From the Institut de Genetique Humaine, CNRS, UPR
1142, 141 Rue de la Cardonille, 34396 Montpellier Cedex 4, France, the
¶ Friedrich Miescher Institut, Maubeerstrasse 66, CH-4056 Basel,
Switzerland, and the
Rockefeller University, Laboratory of
Molecular Cell Biology, New York, New York 10021
Received for publication, June 26, 2000, and in revised form, November 20, 2001
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ABSTRACT |
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Insulin-like growth factors positively regulate
muscle differentiation through activation of the
phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling
pathway. Here, we compare the role of the two closely related Skeletal muscle differentiation is a tightly regulated process
during which proliferative myoblasts escape from the cell cycle, express a battery of muscle-specific genes, and fuse into
multinucleated myotubes. This developmental process is regulated at the
transcriptional level by the basic helix-loop-helix myogenic proteins
including MyoD, Myf-5, myogenin, and MRF4 (1).
Insulin-like growth factors
(IGFI1 and IGFII) are potent
activators of muscle differentiation. An autocrine production of IGFII by myoblasts has been linked to spontaneous differentiation triggered by lowering serum concentration in the culture medium (2, 3). Indeed,
C2 myoblasts overexpressing an IGFII antisense construct fail to
differentiate spontaneously and lose MyoD expression (4).
Recently, Coolican and collaborators (5) showed that activation of
phosphatidylinositol 3 kinases (PI3Ks) is essential for IGF-stimulated
differentiation. Differentiation of L6E9, C2.7, Sol8, and chicken
primary myoblasts was also shown to be dependent on PI3Ks activity
(6-9). Among downstream targets of PI3K, the serine/threonine protein
kinase B/Akt (PKB) was proposed as an essential downstream component
for IGF-induced differentiation (9). Three different isoforms of PKB
have been identified: PKB In this report we have investigated a role for PKB Cell Culture--
C2.7 (4) or L6D2 myoblasts (a subclone of L6
cells; Ref. 20) were grown in proliferation medium (50% Dulbecco's
modified Eagle's medium (Biowhittaker, France), 50% Ham's F-12
supplemented with 10% fetal calf serum (Life Technologies, Inc.). To
induce muscle differentiation, myoblasts were placed in differentiation medium (Dulbecco's modified Eagle's medium, 2% fetal calf serum).
Clonal human myoblasts (CHM) were obtained from needle biopsy sample
taken from the paravertebral muscle of a healthy subject, using an
experimental protocol previously described for rodent (21). To obtain
an homogenous population of myoblasts, cells were grown as a clonal
population and individually tested for their capacity to differentiate
in a low serum containing medium. Selected clones were mixed together.
The population obtained called CHM (for cloned human myoblasts) can be
grown for 6-10 passages. Following transfer into a differentiation
medium, the majority of cells fuse into multinucleated myotubes after
36-48 h, expressing muscle-specific markers such as myogenin and
troponin T.
Western Blot--
50 µg of cellular protein extracts prepared
from proliferating or differentiated C2.7 or L6D2 cells were analyzed
by Western blot as previously described (21). Antibodies used were
rabbit anti-PKB Immunoprecipitation and in Vitro Kinase Assay--
Cells
(myoblasts and myotubes) were resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% w/v
Nonidet P-40, 40 mM
The PKB substrate GST-GRPRTSS*FAEG (the asterisk denotes the
phosphorylated serine) and the mutant substrate GST-GRPKTSSFAEG were
purified from extracts of bacteria (XL1-blue) as described by the
manufacturer of the pGEX-6P-1 plasmid (Amersham Pharmacia Biotech). The
bacteria had been transformed with GST expression plasmids
pGEX-6P-1/GRPRTSSFAEG or pGEX-6P-1/GRPKTSSFAEG, respectively. These
plasmids were constructed by inserting oligonucleotides with the
sequence GATCCGGAAGGCCAAGAACTTCATCGTTCGCAGAGGGTC
(pGEX-6P-1/GRPRTSSFAEG) or GATCCGGAAGGCCAAAAACTTCATCGTTC GCAGAGGGTC
(pGEX-6P-1/GRPKTSSF- AEG) into the BamHI and
XhoI site of pGEX-6P-1.
Cell Fractionation--
Cells were collected in an ice-cold
hypotonic buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 2 µg/ml aprotinin and lysed by 10 strokes
in a Dounce homogenizer. Nuclei were collected by a 10-min
centrifugation at 2000 × g and rinsed once with
hypotonic buffer. Protein concentrations in the cytoplasmic fraction
was determined using the Bradford detection reagent (Bio-Rad). Isolated
nuclei were lysed directly in a volume of 1× Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 8% glycerol, 0.001%
bromphenol blue, and 10 mM dithiothreitol) equal to that of
the cytoplasmic fraction.
Transfection and Luciferase Assay--
Plasmids used for
transfection were: The pECE vector either empty or encoding wild-type
PKB tagged with HA (pECE-HA-PKB
Transfections were carried out using 4 µl of FuGENE and 1 µg
of DNA in 35-mm dishes as described by the manufacturer (Roche Molecular Biochemicals). Transfected cells were kept in proliferation medium for 24 h and transferred to differentiation medium for indicated times before processing for Western blot or
immunofluorescence as described elsewhere (21). Antibodies to HA and
myogenin were used to visualize transfected and differentiated cells, respectively.
For luciferase assay, cells were transfected with 1 µg of total
plasmid DNA composed of pECE/pECE-HA-PKB/MLCK-luc/RL-TK plasmids (ratio
1:1:2:1). Following incubation for 24 h in proliferation medium
and 36 h in differentiation medium, transfected cells were harvested for luciferase assay. Activities of firefly and
Renilla luciferases were measured sequentially using the
luciferase assay kit reagent as described by the manufacturer (Promega,
Charbonnieres, France).
