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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). The level of IRS-1 was relatively low in growth medium (GM, Fig. 1AGo, lane 1), but was induced after 6 and 12 h in DM (Fig. 1AGo, 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. 1AGo, lane 4). Modified IRS-1 abundance dropped at later time points (Fig. 1AGo, lanes 5–7). 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. 1BGo). Significant tyrosine phosphorylation of IRS-1 was detected in GM and after 36 h in DM (Fig. 1BGo, lanes 1, 5–7). Although the level of IRS-1 was reduced after 36 h in DM (Fig. 1AGo, lanes 5–7), 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. 1BGo, lanes 2 and 3) that was increased slightly at 24 h in DM (Fig. 1BGo, 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 2–7) 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).

 
Tyrosine phosphorylation of IRS-1 was necessary for coprecipitation with the PI 3-kinase-regulatory subunit, p85 (Fig. 1CGo, lanes 1, 4–7). 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. 1CGo, lanes 2 and 3). Immunoprecipitation of p85 confirmed the latter observation (Fig. 2Go). Although similar levels of p85 were precipitated from cells at 12 and 48 h in DM (Fig. 2BGo, 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. 2AGo, lanes 1 and 2). Moreover, coprecipitation of IRS-1 and p85 correlated with PI 3-kinase activity (Fig. 1DGo). 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. 3AGo). However, myogenic differentiation, as quantitated by myogenin mRNA accumulation (Fig. 3BGo), 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.

 
PI 3-Kinase-Dependent Serine Phosphorylation of IRS-1 Occurs during Myogenic Differentiation
As shown in Fig. 1AGo, 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. 4AGo, lane 1) was lost after treatment with PP1 (Fig. 4AGo, 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. 4BGo, lanes 1 and 3), were significantly reduced in cells incubated with LY294002 (Fig. 4BGo, 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. 4CGo, lane 2), compared with the cells incubated without LY294002 (Fig. 4CGo, lane 1). After longer incubation of C2C12 cells with LY294002, the level of IRS-1 remained high (Fig. 4CGo, 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. 4CGo, 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).

 
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. 4DGo, 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. 4DGo, 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. 5AGo, lanes 2 and 4) relative to GM (Fig. 5AGo, lane 1). The level of IRS-1 was down-regulated in NFB4 cells treated with exogenous IGF-I (Fig. 5AGo, 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. 5BGo, lanes 1, 2, and 4), which was increased after IGF-I treatment for 12 and 24 h (Fig. 5BGo, lanes 3 and 5). Increased tyrosine phosphorylation of IRS-1 in response to IGF-I was correlated with increased coprecipitation of p85 (Fig. 5CGo, lanes 3 and 5) and with activation of ERKs (Fig. 6AGo, lanes 3 and 5) over that observed in NFB4 cells in DM (Fig. 6AGo, 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. 6BGo, compare lanes 1 and 2), which was reduced in the presence of LY294002 (Fig. 6BGo, lane 3). No change in ERK abundance was apparent (Fig. 6CGo). As expected, treatment with LY294002 resulted in inhibition of PI 3-kinase activity (Fig. 6DGo, lane 3), which was increased in response to IGF-I (Fig. 6DGo, 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 2–5) 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 2–5) were analyzed by Western blot with phospho-MAPK antibody. In panels B–D, 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).

 
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. 7AGo, lane 1) was inhibited by short-term treatment with LY294002 (Fig. 7AGo, lane 2), which was not related to the abundance of ERKs (Fig. 7BGo, lanes 1 and 2). Moreover, 3 h of LY294002 treatment was sufficient to down-regulate the level of myogenin (Fig. 7CGo, 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. 7AGo, lanes 3 and 4), but did not interfere with myogenin expression (Fig. 7CGo, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 0.4–2% 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, {alpha}-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 [{gamma}-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
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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