighed and frozen into liquid nitrogen. Animal
procedures were approved by the French Ministery of Agriculture (authorisation number #75-42).
Mice were sacrificed by cervical dislocation.
Cell cultures - Myoblast primary cultures were derived from gastrocnemia and tibialis anterior
muscles of 4 weeks old male mice, as described (31). Cells were plated on 0.02% gelatin-coated
dishes (Type A from pig skin; Sigma), grown in complete medium composed of DMEM/Ham F12
(Gibco), 2% Ultroser G (Biosepra), 20% fetal calf serum (Gibco), penicillin, streptomycin and L-
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glutamine. To differentiate muscle cells into myotubes, myoblasts were plated on Matrigel-coated
dishes (Becton Dickinson) in complete medium and after 6 h adhesion they were switched to
DMEM/Ham F12 + 2% horse serum. To measure the effect of IGF1 and/or rapamycin, myotubes at
day 2 of differentiation were incubated with 250ng/ml of IGF1-R3 (Sigma) ± 20 nM Rapamycin
(Calbiochem) for a further 2 days in DMEM/Ham F12 + 2% Horse serum. Primary hepatocytes from
12- to 14-week-old male mice were isolated by liver perfusion as described (34). Hepatocytes were
plated at 12 × 104 cells/ cm2 in Primaria dishes (Falcon) in M-199 medium (Invitrogen) supplemented
with 10% fetal bovine serum (FBS, Invitrogen), 0.1% BSA, and 100 nM dexamethasone. After 3 h of
adhesion, cells were incubated in serum-free M-199 medium containing 0.1% BSA for 48 h and then
in amino acid- and glucose-free DMEM + 0.02% BSA for 3 h. Hepatocytes were stimulated for 1 h in
DMEM with growth factors corresponding to 10% FBS + 1 µM insulin (Sigma) + 4 nM EGF (Sigma)
with or without 30 min pre-treatment with 20 nM rapamycin (Calbiochem), 10 µM U0126
(Calbiochem) and 1 µM wortmannin (Calbiochem). All media contained penicillin (100 U/ml),
streptomycin (100 µg/ml), and amphotericin B (Fungizone) (250 ng/ml). To quantify hepatocyte size,
cells were fixed for 10 minutes with 4% paraformaldehyde and stained for 10 minutes with 10%
Giemsa solution in PBS. Cell size was then analysed on a Nikon E800 microscope equipped with a
video camera and Lucia archive software.
Immunoblotting - For immunoblot analysis, cells were washed twice with cold phosphate buffered
saline (PBS), then scraped from the culture dish into lysis buffer B (50 mM Tris pH 8, 1% NP40, 120
mM NaCl, 20 mM NaF, 1 mM benzamidine, 1 mM EDTA, 1 mM EGTA, 15 mM sodium
pyrophosphate, 1 mM PMSF, 2 mM sodium orthovanadate, 2 µg/ml leupeptin, 5 µg/ml aprotinin).
Homogenates were sonicated for 6 min and were spun at 8000 x g for 10 min at 4°C to remove cell
debris. Muscle and liver tissues were frozen in liquid nitrogen and powdered manually with a mortar
and pestle. A volume of glass beads (Sigma G-8772) and a volume of lysis buffer (20 mM Tris pH 8,
5% glycerol, 138 mM NaCl, 2.7 mM KCl, 1% NP40, 20 mM NaF, 18 mM pepstatin A, 5 mM EDTA,
1 mM sodium orthovanadate, 20 mM leupeptin, 1 mM dithiothreitol), equivalent to the tissue volume
were added. The homogenates were vortexed 10 sec and put back on ice for 10 sec; this was repeated
four times. The samples were then agitated for 15 min at 4°C, until complete lysis, and spun at 8000 x
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g for 10 min at 4°C, to remove fat and debris. Protein extracts were resolved by sodium dodecyl
sulfate-PAGE before transfer to nitrocellulose membranes (Protran) and incubation with the following
primary antibodies: anti-phosphorylated Ser235/Ser236 rpS6, anti-phosphorylated Ser422 eIF4B, antieIF4B, anti-phosphorylated Thr56 eEF2, anti-phosphorylated Thr389 p70S6K, anti-phosphorylated
Thr359/Ser363 p90Rsk (Cell Signaling Technology), anti-LC3 (a kind gift from Mizushima N.), antip70S6K (C-18) (Santa Cruz Biochemicals).
Analysis of protein synthesis by [35S]-methionine and [35S]-cysteine incorporation - Muscle cells
(20,000 cells/cm² were plated on matrigel-coated 6 well plates in complete medium. After 4 days of
differentiation in DMEM/HamF12 + 2% horse serum, cells were washed twice with cysteine- and
methionine-free DMEM. Then, 1ml of this DMEM was added, together with 2% dialyzed-horse serum
and 40 µCi of [35S]-methionine and [35S]-cysteine (Promix, Amersham) for 2 h at 37°C. The amount
of radiolabel was saturating, as the amount in the medium did not decrease significantly during the 2 h
incubation. The protein synthesis rate was determined as described (6). Briefly, the cells were washed
twice in ice-cold PBS and radiolabel incorporation into protein was assessed by lysing the cells in
100µl of 0.2% Triton and then removing three aliquots of 10 µl. Two aliquots were counted in a
scintillation counter to evaluate the label in total cell lysate. The third aliquot was used for protein
quantitation by the colorimetric DC Protein Assay (Bio-Rad). 100% ice-cold trichloroacetic acid
(TCA) was added to the remaining 70 µl to a final concentration of 10% to precipitate the protein.
After 10 minutes on ice, the solutions were spun at 12000 x g to pellet the precipitated proteins, and
the amount of free radiolabel was assessed by removing two aliquots of the supernatant and counting
them in a scintillation counter. The amount of radiolabel incorporated into protein was calculated by
subtracting the value of the non-incorporated label from the value of the label in the total cell lysate.
Protein/DNA content - This experiment was done in parallel with the analysis of protein synthesis by
[35S]-methionine and [35S]-cysteine incorporation. Cells (20,000 cells/cm² were plated on matrigelcoated 6 well plates in complete medium. After 4 days of differentiation in DMEM/HamF12 + 2%
horse serum, cells were washed twice in ice-cold PBS, scraped in 400µl of PBS, and spun 3300 x g for
5 min at 4°C. Lysis buffer (100 µl; 0.1% NP-40, 0.5 mM EDTA) was added to the cell pellet and the
samples were frozen into liquid nitrogen, thawed and sonicated for 5 min. The DNA content was
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evaluated using the fluorescent DNA quantitation kit (Biorad). Briefly, Hoechst 33258 dye was added
to an aliquot. The dye bound to dsDNA was measured with a fluorometer (excitation 360 nm;
emission 460 nm). Salmon sperm DNA was used for concentration standard curves. The protein
quantitation was evaluated on an aliquot of the same sample using bovine serum albumin as standard.