Microinjection and Immunofluorescence
Experiments--
Microinjections were carried out in growing myoblasts
before induction of differentiation or in G0-synchronized
REF-52 fibroblasts before serum refeeding, as previously described (26,
27). Injected antibodies were affinity-purified rabbit anti-PKB Expression of PKB
Extracts from proliferative or differentiating myoblasts were probed by
Western blot for expression of PKB isoforms, myogenin as a
differentiation marker, and tubulin as a loading control. As shown in
Fig. 1B, PKB
We have previously shown that, upon induction of differentiation, a
subset of myoblasts escape from the differentiation process and remain
in a quiescent undifferentiated mononucleated state (21). To
investigate if the observed increase in PKB
Taken together, these results show that PKB Activity and Subcellular Distribution of PKB
To characterize further the relative activity of each isoforms during
spontaneous differentiation, we also looked for the subcellular
distribution of PKB
Taken together, these data show that PKB PKB Microinjected anti-PKB
Affinity-purified anti-PKB
To confirm the requirement of PKB
To confirm that the function of PKB
Taken together, these results show that PKB The PI3K/PKB (Akt) pathway has been recently implicated in
IGF-activated muscle differentiation and hypertrophy as well as IGF-induced muscle cell survival (9, 14, 33, 34). Except for the work
by Fujio et al. (Ref. 9, as discussed below), none of these
studies investigated a functional difference between the PKB isoforms.
In this report, we provide evidence that PKB PKB PKB
In summary, we have demonstrated a specific role of PKB (Akt1) and
(Akt2) isoforms of PKB in muscle differentiation. During
differentiation of C2.7 or L6D2 myoblasts, PKB
was up-regulated
whereas expression of PKB
was unaltered. Although the two isoforms
were found active in both myoblasts and myotubes, cell fractionation
experiments indicated that they displayed distinct subcellular
localizations in differentiated cells with only PKB
localized in the
nuclei. In a transactivation assay, PKB
(either wild-type or
constitutively active) was more efficient than PKB
in activating
muscle-specific gene expression. Moreover, microinjection of specific
antibodies to PKB
inhibited differentiation of muscle cells, whereas
control or anti-PKB
antibodies did not. On the other hand,
microinjection of the anti-PKB
antibodies caused a block in cell
cycle progression in both non muscle and muscle cells, whereas
anti-PKB
antibodies had no effect. Taken together, these results
show that PKB
plays a crucial role in the commitment of myoblasts to
differentiation that cannot be substituted by PKB
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Akt1), PKB
(Akt2), and PKB
(Akt3).
All of them are activated by growth factors in a
PI3K-dependent manner. Phosphorylation of
Thr308 (Thr309 in PKB
) in the activation
loop and Ser473 (Ser474 in PKB
) in the
C-terminal activation domain is required for full activation of PKB
and PKB
(reviewed in Refs. 10 and 11). The most studied isoform is
PKB
/Akt1, generally referred to as PKB or Akt. PKB
has been
implicated in several processes stimulated by IGFs such as cell
survival, glucose uptake in myocytes and adipocytes, cell growth, and
differentiation (reviewed in Refs. 12 and 13). Based on a dominant
positive overexpression approach, a role for PKB
has been assigned
in the PI3K-mediated differentiation of avian primary myoblasts (9), as
well as in promoting cell survival when myoblasts escape from the cell
cycle to differentiate (14). Several recent reports suggest that the
and
isoforms of PKB may have distinct functions in cellular
regulation. In adipocytes, PKB
is preferentially involved in
insulin-stimulated glucose transporter 4 (GLUT4) translocation (15, 16)
and is found associated with GLUT4-containing vesicles (17). PKB
appears to be functionally different from PKB
since stress activates PKB
and PKB
, but not PKB
(18). Little is known about PKB
and its potential role in myogenesis. Interestingly, PKB
/Akt2 mRNA is up-regulated during myogenic differentiation and has a tissue-specific distribution in mouse embryos being preferentially expressed in brown fat and muscle tissues, suggesting a preferential role for this isoform in differentiation processes (19).
in skeletal
muscle differentiation. In contrast to the constant expression levels
of PKB
, PKB
protein levels were specifically increased during
differentiation of mouse C2.7 or rat L6D2 myoblasts. The two isoforms
were found active in both myoblasts and myotubes with a
differentiation-dependent increase in PKB
activity
mirroring protein levels. Cell fractionation experiments indicated that the two isoforms differed in their subcellular distributions in differentiated cells with only PKB
localized in the nuclei. When overexpressed in C2.7 cells, PKB
(either wild-type or constitutively active forms) was more efficient than PKB
in enhancing
transactivation of a muscle-specific gene. Finally, inactivation of
PKB
through microinjection of specific antibodies impaired
differentiation of C2.7, L6D2, and human myoblasts, whereas anti-PKB
antibodies had no effect. By contrast, injection of the anti-PKB
antibodies caused a block in cell cycle progression in both nonmuscle
and muscle cells, whereas anti-PKB
antibodies had no effect. Taken together, these results show that the
and
isoforms of PKB play
different roles in myogenesis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(22), sheep anti-PKB
(Upstate Biotechnology),
rabbit anti-myogenin (Santa-Cruz Biotechnology), and mouse anti-tubulin antibodies (Sigma). Secondary antibodies were horseradish
peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Amersham
Pharmacia Biotech). Sheep antibodies were detected using horseradish
peroxidase-conjugated protein A/G (Interchim, France). Blots were
developed by using an Amersham enhanced chemiluminescence reagent.
-glycerophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 4 µM leupeptin, 2 µg/ml aprotinin, 0.1 mM sodium orthovanadate) and homogenized by passages
through a 21-gauge needle. Following a 10-min centrifugation at
12,000 × g, soluble proteins were quantified using a
Bradford method (Bio-Rad). 200-400 µg of cell free extracts were
pre-cleared by incubation with protein G-Sepharose beads (Amersham
Pharmacia Biotech) and incubated overnight at 4 °C with either
rabbit anti-PKB
or sheep anti-PKB
antibodies. 20 µl of protein
G-Sepharose beads were added for 30 min at 4 °C, and beads were
washed three times with lysis buffer containing 0.5 M NaCl, once with lysis buffer, and once with kinase buffer (50 mM
Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 mM
-glycerophosphate). In vitro kinase assay
were performed for 30 min at 30 °C by adding 20 µl of kinase
buffer supplemented with 10 mM MgCl2, 50 µM [
-32P]ATP (Amersham Pharmacia
Biotech, 1,000-2,000 cpm/pmol), 1 µM protein kinase A
inhibitor peptide, and 2 µg of the Crosstide sequence (23, 24) fused
to GST as substrate (see below). Proteins were subsequently separated
on a 12.5% SDS-polyacrylamide gel electrophoresis. The radioactivity
was analyzed by autoradiography.
wt, pECE-HA-PKB
wt), inactive PKB
(pECE-HA-PKB
kd, pECE-HA-PKB
kd), or constitutively active
membrane-targeted PKB (pECE-HA-PKB
m and -
m; 22, 25), pRL-TK
(Promega, Charbonnieres, France), and muscle creatine kinase enhancer
and promoter upstream of firefly luciferase gene (MCK-luc; gift from
S. Leibovitch, Institut Gustave Roussy, Villejuif, France).
antibodies (directed against amino acids 453-466 of human PKB
protein; Ref. 22), affinity-purified sheep anti-PKB
antibodies
(directed against amino acids 455-469 of rat PKB
; Euromedex,
France), or 1 mg/ml anti-hPKB
IgGs (directed against amino acid
454-470 of human PKB
, Ref. 22). Prior to injection, purified
antibodies (0.3-0.5 mg/ml) or sera were dialyzed against 100 mM Hepes, pH 7.4, and mixed when necessary with rabbit
marker antibodies (1 mg/ml). Immunofluorescence analysis was carried
out as previously described (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, but Not PKB
, Is Up-regulated during C2.7
and L6D2 Cell Differentiation--
We have initially determined the
expression profiles of PKB
and PKB
during differentiation of
mouse C2.7 cells. To first ensure that the anti-PKB antibodies were
isoform-specific, C2.7 cells transiently transfected with HA-tagged
membrane-targeted forms of either PKB
or PKB
were examined by
immunoblotting. The rabbit anti-PKB
antibody only immunoblotted the
overexpressed HA-PKB
protein, whereas the sheep anti-PKB
antibody
detected only the overexpressed HA-PKB
(Fig.
1A). In addition, at the exposure shown here, PKB
antibodies only detected HA overexpressed protein whereas anti-PKB
detected both overexpressed
(upper band) and endogenous (lower
faint band) proteins. As comparable amounts of
overexpressed proteins were analyzed (see Fig. 1A,
lane HA), it appears that the anti-PKB
antibodies have a lower affinity (~5-fold) than PKB
antibodies
against their respective isoforms.
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Fig. 1.
Differentiation-dependent
up-regulation of PKB . A, C2.7
cells were transfected with membrane-targeted HA-tagged forms of either
PKB
or PKB
, and comparable amounts of overexpressed proteins (as
probed with anti-HA antibodies) were analyzed by Western blot for
expression of PKB
and PKB
. B, total cellular extracts
were prepared from C2.7 cells before (0d) or after 1, 2, or
3 days (d) being placed in differentiation medium, and
analyzed by Western blot for expression of PKB
, PKB
, myogenin
(myog) as a differentiation marker, and tubulin
(tub) as a loading control. C, C2.7 myotubes were
separated from quiescent myoblasts by limited trypsinization after 3 (3d) or 4 days (4d) in differentiation medium.
Western blot analysis of total cellular extracts from proliferative
myoblasts (P), quiescent myoblasts (Q), and
myotubes (M) was carried out for PKB
, PKB
, troponin T
(TropT) as a differentiation marker, and tubulin
(tub) as a loading control.
protein level remains constant throughout differentiation. In contrast, PKB
expression is up-regulated, as
early as 24 h after induction of differentiation, a time that coincides with appearance of the early differentiation marker myogenin.
A specific up-regulation of PKB
was also seen during differentiation
of rat L6D2 myoblasts (data not shown). Moreover, when compensating for
the different affinities of the anti-PKB
and anti-PKB
antibodies
(see Fig. 1A), PKB
and PKB
proteins may be expressed
at similar levels in myoblasts, but PKB
becomes the major isoform
expressed in myotubes.
level was directly
linked to differentiation, C2.7 myotubes were separated from quiescent
cells, and expression of PKB
was examined by Western blot in these
two subpopulations. PKB
is expressed at similar low levels in
proliferative and nondifferentiated quiescent myoblasts (Fig.
1C, compare lanes P and Q),
whereas it shows a significant increase in differentiated myotubes
(Fig. 1C, lane M). Expression levels
of PKB
are similar in all tested conditions. As expected for a
marker of differentiation, troponin T was detected only in the myotube population.
and PKB
are
differentially expressed during differentiation of muscle cells, with
PKB
being up-regulated in a differentiation-dependent manner.
and PKB
during
Differentiation--
Activation of either PKB
or PKB
occurs
through the same mechanisms. Active PI3Ks allow translocation of PKB to
the plasma membrane, where its PH domain binds to the PI3K lipid
products. Following phosphorylation by two upstream activating kinases, PKB translocates into the nucleus (22, 25). In our culture model,
myoblasts are allowed to differentiate by lowering serum concentration
in the medium. This spontaneous differentiation is possible because of
an autocrine production of IGFs by differentiating myoblasts. We first
estimated the relative activity of each isoform during the process of
spontaneous differentiation by using a kinase assay. PKB
and PKB
were immunoprecipitated from C2.7 myoblasts or myotubes, and their
activity assessed against the Crosstide peptide fused to GST (GST-C) as
a substrate. As shown in Fig. 2A, PKB
and PKB
were found to phosphorylate Crosstide in both myoblasts and myotubes.
