Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Regulate Extracellular Signal-Regulated Kinase-Dependent and -Independent Signaling Pathways during Myogenic Differentiation
Dos D. Sarbassov and
Charlotte A. Peterson
Donald W. Reynolds Department of Geriatrics and the Department of
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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Activation of the insulin-like growth factor (IGF)
autocrine loop is required for myogenic differentiation and results in
sustained activation of extracellular signal-regulated kinases-1 and -2
(ERK-1 and -2). We show here that insulin receptor substrate-1 (IRS-1)
phosphorylation on tyrosine and serine residues and association with
phosphatidylinositol 3-kinase (PI 3-kinase) are also associated with
IGF-dependent myogenic differentiation. Down-regulation of IRS-1 is
linked to its serine phosphorylation dependent on PI 3-kinase activity
and appears required for differentiation to occur, as IRS-1 is not
modified and continues to accumulate in a nondifferentiating myoblast
cell line. Furthermore, inhibition of PI 3-kinase activity with
LY294002 blocks differentiation, as demonstrated by inhibition of
myogenin and myosin heavy chain expression and ERK activation. Blocking
the Raf/MEK/ERK cascade with PD98059 does not block myogenic
differentiation; however, myotubes do not survive. Thus, PI 3-kinase,
in association with IRS-1, is involved in an ERK-independent signaling
pathway in myoblasts required for IGF-dependent myogenic
differentiation and in inducing sustained activation of ERKs necessary
for later stages of differentiation.
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INTRODUCTION
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The insulin-like growth factor II (IGF-II) autocrine loop is
activated during differentiation of C2C12 mouse myoblasts into
postmitotic myotubes and appears absolutely necessary (1, 2).
The level of growth factor in myoblast-conditioned medium is
up-regulated within 24 h in low serum and remains until
differentiation is completed (3), correlating with the expression of
IGF-II mRNA in myoblasts (4). Myogenic differentiation promoted by IGFs
is associated with sustained activation of members of the
mitogen-activated protein kinase (MAPK) family, extracellular
signal-regulated kinase-1 and -2 (ERK-1 and -2) (5), as well as with
induction of the myogenic transcription factor myogenin (6). Inhibition
of myogenin expression by antisense oligonucleotides blocked the
myogenic effect of IGF-I in myoblasts (7). Phosphatidylinositol
3-kinase (PI 3-kinase) activity also appeared to be essential for
terminal differentiation of myoblasts, and inhibition of PI 3-kinase
resulted in suppression of myogenin mRNA induction, implying that PI
3-kinase is a component of IGF signaling during myogenic
differentiation (8, 9, 10).
PI 3-kinase that catalyzes phosphorylation of the D-3 position of
phosphatidylinositol is a key player in a variety of cellular
responses, including mitogenesis, antiapoptosis, differentiation,
receptor trafficking, chemotaxis, membrane ruffling, and glucose
transport (8, 11, 12, 13, 14, 15, 16, 17, 18). Receptor tyrosine kinase-linked growth factors
activate the most common isoform of PI 3-kinase, composed of a 110-kDa
catalytic subunit (p110) linked to an 85-kDa regulatory subunit (p85)
(19, 20, 21, 22, 23). The p85-regulatory subunit functions as a coupling adaptor of
its catalytic subunit to multiple tyrosine kinase signaling elements by
employing phosphotyrosine binding src homology 2 (SH2) domains. After
activation of a receptor tyrosine kinase, the SH2 domains of p85
specifically bind phosphorylated YMXM motifs in the phosphorylated
receptor or intracellular signaling intermediates (24). This
recruitment of PI 3-kinase via the SH2 domain of its regulatory subunit
results in enhanced cellular PI 3-kinase activity by increasing the
catalytic activity of p110 and localizing the p110 catalytic activity
subunit in proximity to its substrate at the membrane (25, 26).
