Role of the p66Shc Isoform in Insulin-like Growth Factor I Receptor Signaling through MEK/Erk and Regulation of Actin Cytoskeleton in Rat Myoblasts*

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.

Skeletal muscle represents an important site of action for the IGFs, 1 because specific high affinity IGF-I receptors are ex-pressed in this tissue (1,2), and both IGF-I and IGF-II stimulate growth and differentiation of skeletal muscle cells (3). Muscle cells also synthesize and secrete IGF-I, IGF-II, and IGF-binding proteins, providing a cell model for integrated autocrine and paracrine control of mitogenic and metabolic actions by the IGFs. IGF signaling in skeletal muscle involves activation of specific cell surface receptors, containing a tyrosine kinase domain within their cytoplasmic portion and undergoing autophosphorylation on specific tyrosine residues upon ligand binding. Receptor autophosphorylation triggers tyrosine phosphorylation of intracellular substrate proteins, including IRS-1, IRS-2, Crk-II, and the Shc proteins (4,5).
The Shc proteins are widely expressed signaling mediators that are tyrosine-phosphorylated by multiple receptor or receptor-associated tyrosine kinases and are capable of stimulating the Ras/MAP kinase pathway after binding to the Grb2 adaptor (6,7). The Shc proteins originate by alternative use of three distinct translation starting points on a longer transcript (p42 Shc , p52 Shc , and p66 Shc ) and of two translation starting points on a shorter transcript (p46 Shc and p52 Shc ); the two mRNA transcripts are generated by alternative splicing from a single gene and are regulated by distinct promoters (8 -10). As a consequence, p66 Shc contains the entire sequence of p46 Shc and p52 Shc , with one phosphotyrosine binding (PTB) domain, one src homology 2 (SH2) domain, and one collagen homology (CH1) region. In addition, p66 Shc has a second CH2 domain of 110 amino acids, located at the NH 2 -terminal portion of the molecule, that is not present in p46 Shc or p52 Shc . Recent reports have established a specific role for the p66 Shc isoform. p46 Shc and p52 Shc are found in every cell type with similar reciprocal relationship, whereas p66 Shc expression varies from cell type to cell type and is also lacking in some cell types (8). Evidence for divergent regulation of p66 Shc versus p46/p52 Shc has emerged from studies that demonstrated specific serine phosphorylation of p66 Shc through an MEK-mediated pathway in response to insulin (11). Knock-out experiments, in which the p66 Shc gene has been selectively inactivated in mice, suggest an important role of this protein in the regulation of cellular responses to oxidative stress, apoptosis, and life span (12,13). However, the intracellular signaling pathways mediating the unique actions of p66 Shc remain to be established.
The mitogenic and survival signals evoked by the IGFs have been extensively studied in multiple cell types, including skeletal muscle cells. Activation of the MEK/Erk pathway in response to the IGFs appears to mediate myoblast growth and differentiation (14,15). IGF-I stimulation of DNA synthesis is blocked by the MEK inhibitor PD98059 (16), and myotubes do not survive or differentiate when the MEK/Erk pathway is similarly blocked by PD98059 (17). Furthermore, augmentation of IGF-I-stimulated cell proliferation in dexamethasonetreated L6 myoblasts is associated with increased Shc and decreased IRS-1 tyrosine phosphorylation (18,19), suggesting that under conditions of glucocorticoid excess the mitogenic response of muscle cells to the IGFs can be modulated by altering the activity of the Shc/Erk pathway. The specific contribution of the p66 Shc isoform to IGF-I action on skeletal muscle cells has not been investigated.
In this study, we show that selective reduction of p66 Shc in L6 skeletal muscle cells results in up-regulation of the MEK/ Erk pathway, leading to increased Erk-1/2 phosphorylation and nuclear localization in the absence of IGF-I stimulation. This is associated with a dramatic perturbation of the actin cytoskeleton, leading to abnormal cell shape, growth, and differentiation of the L6 myoblasts. The abnormalities in both signaling reactions and organization of the actin cytoskeleton are corrected by the MEK inhibitor PD98059, suggesting that in skeletal muscle cells the p66 Shc isoform may physiologically exert an inhibitory role on MEK/Erk, which is necessary for full responsiveness of this signaling pathway to the IGFs and maintenance of normal cell morphology.