PKB
activity was slightly increased in myotubes in comparison to
myoblasts with similar amounts of protein immunoprecipitated at both
stages. PKB
activity was significantly up-regulated in
differentiated myotubes when compared with myoblasts. This increase in
PKB
activity corresponds to an augmentation of immunoprecipitated
proteins. For both isoforms, no activity was detected when a mutated
non-phosphorylatable Crosstide peptide was used as substrate testifying
for specificity of the assay (data not shown).
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Fig. 2.
Subcellular localization and activity of
PKB and PKB
in
myoblasts and myotubes. A, PKB
and PKB
were
immunoprecipitated from proliferative C2.7 myoblasts and myotubes and
their activity assessed against the peptide Crosstide fused to GST
(GST-C) as substrate. The amount of immunoprecipitated
proteins were controlled by Western blot (WB). B,
cytoplasmic and nuclear fractions were prepared from C2.7 myoblasts
(P) and myotubes (M) and analyzed by Western blot
for expression of PKB
and PKB
. Nuclei (N) were lysed
in a volume of Laemmli buffer equivalent to cytosolic fraction
(C) and whole cell extract proteins (WCE).
and PKB
in myoblasts and myotubes, as nuclear
translocation of PKB has been described to follow its activation (22,
25). Cytoplasmic and nuclear extracts of mouse C2.7 or rat L6D2
myoblasts and myotubes were prepared and immunoblotted with anti-PKB
or anti-PKB
antibodies. As shown in Fig. 2B, PKB
and
PKB
were found distributed in both cytoplasm and nuclei at similar
levels in proliferative C2.7 myoblasts. By contrast, only PKB
was
still detected in C2.7 myotube nuclei whereas PKB
was detected only
in the cytoplasm. Similar results have been observed when using L6D2
cell extracts (data not shown).
and PKB
are both active
in myoblasts and myotubes, but differ in their subcellular distribution
in myotubes with only PKB
still detectable in the nucleus. This
difference in subcellular localization strongly suggests a different
function for each isoform during muscle differentiation.
Is More Efficient than PKB
in Transactivating
Muscle-specific Gene in C2.7 Cells--
To examine whether the two PKB
isoforms display similar efficiency in promoting muscle
differentiation, we used a classical transactivation assay with a
luciferase reporter plasmid (MCK-luc) containing the
1256 base pair
upstream region from the muscle creatine kinase, which mediates
differentiation-dependent activity in cultured skeletal
muscle cells (28, 29). Thus, activation of MCK-luc should allow a
quantitative measurement of the extent of differentiation. Wild-type
and mutant (constitutively active or inactive) forms of PKB
and
PKB
were cotransfected with the MCK-luc construct into C2.7 cells.
In each case, plasmid pRL-TK was cotransfected as an internal control
for transfection efficiency. After transfection, cells were placed in
proliferation medium for 24 h, and incubated for an additional
36 h in differentiation medium before harvesting and measurement
of the reporter gene transactivation by luciferase assay. As shown in
Fig. 3, constitutively active PKB
(
m) and PKB
(
m) significantly promoted
MCK-luc activity (up to 3- and 7-fold, respectively) with PKB
m
showing a 2-fold higher efficiency than PKB
m. Significantly,
wild-type PKB
also increased transactivation of the reporter gene to
a level similar to that obtained with the constitutively active PKB
,
whereas wild-type PKB
appeared as inefficient as constitutively inactive mutants, in promoting MCK-luc activity. Expression levels of
the different PKB constructs were checked by Western blot and were
found to be similar in all tested conditions (data not shown). Taken
together, these results show that PKB
(either wild-type or
constitutively active) is more efficient than PKB
in enhancing muscle-specific gene expression.
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Fig. 3.
PKB is more
efficient than PKB
in enhancing
muscle-specific gene expression. C2.7 myoblasts were transfected
with the MCK-luc reporter construct and pRL-TK plasmid, together with
plasmids encoding for wild-type (
wt and
wt), constitutively active (
m and
m), or kinase dead (
kd and
kd) forms of PKB
and PKB
. After 24 h in
proliferation medium followed by 36 h of incubation in
differentiation medium, transfected cells were harvested and luciferase
activity was measured and corrected with respect to pRL-TK activity.
MCK-luc activities are expressed as -fold activation relative to that
obtained in empty pECE cotransfected cells set at 1. Shown are the mean
values of duplicates obtained in two independent experiments.
Antibodies Inhibit Muscle
Differentiation--
To further investigate the differences between
PKB
and PKB
in myogenic cells, we examined whether targeted
inhibition of individual PKB isoforms, through microinjection of
specific antibodies, could interfere with differentiation. Such an
approach has been successfully used to demonstrate the involvement of
key regulatory proteins such as the coactivator p300 (30) or the serum
response factor (26, 31) in skeletal muscle differentiation.
, anti-PKB
antibodies mixed with rabbit
marker antibodies, or marker antibodies alone were injected into both
C2.7 or L6D2 myoblasts. After microinjection cells were switched to the
differentiation medium for 48 h and processed for
immunofluorescence. As shown in Fig. 4,
only microinjection of anti-PKB
antibodies blocked differentiation
of L6D2 and C2.7 cells by 50% and 48%, respectively, whereas neither
anti-PKB
nor marker antibody (data not shown; see also Ref. 26), had a significant effect on myogenin expression and fusion of microinjected cells into myotubes.