Interestingly, the catalytic subunit p110 displays not only lipid but
also serine kinase activities (27). After stimulation, the catalytic
subunit of PI 3-kinase has been shown to phosphorylate its regulatory
subunit p85 and insulin receptor substrate-1 (IRS-1) (28).
The IGF-I receptor, a transmembrane tyrosine kinase, is the primary
mediator of IGF-I and IGF-II signaling in most cell types, including
muscle cells (29), where the IGFs promote both growth and
differentiation (30). Activation of this receptor by binding of IGFs
induces tyrosine phosphorylation of insulin receptor substrate-1
(IRS-1), the major substrate for insulin and IGF-I tyrosine kinase
receptors, and Shc proteins (31). IRS-1 is a 175-kDa protein and
possesses as many as 18 potential phosphotyrosine and 40 phosphoserine
sites (32). Tyrosine-phosphorylated IRS-1 has been shown to recruit a
variety of regulatory proteins including the adaptors Grb2 and Nck,
phosphotyrosine phosphatase Syp, and PI 3-kinase (11, 25, 33, 34, 35).
These proteins contain different SH2 domains that bind to distinct
phosphotyrosine sites on IRS-1. It has been proposed that IRS-1
functions as a "docking" protein for different regulatory proteins,
thereby leading to activation of different signaling cascades (36),
including the phosphorylation cascade that activates ERK-1 and -2, p44
and p42, respectively (31, 33).
IRS-1 is also phosphorylated on serine residues that negatively
regulate signaling (37). Phosphorylation of IRS-1 on serine residues
inhibited its phosphorylation on tyrosine residues and also inhibited
kinase activity of the insulin receptor. Moreover, it induced rapid
degradation of IRS-1 (25, 28, 38, 39). PI 3-kinase has been shown to
phosphorylate IRS-1 on serine residues that required association of
these proteins in a complex that occurred only when IRS-1 was
previously phosphorylated on tyrosine residues (25, 28).
The role of IRS-1 and downstream signaling events in controlling muscle
cell fate are not well understood. Analyses are complicated by the fact
that the IGFs promote both growth and differentiation of myoblasts. In
C2C12 myoblasts, growth stimulated by IGF-I led to IRS-1
phosphorylation and activation of PI 3-kinase activity (11). However,
relatively weak activation of ERK-1 and -2 was associated with
mitogenesis in response to IGF-I compared with other growth factors
(11). It appears that myoblast proliferation induced by IGFs may be
dependent on signaling via heterotrimeric G proteins (5). It has also
been reported that upon serum withdrawal and activation of the IGF
autocrine loop, ERK-2 was inactivated in myoblasts (40). However,
sustained phosphorylation of ERKs, in particular ERK-2, appeared
required for later stages of IGF-dependent differentiation,
i.e. myotube fusion and survival (5, 40, 41). In this study,
we show that IRS-1, through interaction with PI 3-kinase, regulates
myogenic differentiation via ERK-dependent and -independent
pathways.
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RESULTS
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IRS-1 Phosphorylation and Association with PI 3-Kinase Precede ERK
Activation during Myogenic Differentiation
PI 3-kinase activity was shown to be required for myogenic
differentiation (8, 9, 10). We examined the interaction of PI 3-kinase and
IRS-1 to determine whether IRS-1 is involved in transducing the signals
from the IGF-I receptor to PI 3-kinase during myogenic differentiation
of mouse C2C12 myoblasts. Immunoprecipitation and Western analyses
revealed changes in the abundance of IRS-1 in C2C12 cells after
exposure for different lengths of time to differentiation medium (DM,
Fig. 1A
). The level of IRS-1 was
relatively low in growth medium (GM, Fig. 1A
, lane 1), but was induced
after 6 and 12 h in DM (Fig. 1A
, lanes 2 and 3). At 24 h in
DM, the level of this protein remained high with a slight alteration of
its mobility in the gel, suggesting a change in phosphorylation state
(Fig. 1A
, lane 4). Modified IRS-1 abundance dropped at later time
points (Fig. 1A
, lanes 57). To determine whether IRS-1 in C2C12 cells
was phosphorylated on tyrosine residues during myogenic
differentiation, IRS-1 immunoprecipitation products (IP) were analyzed
by phosphotyrosine Western blot (Fig. 1B
). Significant tyrosine
phosphorylation of IRS-1 was detected in GM and after 36 h in DM
(Fig. 1B
, lanes 1, 57). Although the level of IRS-1 was reduced after
36 h in DM (Fig. 1A
, lanes 57), it became highly tyrosine
phosphorylated at later stages of differentiation. Only low level
tyrosine phosphorylation was detected early during differentiation, at
6 and 12 h in DM (Fig. 1B
, lanes 2 and 3) that was increased
slightly at 24 h in DM (Fig. 1B
, lane 4). These results suggest
that activation of the IGF-II autocrine loop during myogenic
differentiation resulted in IRS-1 tyrosine phosphorylation.