EXPERIMENTAL PROCEDURES
Cell Culture-L6 rat skeletal muscle myoblasts were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% donor calf bovine serum (BCS) (both from Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and non-essential amino acids in a 5% CO 2 atmosphere at 37°C. Myoblasts were differentiated into myotubes with 2% horse serum (Invitrogen), 2 nM triiodothyronine (Sigma), and 20 nM insulin. For IGF-I studies, cells were incubated in complete medium containing 0.5% bovine serum albumin (BSA) without calf bovine serum for 16 h, and then stimulated with 100 nM IGF-I (GRO PEP, Adelaide, Australia) for the indicated times or left untreated. To block the MEK/Erk signaling pathway, cells were incubated with 20 M PD98059 (Calbiochem, Merck KGaA) for the indicated times.
Transfection Studies-To generate L6 myoblasts stably expressing reduced levels of the p66 Shc isoform, a 287-bp fragment of the cDNA encoding the p52/p46 Shc proteins (from nucleotide 55 to nucleotide 342), indicated as as1, was generated by amplification using the pMJ/Shc plasmid (kindly provided by Dr. J. Schlessinger, New York, NY) as a template. A 326-nucleotide cDNA fragment corresponding to the NH 2terminal 109 amino acids unique to p66 Shc , indicated as as2, was generated by PCR amplification using the rat p66 Shc cDNA (kindly provided by Dr. J. E. Pessin, New York, NY). To generate stable transfectants, the cDNAs of interest were cloned in antisense orientation into the mammalian expression vector pCR3.1 (Invitrogen), containing a G418 resistance gene, under control of the cytomegalovirus promoter. The as1-and as2-containing plasmids were transfected into L6 myoblasts by liposome-mediated gene transfer using LipofectAMI-NE TM (Invitrogen), and stable transfectants, indicated as L6/Shcas1 and L6/Shcas2, were selected by their ability to grow in neomycincontaining medium. Multiple stable clones of L6 cells hypoexpressing Shc were obtained within 4 weeks. Plasmid integration into the cell genome was confirmed by PCR amplification of genomic DNA with forward and reverse oligonucleotide primers corresponding to the pCR3.1 (5Ј-TAA TAC GAC TCA CTA TAG GG-3Ј) and Shc (5Ј-CTG GCA  CAG TGG CCC TGT CCA TCC-3Ј) sequences, respectively. The amount of Shc protein expressed in transfected L6 myoblasts was determined by immunoblotting total cell lysates with Shc antibodies, as previously described (18).
Immunoprecipitation and Immunoblotting-For immunoprecipitation studies, L6 skeletal muscle cells were washed twice with Ca 2ϩ / Mg 2ϩ -free PBS and then scraped in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10% glycerol, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 2 mM sodium orthovanadate, and 1% Nonidet P-40. Cell lysates were then centrifuged at 12,000 ϫ g for 10 min, and the resulting supernatant was collected and assayed for protein concentration using the Bradford dye binding assay kit with BSA as a standard. Equal amounts of cellular proteins (500 g) were subjected to immunoprecipitation with the indicated antibodies overnight at 4°C. The resulting immune complexes were adsorbed onto 70 l of protein A-Sepharose beads (Amersham Biosciences) for 2 h at 4°C, washed three times with lysis buffer, and then eluted with Laemmli buffer for 5 min at 100°C.