Polysome fractionation - Sucrose density gradient centrifugation was employed to separate the
subpolysomal from the polysomal ribosome fractions. Cells (25000 cells/cm² were plated in matrigelcoated 15 cm plates in complete medium. After 4 days of differentiation in DMEM/HamF12 + 2%
horse serum, myotubes were washed twice in hypotonic buffer (5 mM Tris-HCl pH 7.5; 1.5 mM KCl,
2.5 mM MgCl2). The cells were scraped in 400µl of extraction buffer (5 mM Tris-HCl pH 7.5; 1.5 mM
KCl, 2.5 mM MgCl2, 0.5% Triton X-100, 0.5% sodium deoxycholate, 120 U/ml RNase inhibitor
(Promega), 3mM dithiothreitol). The lysates were spun at 3000 x g for 8 min at 4°C. An aliquot of the
supernatant was removed to measure protein concentration. Heparin was added to the supernatants at a
final concentration of 1 mg/ml. The extracts were rapidly frozen into liquid nitrogen and stored at 80°C. The same amount of protein for each sample (800 µg) was adjusted to the same final volume
(600 µl max) and was layered on a 0.5–1.5 M linear sucrose gradient (20 mM Tris-HCl pH 7.5, 80 mM
NaCl, 5 mM MgCl2, 1 mM dithiothreitol) and centrifuged in a SW41 rotor at 36000 rpm for 2 h at 4
°C. Following centrifugation, using the density gradient fractionation system (Isco), the gradient was
displaced upward through a flow cell recording absorbance at 260 nm.
Quantitative real time PCR - Muscle tissues were frozen in liquid nitrogen and powdered with a
mortar and a pestle. Total RNA from muscle cells was prepared using TriZol reagent according to the
manufacturer’s protocol (Life Technologies, Paisley, UK). Single strand cDNA was synthesized from
400 ng of total RNA with random hexamer primers and the reverse transcriptase Superscript II
(Invitrogen). Quantitative real time PCR (QRT-PCR) was performed using a LightCycler instrument
(Roche Diagnostics) according to the manufacturer’s instructions and using the quantitect SYBR
Green kit (Qiagen). Briefly, the final volume of 20 µl contained 8 ng of cDNA, 50 µM of primer mix,
and 10 µl of quantitect SYBR Green kit. The reactions were carried out in capillaries (Roche
instruments) for 40 cycles. We determined the relative amounts of the mRNAs studied by means of
the second-derivative maximum method, with LightCycler analysis software version 3.5 and beta-
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actin as the housekeeping gene for all studies. The murine primer sequences used were: beta-actin
forward 5’-CTGGCTCCTAGCACCAAGAT-3’, reverse 5’-GGTGGACAGTGAGGCCAGGAT-3’,
atrogin-1
forward
5’-GGCGGACGGCTGGAA-3’,
CAGATTCTCCTTACTGTATACCTCCTTGT-3’,
MuRF-1
reverse
5’-
forward
5’-
ACAACCTGTGCCGCAAGTG-3’, reverse 5’-AGGACAACCTCGTGCCTACAAG-3’. The results
of QRT-PCR were given in arbitrary units and expressed as fold changes in mRNA levels, relative to
wild type controls.
Electron microscopy - For electron microscopy of EDL muscle tissues we used conventional fixationembedding procedures based on glutaraldehyde-osmium fixation and Epon embedding, as previously
described (40). For electron microscopy of myotube cultures, cells were fixed in 2% glutaraldehyde in
0.1M cacodylate buffer, pH7.2, for 2 hours at room temperature. After rinsing with cacodylate buffer,
cells were post-fixed in a mixture of 1% OsO4 and 1% K4Ru(Cn)6 for 2 hours at 4°C, stored overnight
in 70% ethanol and dehydrated through a series of increasing ethanol concentrations. After embedding
in Epon according to the standard protocol, ultrathin sections were obtained and counterstained with
lead citrate and uranyl acetate. At least 25 non-overlapping fields of the sections were photographed at
the final magnification of x10,000. Autophagosome, mitochondria and cytosol volume densities were
quantified by point counting morphometry using a 1 cm grid.
RESULTS
It was previously demonstrated that the growth factor activated mTOR/S6K and MAPK/Rsk
pathways converge on the regulation of rpS6, eIF4B and eEF2K (34; 44; 52). To evaluate whether
S6K deletion was sufficient to affect these putative S6K-translational targets, phosphorylation of rpS6
Ser-235/236, eIF4B Ser-422 and eEF2-Thr56 were measured in mouse tissue in vivo one hour after
administration of insulin and leucine. The phosphorylation state of eEF2-Thr56, the eEF2K site,
provides a readout of eEF2K activity that is negatively controlled by the growth factor pathways (52).
In this study we limited our analysis to skeletal muscles and liver, two major targets of the insulin and
nutrient signaling pathways. The combined deletion of S6K1 and S6K2 genes strongly attenuated the
insulin- and leucine-induced eIF4B phosphorylation and completely abrogated rpS6 phosphorylation
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in gastrocnemius muscle (Fig. 1a). Surprisingly, these conditions were not sufficient to affect eEF2
phosphorylation, precluding the in vivo analysis of this event. These data indicate that S6Ks are the
main kinases responsible for rpS6 and eIF4B phosphorylation in skeletal muscles after leucine and
insulin administration.
The deletion of S6K1 or S6K2 has differential effects on muscle growth that correlates with
S6K1 but not S6K2 activity (31). Therefore we addressed whether the two S6K family members
differentially regulated these putative S6K-translational targets. The effect of the single deletion of
S6K1 or S6K2 on rpS6 and eIF4B was less pronounced than the combined deletion, indicating that
both kinases participate in this regulation (Fig. 1a). The levels of eIF4B phosphorylation were
comparable in S6K1-/- and S6K2-/- muscles while rpS6 phosphorylation was more affected by the S6K2
deletion. Thus the growth responses of the distinct S6K genotypes do not correlate with the
phosphorylation status of rpS6 and eIF4B.
We next evaluated these post-translational modifications in liver from the same mice after
insulin and leucine administration. RpS6 phosphorylation was abolished in liver as well as in muscle
lacking both S6Ks (Fig. 1b). Conversely in liver from S6K1;S6K2-/- mice, the eIF4B phosphorylation
was largely intact. Thus rpS6 is a preferential S6K substrate in both muscle and liver tissues while
eIF4B phosphorylation is predominantly carried by S6K1/S6K2 in skeletal muscles and by an S6Kindependent pathway in liver.
To further strengthen our observations, we set-up primary cultures of skeletal muscle and liver
tissues from wild type and S6K1;S6K2-/- mice, and combined the genetic deletions with
pharmacological inhibitors of the growth factor pathways. Both cell types were stimulated with
nutrients, serum and growth factor peptides to reach a maximal response. In wild type muscle cells
rapamycin treatment inhibited rpS6 and eIF4B phosphorylation, confirming that these translational
targets are under control of the mTOR pathway in muscle (Fig. 2a). In addition rapamycin increased
eEF2 phosphorylation, revealing in cultured cells a negative control of eEFK activity by the mTOR
pathway that was not evident in vivo. In S6K1;S6K2-/- myotubes, basal and growth factor stimulated
rpS6 phosphorylation was abrogated. Although eIF4B phosphorylation was attenuated as compared to
wild type cells, it was not abolished and it remained sensitive to growth factor stimulation (Fig. 2a and
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2b). Of note, rapamycin had no effect on basal and growth factor-induced eIF4B phosphorylation in
S6K1;S6K2-/- myotubes, indicating the involvement of an mTOR-independent pathway that partially
compensates for the loss of S6K activity. In contrast, the sensitivity of eEF2 phosphorylation to
rapamycin did not differ in wild type and S6K1;S6K2-/- myotubes. Taken together, these data
demonstrate that S6 kinases regulate rpS6 and eIF4B phosphorylation in muscle cells whereas eEF2K
activity is under the control of an mTOR-dependent but S6K-independent mechanism. S6K deletion is
sufficient to cause a dramatic loss of rpS6 phosphorylation, while eIF4B phosphorylation is partially
rescued by an mTOR-independent pathway.
In hepatocytes, the phosphorylation of rpS6 was largely but not completely dependent on
mTOR and S6K activities (Fig. 2b). As previously reported (34), the residual rpS6 phosphorylation in
rapamycin treated and S6K deficient cells was blocked by the MAPK inhibitor U0126, suggesting that
Rsk participates to rpS6 phosphorylation but to a lesser extent than S6K. In contrast to muscle cells,
in wild type liver cells rapamycin did not affect the control of eEF2 and eIF4B phosphorylation by
growth factors, suggesting that in liver the mTOR pathway is not predominant over the other pathways
regulating these targets (Fig. 2b; (44)). Consistent with these findings, eIF4B and eEF2 regulation was
not impaired in S6K deficient hepatocytes. Altogether, these findings confirm the in vivo data,
demonstrating that rpS6 is a preferential S6K substrate as compared to eIF4B. Moreover the
contributions of mTOR -dependent and -independent pathways on these phosphorylation events vary
between distinct tissues.
Adult hepatocytes in culture comprised a population of mononucleated and binucleated cells.
S6K deletion significantly decreased size of binucleated cells (3476 ± 192 µm2 surface of wild type vs.
2684 ± 286 µm2 surface of S6K1;S6K2-/- hepatocytes, n=3 independent primary cultures, p<0.05),
while mononucleated cells were less affected (2311 ± 152 µm2 surface of wild type vs. 2088 ± 272
µm2 surface of S6K1;S6K2-/- hepatocytes, n=3 independent primary cultures). Taken together our
findings indicate that the small size of S6K deficient hepatocytes is not accompanied by a defect in
eIF4B and eEF2 phosphorylation.
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To investigate the involvement of other growth factor activated pathways in the
phosphorylation of eIF4B and eEF2, hepatocytes were treated with the MAPK inhibitor U0126, with
the PI3K inhibitor wortmannin and with the mTOR inhibitor rapamycin alone or in combination.
MAPK inhibition was sufficient to block eEF2K activity, as judged by eEF2 phosphorylation, while
rapamycin and wortmannin had no effect (Fig. 2b). Conversely, the combination of PI3K and MAPK
inhibitors was required to completely abrogate eIF4B phosphorylation in wild type hepatocytes,
suggesting that S6K, Rsk and a PI3K-dependent kinase participate in eIF4B phosphorylation in liver.
Since the constitutively active allele of Akt leads to increased eIF4B phosphorylation in a rapamycininsensitive manner (data not shown), and the Akt kinases recognise a similar substrate consensus
sequence as S6K and Rsk, we propose that Akt family members may be the PI3K-dependent kinases
that regulate eIF4B.
To gain further insights into the differential regulation of rpS6 and eIF4B in muscle and liver,
we compared S6K and Rsk activities by using phospho-specific antibodies that recognize S6K1/S6K2
or Rsk1/Rsk3 when they are phosphorylated respectively at a Thr or Ser residues in the hydrophobic
region that are critical for activity. Interestingly, Rsk phosphorylation was higher in hepatocytes than
in skeletal muscle cells, while S6K1 phosphorylation was roughly equivalent in these two cell types
(Fig. 3). Conversely S6K expression was higher in muscle cells than in liver. Although other
mechanisms may also be involved, the differential expression and activity levels of S6K and Rsk in
these two tissues may contribute to the predominance of one pathway over the other in the control of
the translational targets.
Since S6K deletion affected rpS6 and eIF4B phosphorylation in skeletal muscle, we set up to
assess protein synthesis in this cell type. First we assayed the incorporation of radiolabelled
methionine into proteins in cultured wild type and S6K1;S6K2-/- muscle cells at day four of
differentiation, when a difference in rpS6 and eIF4B phosphorylation was clearly observed between
the two genotypes. At this stage muscle cells were differentiated in multinucleated myotubes. The total
amount of protein per myonuclear number was decreased in S6K deficient myotubes (Fig. 4a).
Similarly, the methionine incorporation per myonuclear number was decreased in S6K1;S6K2-/- cells
(Fig. 4b). However when the methionine incorporation was normalised to the total protein content, no
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difference was observed between wild type and mutant cells (Fig. 4c). Since S6K1;S6K2-/- myotubes
displayed approximately a 25% reduction of myotube diameter with no defect in myonuclear number
per cell (31), these data suggest that the reduced incorporation of radiolabelled methionine in S6K
deficient cells is not the primary cause of cell atrophy but rather a consequence of their reduced overall
protein content and cell size. To more precisely assess translation initiation, we fractionated
polysomes, monosomes and free ribosomal subunits over a sucrose gradient, as the amount of
ribosomes engaged in polysomes versus monosomes reflects the global translational rates. As
observed in fibroblasts, hepatocytes and embryonic stem cells, S6K1;S6K2-/- muscle cells displayed a
similar polysomal profile in resting conditions as compared to wild type, and rapamycin produced a
decrease in the polysomal fraction in both genotypes (data not shown and Ref.(34)). Thus our analysis
does not reveal a major defect of global translation initiation as a consequence of S6K inactivation,
though we cannot rule out a regulation of specific mRNA classes. Of note S6K deletion mimics the
rapamycin effect on muscle cell size, but not on global translation initiation.
Since S6K deficient muscles are atrophic, and nutrient deprivation does not cause further
reduction in skeletal muscle mass (31), we reasoned that an atrophic program leading to protein
degradation may be either constitutively turned on in S6K1;S6K2-/- muscle cells or its induction may
be blunted. Starvation initiates two distinct mechanisms, the proteasome-mediated degradation of
ubiquitinated proteins and the autophagosome-mediated digestion of cellular components. Both
processes release amino acids that are used by muscles and other tissues as mitochondrial energy
substrates. The onset of these atrophy programs may be followed by appropriate molecular markers.
First we assessed the induction of autophagy by detecting the post-translational modifications of LC3
protein that is cleaved and conjugated to phospholipids during the early steps of autophagy (21). These
events increase the electrophoretic mobility of LC3 and can be revealed by immunoblot analysis with
anti-LC3 antibodies. As shown in figure 5a, starvation led to the decrease in the slower migrating full
lenght LC3 (LC3 I) and the appearance of a faster migrating band corresponding to cleaved LC3 (LC3
II). This marker of autophagy was also detected in starved S6K1;S6K2-/- muscles suggesting that
autophagy does not require S6K activity.
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To directly follow autophagosome formation, electron microscopy was performed in cultured
myotube cells. The volume densities of early and late autophagosomes as well as mitochondria were
quantified by morphometric analysis of differentiated myotubes in culture and normalized to the total
cytosolic volume (Fig. 5c). The early autophagosomes were recognized as double membrane vesicles
while late autophagosomes contained electron dense lysosomal degradation products. As expected, a
long-term rapamycin treatment increased autophagosome formation in wild type myotubes. However
S6K deletion had no effect on autophagy. Of note, S6K1;S6K2-/- muscle cells displayed an increased
mitochondrial mass, as previously reported for skeletal muscles (50). These data suggest that the small
size of S6K1;S6K2-/- muscle fibers is not a consequence of increased autophagy.
Since the expression of atrogin1 and MuRF1, two E3 ubiquitin ligases that target specific
proteins to the proteasome, is rapidly up-regulated in all forms of muscle atrophy and is required for
muscle wasting (3; 41; 47), we assayed their mRNA levels by QRT-PCR after starvation of wild type
and S6K deficient mice. As expected, in wild type gastrocnemius and tibialis anterior muscles a twoday starvation period induced a dramatic up-regulation of atrogin1 and MuRF1 mRNAs that was
suppressed by two-day refeeding (Fig. 6). Strikingly, S6K deficient muscles exhibited the same pattern
of atrogin1 and MuRF1 mRNA expression as compared to wild type. Thus S6K1;S6K2-/- muscles are
smaller and their mass is not decreased by starvation, despite a normal regulation of atrogin1 and
MuRF1.
To evaluate whether S6K deficient muscles displayed differential responses depending on the
atrophic conditions, we measured the effect of denervation on muscle mass and atrophy-related E3
ubiquitin ligases. Sciatic nerve section led to MuRF1 expression in gastrocnemius muscles of both
wild type and S6K1;S6K2-/- genotypes (Fig. 7a). While the mass of S6K deficient gastrocnemius
muscles was largely resistant to starvation (31), denervation reduced by approximately 20% and 30%
the weight of S6K1;S6K2-/- gastrocnemius muscles, respectively, at day 7 and 12 after surgery (Fig.
7b). Thus S6K deletion does not lead to a minimal muscle cell size, as S6K1;S6K2-/- fibers are
shrunken after denervation similar to wild type controls. In conclusion the S6K inactivation confers a
small size phenotype that is selectively resistant to nutrient deprivation and does not stem from
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deregulation of two main pathways involved in starvation-induced muscle wasting, autophagy and
atrophy-related E3 ubiquitin ligases.
DISCUSSION
In all eukaryotic cells TOR integrates nutrient signals and coordinate cell growth, proliferation
and metabolism (53). The central role of mammalian TOR is underlined by the striking phenotype of
mTOR-null mouse embryos that do not develop after the blastocyst stage (8; 28). mTOR resides in
two large complexes (mTORC), associated with either raptor protein in mTORC1 or rictor protein in
mTORC2 (53). The functions of mTORC1 have been characterized by using the antibiotic rapamycin,
a specific inhibitor of mTOR/raptor that has cytostatic actions and reduces cell size. It is well
documented that mTORC1 interacts with the translational machinery. mTOR/raptor can associate with
eIF3 in a nutrient- and growth factor-dependent manner and regulate the phosphorylation of 4EBP,
eIF4G and S6K (15; 16). Rapamycin affects protein turnover at multiple steps, from the ribosome
biogenesis (19; 25), to the translation initiation and elongation (1; 5), to the protein degradation by
increasing autophagy (45) and the expression of proteasome subunits and ubiquitin ligases (10; 39).
However it is unclear i) whether these processes are coordinated, ii) what are the molecular targets of
mTOR regulating these events, and iii) how growth is dependent on this control.
Genetic inactivation of S6 kinases represents an attractive model to address these questions for
the following reasons. Mice carrying S6K1, S6K2 and the combined gene deletions are viable, and
S6K1
-/-
mice, but not S6K2-/-, present a growth defect (34). Moreover S6K1 deletion in skeletal
muscles is sufficient to mimic the inhibitory effect of rapamycin on cell growth but not proliferation
(31). Interestingly the regulation of translational targets is tissue specific. We show that S6K deficient
muscles present an impaired rpS6 and eIF4B phosphorylation, while in liver the MAPK/Rsk and
PI3K/Akt pathways can compensate for the loss of S6K and partly or completely rescue the
phosphorylation of rpS6 and eIF4B (Fig. 1 and 2). The predominance of the mTOR pathway in
15
Page 16 of 37
skeletal muscle is also demonstrated by the control of EF2 phosphorylation that is rapamycin sensitive
in muscle cells but not in hepatocytes (Fig. 1 and 2). However we also show that EF2 kinase activity is
not affected by S6K signaling, in contrast to what was previously proposed (52). The tissue specific
difference of translational target phosphorylation may stem from a change in expression and/or
activity of distinct signal transduction elements (Fig. 3). This regulation may contribute to the different
physiological responses of liver and muscle to nutrient availability, as the mTOR pathway depends on
nutrient levels while MAPK and Akt activities are not modulated by these stimuli.
Here we conclude that the atrophy of S6K deficient muscles is not causally linked to a defect
in rpS6 and eIF4B phosphorylation. In muscle S6K2 activity contributes to the phosphorylation of
these factors without affecting the muscle growth responses (Fig. 2 and Ref. (31)), suggesting that
muscle growth is controlled by an S6K1-specific mechanism independent of rpS6 and eIF4B
phosphorylation. Consistent with these findings, S6K deficient hepatocytes are smaller than wild type
and eIF4B phosphorylation is not affected (Fig. 1). Moreover mice carrying a non-phosphorylatable
rpS6 allele display a milder phenotype as compared to the S6K1 deficient mice (38). The rpS6 mutant
mice in the adult stage do not present a reduction of total body size and the defect of cell growth is
limited to pancreatic beta cells. It is possible that rpS6 phosphorylation is required for growth of
specific tissues that do not include skeletal muscle. Alternatively the defect of pancreatic beta cell
growth in rpS6 mutant mice is a consequence of metabolic adaptations as these mice present increased
insulin sensitivity. S6 kinases have additional substrates that are not implicated in the translational
machinery, such as SKAR (36), mTOR (17), IRS1 (14), BAD (13). Of note some of these proteins
may be S6K1-specific targets (36). Future studies should address their direct implication in muscle
cell growth.
Recent studies have suggested that a change in the balance between protein synthesis and
degradation determines the rate of muscle growth (9). During cachexia, nutrient deprivation and
denervation, the E3 ubiquitin ligases atrogin-1 and MuRF1 are regulated by Akt/Foxo signalling, and
this response precedes and contributes to rapid atrophy (3; 41; 47). In addition, the degradation of
long-lived proteins by the formation of autophagic vacuoles accompanies several myopathy diseases
(29; 30; 51). mTOR plays a major role in muscle growth, as rapamycin is a potent inhibitor of muscle
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Page 17 of 37
hypertrophy and affect autophagy as well as ubiquitin ligase expression (4; 7; 32; 37; 39). Moreover
the rapamycin insensitive mTORC2 regulates Akt (42), and should therefore affect Foxo-mediated
atrogin-1 and MuRF1 gene expression. The muscle atrophy model triggered by the inactivation of the
mTOR substrate S6K1 in mice is characterized by thin size and complete resistance to nutrient
deprivation (31). Surprisingly, here we show that S6 kinases do not mediate the regulation of global
translation, expression of atrophy-related genes, and autophagy by nutrient/mTOR signaling (Fig. 4, 5,
6 and 7), although S6K1 is permissive for cell growth depending on nutrient availability.
Our results indicate that muscle atrophy upon S6K deletion is distinct from the atrophy
triggered by inactivating other mTOR effectors. It is possible that S6K1 controls the expression of a
specific subset of proteins that function as a size checkpoint in muscle. The existence of such factors
has been proposed after genetic screening for size mutants in yeast and Drosophila cells (11; 20). In
both organisms, the majority of genes affecting cell size encode nucleolar proteins suggesting that the
rate of ribosome biogenesis may set cell size. Although S6K1 deficient cells do not present defects in
5’TOP mRNA translation coding for ribosomal proteins (34), we cannot exclude that rDNA
transcription, rRNA processing or other activities required for ribosome biogenesis may be affected.
Alternatively S6K1 inactivation may decrease cellular components other than proteins. We have
recently demonstrated that S6K1;S6K2-/- muscles display a sharp reduction in lipid content (V.
Aguilar, A.S., M.P, unpublished data), possibly due to increased fatty acid beta oxidation (50). Future
studies should address whether the amount of intracellular lipids participates in the cell volume
control. Of note a switch in energy substrate utilization is known to affect muscle size and sensitivity
to nutrient availability, as oxidative fibers tend to be smaller and resistant to starvation as compared to
glycolytic fibers (26). As shown in Figure 5 and previously reported (50), S6K deficient muscles
display increased mitochondrial mass. Since S6K1;S6K2-/- muscle size is resistant to nutrient
deprivation while maintaining sensitivity to denervation (Fig. 7 and Ref. (31)), S6K may have the
specific function in the mTOR pathway to regulate a metabolic program adapting cell size to nutrient
availability. Defining the distinct types of atrophy resulting from inactivation of mTOR effectors has
the potential to target specific functions in human disease.
17
Page 18 of 37
ACKNOWLEDGMENTS
We thank the Novartis Foundation and George Thomas laboratory for the use of S6K mutant mice.
We are grateful to the members of INSERM-U810 for support and to G. Posthuma (UMC Utrecht) for
his advice on the morphometric analyses.
GRANTS
This work was supported by a grant to M.P. from the INSERM Avenir program (R01131KS), from
Fondation de la Recherche Medicale (DEQ20061107956), from Fondation Schlumberger pour
l’Education et la Recherche, and to A.S. and M.P. from the Association Française contre les
Myopathies (9971). V.M. is a recipient of a fellowship from the Association Française contre les
Myopathies.
Reference List
1. Beretta L, Gingras AC, Svitkin YV, Hall MN and Sonenberg N. Rapamycin blocks
the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.
EMBO J 15: 658-664, 1996.
2. Blommaart EF, Luiken JJ, Blommaart PJ, Van Woerkom GM and Meijer AJ.
Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat
hepatocytes. J Biol Chem 270: 2320-2326, 1995.
3. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou
WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM,
18
Page 19 of 37
Stitt TN, Yancopoulos GD and Glass DJ. Identification of ubiquitin ligases required
for skeletal muscle atrophy. Science 294: 1704-1708, 2001.
4. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R,
Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ and Yancopoulos GD.
Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can
prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014-1019, 2001.
5. Browne GJ and Proud CG. A novel mTOR-regulated phosphorylation site in
elongation factor 2 kinase modulates the activity of the kinase and its binding to
calmodulin. Mol Cell Biol 24: 2986-2997, 2004.
6. Conlon I and Raff M. Differences in the way a mammalian cell and yeast cells
coordinate cell growth and cell-cycle progression. J Biol 2: 7, 2003.
7. Dennis PB, Fumagalli S and Thomas G. Target of rapamycin (TOR): balancing the
opposing forces of protein synthesis and degradation. Curr Opin Genet Dev 9: 49-54,
1999.
8. Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, Spetz JF, Um SH,
Brown EJ, Cereghini S, Thomas G and Kozma SC. Disruption of the mouse mTOR
gene leads to early postimplantation lethality and prohibits embryonic stem cell
development. Mol Cell Biol 24: 9508-9516, 2004.
9. Glass DJ. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy.
Nat Cell Biol 5: 87-90, 2003.
19
Page 20 of 37
10. Grolleau A, Bowman J, Pradet-Balade B, Puravs E, Hanash S, Garcia-Sanz JA
and Beretta L. Global and specific translational control by rapamycin in T cells
uncovered by microarrays and proteomics. J Biol Chem 277: 22175-22184, 2002.
11. Guertin DA, Guntur KV, Bell GW, Thoreen CC and Sabatini DM. Functional
genomics identifies TOR-regulated genes that control growth and division. Curr Biol
16: 958-970, 2006.
12. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C and Avruch J. Amino
acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a
common effector mechanism. J Biol Chem 273: 14484-14494, 1998.
13. Harada H, Andersen JS, Mann M, Terada N and Korsmeyer SJ. p70S6 kinase
signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD.
Proc Natl Acad Sci U S A 98: 9666-9670, 2001.
14. Harrington LS, Findlay GM, Gray A, Tolkacheva T, Wigfield S, Rebholz H,
Barnett J, Leslie NR, Cheng S, Shepherd PR, Gout I, Downes CP and Lamb RF.
The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS
proteins. J Cell Biol 166: 213-223, 2004.
15. Harris TE, Chi A, Shabanowitz J, Hunt DF, Rhoads RE and Lawrence JC, Jr.
mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin. EMBO
J 25: 1659-1668, 2006.
20
Page 21 of 37
16. Holz MK, Ballif BA, Gygi SP and Blenis J. mTOR and S6K1 mediate assembly of
the translation preinitiation complex through dynamic protein interchange and ordered
phosphorylation events. Cell 123: 569-580, 2005.
17. Holz MK and Blenis J. Identification of S6 kinase 1 as a novel mammalian target of
rapamycin (mTOR)-phosphorylating kinase. J Biol Chem 280: 26089-26093, 2005.
18. Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB and Thomas G.
Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO
J 16: 3693-3704, 1997.
19. Jefferies HB, Reinhard C, Kozma SC and Thomas G. Rapamycin selectively
represses translation of the "polypyrimidine tract" mRNA family. Proc Natl Acad Sci
U S A 91: 4441-4445, 1994.
20. Jorgensen P, Rupes I, Sharom JR, Schneper L, Broach JR and Tyers M. A
dynamic transcriptional network communicates growth potential to ribosome synthesis
and critical cell size. Genes Dev 18: 2491-2505, 2004.
21. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami
E, Ohsumi Y and Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is
localized in autophagosome membranes after processing. EMBO J 19: 5720-5728,
2000.
21
Page 22 of 37
22. Kozma SC, Ferrari S, Bassand P, Siegmann M, Totty N and Thomas G. Cloning
of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second
messenger subfamily. Proc Natl Acad Sci U S A 87: 7365-7369, 1990.
23. Kozma SC and Thomas G. Regulation of cell size in growth, development and
human disease: PI3K, PKB and S6K. Bioessays 24: 65-71, 2002.
24. Krieg J, Hofsteenge J and Thomas G. Identification of the 40 S ribosomal protein
S6 phosphorylation sites induced by cycloheximide. J Biol Chem 263: 11473-11477,
1988.
25. Mayer C, Zhao J, Yuan X and Grummt I. mTOR-dependent activation of the
transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev
18: 423-434, 2004.
26. Mendell JR and Engel WK. The fine structure of type II muscle fiber atrophy.
Neurology 21: 358-365, 1971.
27. Montagne J, Stewart MJ, Stocker H, Hafen E, Kozma SC and Thomas G.
Drosophila S6 kinase: a regulator of cell size. Science 285: 2126-2129, 1999.
28. Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, Edenhofer F, Kiyama H,
Yonezawa K and Yamanaka S. mTOR is essential for growth and proliferation in
early mouse embryos and embryonic stem cells. Mol Cell Biol 24: 6710-6718, 2004.
22
Page 23 of 37
29. Nishino I. Autophagic vacuolar myopathies. Curr Neurol Neurosci Rep 3: 64-69,
2003.
30. Nishino I, Fu J, Tanji K, Yamada T, Shimojo S, Koori T, Mora M, Riggs JE, Oh
SJ, Koga Y, Sue CM, Yamamoto A, Murakami N, Shanske S, Byrne E, Bonilla E,
Nonaka I, DiMauro S and Hirano M. Primary LAMP-2 deficiency causes X-linked
vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406: 906-910, 2000.
31. Ohanna M, Sobering AK, Lapointe T, Lorenzo L, Praud C, Petroulakis E,
Sonenberg N, Kelly PA, Sotiropoulos A and Pende M. Atrophy of S6K1(-/-)
skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control.
Nat Cell Biol 7: 286-294, 2005.
32. Pallafacchina G, Calabria E, Serrano AL, Kalhovde JM and Schiaffino S. A
protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle
growth but not fiber type specification. Proc Natl Acad Sci U S A 99: 9213-9218,
2002.
33. Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, Marchand-Brustel Y,
Klumperman J, Thorens B and Thomas G. Hypoinsulinaemia, glucose intolerance
and diminished beta-cell size in S6K1-deficient mice. Nature 408: 994-997, 2000.
34. Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J, Mueller M,
Fumagalli S, Kozma SC and Thomas G. S6K1(-/-)/S6K2(-/-) Mice Exhibit Perinatal
Lethality and Rapamycin-Sensitive 5'-Terminal Oligopyrimidine mRNA Translation
23
Page 24 of 37
and Reveal a Mitogen-Activated Protein Kinase-Dependent S6 Kinase Pathway. Mol
Cell Biol 24: 3112-3124, 2004.
35. Raught B, Peiretti F, Gingras AC, Livingstone M, Shahbazian D, Mayeur GL,
Polakiewicz RD, Sonenberg N and Hershey JW. Phosphorylation of eucaryotic
translation initiation factor 4B Ser422 is modulated by S6 kinases. EMBO J 23: 17611769, 2004.
36. Richardson CJ, Broenstrup M, Fingar DC, Julich K, Ballif BA, Gygi S and Blenis
J. SKAR is a specific target of S6 kinase 1 in cell growth control. Curr Biol 14: 15401549, 2004.
37. Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, Yancopoulos
GD and Glass DJ. Mediation of IGF-1-induced skeletal myotube hypertrophy by
PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol 3: 1009-1013,
2001.
38. Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Nir T, Dor Y,
Zisman P and Meyuhas O. Ribosomal protein S6 phosphorylation is a determinant
of cell size and glucose homeostasis. Genes Dev 19: 2199-2211, 2005.
39. Sacheck JM, Ohtsuka A, McLary SC and Goldberg AL. IGF-I stimulates muscle
growth by suppressing protein breakdown and expression of atrophy-related ubiquitin
ligases, atrogin-1 and MuRF1. Am J Physiol Endocrinol Metab 287: E591-E601,
2004.
24
Page 25 of 37
40. Sandri M, Podhorska-Okolow M, Geromel V, Rizzi C, Arslan P, Franceschi C
and Carraro U. Exercise induces myonuclear ubiquitination and apoptosis in
dystrophin-deficient muscle of mice. J Neuropathol Exp Neurol 56: 45-57, 1997.
41. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K,
Schiaffino S, Lecker SH and Goldberg AL. Foxo transcription factors induce the
atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117:
399-412, 2004.
42. Sarbassov DD, Guertin DA, Ali SM and Sabatini DM. Phosphorylation and
regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101, 2005.
43. Scott RC, Schuldiner O and Neufeld TP. Role and regulation of starvation-induced
autophagy in the Drosophila fat body. Dev Cell 7: 167-178, 2004.
44. Shahbazian D, Roux PP, Mieulet V, Cohen MS, Raught B, Taunton J, Hershey
JW, Blenis J, Pende M and Sonenberg N. The mTOR/PI3K and MAPK pathways
converge on eIF4B to control its phosphorylation and activity. EMBO J 2006.
45. Shigemitsu K, Tsujishita Y, Hara K, Nanahoshi M, Avruch J and Yonezawa K.
Regulation of translational effectors by amino acid and mammalian target of
rapamycin signaling pathways. Possible involvement of autophagy in cultured
hepatoma cells. J Biol Chem 274: 1058-1065, 1999.
25
Page 26 of 37
46. Shima H, Pende M, Chen Y, Fumagalli S, Thomas G and Kozma SC. Disruption
of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional
S6 kinase. EMBO J 17: 6649-6659, 1998.
47. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M,
Yancopoulos GD and Glass DJ. The IGF-1/PI3K/Akt pathway prevents expression
of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors.
Mol Cell 14: 395-403, 2004.
48. Thomas G, Martin-Perez J, Siegmann M and Otto AM. The effect of serum, EGF,
PGF2 alpha and insulin on S6 phosphorylation and the initiation of protein and DNA
synthesis. Cell 30: 235-242, 1982.
49. Thomas G, Siegmann M, Kubler AM, Gordon J and Jimenez dA. Regulation of
40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells. Cell 19: 10151023, 1980.
50. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fumagalli S,
Allegrini PR, Kozma SC, Auwerx J and Thomas G. Absence of S6K1 protects
against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431:
200-205, 2004.
51. Wang X, Blagden C, Fan J, Nowak SJ, Taniuchi I, Littman DR and Burden SJ.
Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal
muscle. Genes Dev 19: 1715-1722, 2005.
26
Page 27 of 37
52. Wang X, Li W, Williams M, Terada N, Alessi DR and Proud CG. Regulation of
elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 20: 4370-4379,
2001.
53. Wullschleger S, Loewith R and Hall MN. TOR signaling in growth and metabolism.
Cell 124: 471-484, 2006.
27
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FIGURE LEGENDS
Figure 1. The control of eIF4B phosphorylation is tissue specific. Western blot analysis of protein
extracts from wild-type, S6K1-/-, S6K2-/- and S6K1;S6K2-/- gastrocnemius muscle (A ) or liver tissue
(B ) was carried out with anti-phosphorylated S6 (Ser 235/236), anti-phosphorylated eIF4B (Ser 422),
anti-phosphorylated eEF2 (Thr 56) and total eIF4B, the latter as a loading control. Five weeks old
male mice of the indicated genotype were sacrificed after 48 h starvation (starved) or after 48 h
starvation and 1 h stimulation with 1.4 g/kg leucine and 1 U/kg insulin (Leu+Ins). The phosphoeIF4B/total eIF4B ratio of signal intensity is indicated, as assessed by the ImageJ software.
Figure 2. Effect of S6Ks deletion on the phosphorylation of rpS6, eIF4B and eEF2 in skeletal muscle
cells and hepatocytes. Western blot analysis of protein extracts from wild-type and S6K1; S6K2-/myotubes (A ) was carried out with the indicated antibodies. Myotubes from the indicated genotypes
were allowed to differentiate for 2 days in 2% horse serum and then incubated for a further 2 days in a
presence of either vehicle (none) or 250 ng/ml IGF1-R3 (IGF) or 20 nM Rapamycin (Rap) or both
(IGF+Rap). The phospho-eIF4B/total eIF4B ratio of signal intensity is indicated, as assessed by the
ImageJ software. B, Histogram shows the average ± SEM of the phospho-eIF4B/total eIF4B ratio from
three distinct experiments. Myotubes were treated with vehicle, or 250 ng/ml IGF1-R3 in presence or
absence of 20 nM rapamycin as described in A, and immunoblot signals were quantified using the
ImageJ software. C, Western blot analysis of protein extracts from wild-type and S6K1; S6K2-/hepatocytes was carried out with the indicated antibodies. Hepatocytes from the indicated genotypes
were grown in amino acids-, glucose- and serum-free DMEM + 0.2% BSA for 3 h (None) and then
stimulated for 1 h in DMEM supplemented with growth factors (GF; 10% FBS + 1 µM insulin + 4 nM
EGF), in the presence or in absence of 20 nM rapamycin (Rap ) ± 10 µM U0126 (U0) ± 1 µM
wortmannin (Wort).
Figure 3. Differential expression and activity levels of S6K and Rsk in muscle and liver tissue.
Western blot analysis of protein extracts from wild-type and S6K1;S6K2-/- muscle and liver tissues or
myotubes and hepatocytes were carried out with anti-phosphorylated p90Rsk (Thr 359/ Ser 363), anti-
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p70S6K, anti-phosphorylated p70S6K (Thr 389) and anti-tubulin, the latter as loading control. Muscle
and liver tissue from the indicated genotypes were extracted after 48 h starvation and 1 h stimulation
with 1.4 g/kg leucine and 1 U/kg insulin (Leu+Ins); myotubes from the indicated genotypes were
allowed to differentiate for 2 days in 2% horse serum and then incubated for a further 2 days in
presence of 250 ng/ml IGF1-R3 (IGF); hepatocytes from the indicated genotypes were grown in amino
acids-, glucose- and serum-free DMEM + 0.2% BSA for 3 h and then stimulated for 1 h in DMEM
supplemented with growth factors (GF; 10% FBS + 1 µM insulin + 4 nM EGF).
Figure 4. Rate of protein synthesis in S6K deficient cells. Myotubes from the indicated genotypes
were allowed to differentiate for 4 days in 2% horse serum A, Total amount of protein per myonuclar
number. The assay was performed by measuring protein and DNA concentrations and calculating the
ratio protein/DNA. Each sample was run in triplicates and histograms are the mean ± SEM of three
independent experiments. B, Radiolabelled-amino acid incorporation per myonuclear number. The
assay of protein synthesis rates was performed by measuring the amount of [35S]-methionine and [35S]cysteine incorporation into cellular proteins over 2 h and normalized either to total DNA content. C,
Radiolabelled amino acid incorporation per total protein content. The assay was performed as
described in B, though the data of incorporated radiolabelled amino acids were normalized to total
protein. Each experiment was done in triplicates and histograms are means ± SEM of 3 independent
experiments.
Figure 5. Autophagy is not affected by S6K inactivation. A, Western blot analysis of protein extracts
from wild-type and S6K1; S6K2-/- gastrocnemius muscle was carried out with anti-LC3 and anti
eIF4B antibodies, the latter as a loading control. Five weeks old male mice were either starved 48 h
(Starved) or starved 48 h and then refed with normal chow for 1 h (Refed). B, Detection of
autophagosome by electron microscopy analysis of myotube cultures. Early (arrowhead) and late
(arrows) autophagosomes, and mitochondria (asterisks) in a representative electron microscopy image
of 48 h rapamycin-treated wild type myotubes. The histograms are the ratio of autophagosome volume
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density vs. total cytosolic volume and mitochondrial volume density vs. total cytosolic volume. At
least 25 non overlapping fields were analysed in one culture.
Figure 6. Atrophic S6K deficient muscles exhibit a normal regulation of Atrogin1 and MuRF1. QRTPCR on gastrocnemia (GC, left panels) and tibialis anterior (TA, right panels) muscles from wild-type
and S6K1; S6K2-/- mice was carried out with Atrogin-1 primer (upper panel) or MuRF-1 primer
(lower panel). Five weeks old male mice were randomly fed (control), starved for 48 h (starved) or
starved for 48 h and then refed with normal chow for 48 h (refed). Each sample was run in triplicates
and histograms are means ± SEM of 5 animals per condition.
Figure 7. Size of S6K deficient muscles is reduced by denervation. A, QRT-PCR was carried out with
MuRF-1 primers and gene expression in denervated muscles was compared to the controlateral
muscle. The right hindlimb muscles from two months old male mice were denervated by severing the
right sciatic nerve in the mid-tight. The left hindlimb from each animal serves as its own control. At
days 3, 7 and 12 post-denervation, the right and the left gastrocnemia muscles were weighed and RNA
samples prepared. Each sample was run in triplicates and histograms are means ± SEM of 3 animals
for each condition. B, Muscle weight during denervation. Results are expressed as percentage of
controlateral muscle weight and are average ± SEM of at least three mice of the indicated genotype at
each time point.
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Figure 1
Mieulet et al.
b
0.05
0.8 0.05 0.6 0.01 0.5 0.05 0.3
starved
Leu+Ins
Leu+Ins
starved
Leu+Ins
starved
Liver
starved
Leu+Ins
starved
Leu+Ins
starved
Leu+Ins
starved
starved
Leu+Ins
Muscle
Leu+Ins
a
P-S6
P-S6
P-eIF4B
P-eIF4B
Total eIF4B
Total eIF4B
P-eIF4B/eIF4B
0.01
0.8 0.02 0.8 0.01 0.9 0.02 0.7
P-eEF2
S6K1;S6K2-/-
S6K2-/-
S6K1-/-
+/+
S6K1;S6K2-/-
S6K2-/-
S6K1-/-
P-eEF2
+/+
P-eIF4B/eIF4B
Page 32 of 37
Mieulet et al.
Figure 2
b
IGF+Rap
Rap
IGF
None
Rap
IGF
None
IGF+Rap
Myotubes
a
P-eIF4B/eIF4B
+/+
S6K1;S6K2-/-
150
49
80
58
90
P-eIF4B/eIF4B
P-eEF2
50
0
eEF2
IGF
56
IGF+Rap
35
None
111
IGF+Rap
100
100
IGF
Total eIF4B
None
% of control
P-eIF4B
P-S6
tubulin
+/+
S6K1;S6K2-/-
GF+Rap+Wort+U0
GF+Wort+U0
GF+Rap+Wort
GF+Wort
GF+Rap+U0
GF+U0126
GF+Rap
GF
None
GF+Wort+U0
GF+Rap+Wort
GF+Wort
GF+Rap+U0
GF+U0126
GF+Rap
GF
None
GF+Rap+Wort+U0
Hepatocytes
b
P-eIF4B
Total eIF4B
P-eEF2
eEF2
P-S6
P-Rsk
+/+
S6K1;S6K2-/-
+/+
Ins+Leu Ins+Leu
IGF
S6K1;S6K2-/-
+/+
S6K1;S6K2-/-
+/+
S6K1;S6K2-/-
+/+
S6K1;S6K2-/-
Hepatocytes
Myotubes
Liver
Muscle
Page 33 of 37
Figure 3
Mieulet et al.
P-Rsk
S6K
P-S6K
tubulin
GF
Page 34 of 37
Figure 4
Mieulet et al.
b
c
120
100
6
*
4
2
0
+/+
S6K1;S6K2-/-
80
60
40
20
0
35S
met / DNA
% of wt
protein / DNA
8
% of wt
Protein (0g)/ DNA (0g)
a
+/+
S6K1;S6K2-/-
140
120
100
80
60
40
20
0
35S
met / protein
+/+
S6K1;S6K2-/-
*
*
** *
*
* *
*
0,016
0,014
0,012
0,01
0,008
0,006
0,004
0,002
0
S6K1;S6K2-/-
+/+
0,1
0,08
0,06
0,04
0,02
0
S6K1;S6K2-/-
Refed
Refed
Starved
Starved
Refed
Refed
Figure 5
+/+
Mitochondria/cytosol ratio
rapamycin
b
control
*
control
+/+
Autophagosome/cytosol ratio
Starved
Starved
Page 35 of 37
Mieulet et al.
a
LC3 II
LC3 I
Total eIF4B
S6K1;S6K2-/-
MuRF-1 expression in GC muscle
S6K1;S6K2-/40
0
+/+
refed
0
control
refed
starved
control
refed
+/+
starved
20
starved
Figure 6
control
40
refed
60
control
80
50
starved
S6K1;S6K2-/-
Fold induction over +/+ control
refed
starved
Atrogin-1 expression in GC muscle
Fold induction over +/+ control
refed
starved
+/+
control
refed
starved
+/+
control
70
60
50
40
30
20
10
0
refed
control
Fold induction over +/+ control
100
starved
control
Fold induction over +/+ control
Page 36 of 37
Mieulet et al.
Atrogin-1 expression in TA muscle
S6K1;S6K2-/-
40
30
20
10
0
MuRF-1 expression in TA muscle
S6K1;S6K2-/-
30
20
10
Page 37 of 37
Figure 7
a
Mieulet et al.
+/+
14
b
S6K1;S6K2-/-
110
Gastroc weight
(% of control)
12
10
8
6
4
2
100
90
80
70
+/+
S6K1;S6K2-/-
60
denervated
control
denervated
50
0
control
Fold induction over +/+ control
MuRF-1 expression in GC muscle
0
2
4
6
8
10
12
days