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Fig. 4.
Microinjection of anti-PKB
but not anti-PKB
antibodies prevents
differentiation of C2.7 and L6D2 cells. A,
proliferative myoblasts were microinjected with affinity purified
anti-PKB
or anti-PKB
antibodies. Cells were placed in
differentiation medium and 48 h later were fixed and stained for
the injected antibodies and myogenin. Shown are fluorescence
micrographs of cells injected with anti-PKB
(panels
A-C) or anti-PKB
(panels D-F), and stained
for the injected antibodies (panels A and
D), myogenin (panels B and
E), and the superimposition of the two (panels
C and F). The histogram summarizes the data
obtained from two different sets of microinjection experiments done in
both C2.7 and L6D2 cells. Percent of myogenin expression refers to the
percentage of microinjected cells expressing myogenin. The
number (n) of cells injected with anti-PKB
(
) and
anti-PKB
(
) antibodies is indicated.
for muscle differentiation, we
performed a second set of microinjection experiments. We used the
rabbit anti-hPKB
antibodies directed against amino acids 454-470 of
the human protein (22). Because these antibodies recognized with high
affinity the human protein, injection was carried out in CHM (see
"Materials and Methods") as described above. As shown in Fig.
5, microinjection of anti-hPKB
IgGs
into CHM efficiently blocked differentiation. The majority of injected cells remained mononucleated with ~20% expressing myogenin.
Inversely, in control experiments in which myoblasts were microinjected
with pre-immune serum, 60% of injected cells were either found in
multinucleated myotubes or in mononucleated myocytes expressing
myogenin. This percentage of differentiation was similar to that of
surrounding noninjected cells.
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Fig. 5.
Microinjection of anti-human
PKB antibodies blocks differentiation of human
myoblasts. CHM cells were injected with anti-hPKB
IgGs or
pre-immune serum as control and incubated in differentiation medium for
36-48 h. Cells were analyzed by immunofluorescence as described in
Fig. 4. Shown are fluorescence micrographs of cells injected with
pre-immune serum (panels A-C) or anti-hPKB
antibodies (panels D-F), and stained for the injected
antibodies (panels A and D), myogenin
(panels B and E) and the
superimposition of the two (panels C and
F). The histogram summarizes the data obtained in two
independent experiments with the percentage of myogenin expression in
surrounding noninjected cells (indicated as 0), cells injected with the
pre-immune serum (PI), or cells injected with anti-hPKB
IgGs. n = number of injected cells.
is not necessary for the
transition from myoblast to myotube, we further performed
microinjection experiments to prove that anti-PKB
antibodies were
inhibitory to PKB
. For that purpose, we monitored the effect of
microinjected anti-PKB antibodies on cell proliferation of REF-52
fibroblasts, which express both PKB
and PKB
as probed by
immunoblotting (data not shown). Indeed, a recent report described that
PKB
is part of a Ras-PI3K-glycogen synthase kinase-3
transduction pathway that positively regulates cyclin D1, a key
component of the cell cycle involved in progression of cells through
G1 phase (32). Serum-starved REF-52 fibroblasts were
injected with anti-PKB
or anti-PKB
antibodies, refed with serum
to enter the cell cycle and incubated in a medium containing BrdUrd for
20-22 h to follow passage through S phase via DNA synthesis. As
shown in Fig. 6, BrdUrd incorporation
was inhibited in 90% of anti-PKB
injected cells, whereas
anti-PKB
had no significant effect on S phase entry with 73% of
injected cells having passed through S phase. These data indicate that
anti-PKB
antibodies are active in inhibiting PKB
. Similarly,
injection of anti-PKB
antibodies blocked proliferation of C2.7
cells, whereas injected anti-PKB
antibodies had no effect (data not
shown).
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Fig. 6.
Microinjection of anti-PKB
but not anti-PKB
antibodies impedes
proliferation. G0-synchronized REF-52 fibroblasts were
microinjected with anti-PKB
(A) or anti-PKB
(B) antibodies and following serum refeeding, incubated with
BrdUrd for 20-22 h to follow S phase completion. Shown are
fluorescence micrographs of cells double-stained for the injected
antibodies (red) and for incorporation of BrdUrd
(green). The histogram summarizes the data obtained from two
different microinjection experiments. Percent of DNA synthesis refers
to the percentage of microinjected cells that have incorporated BrdUrd.
The number of cells injected with anti-PKB
(
) or anti-PKB
(
) is indicated.
plays a specific
function in triggering differentiation of myoblasts, a role for which
PKB
appears unable to substitute.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and PKB
play
distinct roles in myogenesis and demonstrate that PKB
is necessary
to trigger muscle cell differentiation.
and -
Differ in Their Expression and Subcellular
Localizations during Differentiation--
The first difference between
PKB
and PKB
in muscle cells can be seen at the expression level,
with only PKB
protein being up-regulated when C2.7 and L6D2
myoblasts are induced to differentiate and maintained at high level in
differentiated cells. Inversely, PKB
protein levels remain constant
during differentiation. An increase in PKB
protein and/or mRNA
level was previously reported in differentiating Sol8 (6), C2C12
myoblasts (19), and MyoD-converted 10T1/2 fibroblasts (35), showing
that up-regulation of PKB
is not restricted to our cell models, and
most likely occurs at the transcriptional level. In this context, it is
quite surprising that Fujio et al. (9) described an
induction of PKB
protein during differentiation of C2C12 cells. This
may be explained by insufficient specificity of the anti-PKB
antibody used in that study. We found both PKB
and PKB
active in
phosphorylating the Crosstide peptide in both myoblasts and myotubes.