For immunoblotting studies, equal amounts of cellular proteins were resolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, as appropriate, directly or following immunoprecipitation with the specific antibodies, as indicated. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) using a transfer buffer containing 192 mM glycine, 20% (v/v) methanol, and 0.02% SDS. To reduce nonspecific binding, the membranes were incubated in TNA buffer (10 mM Tris-HCl, pH 7.8, 0.9% NaCl, 0.01% sodium azide) supplemented with 5% BSA and 0.05% Nonidet P-40 for 2 h at 37°C, or in phosphate-buffered saline (PBS) supplemented with 3% nonfat dry milk for 2 h at room temperature, as appropriate, and then incubated overnight at 4°C with the indicated antibodies. The proteins were visualized by enhanced chemiluminescence (ECL) using horseradish peroxidase-labeled anti-rabbit or antimouse IgG (Amersham Biosciences) and quantified by densitometric analysis using Optilab® image analysis software (Graftek SA, Mirmande, France).
Immunofluorescence Analyses-To visualize the actin cytoskeleton, L6 cells were grown on coverslips in complete medium for the indicated times, then fixed with 4% paraformaldehyde and permeabilized at Ϫ20°C with 100% methanol. Fixed cells were incubated with fluorescein isothiocyanate-conjugated (FITC) phalloidin (purchased from Sigma-Aldrich) for 30 min and subsequently washed three times with PBS. Coverslips were mounted on glass slides with Gel mount (Biomeda, Foster City, CA). Fluorescence signals were detected by conventional epifluorescence microscopy (Leica DMLB microscope with a Sensicam 12 Bitled charge-coupled device camera, Bansheim, Germany), and all images were captured at the same magnification.
To examine vinculin-containing focal adhesions, L6 cells grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Incubation with primary antibody against vinculin (1:400) was conducted at room temperature for 16 h, followed by incubation with secondary Alexa 488 Fluor anti-mouse goat antibody (1:500, Molecular Probes, Eugene, OR) for 1 h and washing three times with PBS. Cells were finally stained with TO-PRO-3 (1:10,000, Molecular Probes) to visualize nuclei, and coverslips were mounted using Gel Mount (Biomeda). Images were acquired on a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems, Heerbrugg, Switzerland), and all images were taken at the same magnification.
To study the intracellular localization of Erk-1/2, L6 cells were grown on glass slides, arrested at 50% confluence in serum-free medium for 24 h, and then incubated in the absence or presence of 100 nM IGF-I for 30 min. To assess total Erk-1/2, control and IGF-I-treated cells were fixed with methanol/acetone (70:30, v/v) for 10 min at Ϫ20°C. After a 10-min rehydration with multiple PBS washes at 25°C, the cells were blocked with PBS/10% BCS for 45 min and then incubated with the monoclonal MAP kinase antibody in PBS/10% BCS (1:100) for 60 min at 25°C. Cells were then washed three times with PBS and incubated with FITC-conjugated anti-mouse antibodies in PBS/10% BCS (1:100) for 60 min at 25°C in the dark. To evaluate the localization of phospho-Erk-1/2, control and IGF-I-treated cells were fixed with 3% paraformaldehyde for 20 min and permeabilized with 100% methanol for 5 min at Ϫ20°C. After a 10-min rehydration with multiple PBS washes at 25°C, cells were blocked with blocking buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100 (Tris-buffered saline 1ϫ, TBST) supplemented with 5.5% BCS for 45 min, and then incubated with the polyclonal phospho-p44/p42 MAP kinase (Thr-202/Tyr-204) antibody in Tris-buffered saline 1ϫ (TBS) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl/3% BSA (1:100) for 60 min at 25°C. Cells were then washed three times with TBST and incubated with FITC-conjugated anti-rabbit antibodies in TBST/3% BSA (1:100) for 60 min at 25°C in the dark. Finally, cells were washed three times with PBS, mounted under glass coverslips with CITIFLUOR medium, examined under epifluorescent illumination with excitation-emission filters for FITC by epifluorescence microscopy (Leica DMRXA microscope), and photographed with an Olympus inverted microscope equipped with phase-contrast and UV illumination through an FITC filter.