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Figure 1. Analysis of IRS-1 and Associated PI 3-Kinase in
C2C12 Cells during Myogenic Differentiation
IRS-1 IP from C2C12 cells maintained in GM (lane 1) and after exposure
for the indicated times to DM (lanes 27) were analyzed by Western
blot with IRS-1 (A), phosphotyrosine PY20 (B), and p85 (C) antibodies.
In panel D, PI 3-kinase assays were performed on IRS-1 IP. PIP
indicates phosphorylated lipid analyzed by ascending chromatography
from the origin (Ori).
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Tyrosine phosphorylation of IRS-1 was necessary for coprecipitation
with the PI 3-kinase-regulatory subunit, p85 (Fig. 1C
, lanes 1, 47).
Relatively little p85 coprecipitated with IRS-1 early during
differentiation, after 6 and 12 h in DM, when IRS-1 was abundant
but unphosphorylated (Fig. 1C
, lanes 2 and 3). Immunoprecipitation of
p85 confirmed the latter observation (Fig. 2
). Although similar levels of p85 were
precipitated from cells at 12 and 48 h in DM (Fig. 2B
, lanes 1 and
2), coprecipitated IRS-1 was not readily detectable after 12 h in
DM but increased dramatically later during differentiation at 48 h
in DM (Fig. 2A
, lanes 1 and 2). Moreover, coprecipitation of IRS-1 and
p85 correlated with PI 3-kinase activity (Fig. 1D
). Tyrosine
phosphorylation of IRS-1 and association with PI 3-kinase preceded the
sustained activation of ERKs, particularly ERK-2 (p42), detected after
36 h in DM by Western blot analysis with a phospho-MAPK antibody
(Fig. 3A
). However, myogenic
differentiation, as quantitated by myogenin mRNA accumulation (Fig. 3B
), appeared to precede not only ERK phosphorylation, but also IRS-1
phosphorylation, association with p85, and increased PI 3-kinase
activity. This is consistent with reports that upon withdrawal of
serum, activation of the myogenin gene occurred before the increase in
IGF-II in C2C12 myoblasts (4, 42).

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Figure 2. Abundance of IRS-1 Associated with the Regulatory
Subunit of PI 3-Kinase
p85 IP from C2C12 cells after exposure to DM for 12 h (lane 1) and
48 h (lane 2) were analyzed by Western blot with IRS-1 (A) and p85
(B) antibodies.
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Figure 3. ERK Phosphorylation during Myogenic Differentiation
Extracts from C2C12 cells in GM and after exposure for the indicated
times to DM were analyzed by Western blot with phospho-MAPK (A) and
myogenin (B) antibodies.