Whereas PKB
was slightly activated during differentiation, PKB
activity was up-regulated in a manner corresponding to the increase in
protein levels. This is consistent with the fact that both isoforms are activated in a PI3K-dependent manner (22, 25). In fact, a major difference between the two isoforms lies in their subcellular distribution in differentiated cells, with only PKB
maintained in
the nuclear compartment. Both PKB
and PKB
were found in myoblast nuclei and cytoplasm, but in contrast to PKB
, PKB
was no more detectable in myotube nuclei. The mechanisms by which PKB kinases translocate to the nucleus following membrane activation have been
poorly studied. The oncogene for Tcl1, which is involved in the
development of mature T-cell leukemia, was recently shown to be a
cofactor of PKB. Tcl1, by interacting with PKB, enhances its activity
by promoting its nuclear translocation (36). Because Tcl1 is restricted
to certain lymphoid cells, other molecules, perhaps related to Tcl1,
responsible for PKB nuclear translocation may exist in differentiated
cells and remain to be identified. The unique nuclear localization of
PKB
in myotubes suggests that it may stimulate differentiation by
phosphorylating nuclear substrates. In this context, it may appear
puzzling that membrane-targeted forms, albeit incapable to translocate
to the nucleus, display activation of muscle-specific genes.
Constitutively active mutants may be able to activate endogenous PKB.
Indeed, activation of PKB appears to involve oligomerization of PKB
molecules (37, 38). In addition, PKB may be responsible for its
auto-activation by phosphorylating Ser473
(Ser474 in PKB
) referred to as hypothetical PDK2 site
(39). Constitutive membrane-targeted mutants may be able to oligomerize
with and phosphorylate endogenous PKB, thus explaining an activation
effect that would involve nuclear localization of endogenous PKB.
Interestingly, PI3K was recently shown to positively regulate the
transcriptional activity of myocyte enhancer factor 2 family members,
which cooperate with myogenic factors in the regulation of
muscle-specific genes including myogenin (40). As such, it will be
interesting to determine whether myocyte enhancer factor 2 proteins
constitute a direct target of PKB
.
and PKB
Have Distinct Functions in Myogenesis--
The
high specificity of the antibodies directed against PKB
and PKB
(see Fig. 1) prompted us to use a microinjection assay to discriminate
between the role played by each isoform in muscle differentiation. The
antibodies used are directed against the C-terminal regulatory domain
of PKB
and PKB
, and, as such, upon binding they may interfere
with the kinase activation. Microinjection of two different anti-PKB
antibodies efficiently blocked differentiation of C2.7, L6D2, and human
myoblasts, respectively, an effect that was not observed after
injection of anti-PKB
antibodies. The efficiency of injected PKB
antibodies in inhibiting endogenous PKB
was attested by its
inhibitory effect on the G1 to S transition of REF-52
fibroblasts and C2.7 myoblasts. Indeed, PKB
was recently shown to
control expression of cyclin D1, a key component of G1 progression (32, 41). Moreover, the tumor suppressor MMAC/PTEN, a lipid
phosphatase that dephosphorylates PI3K-generated
3'-phosphatidylinositides, was shown to modulate G1 to S
phase progression by regulating PKB signaling pathway (42, 43).
Inversely, anti-PKB
antibodies had no effect on cell proliferation,
indicating that inhibition of differentiation by these antibodies is
not dependent on cell-cycle events. In accordance with our results,
inhibition of PKB
protein expression was shown to suppress the
tumorigenicity of human pancreatic cancer cells without affecting
proliferation as estimated by the population doubling time in culture
(44). In conclusion, our results strongly suggest that PKB
and
PKB
isoforms play specific and distinct roles in myogenesis. First,
we show that PKB
is necessary for differentiation of either, mouse,
rat, or human myoblasts, implying that the role for PKB
/Akt2 in
myogenesis is conserved in mammalian cells. Second, our results tend to
indicate that PKB
cannot directly regulate the commitment to
differentiate but is more likely involved in proliferation.
Interestingly, recent reports have implicated PKB
in myocyte
viability (9, 34). We have observed a loss in anti-PKB
injected
cells when placed into differentiation medium, in agreement with the
fact that PKB
may be the isoform involved in anti-apoptotic
signaling. The isoform-specific function of PKB
in inducing muscle
differentiation may appear contradictory to previous reports showing a
role for PKB
in the myogenic-inducing activity of IGF. This
discrepancy highlights inherent problems of overexpression assays. In
these reports, a role for PKB
in IGF activated differentiation (9)
or hypertrophy (33) has been demonstrated by overexpressing
constitutively active forms of the kinase. Accordingly, our results
show that constitutively active forms of PKB
and PKB
were both
able to enhance muscle-specific gene transactivation, although PKB
appeared 2-fold more effective. However, when assessing wild-type
isoforms, only PKB
displayed a myogenic activity (see Fig. 3). The
constitutive active forms may escape from accurate regulation such as
association with regulatory partners. This notion is exemplified by the
two related isoforms, p110
and p110
catalytic subunits of class IA PI3Ks. Studies based on overexpression assay of an activated mutant
of the p110 subunits or dominant negative p85 regulatory subunit (which
binds both p110
and p110
) could not discriminate between the two
isoforms that were assumed to play similar roles in cellular
regulation. However, by using an antibody microinjection approach,
recent reports clearly show that p110
and p110
have different
functions in either platelet-derived growth factor- and
insulin-activated actin reorganization in aortic endothelial cells
(45), insulin-responsive glucose transport activity in adipocytes (46),
or epidermal growth factor-stimulated actin nucleation in breast cancer
cells (47). Interestingly, p110
but not p110
was shown to
associate with the GLUT4 glucose transporter compartment (48) and play
a crucial role in transmitting the signals to translocate GLUT4 (46), a
biological process in which PKB
is specifically involved (15, 16).
