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Departments of Medicine and Biochemistry and Molecular Biology University of Arkansas for Medical Sciences and the Geriatric Research, Education, and Clinical Center McClellan Veterans Hospital Little Rock, Arkansas 72205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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i2
were detected in NFB4 cells and IGF-I treated NFB4 cells, which
correlated with responsive Gi signaling.
Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or
treatment with IGF-II was associated with loss of
Gi2 and inhibition of
Gi-dependent signaling. Thus, IGF-I and IGF-II
activate distinct signaling cascades, with IGF-II eliciting a stronger
differentiation effect correlated with down-regulation of
Gi2 protein. Short-term stimulation of NFB4
cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1
and -2 activation. Transient stimulation of NFB4 cells with IGF-I while
blocking activation of Gi-proteins is with
pertussis toxin resulted in preferential activation of ERK-2
characteristic of differentiated C2C12 cells, suggesting that
proliferation induced by IGF-I is Gi-dependent
and separable from the IGF-I-signaling pathway that leads to
differentiation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Seven membrane-spanning receptors that interact with Bordella pertussis
toxin (PT)-sensitive heterotrimeric G proteins also are able to mediate
p21ras-dependent activation of ERKs (12, 13).
Receptor activation stimulates nucleotide exchange and
dissociation of the G protein, releasing the Gi subunit
in its GTP-bound state from the Gß
complex.
Gß complex mediates src-dependent phosphorylation of
the epidermal growth factor receptor and thereby activation of the
p21ras pathway (14). Employing this mechanism, G
protein-coupled receptors can also contribute to
p21ras-dependent cellular response.
The role of ERK activity in myogenic growth and differentiation is not well understood. The formation of skeletal muscle in embryogenesis proceeds through commitment of mesodermal progenitors to the myogenic lineage and subsequent differentiation of skeletal myoblasts into terminally differentiated myotubes. Growth factors, such as basic fibroblast growth factor and transforming growth factor-ß, play a central role in maintenance of myoblasts in the proliferative state that is nonpermissive for the expression of muscle-specific genes (15, 16). Basic fibroblast growth factor-stimulated proliferation is accompanied by robust and transient ERK activation (17). Exit from the cell cycle that can be forced by serum withdrawal from the medium induces terminal differentiation. Transition of myoblasts to myotubes is accompanied by activation of the IGF-II autocrine loop and appears absolutely required (Ref. 18; reviewed in Ref.19). The IGF-I receptor is the main mediator of both IGF-I and IGF-II signaling in myoblasts (20), where IGF-I promoted proliferation followed by differentiation, whereas IGF-II demonstrated less mitogenic effect (21). Activation of the IGF-I receptor by binding of IGFs induces tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the major substrate for insulin and type I IGF tyrosine kinase receptors, and Shc proteins. The Grb-2 adaptor protein links IRS-1, and alternatively Shc proteins, to the p21ras-signaling pathway, resulting in activation of ERK-1 and -2 (22, 23). Recently it was shown that the ERK pathway mediates primarily the proliferative effects of IGF-I on myoblasts (24). In our previous study, we characterized the IGF signal transduction pathway during myogenic differentiation of C2C12 myoblasts and nondifferentiating mutant NFB4 cells that fail to activate the IGF-II autocrine loop and require exogenous IGFs to induce differentiation (25). Exogenous IGF-I partially rescued the mutant phenotype, making these cells useful tools for manipulating the IGF pathway and analyzing downstream signaling components.