Thymidine Incorporation-Thymidine incorporation was carried out as previously described (18). Briefly, L6 cells were grown in 35-mm multiwell plates to ϳ50% confluence in MEM with 10% BCS. Following incubation in serum-free MEM for 16 h, L6 myoblasts were incubated with or without 100 nM IGF-I for 16 h at 37°C. The cells were then incubated for 1 h at 37°C in fresh MEM containing 0.2% BSA, 25 mM HEPES (pH 7.6), and 1 Ci/ml [ 3 H]thymidine. The medium was removed, and the cells were washed twice with ice-cold PBS, left in 10% trichloroacetic acid for 30 min on ice, and washed twice with ice-cold 10% trichloroacetic acid. The cells were then solubilized in 0.1 N NaOH for 30 min at 37°C, and the amount of 3 H was quantitated by liquid scintillation counting. For each condition, experiments were carried out in triplicate.
Statistical Analyses-All data are expressed as mean Ϯ S.E. Data are expressed as percentage of control or basal control values, as appropriate. Statistical analyses were performed by unpaired Student's t tests.

Generation of L6 Skeletal Muscle Myoblasts with Reduced
p66 Shc Protein Levels-To determine the specific contribution of the p66 Shc isoform to IGF-I action and signaling in skeletal muscle cells, the cellular levels of this IGF-I receptor substrate were selectively reduced by expressing specific Shc antisense RNA sequences. For this purpose, undifferentiated L6 skeletal muscle cells were stably transfected with two independent Shc antisense sequences, designated as1 and as2. as1 represents a 287-nucleotide cDNA fragment corresponding to both the p46/ p52 Shc and p66 Shc mRNA transcripts, whereas as2 represents a 326-nucleotide cDNA fragment specific to the NH 2 -terminal 109 amino acids unique to the p66 Shc mRNA transcript (Fig.  1A). Independent clones of L6 cells showing decreased expression of p66 Shc were identified by immunoblotting with anti-Shc antibodies and selected for further analyses.
Both as1 and as2 induced a marked and selective reduction in p66 Shc protein levels in multiple clones of L6 myoblasts (Fig.  1, B and C). as1 decreased the p66 Shc protein ϳ90% compared with untransfected wild-type L6 myoblasts (p Ͻ 0.05) or L6/ Neo myoblasts transfected with the empty pCR3.1 vector. By contrast, the protein levels of the other Shc isoforms, i.e. p52 Shc and p46 Shc , were not significantly different in L6/Shcas1, L6/ Neo, and untransfected L6 myoblasts (Fig. 1B). A 65% reduction in p66 Shc was obtained following overexpression of the as2 FIG. 1. Antisense-mediated inhibition of p66 Shc protein expression in L6 myoblasts. A, structure of p46/p52 Shc and p66 Shc mRNAs. The phosphotyrosine binding (PTB), collagen homology 1 (CH1), and src homology 2 (SH2) domains common to all Shc isoforms, and the collagen homology 2 (CH2) domain specific to p66 Shc are indicated. Antisense cDNA fragments corresponding to nucleotides 381-668 of the p66 Shc cDNA (indicated as as1) or nucleotides 1-326 of the p66 Shc cDNA (indicated as as2) were cloned in pCR3. 1  p66 Shc Signaling in Skeletal Muscle Cells cDNA construct (p Ͻ 0.05), which also did not affect p52 Shc or p46 Shc protein levels (Fig. 1C). Therefore, L6 skeletal muscle cell lines with marked reduction of p66 Shc were established by stable transfection of as1 or as2. The selective inhibition of p66 Shc expression in the absence of changes in expression levels of the other Shc isoforms may be potentially explained by different sensitivity to antisense-mediated inhibition of protein translation of the two mRNA transcripts encoding the Shc proteins (8 -10). Therefore, both as1 and as2 interfere with the mRNA transcript encoding p66 Shc , whereas the mRNA transcript encoding p46/52 Shc may be resistant to interference by as1 (Fig. 1A).
Erk-1/2 Signaling in L6/Shcas Myoblasts-The Shc proteins regulate cellular responses through activation of the Grb2/Sos/Ras signaling cascade, leading to phosphorylation and activation of the MAP kinase family members Erk-1 and Erk-2. To verify whether selective reduction of the p66 Shc protein levels could modify this signaling pathway, we analyzed basal and IGF-I-induced Erk-1/2 phosphorylation in L6/Shcas1 and L6/Shcas2 myoblasts with decreased p66 Shc protein levels. Activation of Erk-1 and Erk-2 was evaluated by immunoblotting with phospho-Erk-1/2 (Thr-202/Tyr-204) antibodies in two independent clones of L6/Shcas1 myoblasts (C6 and D28) and two independent clones of L6/Shcas2 myoblasts (E15 and E21). In the L6/Shcas1 clones, basal Erk-1 and Erk-2 phosphorylation was increased 411 and 360% of control, respectively (p Ͻ 0.05) ( Fig. 2A). By contrast, basal phosphorylation of Erk-1 and Erk-2 was similar in L6 myoblasts transfected with the empty vector (L6/Neo, clones N1 and N5) and in untransfected wildtype L6 myoblasts (respectively, p ϭ 0.32 and p ϭ 0.97), indicating that plasmid transfection and clonal selection in neomycin-containing medium did not affect the level of Erk phosphorylation. No change in total Erk-1 or Erk-2 protein content was evident in wild-type L6, L6/Neo, and L6/Shcas1 myoblasts ( Fig. 2A). Increased phosphorylation of Erk isoforms in the basal state was also evident in two independent clones of L6/Shcas2 myoblasts compared with control ( Fig. 2B; p Ͻ 0.05), although this change was of lower magnitude (i.e. 280 and 200% increases of Erk-1 and Erk-2 phosphorylation, respectively, in clones E15 and E21 versus control) as compared with that seen in L6/Shcas1 myoblasts. Total protein content of Erk-1 and Erk-2 was slightly and not significantly increased in L6/Shcas2 compared with control myoblasts (Fig. 2B). Therefore, stable overexpression of either as1 or as2 in L6 myoblasts results in decreased p66 Shc content and increased phosphorylation of Erk-1 and Erk-2 not due to changes in total Erk protein content. L6/Shcas1 myoblasts were used in subsequent studies, because they showed greater decrease in p66 Shc protein levels and more prominent increase in Erk-1/2 phosphorylation as compared with L6/Shcas2 myoblasts.
To assess the responsiveness of Erk-1/2 phosphorylation to IGF-I stimulation, control and L6/Shcas myoblasts were treated with 100 nM IGF-I for various times and subjected to immunoblotting with phospho-Erk-1/2 antibodies. In control L6/Neo cells, IGF-I markedly increased phosphorylation of both Erk isoforms, which was evident after 3 min of stimulation, peaked at 5 min, and remained sustained up to 30 min (Fig. 2C). By contrast, Erk-1/2 phosphorylation was elevated in the basal state in L6/Shcas myoblasts and showed no further increase during IGF-I stimulation (Fig. 2C).
Activation of Erk-1/2 following phosphorylation on Thr-202/ Tyr-204 results in translocation of the phosphorylated kinases from the cytoplasm, where they are normally retained presumably via a cytoplasmic anchoring complex, to the nucleus (20). To investigate the intracellular localization of activated Erk-1/2 in myoblasts with reduced p66 Shc levels, L6/Shcas cells were studied by immunofluorescence using antibodies to total or phosphorylated Erk-1/2 and compared with control. In control L6 myoblasts, Erk-1/2 appeared uniformly distributed in the cell cytoplasm under basal conditions. IGF-I stimulation induced an increase in the Erk-1/2 signal in the cell nucleus and perinuclear region (Fig. 3A). Increased nuclear fluorescence in IGF-I-treated cells was also observed using antibodies to phosphorylated Erk-1/2 (Fig. 3B), indicating IGF-I-dependent nuclear translocation of the phosphorylated form of Erk-1/2. As compared with control cells, L6/Shcas myoblasts showed a greater amount of Erk-1/2 in their nucleus already in the basal state, which did not augment upon IGF-I stimulation (Fig. 3A). The nuclear Erk-1/2 in the unstimulated L6/Shcas myoblasts was constitutively phosphorylated, as demonstrated by immunofluorescence with phospho-Erk-1/2 antibodies (Fig.  3B). Thus, L6/Shcas myoblasts show constitutive activation and nuclear translocation of Erk-1/2 proteins in the absence of IGF-I stimulation.