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PI 3-Kinase-Dependent Serine Phosphorylation of IRS-1 Occurs during
Myogenic Differentiation
As shown in Fig. 1A
, the mobility of IRS-1 in C2C12 cells at
24 h in DM was altered compared with early time points; however,
this did not correlate well with tyrosine phosphorylation, suggesting a
different type of modification. To determine whether the slower
migrating form of IRS-1 is related to serine/threonine phosphorylation,
IRS-1 IP were treated with recombinant protein phosphatase 1 (PP1), a
specific phosphoserine/phosphothreonine phosphatase. The slower
migrating form of IRS-1 (Fig. 4A
, lane 1)
was lost after treatment with PP1 (Fig. 4A
, lane 2), supporting the
idea that IRS-1 was phosphorylated on serine/threonine residues after
exposure to DM, and this phosphorylation was primarily responsible for
the altered protein mobility in the gel. PI 3-kinase is required for
myogenic differentiation (8, 9, 10), and this kinase has been shown to
phosphorylate IRS-1 on serine residues in other cell types, leading to
IRS-1 degradation (39, 43). We used the stable and cell-permeant
inhibitor of PI 3-kinase, LY294002 (8), to affect IRS-1
phosphorylation. Myogenic differentiation of C2C12 cells was inhibited
by LY294002. The myogenic markers, myogenin and myosin heavy chain
(MyHC), normally expressed at 24 and 48 h in DM (Fig. 4B
, lanes 1
and 3), were significantly reduced in cells incubated with LY294002
(Fig. 4B
, lanes 2 and 4). Incubation of C2C12 cells in DM for 24 h
with the PI 3-kinase inhibitor abolished phosphorylation of IRS-1,
evidenced by more rapid migration of IRS-1 in the gel (Fig. 4C
, lane
2), compared with the cells incubated without LY294002 (Fig. 4C
, lane
1). After longer incubation of C2C12 cells with LY294002, the level of
IRS-1 remained high (Fig. 4C
, lane 4), suggesting that modification of
IRS-1 on serine residues dependent on PI 3-kinase activity is critical
for IRS-1 degradation in myoblasts (Fig. 4C
, lane 3).

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Figure 4. Effects of Protein Phosphatase 1 (PP1) and the PI
3-Kinase Inhibitor, LY294002, on Mobility and Degradation of IRS-1
IRS-1 IP from C2C12 cells 24 h in DM were incubated ± PP1
and analyzed by Western blot with IRS-1 antibody (A). Extracts isolated
from C2C12 cells incubated in DM ± PI 3-kinase inhibitor LY294002
for 24 h (+LY, lane 2) or 48 h (+LY, lane 4) were analyzed by
Western blot with MyHC and myogenin (B) and IRS-1 (C) antibodies. IRS-1
IP from C2C12 cells incubated ± LY294002 for 24 h in DM were
analyzed by Western blot with phosphotyrosine PY-20 (upper
panel) and p85 (lower panel) antibodies (D).
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To determine whether tyrosine phosphorylation of IRS-1 is affected by
PI 3-kinase inhibition, IRS-1 was immunoprecipitated from C2C12 cells
incubated with and without LY294002 and analyzed by phosphotyrosine
Western blot. Inhibition of PI 3-kinase did not interfere with tyrosine
phosphorylation of IRS-1 and, in fact, was enhanced in the cells
treated with LY294002 (Fig. 4D
, upper panel). The regulatory
subunit of PI 3-kinase, p85, coprecipitated with IRS-1 in the cells
incubated with and without LY294002 and was more abundant in
LY294002-treated cells (Fig. 4D
, lower panel). These data
indicate that inhibition of PI 3-kinase activity blocked serine
phosphorylation of IRS-1 responsible for its down-regulation but did
not interfere with tyrosine phosphorylation of IRS-1 and its
association with p85.
Direct Analysis of Signaling Events Occurring in Response to
IGFs
That IGF signaling results in tyrosine phosphorylation of IRS-1,
association with PI 3-kinase, increased PI 3-kinase activity, and
down-regulation of IRS-1 was directly demonstrated using the
nondifferentiating mutant NFB4 cell line (44). NFB4 cells fail to
activate the IGF-II autocrine loop upon exposure to DM, allowing us to
manipulate the IGF-signaling pathway directly. Treatment of NFB4 cells
with exogenous IGF-I results in myogenic differentiation (5, 42). The
level of IRS-1 in NFB4 cells appeared high at all time points in DM
(Fig. 5A
, lanes 2 and 4) relative to GM
(Fig. 5A
, lane 1). The level of IRS-1 was down-regulated in NFB4 cells
treated with exogenous IGF-I (Fig. 5A
, lanes 3 and 5), suggesting that
IGFs influence IRS-1 abundance. Basal tyrosine phosphorylation of IRS-1
was observed in NFB4 cells incubated in GM and in DM without growth
factor (Fig. 5B
, lanes 1, 2, and 4), which was increased after IGF-I
treatment for 12 and 24 h (Fig. 5B
, lanes 3 and 5). Increased
tyrosine phosphorylation of IRS-1 in response to IGF-I was correlated
with increased coprecipitation of p85 (Fig. 5C
, lanes 3 and 5) and with
activation of ERKs (Fig. 6A
, lanes 3 and
5) over that observed in NFB4 cells in DM (Fig. 6A
, lanes 2 and 4).
Moreover, ERK phosphorylation in response to IGF treatment in NFB4
cells was dependent on PI 3-kinase activity. After treatment with a
relatively high dose of IGF-I, ERK phosphorylation increased
dramatically (Fig. 6B
, compare lanes 1 and 2), which was reduced in the
presence of LY294002 (Fig. 6B
, lane 3). No change in ERK abundance was
apparent (Fig. 6C
). As expected, treatment with LY294002 resulted in
inhibition of PI 3-kinase activity (Fig. 6D
, lane 3), which was
increased in response to IGF-I (Fig. 6D
, lane 2). Overall, these data
demonstrated that in response to activation of the IGF-II autocrine
loop in C2C12 cells and in NFB4 cells in response to exogenous IGF-I,
IRS-1 was tyrosine phosphorylated, associated with the regulatory
subunit of PI 3-kinase, and degraded. Furthermore, ERK activation
required PI 3-kinase activity.

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Figure 5. Abundance and Tyrosine Phosphorylation of IRS-1 in
NFB4 Mutant Cells and Its Association with the Regulatory Subunit of PI
3-Kinase in Response to IGF-I
IRS-1 IP from NFB4 cells incubated in GM (lane 1) or DM ±
exogenous IGF-I (50 ng/ml) for the indicated times (lanes 25) were
analyzed by Western blot with IRS-1 (A), phosphotyrosine (B), and p85
(C) antibodies.
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Figure 6. ERK Phosphorylation in NFB4 Cells after IGF-I and
LY294002 Treatment
A, Extracts from NFB4 cells in GM (lane 1) or DM ± exogenous
IGF-I (50 ng/ml) for the indicated times (lanes 25) were analyzed by
Western blot with phospho-MAPK antibody. In panels BD, extracts from
NFB4 cells cultured 24 h in DM alone (lane 1) or after stimulation
for 20 min with IGF-I (150 ng/ml, lane 2) were analyzed. In lane 3,
cultures were pretreated for 1 h before IGF-I stimulation with
LY294002. Western blots were performed with phospho-MAPK (B) or ERK1/2
(C) antibodies. PI 3-kinase assays were performed on IRS-1 IP from the
same extracts and phosphorylated lipid (PIP) is indicated (D).