In this study we analyzed ERK activity in C2C12 cells during myogenic differentiation and also in nondifferentiating mutant NFB4 cells derived from them by chemical mutagenesis. After exposure of cells to low serum, ERK-1 and -2 in NFB4 cells remained responsive to stimulation by mitogens whereas they were much less responsive in C2C12 cells. Reactivation of ERKs occurred in response to different mitogenic signals and appeared dependent on signaling through Gi proteins. Furthermore, induction of differentiation in both cell types was correlated with preferential and sustained activation of ERK-2. Activation of ERK-1 and -2 was an early response to IGF-I, which could be blocked by PT leading to sustained activation of ERK-2. Thus, the biphasic response of myoblasts to IGF-I, proliferation followed by differentiation, was correlated with specific ERK activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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)). Both
C2C12 and NFB4 cell lines demonstrated low kinase activity in the mol
wt range of ERK-1 and -2, indicated as p44 and p42, in differentiation
medium (DM) low in serum (Fig. 1A, lanes 1 and 3). After exposure to DM
for 24 h, stimulation of NFB4 cells by medium containing high
serum (growth medium, GM) induced activation of the p44 and p42, as
well as a 97-kDa kinase (Fig. 1A, lane 4; p42, p44, and p97) that might
represent a novel kinase of this family, ERK-5 (26). By contrast, ERK
activation in response to serum was significantly reduced in C2C12
cells (Fig. 1A, lane 2). Similar to NFB4 cells, C2C12 cells blocked
from differentiation by stable expression of constitutively active Ras
also demonstrated highly inducible ERK activity after stimulation by GM
(data not shown).
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, lanes 1
and 2). A small proportion of ERK-2 appeared to be phosphorylated as
detected by a shift in mobility, and this was relatively unaffected by
serum stimulation, consistent with in-gel kinase results. ERK-2
abundance in NFB4 cells was comparable to C2C12s (1.5-fold), whereas
ERK-1 was 4 times more abundant in NFB4 cells, as abundant as ERK-2
(Fig. 1B, lanes 3 and 4). After stimulation by GM, both kinases became
phosphorylated in NFB4 cells but to different extents. ERK-1 became
completely phosphorylated whereas approximately half of the ERK-2
demonstrated altered mobility.
Phosphorylation of ERK-1 and -2 in C2C12 compared with NFB4 cells was
quantitated using a phospho-MAPK antibody specifically selected to
recognize only activated (phosphorylated) forms of ERK-1 and -2 (Fig. 1C). This antibody reacted strongly with ERK-1 and -2 only in NFB4
cells stimulated by GM (Fig. 1C, lane 4). Much less reactivity
(
20-fold less) was detectable in unstimulated cells (Fig. 1C, lane
3). ERKs were weakly recognized in C2C12 cells, which increased
slightly after stimulation by GM (Fig. 1C, lanes 1 and 2). This was
most apparent for ERK-2 due to its abundance (see Fig. 1B). Overall,
phosphorylation of ERK-1 and -2 was increased 5-fold in NFB4 compared
with C2C12 cells in response to serum stimulation. These data indicated
that kinase activity of ERK-1 and -2 was correlated with their
phosphorylation state, and that NFB4 myoblasts that continued to
proliferate in low serum had ERK activity that was strongly serum
inducible.
Sustained Activation of ERKs during Myogenic
Differentiation Is Dependent on Activation of the IGF-II Autocrine Loop
(
Figs. 2–4)
It was shown above that NFB4 cells that do not differentiate upon
serum withdrawal retained highly inducible ERK-1 and -2. Moreover,
activated ERK-2 was detectable in differentiated C2C12 cells (Fig. 1, B
and C, lane 1), suggesting that sustained ERK activation may be
involved in myogenic differentiation. This was examined by performing a
time course of ERK phosphorylation during differentiation of C2C12
cells by phospho-MAPK Western blot (Fig. 2). C2C12 cells have been well
characterized, and IGF-II is secreted into the medium within 24 h
of exposure to DM, and fully formed myotubes are present within 72
h (19, 20, 25). In GM and early during differentiation of C2C12 cells,
low level ERK phosphorylation was observed (Fig. 2, top
panel, lanes 1 and 2). Phosphorylation of ERK-2 was increased
slightly (1.5-fold) by 12 h and within 36 h of incubation in
DM, ERK-2 became preferentially phosphorylated (4-fold over GM levels)
that was sustained (Fig. 2, top panel, lanes 5–7),
indicating that activation of ERK-2 was associated with myogenic
differentiation of C2C12 cells. ERK-1 phosphorylation also increased
slightly with differentiation, although this was less apparent due to
low protein abundance. Neither ERK-1 nor ERK-2 abundance changed during
differentiation demonstrated with a p44/42 MAPK antibody recognizing
phosphorylated and nonphosphorylated ERKs (Fig. 2, bottom
panel). ERK-2 phosphorylation correlated with tyrosine
phosphorylation of IRS-1 in C2C12 cells after 36 h of incubation
in DM, dependent on the IGF-II autocrine loop (D. D. Sarbassov and C.