Although in this case there is no direct evidence that PI3K
activates PKB
, this example illustrates how cellular functions may
be influenced by subcellular localization.
in
triggering muscle differentiation. The characterization of substrates that are specifically phosphorylated by PKB
during differentiation is critical to understand how the kinase is involved in this process. A
good candidate is glycogen synthase kinase-3
, which, in
addition to being a target of PKB, belongs to the Wnt signaling pathway known to positively regulate differentiation (reviewed in Ref. 49).
Other potential in vivo downstream targets of PKB
include p70S6 kinase and transcription factors such as the forkhead family member FHRK1 (50). We are currently investigating which of these signaling molecules may be modulated by PKB
and as such involved in
the control of myogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Patric Turowski for technical advice and helpful discussions and Olivier Zugasti for help in PKB kinase assay. We are grateful to Yegor Vassetzky for comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from Association Francaise contre les Myopathies, by Association pour la Recherche contre le Cancer Grant 9484, and by Human Frontiers Science Program Organisation Collaborative Grant 533/1996-M.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 33-499-61-99-66; Fax: 33-499-61-99-69; E-mail: ned.lamb@igh.cnrs.fr.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005587200
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ABBREVIATIONS |
---|
The abbreviations used are: IGF, insulin-like growth factor; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; GLUT4, glucose transporter 4; GST, glutathione S-transferase; BrdU, bromodeoxyuridine; CHM, clonal human myoblasts.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Molkentin, J. D., and Olson, E. N. (1996) Curr. Opin. Genet. Dev. 6, 445-453[CrossRef][Medline] [Order article via Infotrieve] |
2. | Florini, J. R., Ewton, D. Z., Magri, K. A., and Mangiacapra, F. J. (1994) in Current Directions in Insulin-like Growth Factor Research (Leroith, D. , and Raizada, M. K., eds) , p. 319, Plenum Press, New York |
3. | Florini, J. R., Magri, K. A., Ewton, D. Z., James, P. L., Grindstaff, K., and Rotwein, P. S. (1991) J. Biol. Chem. 24, 15917-15923 |
4. |
Montarras, D.,
Aurade, F.,
Johnson, T.,
Ilan, J.,
Gros, F.,
and Pinset, C.
(1996)
J. Cell Sci.
109,
551-560 |
5. |
Coolican, S. A.,
Samuel, D. S.,
Ewton, D. Z.,
McWade, F. J.,
and Florini, J. R.
(1997)
J. Biol. Chem.
272,
6653-6662 |
6. | Calera, M. R., and Pilch, P. F. (1998) Biochem. Biophys. Res. Commun. 251, 835-841[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Kaliman, P.,
Vinals, F.,
Testar, X.,
Palacin, M.,
and Zorzano, A.
(1996)
J. Biol. Chem.
271,
19146-19151 |
8. | Pinset, C., Garcia, A., Rousse, S., Dubois, C., and Montarras, D. (1997) C. R. Acad. Sci. 320, 367-374 |
9. |
Jiang, B. H.,
Aoki, M.,
Zheng, J. Z.,
Li, J.,
and Vogt, P. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2077-2081 |
10. | Meier, R., and Hemmings, B. A. (1999) J. Recept. Signal Transduct. Res. 19, 121-128[Medline] [Order article via Infotrieve] |
11. | Peterson, R. T., and Schreiber, S. L. (1999) Curr. Biol. 15, 521-524 |
12. | Alessi, D. R., and Cohen, P. (1998) Curr. Opin. Genet. Dev. 8, 55-82[CrossRef][Medline] [Order article via Infotrieve] |
13. | Coofer, P. J., Jin, J., and Woodgett, R. (1998) Biochem. J. 335, 1-13[Medline] [Order article via Infotrieve]. |
14. |
Fujio, Y.,
Guo, K.,
Mano, T.,
Mitsuuchi, Y.,
Testa, J. R.,
and Walsh, K.
(1999)
Mol. Cell. Biol.
19,
5073-5082 |
15. |
Hill, M. M.,
Clarck, S. F.,
Tucker, D. F.,
Birnbaum, M. J.,
James, D. E.,
and Macaulay, S. L.
(1999)
Mol. Cell Biol.
19,
7771-7781 |
16. |
Summers, S. C,
Whiteman, E. L.,
Cho, H.,
Lipfert, L.,
and Birnbaum, J.
(1999)
J. Biol. Chem.
274,
23858-23867 |
17. |
Calera, M. R.,
Martinez, C.,
Liu, H.,
Jack, A. K.,
Birnbaum, M. J.,
and Pilch, P. F.
(1998)
J. Biol. Chem.
273,
7201-7204 |
18. | Shaw, M, Cohen, P, and Alessi, D. R. (1998) Biochem. J. 336, 241-246[Medline] [Order article via Infotrieve] |
19. | Altomare, D. A., Lyons, G. E., Mitsuuchi, Y., Cheng, J. Q., and Testa, J. R. (1997) Oncogene 16, 2407-2411 |
20. | Yaffe, D. (1968) Proc. Natl. Acad. Sci. U. S. A. 61, 477-483[Medline] [Order article via Infotrieve] |
21. |
Kitzmann, M.,
Carnac, G.,
Vandromme, M.,
Primig, M.,
Lamb, N. J. C.,
and Fernandez, A.