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Sustained ERK Activation and Myogenic Differentiation Require PI
3-Kinase Activity
To determine whether sustained activation of ERKs in response to
the IGF-II autocrine loop is dependent on PI 3-kinase activity and
required for differentiation of C2C12 cells, LY294002 was added after
exposure to DM, and activity of ERKs was analyzed by phospho-MAPK
antibody. ERK activation during myogenic differentiation (Fig. 7A
, lane 1) was inhibited by short-term
treatment with LY294002 (Fig. 7A
, lane 2), which was not related to the
abundance of ERKs (Fig. 7B
, lanes 1 and 2). Moreover, 3 h of
LY294002 treatment was sufficient to down-regulate the level of
myogenin (Fig. 7C
, lanes 1 and 2), indicating that PI 3-kinase activity
is required for sustained ERK activation and muscle-specific gene
expression. This is in contrast to results obtained with an inhibitor
of MEK1-dependent ERK activation, PD98059. Blocking MEK1 activity
resulted in decreased activation of ERKs (Fig. 7A
, lanes 3 and 4),
but did not interfere with myogenin expression (Fig. 7C
, lanes 3 and
4). However, myotubes did not survive (5), suggesting that the
ERK-signaling pathway, dependent on PI 3-kinase activity, is involved
in relatively late stages of myogenic differentiation.

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Figure 7. Inhibition of PI 3-Kinase and MEK1 Activity during
Myogenic Differentiation
Extracts isolated from C2C12 cells incubated 30 h in DM treated
for the final 3 h with LY294002 (+LY, lane 2) or PD98059 (+PD,
lane 4) were analyzed by Western blot with phospho-MAPK (A), ERK-1/2
(B), and myogenin (C) anti-bodies.
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DISCUSSION
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This work demonstrates a link between IRS-1 phosphorylation,
PI 3-kinase activity, and sustained activation of ERKs in response to
IGFs and that these signaling events appear essential during
relatively late stages of myogenic differentiation. During
differentiation of C2C12 myoblasts, IRS-1 abundance and phosphorylation
state changed. IRS-1 was present at low levels in GM but was tyrosine
phosphorylated, possibly due to the presence of insulin or cytokines
contained in high serum (45, 46). IRS-1 was up-regulated in an
unphosphorylated state upon exposure to DM, coincident with increased
expression of myogenin, an early marker of myogenic differentiation.
Activation of the IGF-II autocrine loop after exposure to DM for
approximately 24 h (3) resulted in IRS-1 tyrosine phosphorylation.
Therefore, amplification of IGF signaling during differentiation
appears to involve accumulation of IGF-II in the medium, up-regulation
of the IGF-I receptor (47), and up-regulation of IRS-1. The subsequent
drop in IRS-1 abundance appears to be required for differentiation to
proceed. Nondifferentiating NFB4 cells that failed to activate the
IGF-II autocrine loop continued to accumulate high levels of IRS-1 in
DM. IRS-1 was tyrosine phosphorylated in response to exogenous IGF-I,
resulting in loss of IRS-1 and differentiation of the mutant cells
(42). The level of IRS-1 has been shown to be critical for cellular
fate in other cell types, with overexpression resulting in cellular
transformation (48, 49).
After tyrosine phosphorylation of IRS-1 in C2C12 and NFB4 cells, the
p85- regulatory subunit of PI 3-kinase was associated with IRS-1, and
PI 3-kinase activity was increased, indicating that recruitment and
activation of PI 3-kinase by the IGF-signaling pathway occurred during
myogenic differentiation. Previous work demonstrated that IRS-1 and PI
3-kinase were also activated during the mitogenic response of myoblasts
to IGF-I (11). The association of PI 3-kinase and IRS-1 resulted in
serine phosphorylation of IRS-1 that was responsible for altered
protein mobility during electrophoresis and appeared critical for its
down-regulation. Tyrosine phosphorylation of IRS-1 in NFB4 cells by
exogenous IGF-I occurred before its down-regulation, supporting reports
that tyrosine phosphorylation is required for recruitment of PI
3-kinase and subsequent serine phosphorylation (25, 28). Most
importantly, as shown here and by others (8, 9, 10), PI 3-kinase appeared
required for myogenic differentiation. PI 3-kinase inhibitors blocked
myogenic differentiation, demonstrated by inhibition of myogenin and
MyHC gene expression. Paradoxically, initiation of differentiation,
monitored by myogenin expression, occurred before significant IRS-1
phosphorylation and associated PI 3-kinase activity. Although it is
possible that a PI 3-kinase, independent of IRS-1, may be involved in
the early steps of differentiation, it appears more likely that
initiation of myogenic differentiation is negatively controlled by
mitogens in serum that inhibit differentiation from occurring.
Withdrawal of serum results in activation of myogenin expression that
is detected before IGF-II accumulation (4, 42). IGF signaling then
appears required for myogenic differentiation to proceed, dependent
upon serine phosphorylation by PI 3-kinase leading to IRS-1
down-regulation.
IRS-1 tyrosine phosphorylation and PI 3-kinase activity in
response to IGFs were required for sustained activation of ERKs during
myogenic differentiation. Prolonged activation of ERKs is associated
with differentiation in other cell types (50, 51), and activation of
ERKs by PI 3-kinase has been demonstrated but appears to be cell type
specific (52). ERK activation in myoblasts was also dependent on MEK1
activity, but blocking the Raf/MEK/ERK cascade by PD98059 did not
affect myogenin gene expression. This is consistent with reports that
initiation of myogenic differentiation was independent of ERK
activation (10) and may even be enhanced with decreased ERK activity
(40, 53). However, PD98059 interfered with survival of differentiating
myoblasts in low serum (5), suggesting that sustained ERK activity may
be required for differentiation to proceed and may even be required for
myoblast fusion (40), a relatively late event in myogenic
differentiation. This may be directly dependent on MyoD
expression, which appears to be down-regulated by PD98059 (41).
Although both PD98059, the MEK1 inhibitor, and LY294002, the PI
3-kinase inhibitor, interfered with the sustained activation of ERKs in
response to IGFs, only LY294002 resulted in inhibition of myogenin gene
expression, suggesting that PI 3-kinase also regulates ERK-independent
signaling pathways in myoblasts required to maintain expression of
muscle-specific genes during myogenic differentiation. This pathway
likely involves p70S6k, as Coolican et al. (53)
showed that LY294002 completely abolished p70S6k activity
but had relatively little effect on ERK phosphorylation in L6A1
myoblasts. In addition, the inhibition of myogenin expression by the PI
3-kinase inhibitor might be explained by up-regulation of the dominant
negative basic helix-loop-helix protein Id observed in
LY294002-treated 10T1/2-MyoD cells (8). Overall, IRS-1 and PI
3-kinase appeared to be crucial components of IGF signaling leading to
sustained activation of ERKs during myogenic differentiation.
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MATERIALS AND METHODS
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Cell Culture
NFB4 is a subclone of the nondifferentiating NFB cell line
originally derived from the C2C12 mouse muscle cell line (42, 44). Both
cell lines were grown in serum-rich GM: DMEM (GIBCO BRL, Gaithersburg,
MD) supplemented with 10% FBS and 10% defined bovine serum (all serum
from Hyclone, Logan, UT) at 37 C in a humidified 10% CO2,
90% air atmosphere. Confluent cells were washed with serum-free DMEM
and maintained in DM: DMEM plus 0.42% horse serum. PI 3-kinase
inhibitor, LY294002 (BioMol Research Laboratories, Plymouth Meeting,
PA) was used at a concentration of 20 µM in DM. PD98059
(New England Biolabs, Inc., Beverly, MA) was applied in DM at a
concentration of 50 µM. For IGF-I treatment (provided by
Elena Moerman, University of Arkansas for Medical Sciences, Little
Rock, AR), cells were maintained in DM plus IGF-I (50 ng/ml) added
fresh daily as described (5).
Western Blot Analysis
Dishes (100 mm) were washed twice with cold PBS and lysed in 0.5
ml of 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-1/2 antibodies
(New England Biolabs) were diluted 1:1000 in PBST containing 3% BSA
overnight at 4 C. Polyclonal IRS-1 and p85 antibodies (provided by Dr.
Morris F. White, Joslin Diabetes Center, Boston, MA) were diluted
1:3000 in PBST containing 5% milk for 1 h. Blots were washed five
times with PBST for a total of 30 min, before and after incubating with
goat antirabbit horseradish peroxidase-conjugated secondary antibody
(Pierce, Rockford, IL) diluted 1:4000 in PBST containing 4% milk for
1 h. Monoclonal antibodies against MyHC [A4.1025 (42)], myogenin
(provided by Dr. Woody Wright, University of Texas Southwestern Medical
Center, Dallas, TX), and phosphotyrosine (PY20, Transduction
Laboratories, Lexington, KY) were used as described previously (5, 42).
Renaissance Chemiluminescence Reagent (DuPont/NEN, Wilmington, DE) was
used as the detection system. Blots were stripped and reprobed as
described (54).
Immunoprecipitation
Cell extracts (500 µg protein) were adjusted to 1 ml with
buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1
mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride,
0.2 mM Na3VO4, 1% Triton X-100)
and incubated with undiluted IRS-1 or p85 antibody (2 µl) for 80 min
at 4 C. Protein A conjugated to agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) was added for 40 min. Beads were washed
three times with the same buffer and boiled for 2 min before
electrophoresis as described. For PP1 treatment (recombinant protein
phosphatase 1,
-isoform, from Calbiochem, San Diego, CA), the IRS-1
IP were incubated with 4 U of phosphatase for 30 min at 30 C in a
buffer containing 40 mM HEPES, pH 7.0, 5 mM
dithiothreitol, 400 µM MnCl2, and 200 µg/ml
BSA, followed by boiling in sample buffer.
PI 3-Kinase Assay
PI 3-kinase assays were performed essentially as described (55).
Briefly, IRS-1 IP were washed in buffer (20 mM HEPES, pH
7.5, 100 mM NaCl, 0.5 mM EGTA) and 300 µg of
each lipid (phosphatidylinositol and phosphatidylserine, sonicated)
were added in 40 µl of the same buffer. The PI 3-kinase reaction was
initiated by addition of 10 µl of buffer containing
[
-32P]ATP, 30 mM MgCl2, and
250 µM ATP. Samples were incubated for 10 min at 30 C,
and reactions were terminated by adding 100 µl of 1 N HCl.
Phospholipids were extracted with 200 µl chloroform-methanol (1:1),
washed with 200 µl methanol-1 N HCl (1:1), and lyophilized to
dryness. Phospholipids were resuspended in 20 µl chloroform-methanol
(95:5), spotted onto a TLC plate (Merck, Darmstadt, Germany)
impregnated with 1% potassium oxalate, and resolved by ascending
chromatography (chloroform-methanol-acetone-glacial acetic
acid-H2O, 40:13:15:12:7). Phospholipid standards were
visualized by exposure to I2 vapor, and radiolabeled lipids
were detected by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Morris F. White for providing IRS-1 and
p85 antibodies, Dr. Woody Wright for myogenin antibody, Elena Moerman
for IGF-I, Drs. Deborah Burks and Sebastian Pons for helpful
discussions and advice, and Jane Taylor-Jones for help with manuscript
preparation.
 |
FOOTNOTES
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Address requests for reprints to: Charlotte A. Peterson, McClellan Veterans Hospital, Research 151, 4300 West 7th Street, Little Rock, Arkansas 72205. E-mail:
petersonchar-lottea{at}exchange.uams.edu
This work was supported by grants from the National Institutes of
Health-National Institute on Aging to C.A.P. and a grant from
the University of Arkansas for Medical Sciences, Committee for
Allocation of Graduate Student Research Funds (CAGSRF) to
D.D.S.
Received for publication February 4, 1998.
Revision received August 24, 1998.
Accepted for publication September 1, 1998.
 |
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