A. Peterson, unpublished observations), implying involvement of the IGF
signal transduction pathway in sustained activation of ERK-2. To test
this idea, ERK activation in C2C12 cells in which the IGF-II autocrine
loop was inhibited by IGF-II antisense expression was analyzed (Fig. 3). It has been shown that expression of
IGF-II antisense oligonucleotides in C2C12 cells blocked myogenic
differentiation and induced apoptosis (18). In our study, expression of
IGF-II antisense oligonucleotides in C2C12 cells resulted in loss of
accumulation of IGF-II precursor protein (Fig. 3A, lane 4) compared
with control (Fig. 3A, lane 3) and vector transfected (Fig. 3A, lane 1)
cells, similar to the NFB4 phenotype (Fig. 3A, lane 2). Furthermore,
IGF-II antisense-expressing cells differentiated poorly as indicated by
inhibition of myogenin and myosin heavy chain (MyHC) accumulation after
exposure to DM (Fig. 3B, lane 4), again, similar to the NFB4 phenotype
(Fig. 3B, lane 2). Treatment of IGF-II antisense-expressing cells with
exogenous IGFs restored the differentiated phenotype (Fig. 3B, lane 5),
as in control C2C12 cells (Fig. 3B, lane 3) and vector-transfected
cells (Fig. 3B, lane 1). Moreover, C2C12 cells expressing IGF-II
antisense oligonucleotides were not able to activate ERK-2 in DM (Fig. 3C, lanes 1 and 3). Exogenous IGF-I induced a 3-fold increase in
phosphorylation of ERK-2 (Fig. 3C, lane 4) that required greater than
24 h incubation with the growth factor (Fig. 3C, lane 2). Thus,
the IGF signal transduction pathway was necessary for activation of
ERK-2 during myogenic differentiation of C2C12 cells.
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).
After 36 h exposure to DM + PD98059, ERK phosphorylation was
significantly inhibited in C2C12 cells (Fig. 4A, top panel),
whereas ERK accumulation was unaffected by the inhibitor (data not
shown). PD98059 treatment appeared to be detrimental to the
differentiation process. Although the expression of myogenin was
unaffected (Fig. 4A, bottom panel) and myotubes did form,
they did not survive (Fig. 4B). In the presence of the inhibitor, the
cells rounded up and detached from the plate compared with untreated
cells, suggesting that sustained ERK phosphorylation is required for
cell survival during differentiation.
Differential ERK Activation in Response to
Gi-Dependent Signaling Pathways (
Figs. 5–7)
ERK activation in NFB4 cells in response to serum was also
produced by specific signaling molecules. Lysophosphatidic acid (LPA)
acts through its cognate receptor that interacts with PT-sensitive
heterotrimeric G proteins (13, 28, 29, 30). LPA activated ERKs in NFB4
cells but not in C2C12 cells assayed by the in-gel kinase assay (Fig. 5, compare lanes 1 and 2). Under these
conditions activation of ERK-1 (p44) was more pronounced. Blocking the
Gi-dependent pathway by PT in NFB4 cells inhibited
activation of ERKs by LPA (Fig. 5, lane 3). These data suggested that
activation of ERKs in NFB4 cells occurred through a
Gi-dependent pathway. Taken together with the results
described above, it appears that increased phosphorylation of ERK-1
relative to ERK-2 is associated with proliferation, whereas high levels
of phosphorylated ERK-2 relative to ERK-1 is characteristic of
differentiation.
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, lanes 1, 4, and 7). Stimulation of
IGF-I treated NFB4 cells by either GM or LPA strongly induced
activation of ERKs (Fig. 6, lanes 2 and 3), whereas ERKs in
NFB4/myogenin and NFB4/MyoD cells were responsive only to GM (Fig. 6, lanes 5 and 8) but not to LPA (Fig. 6, lanes 6 and 9). Thus, NFB4 cells
rescued by direct IGF-I treatment and those rescued by myogenin or MyoD
expression differed in their response to LPA. In IGF-I-rescued NFB4
cells, ERKs, in particular ERK-1, were still responsive to the
Gi-dependent pathway, whereas in myogenin or
MyoD-transfected cells this pathway was inhibited.
The mitogenic effect of LPA involving activation of ERKs is mediated
through its receptor coupled to Gi2 protein. It might be
that activation of ERKs through a Gi-dependent pathway was
dependent on the abundance of Gi2 protein in C2C12 and
NFB4 cells. Similar levels of Gi1 protein were detected
in C2C12, NFB4, and rescued NFB4 cells, whereas the levels of
Gi2 were different (Fig. 7A). A low level of Gi2
was found in C2C12 cells (Fig. 7A, lane 1), and a 10-fold higher level
of this protein was detected in NFB4 cells relative to
Gi1 (Fig. 7A, lane 2). Northern analysis also revealed
overexpression of Gi2 mRNA in NFB4 cells compared with
C2C12 cells (data not shown). IGF-I-rescued NFB4 cells (Fig. 7A, lane
3) continued to accumulate Gi2 whereas the protein was 3
times less abundant in NFB4/myogenin and NFB4/MyoD cells relative to
Gi1 (Fig. 7A, lanes 4 and 5). Thus, the abundance of
Gi2 correlated with the ERK response to LPA
stimulation.
We and others showed that IGF-I treatment inhibits activation of the
IGF-II autocrine loop in myoblasts (25, 31). Activation of the IGF-II
autocrine loop is normally associated with myogenic differentiation of
C2C12 cells and was also detected in NFB4/myogenin and NFB4/MyoD cells
(25), implying that the IGF-II-signaling pathway may lead to
down-regulation of Gi2. This appears to be the case as
incubation of NFB4 cells with exogenous IGF-II induced down-regulation
of Gi2 by 4-fold relative to Gi1 (Fig. 7A, lane 6). These results suggest that NFB4 cells respond differently
to long-term exposure to IGF-I vs. IGF-II.
To determine whether the different effects of IGF-I and IGF-II on the
level of Gi2 protein correlated with induction of
myogenic differentiation in NFB4 cells, we analyzed expression of
myogenin. Myogenin accumulated to very low levels in NFB4 cells (Fig. 7, lane 1). Incubation of NFB4 cells with IGF-I induced expression of
myogenin (Fig. 7B, lane 2), whereas a higher level of myogenin was
detected in NFB4 cells incubated with IGF-II (Fig. 7B, lane 3). Thus,
IGF-II appeared to be a stronger myogenic factor, compared with IGF-I,
consistent with previous reports (21, 25).
PT Alters ERK Activity in Response to IGF-I (Fig. 8)
Rapid phosphorylation of ERKs in response to serum stimulation and
LPA treatment that was preferential for ERK-1 was characteristic of the
proliferative phenotype of the mutant NFB4 cells, whereas sustained
activation of ERK-2 was associated with IGF-dependent myogenic
differentiation. These observations suggest that ERK activity in NFB4
cells may be an indicator of the biphasic effects of IGF-I on
myoblasts: proliferation is the early response followed by
differentiation (21). This idea was tested by analyzing ERK activity in
NFB4 cells incubated in DM and stimulated by IGF-I for 10 min. Low
level phosphorylation of ERKs was detected in DM by the phospho-MAPK
antibody (Fig. 8, lane 1). Short-term
IGF-I stimulation induced activation of both ERK-1 and -2, whereas LPA
stimulation resulted in preferential activation of ERK-1 (Fig. 8, compare lanes 2 and 4; see also Fig. 5). This effect was abrogated by
preincubation with PT (Fig. 8, lane 5). Surprisingly, in cells
preincubated with PT, IGF-I induced preferential phosphorylation of
ERK-2 relative to ERK-1 (Fig. 8, lane 3). Thus, in response to IGF-I,
high levels of Gi2 protein in NFB4 cells were linked
with activation of ERK-1 and -2. Blocking Gi signaling by
PT resulted in preferential activation of ERK-2, similar to the pattern
of ERK phosphorylation in differentiated C2C12 cells treated with IGF-I
(Fig. 8, lane 6). These results suggest that early IGF-I signaling
events associated with proliferation can be altered by blocking
activation of Gi proteins, thereby mimicking signaling
events occurring during myogenic differentiation dependent on the
IGF-II autocrine loop.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Activation of the IGF-II autocrine loop is required for myogenic
differentiation of C2C12 cells, and IGFs are survival factors for
myoblasts (18, 19). NFB4 cells fail to activate IGF-II gene expression
upon serum withdrawal, are unable to differentiate, and do not
demonstrate activation of ERK-2 in DM. Inhibition of IGF-II
accumulation in C2C12 cells also resulted in a nondifferentiated
phenotype and no ERK-2 activation. Exogenous long-term IGF-I treatment
was able to induce differentiation and concomitant activation of ERK-2.