(1998)
J. Cell Biol.
142,
1447-1459 |
22. |
Meier, R.,
Alessi, D. R.,
Cron, P.,
Andjelkovic, M.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
30491-30497 |
23. | Cross, D. A., Alessi, D. R,., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve] |
24. | Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A., and Cohen, P. (1996) FEBS Lett. 399, 333-338[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Andjelkovic, M.,
Alessi, D. R.,
Meier, R.,
Fernandez, A.,
Lamb, N. J. C.,
Frech, M.,
Cron, P.,
Cohen, P.,
Lucocq, J. H. M.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
31515-31524 |
26. | Vandromme, M., Gauthier-Rouviere, C., Carnac, G., Lamb, N., and Fernandez, A. (1992) J. Cell Biol. 118, 1489-1500[Abstract] |
27. | Girard, F., Strausfeld, U., Fernandez, A., and Lamb, N. J. C. (1991) Cell 67, 1169-1179[Medline] [Order article via Infotrieve] |
28. | Lassar, A. B., Buskin, J. N., Lockshon, D., Davis, R. L., Apone, S., Hauschka, S. D., and Weintraub, H. (1989) Cell 58, 823-831[Medline] [Order article via Infotrieve] |
29. | Shiel, M. A., Hugen, H. S., Clegg, C. H., and Hauschka, S. D. (1996) Mol. Cell. Biol. 16, 5058-5068[Abstract] |
30. |
Puri, P. L.,
Avantaggiati, M. L.,
Balsano, C.,
Sang, N.,
Graessmann, A.,
Giordano, A.,
and Levrero, M.
(1997)
EMBO J.
16,
369-383 |
31. | Gauthier-Rouviere, C., Vandromme, M., Tuil, D., Lautredou, N., Morris, M., Soulez, M., Kahn, A., Fernandez, A., and Lamb, N. (1996) Mol. Biol. Cell 7, 719-729[Abstract] |
32. |
Diehl, J. A.,
Cheng, M.,
Roussel, M. F.,
and Sherr, C. J.
(1998)
Genes Dev.
12,
3499-3511 |
33. |
Rommel, C.,
Clarke, B. A.,
Zimmermann, S.,
Nunez, L.,
Rossman, R.,
Reid, K.,
Moelling, K.,
Yancopoulos, G. D.,
and Glass, D. J.
(1999)
Science
286,
1738-1741 |
34. |
Lawlor, M. A.,
Feng, X.,
Everding, D. R.,
Sieger, K.,
Stewart, C. E.,
and Rotwein, P.
(2000)
Mol. Cell. Biol.
20,
3256-3265 |
35. | Altomare, D. A., Guo, K., Cheng, J. Q., Sonoda, G., Walsh, K., and Testa, J. R. (1995) Oncogene 11, 1055-1060[Medline] [Order article via Infotrieve] |
36. |
Pekarsky, Y.,
Koval, A.,
Hallas, C.,
Bichi, R.,
Tresini, M.,
Malstrom, S.,
Russo, G.,
Tsichlis, P.,
and Croce, C. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3028-33 |
37. | Datta, K., Franke, T. F., Chan, T. O., Makris, A, Yang, S. I., Kaplan, D. R., Morrison, D. K., Golemis, E. A., and Tsichlis, P. N. (1995) Mol. Cell. Biol. 15, 2304-2310[Abstract] |
38. | Laine, J., Kunstle, G., Obata, T., Sha, M., and Noguchi, M. (2000) Mol. Cell. 6, 395-407[Medline] [Order article via Infotrieve] |
39. |
Toker, A.,
and Newton, A. C.
(2000)
J. Biol. Chem.
275,
8271-8274 |
40. |
Tamir, Y.,
and Bengal, E.
(2000)
J. Biol. Chem.
275,
34424-34432 |
41. |
Muise-Helmericks, R. C.,
Grimes, H. L.,
Bellacosa, A.,
Malstrom, S. E.,
Tsichlis, P. N.,
and Rosen, N.
(1998)
J. Biol. Chem.
273,
29864-29872 |
42. |
Ramaswamy, S.,
Nakamura, N.,
Vazquez, F.,
Batt, D. B.,
Perera, S.,
Roberts, T. M.,
and Sellers, W. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2110-2115 |
43. |
Sun, H.,
Lesche, R.,
Li, D. M.,
Liliental, J.,
Zhang, H.,
Gao, J.,
Gavrilova, N.,
Mueller, B.,
Liu, X.,
and Wu, H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6199-6204 |
44. |
Cheng, J. Q.,
Ruggeri, B.,
Klein, W. M.,
Sonoda, G.,
Altomare, D. A.,
Watson, D. K.,
and Testa, J. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3636-3641 |
45. |
Hooshmand-Rad, R.,
Hajkova, L.,
Klint, P.,
Karlsson, R.,
Vanhaesebroeck, B.,
Claesson-Welsh, L.,
and Heldin, C. H.
(2000)
J. Cell Sci.
113,
207-214 |
46. |
Asano, T.,
Kanda, A.,
Katagiri, H.,
Nawano, M.,
Ogihara, T.,
Inukai, K.,
Anai, M.,
Fukushima, Y.,
Yazaki, Y.,
Kikuchi, M.,
Hooshmand-Rad, R.,
Heldin, C. H.,
Oka, Y.,
and Funaki, M.
(2000)
J. Biol. Chem.
275,
17671-17676 |
47. |
Hill, K.,
Welti, S., Yu, J.,
Murray, J. T.,
Yip, S. C.,
Condeelis, J. S.,
Segall, J. E.,
and Backer, J. M.
(2000)
J. Biol. Chem.
275,
3741-3744 |
48. | Wang, Q., Bilan, P. J., Tsakiridis, T., Hinek, A., and Klip, A. (1998) Biochem. J. 331, 917-928[Medline] [Order article via Infotrieve] |
49. | Moon, R. T., Brown, J. D., and Torres, M. (1997) Trends Genet. 13, 157-162[CrossRef][Medline] [Order article via Infotrieve] |
50. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |