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MOLECULAR REPRODUCTION AND DEVELOPMENT 75:810–817 (2008)

Insulin-Like Growth Factor-1 (IGF-1) and Leucine


Activate Pig Myogenic Satellite Cells Through
Mammalian Target of Rapamycin (mTOR) Pathway
BING HAN,1,2 JUNFENG TONG,2 MEI J. ZHU,2 CHANGWEI MA,1** AND MIN DU1,2*
1
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing,
People’s Republic of China
2
Department of Animal Science, University of Wyoming, Laramie, Wyoming

ABSTRACT Myogenic satellite cells are adult 4E-binding protein 1 (4E-BP1) and ribosomal protein
stem cells and have important roles in skeletal muscle S6 kinase (S6K) (Schmelzle and Hall, 2000; Hara
growth, repair, and regeneration. Both insulin-like growth et al., 2002; Shen et al., 2005). A small GTP-binding
factor-1 (IGF-1) and leucine stimulate skeletal muscle protein Ras-homologue expressed in brain (Rheb) is a
growth, which link to the activation and proliferation of direct upstream activator of mTOR. Rheb is a down-
myogenic satellite cells in skeletal muscle. Mammalian stream target of the GTPase-activating protein (GAP)
target of rapamycin (mTOR) signaling is one of the domain of tuberous sclerosis complex 2 (TSC2)
main signaling pathways controlling protein synthesis (Inoki et al., 2003). The proteins TSC1 and TSC2 form
and cell proliferation. Thus, IGF-1 and leucine may a functional complex which promotes the conversion
stimulate activation of myogenic satellite cells through of Rheb-GTP into Rheb-GDP and, thus, negatively
mTOR signaling. In this study, myogenic satellite cells regulates mTOR signaling and cell growth (McManus
were isolated from 6-month-old pigs and subjected to and Alessi, 2002; Inoki et al., 2003). TSC2 is phosphory-
IGF-1 and leucine treatments. Both IGF-1 and leucine lated by Akt and thereby inhibits the function of TSC1/
upregulated mTOR signaling in myogenic satellite cells. TSC2 complex, leading to the activation of Rheb and
The phosphorylation of mTOR at Ser2448 increased phosphorylation of mTOR (Inoki et al., 2003).
83.8  7.7% by IGF-1 (P < 0.05) and 83.4  5.7% It is well documented that insulin-like growth factor-1
by leucine (P < 0.05). The downstream targets of (IGF-1) and leucine stimulates skeletal muscle growth.
mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) Both IGF-1 and leucine stimulate mTOR signaling
were also phosphorylated due to IGF-1 and leucine in skeletal muscle, which may be responsible for the
treatments. Treatment with IGF-1 and leucine induced enhanced muscle growth. There are two pathways
the phosphorylation of tuburin (TSC2), a key mediator contributing to skeletal muscle growth: (1) enhanced
upstream of mTOR signaling, by 272.8  26.4% protein synthesis in existing muscle fibers; (2) activation
and 94.2  28.7%, respectively. Treatment of cells with and proliferation of myogenic satellite cells which infuse
both IGF-1 and leucine did not show synergistic effect into muscle fibers. Accumulating data show the impor-
on mTOR signaling. Inhibition of mTOR by rapamycin tance of mTOR signaling in the control of skeletal
abolished the protein synthesis and cell proliferation muscle growth (Lawrence, 2001; Deldicque et al., 2005;
stimulated by both IGF-1 and leucine. In summary, Nader et al., 2005). In cell culture, activation of mTOR
our data showed that in preliminary cultured myogenic leads to myotube hypertrophy (Nader et al., 2005; Park
satellite cells mTOR signaling was activated due to IGF-1 et al., 2005). Exercise, insulin, IGF-1, and amino acids
and leucine treatments, and this mTOR activation is stimulate muscle protein synthesis partially through
necessary for the activation of myogenic satellite cells. the activation of the mTOR pathway (Bolster et al.,
Mol. Reprod. Dev. 75: 810–817, 2008.
ß 2007 Wiley-Liss, Inc.
Bing Han and Junfeng Tong have equal contributions.
Grant sponsor: National Natural Science Foundation of China; Grant
Key Words: leucine; IGF-1; myogenic satellite cell; sponsor: USDA Cooperative State Research, Education, and Exten-
mTOR; skeletal muscle sion Service; Grant numbers: 2007-35203-18065, 2006-55618-16914.
*Correspondence to: Min Du, Department of Animal Science,
University of Wyoming, Laramie, WY 82071.
INTRODUCTION E-mail: mindu@uwyo.edu
The mammalian target of rapamycin (mTOR) involves **Correspondence to: Changwei Ma, College of Food Science and
the sensing of nutritional status in cells and coordinates Nutritional Engineering China Agricutural University Beijing 100083
China. E-mail: chwma@cau.edu.cn
nutrient status with protein synthesis (Bodine et al.,
Received 24 February 2007; Accepted 24 July 2007
2001; Bolster et al., 2003; Sakamoto et al., 2003). Published online 21 November 2007 in Wiley InterScience
Activation of mTOR upregulates protein translation (www.interscience.wiley.com).
through phosphorylating eukaryotic initiation factor DOI 10.1002/mrd.20832