By contrast, a Gi-signaling pathway was activated in NFB4
cells but not in C2C12 cells in response to LPA leading to preferential
ERK-1 activation. LPA acts through its cognate receptor that is coupled
with Gi2 protein, and the level of Gi2
protein correlated with PT-sensitive activation of ERK-1. A low
abundance of this protein was demonstrated in C2C12 and a high
abundance in NFB4 cells, suggesting that Gi2-signaling
contributes to the proliferative phenotype of NFB4 cells. Thus, a high
ratio of phosphorylated ERK-2/ERK-1 is associated with myogenic
differentiation, whereas a high ratio of phosphorylated ERK-1/ERK-2 is
associated with proliferation. These results suggest that ERK-1 and -2
may be functionally distinct. The mechanism whereby these kinases may
participate in different cellular processes is unknown but may involve
different substrates or different subcellular localization.
Although the IGF-I receptor is the main mediator of IGF-I and IGF-II,
the effects of these factors on myogenic differentiation were not
identical. It has been reported previously that IGF-I has a greater
mitogenic effect and IGF-II is more myogenic (21). IGF-I-treated NFB4
cells remained responsive to LPA stimulation, whereas NFB4/myogenin and
NFB4/MyoD cells, able to activate the IGF-II autocrine loop,
demonstrated nonresponsiveness to stimulation by LPA. Thus, rescue of
the mutant phenotype by IGF-I treatment or by indirect activation of
the IGF-II autocrine loop by myogenin or MyoD expression were distinct.
This is confirmed by the fact that in NFB4/myogenin, NFB4/MyoD, and
NFB4 cells directly treated with IGF-II (but not IGF-I),
Gi2 protein was down-regulated. Specific coupling of the
IGF-II receptor with Gi2 protein has been reported (33).
Long-term activation of Gi2 signaling by the IGF-II
receptor may result in down-regulation of Gi2 protein.
IGF-I was not able to down-regulate Gi2 protein possibly
due to its low affinity to the IGF-II receptor. This effect of IGF-II
may also be linked to the putative atypical IGF-I receptor associated
only with myogenic differentiation of myoblasts that preferentially
binds IGF-II (34). Thus, activation of the IGF-II autocrine loop
associated with myogenic differentiation of C2C12 cells and NFB4 cells
by expression of myogenin or MyoD demonstrated low-level
Gi2 protein accumulation correlated with
nonresponsiveness of these cells to PT-sensitive activation of ERKs by
LPA. The stronger myogenic effect of IGF-II vs. IGF-I might
be linked with the ability of IGF-II to down-regulate one of the
proliferative pathways associated with
Gi2-signaling.
Signaling through the IGF-I receptor was altered by PT, suggesting that
Gi-proteins are involved in this process. It has been shown
in other cell types that blocking Gi-dependent signaling
interfered with activation of ERKs by the IGF-I signal transduction
pathway (33, 35). Stimulation of NFB4 cells that overexpress
Gi2 protein by IGF-I induced activation of ERK-1 and -2.
Similar stimulation of NFB4 cells while blocking the activity of
Gi proteins with PT altered IGF signaling, resulting in
preferential activation of ERK-2 that mimicked ERK-2 activation during
normal differentiation of C2C12 cells. Thus, the dual effects of IGF-I
on myoblasts, induction of proliferation and differentiation, appear to
be mediated by different signaling pathways that result in distinct
patterns of ERK activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Introduction of a pEMSVscribe2/IGF-II antisense expression plasmid
(18) or empty vector into C2C12 cells was performed by the calcium
phosphate coprecipitation method as described (36). C2C12 cells were
plated at 1.5 x 105 cells/100-mm tissue culture plate
and 24 h later, cells were washed and cotransfected with 10 µg
of a pEMSVscribe2/IGF-II antisense expression plasmid together with
1 µg pSV2neo. The DNA was removed 24 h later with the addition
of fresh GM. After an additional 24 h, cells were split 1:4 and
refed with medium to which 400 µg/ml of G418 (Geneticin, GIBCO/BRL,
Gaithersburg, MD) had been added. Selection proceeded for 14 days and
G418-resistant clones were picked randomly. Pooled clones of NFB4 cells
constitutively expressing MyoD or myogenin have been described
previously (25).
Western Blot Analysis
After incubation and stimulation of cells, 100-mm dishes were
washed twice with cold PBS and lysed in 0.5 ml cold lysis buffer (50
mM HEPES, pH 7.4, 150 mM NaCl, 1.5
mM MgCl2, 1 mM EGTA, 100
mM NaF, 10 mM sodium pyrophosphate, 1
mM phenylmethylsulfonyl fluoride, 3 µg/ml of each
leupeptin and aprotinin, 6 µg/ml of antipain, 30 µg/ml of
benzamidine, 1 mM Na3VO4, 1%
Triton X 100) for 10 min by shaking. All manipulations of cell lysates
were at 4 C. Lysates were scraped into microcentrifuge tubes and
cleared of nuclei and detergent-insoluble material by centrifugation
for 10 min at 14,000 rpm. Samples (35 µg of protein) were resolved by
discontinuous electrophoresis through 7.5% SDS polyacrylamide gels and
electrophoretically transferred to PVDF membrane (Immobilon P,
Millipore, Bedford, MA). Blots were blocked for 1 h in 5% milk in
PBS plus 0.5% Tween-20 (PBST). Phospho-MAPK and ERK-1 and -2
antibodies (New England Biolabs) were applied at a 1:1000 dilution in
PBST containing 3% BSA (heat shock treated, Fisher, Pittsburgh, PA)
overnight at 4 C. Gi1 and Gi2
(Calbiochem, San Diego, CA), myogenin (hybridoma F5D, Dr. Woodring
Wright, University of Texas Southwestern Medical Center, Dallas, TX),
MyHC (A4.1025, 25), and IGF-II (sc-1417, Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) antibodies were applied at 1:1000 dilution in
PBST containing 3% milk and incubated for 1 h. Blots were washed
5 times with PBST for a total of 30 min, before and after incubating
with horseradish peroxidase-conjugated secondary antibody (Pierce,
Rockford, IL) diluted 1:4000 in PBST containing 4% milk for 1 h.
Renaissance Chemiluminescence Reagent (DuPont/NEN, Wilmington, DE) was
used as the detection system. Signals were quantitated using a
computing densitometer with LasarQuant software (Molecular Dynamics,
Sunnyvale, CA). Blots were stripped and reprobed as described (37).
Immunocytochemistry
Immunocytochemical analyses were performed essentially as
described (25). Briefly, cells were washed in PBS, fixed with 1%
paraformaldehyde, and then treated with ice-cold methanol. Cells were
incubated with undiluted MyHC A4.1025 hybridoma tissue culture
supernatant for 1 h at room temperature followed by incubations
with horseradish peroxidase-conjugated anti-mouse IgG. Peroxidase
reactivity was visualized using the DAB substrate kit (Vector
Laboratories, Burlingame, CA).
In-Gel Kinase Assay
The in-gel kinase assay was performed essentially as described
(38). Cell lysates were boiled in Laemmli sample buffer for 2 min and
resolved by discontinuous electrophoresis through 7.5% SDS
polyacrylamide gel containing 0.5 mg/ml myelin basic protein (Sigma).
The gel was fixed by four washes with 20% 2-propanol in buffer A (50
mM Tris-HCl buffer, pH 8.0, containing 2 mM
dithiothreitol) for 2 h, and SDS was removed by washing the gel
for 2 h in several volumes of buffer A, changing the solution
every 15 min. Proteins in the gel were denatured in 6 M
guanidine HCl for 2 h in buffer A and then renatured overnight at
4 C in buffer A containing 0.04% Tween 40 (Sigma). After preincubation
of the gel for 1 h in 5 ml of 40 mM HEPES, pH 8.0,
containing 2 mM dithiothreitol and 10 mM
MgCl2, the kinase reaction was carried out by incubation of
the gel for 1 h at 25 C in 5 ml of 40 mM HEPES, pH
8.0, containing 25 µCi of [-32P]ATP, 40
µM ATP, 0.5 mM EGTA, 2 mM
dithiothreitol, and 10 mM MgCl2. After the
kinase reaction the gel was washed several times in 5% (wt/vol)
trichloroacetic acid containing 1% pyrophosphate until the
radioactivity reached background levels. After washes, the gel was
dried and exposed to x-ray film.
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ACKNOWLEDGMENTS |
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2/IGF-II
antisense expression plasmid, Dr. Woodring Wright for the myogenin
hybridoma, Elena Moerman for IGF-I, and Jane Taylor-Jones for
assistance with artwork. |
FOOTNOTES |
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This work was supported by grants from the National Institutes of Health-National Institute on Aging (to C.A.P).
Received for publication May 27, 1997. Revision received September 8, 1997. Accepted for publication September 23, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2ß2 IGF-II receptor type C2
myoblasts. Eur J Biochem 208:273–279[Medline]
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S. Adi, B. Bin-Abbas, N.-Y. Wu, and S. M. Rosenthal Early Stimulation and Late Inhibition of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation by IGF-I: A Potential Mechanism Mediating the Switch in IGF-I Action on Skeletal Muscle Cell Differentiation Endocrinology, February 1, 2002; 143(2): 511 - 516. [Abstract] [Full Text] [PDF]
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J. Tureckova, E. M. Wilson, J. L. Cappalonga, and P. Rotwein Insulin-like Growth Factor-mediated Muscle Differentiation. COLLABORATION BETWEEN PHOSPHATIDYLINOSITOL 3-KINASE-Akt-SIGNALING PATHWAYS AND MYOGENIN J. Biol. Chem., October 12, 2001; 276(42): 39264 - 39270. [Abstract] [Full Text] [PDF]
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 E. V. Thrane, M. Refsnes, G. H. Thoresen, M. Lag, and P. E. Schwarze Fluoride-Induced Apoptosis in Epithelial Lung Cells Involves Activation of MAP Kinases p38 and Possibly JNK Toxicol. Sci., May 1, 2001; 61(1): 83 - 91. [Abstract] [Full Text] [PDF]
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 M. A. Lawlor and P. Rotwein Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways J. Cell Biol., December 11, 2000; 151(6): 1131 - 1140. [Abstract] [Full Text] [PDF]
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M. A. Lawlor and P. Rotwein Insulin-Like Growth Factor-Mediated Muscle Cell Survival: Central Roles for Akt and Cyclin-Dependent Kinase Inhibitor p21 Mol. Cell. Biol., December 1, 2000; 20(23): 8983 - 8995. [Abstract] [Full Text] [PDF]
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 C. M. Boney, P. A. Gruppuso, R. A. Faris, and A. R. Frackelton Jr. The Critical Role of Shc in Insulin-Like Growth Factor-I-Mediated Mitogenesis and Differentiation in 3T3-L1 Preadipocytes Mol. Endocrinol., June 1, 2000; 14(6): 805 - 813. [Abstract] [Full Text]
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Z. Wu, P. J. Woodring, K. S. Bhakta, K. Tamura, F. Wen, J. R. Feramisco, M. Karin, J. Y. J. Wang, and P. L. Puri p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps Mol. Cell. Biol., June 1, 2000; 20(11): 3951 - 3964. [Abstract] [Full Text] [PDF]
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M. A. Lawlor, X. Feng, D. R. Everding, K. Sieger, C. E. H. Stewart, and P. Rotwein Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways Mol. Cell. Biol., May 1, 2000; 20(9): 3256 - 3265. [Abstract] [Full Text] [PDF]
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A. F. Valledor, M. Comalada, J. Xaus, and A. Celada The Differential Time-course of Extracellular-regulated Kinase Activity Correlates with the Macrophage Response toward Proliferation or Activation J. Biol. Chem., March 15, 2000; 275(10): 7403 - 7409. [Abstract] [Full Text] [PDF]
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H. Hallak, A. E. M. Seiler, J. S. Green, B. N. Ross, and R. Rubin Association of Heterotrimeric Gi with the Insulin-like Growth Factor-I Receptor. RELEASE OF Gbeta gamma SUBUNITS UPON RECEPTOR ACTIVATION J. Biol. Chem., January 28, 2000; 275(4): 2255 - 2258. [Abstract] [Full Text] [PDF]
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K. L. Haugk, H.-M. P. Wilson, K. Swisshelm, and L. S. Quinn Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF Endocrinology, January 1, 2000; 141(1): 100 - 110. [Abstract] [Full Text] [PDF]
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 J. C. Aliaga, C. Deschenes, J.-F. Beaulieu, E. L. Calvo, and N. Rivard Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells Am J Physiol Gastrointest Liver Physiol, September 1, 1999; 277(3): G631 - G641. [Abstract] [Full Text] [PDF]
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 A. F. Valledor, J. Xaus, L. Marques, and A. Celada Macrophage Colony-Stimulating Factor Induces the Expression of Mitogen-Activated Protein Kinase Phosphatase-1 Through a Protein Kinase C-Dependent Pathway J. Immunol., September 1, 1999; 163(5): 2452 - 2462. [Abstract] [Full Text] [PDF]
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 D. J. Peterson, H. Ju, J. Hao, M. Panagia, D. C. Chapman, and I. M.C. Dixon Expression of Gi-2{alpha} and Gs{alpha} in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction Cardiovasc Res, March 1, 1999; 41(3): 575 - 585. [Abstract] [Full Text] [PDF]
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D. D. Sarbassov and C. A. Peterson Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Regulate Extracellular Signal-Regulated Kinase-Dependent and -Independent Signaling Pathways during Myogenic Differentiation Mol. Endocrinol., December 1, 1998; 12(12): 1870 - 1878. [Abstract] [Full Text]
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E. Chevillotte, J. Rieusset, M. Roques, M. Desage, and H. Vidal The Regulation of Uncoupling Protein-2 Gene Expression by omega -6 Polyunsaturated Fatty Acids in Human Skeletal Muscle Cells Involves Multiple Pathways, Including the Nuclear Receptor Peroxisome Proliferator-activated Receptor beta J. Biol. Chem., March 30, 2001; 276(14): 10853 - 10860. [Abstract] [Full Text] [PDF]
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M. Das, D. M. Bouchey, M. J. Moore, D. C. Hopkins, R. A. Nemenoff, and K. R. Stenmark Hypoxia-induced Proliferative Response of Vascular Adventitial Fibroblasts Is Dependent on G Protein-mediated Activation of Mitogen-activated Protein Kinases J. Biol. Chem., May 4, 2001; 276(19): 15631 - 15640. [Abstract] [Full Text] [PDF]
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L. L. Tortorella, D. J. Milasincic, and P. F. Pilch Critical Proliferation-independent Window for Basic Fibroblast Growth Factor Repression of Myogenesis via the p42/p44 MAPK Signaling Pathway J. Biol. Chem., April 20, 2001; 276(17): 13709 - 13717. [Abstract] [Full Text] [PDF]
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