ß 2007 WILEY-LISS, INC.


Molecular Reproduction and Development

IGF-1 AND LEUCINE IN mTOR OF MYOGENIC SATELLITE CELLS 811

2004; Deldicque et al., 2005). In addition, leucine is a 2.5 mM EDTA, 1% protease inhibitor cocktail (Sigma).
potent activator of mTOR signaling pathway (Anthony Lysate was centrifuged at 12,000g for 15 min at 48C
et al., 2001; Du et al., 2007). However, despite numerous and protein concentration in the supernatant was
studies on the mTOR signaling of skeletal muscle as determined by the bicinchoninic acid method (Bio-rad,
a whole, few studies were conducted on the mTOR Hercules, CA) (Zhu et al., 2006).
signaling of myogenic satellite cells due to IGF-1 and
leucine treatments. Existing studies on myogenic cells Electrophoresis and Immunoblotting
uses C2C12 cells (Park and Chen, 2005) and no study Cell lysates containing equivalent amounts of pro-
was conducted in the primary cultured myogenic teins (30 mg) were boiled in Laemmli sample buffer
satellite cells. The objective of this study is to evaluate with 5% mercaptoethanol at 958C for 5 min. A 5–20%
mTOR signaling in cultured myogenic satellite cells as gradient gel was used for SDS–PAGE separation of
affected by IGF-1 and leucine. proteins. A Hoefer mini-gel system was used for
casting gels and running electrophoresis. Following
MATERIALS AND METHODS electrophoresis, proteins on the gel were transferred to
Antibodies and Chemicals for Cell Culture nitrocellulose membrane in a transfer buffer containing
20 mM Tris-base, 192 mM glycine, 0.1% SDS, and 20%
Antibodies against phospho-mTOR at Ser2448, phospho-
methanol.
S6 kinase at Thr389, phospho-Akt at Ser473, phospho-TSC2
Membranes were incubated in a blocking solu-
at Thr1462, phospho-4E-binding protein 1 (4E-BP1) at
tion consisting of 5% nonfat dry milk in TBS/T (0.1%
Thr37/46 were purchased from Cell Signalling Technology
Tween-20, 50 mM Tris-HCl pH 7.6, and 150 mM NaCl)
Inc. (Beverly, MA). Antibody against actin was obtained
for 1 hr followed by overnight incubation at 48C in
from Developmental Studies Hybridoma Bank (Iowa city,
primary antibodies 1:1,000 diluted with TBS/T contain-
IA). Cell culture medium (DMEM) and fetal bovine serum
ing 1% nonfat dry milk. At the end of the primary
(FBS) were obtained from Sigma (St. Louis, MO). IGF-1
antibody incubation, the membranes were washed
was purchased from BD Biosciences (San Jose, CA).
three times for 5 min each with 20 ml of TBS/T. After
Rapamycin was purchased from EMD Biosciences, Inc
that, membranes were incubated with horseradish
(San Diego, CA). L-leucine, Long-R3-IGF-1, and other
peroxidase-conjugated secondary antibodies at 1:1,000
chemicals were purchased from Sigma.
dilution for 1 hr in TBS/T with gentle agitation.
Following three 10-min washes, membranes were visua-
Myogenic Satellite Cells
lized using ECL Western blotting reagents (Amersham
Primary myogenic stem cells were obtained from the Bioscience, Piscataway, NJ) and exposure to film (MR,
semitendinosus muscle of three different 6-month-old Kodak, Rochester, NY). The density of bands was
pigs. Skeletal muscle was minced in DMEM medium quantified by using an Imager Scanner II and Image-
after trimming off all visible fat and connective tissue, Quant TL software (Amersham Bioscience) (Du et al.,
and then incubated in 1 mg/ml protease (Sigma) at 378C 2004). To reduce the variation between blots, cell lysates
for 1 hr. The tissue slurry was centrifuged at 1,500g for of all treatments were run in a single gel. Band density
10 min and the pellet was suspended in DMEM medium among different blots was normalized according to the
and centrifuged at 400g for 5 min. The supernatant was density of the reference band. Band density was also
transferred to another tube and centrifuged at 1,500g for normalized according to the actin content (Yang et al.,
10 min. The resulting pellet containing myoblasts 2005).
was used for cell culture (Doumit and Merkel, 1992).
Cells (1  105/flask) were cultured in DMEM medium Measurement of Protein Synthesis
containing 10% heat-inactivated FBS, plus 50 U/ml Protein synthesis was measured by the rate of
penicillin, and 50 g/ml streptomycin in an incubator incorporation of (3H( phenylalanine into total mixed
under a humidified atmosphere of 5% CO2/95% air at proteins. Two mCi L-[2,3,4,5,6-3H] phenylalanine
378C. Then, cells were treated in the same medium (120 Ci/mmol) (Amersham Bioscience, Buckingham-
without FBS for 4 hr and subjected to treatments shire, UK) was added to each flask of myogenic cells
including 2 mM L-leucine, 250 ng/ml IGF-1, and 20 nM (60% confluence) and incubated for 30 min. Cells were
rapamycin as appropriate. washed with ice-cold PBS. Then, cells were collected and
The purity of the cultured myogenic satellite cells mixed with equal volume of ice-cold 20% trichloroacetic
were assessed by immunohistochemical staining with acid. The resultant precipitate was solubilized with
anti-myosin heavy chain antibody (DSHB, University of 200 ml 1 M NaOH for 30 min and mixed with 5 ml
Iowa, IA) after inducing fusion by 2% horse serum as scintiverse (Fisher Scientific, Hanover Park, IL). The
previously described (Blanton et al., 1999; Park et al., incorporation of the radioisotopes was measured by
2005). Cells with more than 80% purity were used. liquid scintillation counting (Du et al., 2007).

Preparation of Cell Lysates Thymidine Assay for Proliferation Analysis


Cells were lysed in a buffer containing 50 mM HEPES Proliferation assay of thymidine was performed by
pH 7.4, 137 mM NaCl, 1% NP-40, 10% glycerol, 2 mM measuring the incorporation of thymidine into nuclei
Na3VO4, 100 mM NaF, 1 mM MgCl2, 1 mM CaCl2, DNA. Briefly, myogenic cells (1  105/disk, doubling time
Molecular Reproduction and Development

812 B. HAN ET AL.

approximately 24 hr) were cultured to 60% confluence in (Fig. 4). To show the role of mTOR in cell proliferation
6-well plates. [3H]Thymidine, 1.0 mCi (PerkinElmer Life and protein synthesis stimulated by IGF-1 and leucine,
Sciences) was added to each well and cells were incubated rapamycin was used to inhibit mTOR. Applying rapa-
for 4 hr at 378C. At the end of this period, plates were mycin abolished the proliferation and protein synthesis
placed on ice, and cells were washed with ice-cold PBS, stimulated by IGF-1 and leucine (Fig. 4), showing
collected, and precipitated with ice-cold 20% trichloro- that mTOR signaling is essential for the activation
acetic acid. The resultant precipitate was used for liquid of myogenic satellite cells stimulated by IGF-1 and
scintillation counting (Yang et al., 2005). leucine.
Statistics DISCUSSION
Data were analyzed as a complete-randomized design IGF-1 stimulates skeletal muscle growth mainly
using GLM (General Linear Model of Statistical Ana- through activation of phosphoinositide-3 kinase (PI3K/
lysis System, SAS, 2000). Three separate experiments Akt)-signaling pathway (Bush et al., 2003; Latres et al.,
were conducted and each experiment was regarded as a 2005; Park et al., 2005; Song et al., 2005; Subramaniam
replicate (n ¼ 3). The differences in the mean values et al., 2005; Vary, 2006). One of the major targets of
were compared by Student’ t-test, and mean values and PI3K/Akt signaling is mTOR. Accumulating data show
standard error of means were reported (P < 0.05). the importance of mTOR signaling in the control of
skeletal muscle growth (Lawrence, 2001; Deldicque
RESULTS et al., 2005; Nader et al., 2005). The mTOR protein is
Figure 1 shows the time courses of mTOR phosphory- a large protein with a molecular weight of around
lation at Ser2448 and Akt at Ser473 following 250 ng/ml 290 kD. It is involved in sensing nutritional status in
IGF-1, 100 ng/ml Long-R3-IGF-1, and leucine treat- cells and coordinating nutrient status with protein
ments. The phosphorylation of Akt reached maximal synthesis (Bodine et al., 2001; Bolster et al., 2003;
at around 15 min following treatment (Fig. 1A–D) and Sakamoto et al., 2003). mTOR functions in vivo by
mTOR reached maximal at around 30 min (Fig. 1A, forming either mTOR complex 1 (mTORC1) or complex
D–F). Since the objective of this study is to investigate 2, with mTORC1 having a key role in controlling protein
the activation of mTOR signaling, 30 min of IGF-1 and synthesis (Martin and Hall, 2005). However, mTOR
leucine treatments were chosen for further studies. complex 2 is insensitive to rapamycin and relates to
Both IGF-1 and leucine treatment stimulated mTOR cytoskeletal rearrangement (Martin and Hall, 2005).
signaling (Fig. 2). IGF-1 (250 ng/ml) treatment in- Since mTOR complex 2 does not relate to protein
creased mTOR phosphorylation for 83.8  7.7% and synthesis, it will not be discussed further. The mTORC1
leucine for 83.4  5.7% (Fig. 2A,B). No synergistic consists of regulatory-associated protein of mTOR
effect of IGF-1 and leucine on mTOR phosphorylation (raptor), GbL, and mTOR. GbL binds in a constitutive
was observed (Fig. 2A,B). The phosphorylation of interaction to stimulate the kinase activity of the TOR
downstream effector of mTOR, ribosomal protein catalytic domain (Kim et al., 2003). Raptor has a role in
S6K, was also increased for 148.6  6.5% and sensing amino acids and coordinating with mTOR
191.0  7.7% due to 250 ng/ml IGF-1 and leucine activation (Hara et al., 2002). mTOR controls the rate
treatment, respectively (Fig. 2C). The phosphorylation of phosphorylation of key proteins involved in protein
of another downstream effector of mTOR, 4E-BP1 was synthesis, including 4E-BP1 and ribosomal protein S6K
also increased due to IGF-1 and leucine treatments (Hara et al., 2002). Phosphorylation of 4E-BP1 by mTOR
(Fig. 2D). No synergistic effect of IGF-1 and leucine releases eukaryotic initiation factor-4E (eIF4E) which
treatments on mTOR signaling was observed (Fig. 2). binds to eIF4G and initiates translation. mTOR directly
Since 250 ng/ml of IGF-1 is relatively high compared phosphorylates S6K and S6K phosphorylates ribosomal
to the physiological level and also IGF-binding pro- protein S6 (Jefferies et al., 1997; Bodine et al., 2001).
teins may interfere with IGF-1, a truncated IGF-1 Phosphorylation of ribosomal protein S6 drives trans-
receptor agonists that does not interact with the lation of a small family of abundant transcripts that
IGF-binding proteins, Long-R3-IGF-1, was also used. encode primarily ribosomal proteins and components
Long-R3-IGF-1 treatment (100 ng/ml) enhanced the of the translational apparatus (Jefferies et al., 1997;
mTOR phosphorylation at a level similar to 250 ng/ml Schmelzle and Hall, 2000). Thus, the activation of
IGF-1 treatment. Again, no synergistic effect of Long- mTOR upregulates the translational machinery and
R3-IGF-1 and leucine on mTOR phosphorylation was promotes protein translation (Schmelzle and Hall, 2000;
detected (Fig. 2E). Because of the similar effects of Shen et al., 2005). In this study, both IGF-1 and leucine
250 ng/ml of IGF-1 and 100 ng/ml of Long-R3-IGF-1 on enhanced the phosphorylation of mTOR and its down-
mTOR phosphorylation (Fig. 2B,E), only 250 ng/ml of stream effectors, S6K and 4E-BP1, showing that mTOR
IGF-1 was used for the remaining measurements. signaling was activated due to IGF-1 and leucine
TSC2 is a key mediator between Akt and mTOR. Both treatments in myogenic satellite cells. The upregulation
250 ng/ml IGF-1 and leucine treatments increased the of mTOR signaling should be responsible for the
phosphorylation of TSC2 (Fig. 3). enhanced protein synthesis in myogenic cells stimu-
Both 250 ng/ml IGF-1 and leucine enhanced the lated by IGF-1 and leucine. These results are consistent
proliferation and protein synthesis in myogenic cells with previous reports. Knockout S6K does not affect
Molecular Reproduction and Development

IGF-1 AND LEUCINE IN mTOR OF MYOGENIC SATELLITE CELLS 813

Fig. 1. Time course of mTOR activation due to IGF-1 and leucine phosphorylation due to 250 ng/ml IGF-1 treatment. Panel D: Akt
supplements. Myogenic satellite cells were stimulated by 2 mM leucine, phosphorylation due to 100 ng/ml Long-R3-IGF-1 treatment. Panel E:
250 ng/ml IGF-1, and 100 ng/ml Long-R3-IGF-1. Cells were collected at mTOR phosphorylation due to leucine treatment. Panel F: mTOR
0, 15, 30, 60, and 120 min following IGF-1 or leucine supplement for the phosphorylation due to 250 ng/ml IGF-1 treatment. Panel G: mTOR
analyses of mTOR phosphorylation at Ser2448 and Akt phosphorylation phosphorylation due to 100 ng/ml Long-R3-IGF-1 treatment. Mean 
at Ser473 by immunoblotting. Panel A: representative immunoblots; SEM. *P < 0.05 versus Control, n ¼ 3.
Panel B: Akt phosphorylation due to leucine treatment. Panel C: Akt
Molecular Reproduction and Development

814 B. HAN ET AL.

Fig. 2. Effect of IGF-1 and leucine on mTOR signaling in satellite phosphorylation due to 100 ng/ml IGF-1 or leucine treatments. Panel
cells. Cells were incubated in the presence of 250 ng/ml IGF-1, 100 ng/ D: 4E-BP1 phosphorylation due to 100 ng/ml IGF-1 or leucine
ml Long-R3-IGF-1, or 2mM leucine for 30 min. Panel A: Representa- treatments. Panel E: Representative immunoblots and mTOR
tive blots showing the phosphorylation of mTOR, S6K, and 4E-BP1 due phosphorylation due to 100 ng/ml Long-R3-IGF-1 treatment or Long-
to 100 ng/ml IGF-1 or leucine treatments. Panel B: mTOR phosphor- R3-IGF-1 plus leucine treatments. Mean  SEM. *P < 0.05 versus
ylation due to 100 ng/ml IGF-1 or leucine treatments. Panel C: S6K Control, n ¼ 3.
Molecular Reproduction and Development

IGF-1 AND LEUCINE IN mTOR OF MYOGENIC SATELLITE CELLS 815

(McManus and Alessi, 2002; Inoki et al., 2003). TSC2 is


phosphorylated by Akt and thereby inhibits the function
of TSC1/TSC2 complex, leading to the activation of
Rheb and phosphorylation of mTOR (Inoki et al., 2003).
Our data showed that IGF-1 and leucine enhanced the
phosphorylation of Akt and TSC2, consistent with the
enhanced mTOR signaling observed in these cells.
Besides enhancing protein synthesis, activation of
mTOR signaling also promotes cell proliferation and
plays an essential role in skeletal myogenesis (Erbay
et al., 2003). In C2C12 cell culture, activation of mTOR
leads to myotube hypertrophy (Nader et al., 2005b; Park
et al., 2005). Inhibition of mTOR leads to arrest of cells in
the G1-phase of the cell cycle (Hashemolhosseini
et al., 1998; Gaben et al., 2004). In the current study,
the proliferation of myogenic cells was inhibited by
rapamycin, a specific inhibitor of mTOR, eliciting that
mTOR signaling has a crucial role in the proliferation of
myogenic cells.
Our data showed that leucine stimulates mTOR
signaling (Du et al., 2007). This stimulation might be
associated with the inhibition of AMP-activated protein
kinase (AMPK). AMPK is a key mediator of energy
Fig. 3. Effect of IGF-1 and leucine on p-TSC2 in satellite cells. Cells metabolism (Hardie, 2004; Kim et al., 2004). AMPK is
were incubated in the presence of 250 ng/ml IGF-1 or 2mM leucine for
switched on by an increase in the AMP/ATP ratio (Kim
30 min. Panel A: Representative blots showing the phosphorylation of
TSC2. Panel B: Mean  SEM for the phosphorylation of TSC2. et al., 2004; Hawley et al., 2005; Woods et al., 2005).
*P < 0.05 versus Control, n ¼ 3. Leucine and other branched chain amino acids (BCAA)
have a distinct metabolic pathway compared to other
amino acids. Other amino acids are mainly degraded in
myoblast cell proliferation but reduces myoblast size liver. However, due to the absence of BCAT in liver,
(Ohanna et al., 2005), suggesting that mTOR signaling BCAA are degraded primarily in skeletal muscle
mediated by S6K is crucial for protein synthesis. (Sweatt et al., 2004) and used for ATP synthesis (Lynch
Recent studies showed that a small GTP-binding et al., 2003; Tokunaga et al., 2004), which inhibits
protein Rheb is a direct upstream activator of mTOR. AMPK activity (Du et al., 2007). AMPK controls the
Rheb is a downstream target of the GAP domain of activation of mTOR and protein synthesis through
TSC2 (Inoki et al., 2003). Rheb binds directly to the several possible mechanisms. First, AMPK phosphor-
amino-terminal lobe of the mTOR catalytic domain, ylates TSC2 directly, thereby enhancing the activity of
and the kinase activity of TOR is determined by the state TSC1/TSC2 complex (Inoki et al., 2003). Activated TSC2
of Rheb nucleotide charging. The proteins TSC1 and acts through Rheb to inhibit mTOR function (Gao et al.,
TSC2 form a functional complex which promotes the 2002). Second, AMPK may also inhibit the activity
conversion of Rheb-GTP into Rheb-GDP and, thus, of mTOR directly by phosphorylation at Thr2446. The
negatively regulates mTOR signaling and cell growth phospohorylation at Thr2446 and Ser2448 is mutually

Fig. 4. Effect of IGF-1 and leucine on proliferation and protein synthesis of satellite cells. Cells were
incubated in the presence of 250 ng/ml IGF-1, 2mM leucine, or 20 nM rapamycin (Rap) for 30 min.
Mean  SEM. *P < 0.05 versus Control, n ¼ 3.
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816 B. HAN ET AL.

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ACKNOWLEDGMENTS mediating metabolic responses to exercise. Med Sci Sports Exerc
This work was supported by National Natural Science 36:28–34.
Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivieres S, Mercep L,
Foundation of China (Project number: 30540420523) Ferrari S. 1998. Rapamycin inhibition of the G1 to S transition is
and Research Initiative Grant 2007-35203-18065 and mediated by effects on cyclin D1 mRNA and protein stability. J Biol
2006-55618-16914 from the USDA Cooperative State Chem 273:14424–14429.
Research, Education, and Extension Service. The mono- Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM,
Frenguelli BG, Hardie DG. 2005. Calmodulin-dependent protein
clonal antibody of actin developed by Jim Jung-Ching
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Lin was obtained from the Developmental Studies activated protein kinase. Cell Metab 2:9–19.
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NICHD and maintained by the University of Iowa, response to control cell growth and survival. Cell 115:577–590.
Department of Biological Sciences, Iowa City, IA 52242. Jakobsen SN, Hardie DG, Morrice N, Tornqvist HE. 2001. 50 -AMP-
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