IGF-I exerts a dual effect on proliferation and differentiation of skeletal muscle cells, which relies upon an intact Erk-1/2 responsiveness to this growth factor (16,17,21). Because Erk activation was altered in L6/Shcas compared with control L6 myoblasts, IGF-I effects on DNA synthesis and cell differentiation were next determined. In untransfected L6 and L6/Neo myoblasts IGF-I stimulation resulted in a 3-fold increase in DNA synthesis, measured by determining the rates of [ 3 H]thymidine incorporation into DNA (p Ͻ 0.05 versus basal) (Fig.  4A). By contrast, in L6/Shcas myoblasts the IGF-I effect on DNA synthesis was modest and statistically not significant (Fig. 4A). Differentiation into myotubes, which is largely mediated by IGFs secreted in an autocrine manner (3), was also impaired in the L6/Shcas myoblasts. Both wild-type L6 and L6/Neo myoblasts became elongated and fused to form the characteristic multinucleated myotubes when grown in the differentiation medium (Fig. 4B). By contrast, L6/Shcas myoblasts showed elongation and some degree of alignment, but did not develop into myotubes (Fig. 4B).
IGF-I Receptor Signaling in L6/Shcas Myoblasts-The constitutive activation of Erk in the absence of IGF-I stimulation in myoblasts with reduced p66 Shc levels could potentially result from increased activity of steps in the IGF-I signaling cascade upstream of Erk. To explore this possibility, tyrosine phosphorylation and total protein levels of the IGF-I receptor were measured in control and L6/Shcas myoblasts. As shown in Fig.  5A, IGF-I receptor protein levels were similar in wild-type L6, L6/Neo, and L6/Shcas myoblasts. Tyrosine phosphorylation of the IGF-I receptor was very low under basal conditions and was increased severalfold following incubation with 100 nM IGF-I for 10 min in wild-type L6, L6/Neo, and L6/Shcas myoblasts (Fig. 5A). Tyrosine phosphorylation of p52 Shc , the predominant Shc isoform undergoing tyrosine phosphorylation in response to IGF-I stimulation in L6 cells (18), was also low in the absence of IGF-I in wild-type L6, L6/Neo, and L6/Shcas myoblasts, and was increased to a similar extent in all cell lines after stimulation with IGF-I (Fig. 5B). Tyrosine phosphorylation of p66 Shc was induced by IGF-I in wild-type L6 and L6/Neo myoblasts but not, as expected, in the L6 myoblasts expressing low levels of this Shc isoform following transfection of the p66 Shc antisense (Fig. 5B). These results indicate that the constitutive activation of Erk in myoblasts with reduced p66 Shc is not the consequence of increased tyrosine phosphorylation of the IGF-I receptor or Shc proteins.
Erk-1/2 phosphorylation and activation in response to growth factor stimulation is mediated by the serine kinase MEK. To further investigate the mechanisms responsible for the increased activity of Erk in the L6/Shcas myoblasts, the activation of MEK was measured by immunoblotting with specific phospho-MEK (Ser-217/Ser-221) antibodies. In wild-type L6 and L6/Neo myoblasts, MEK phosphorylation was low in the basal state and increased 7-fold after IGF-I stimulation (Fig. 5C). In contrast, MEK phosphorylation was already augmented in the basal state in L6/Shcas myoblasts (5-fold versus control, p Ͻ 0.05), and showed no significant changes when cells were stimulated with IGF-I. These findings parallel the changes in Erk-1/2 phosphorylation observed in L6/Shcas myoblasts (Fig. 2, A-C). The increased MEK phosphorylation was not explained by changes in MEK protein levels, which were similar in wild-type L6, L6/Neo, and L6/Shcas myoblasts (Fig.   5C). Thus, constitutive MEK activation in the absence of IGF-I stimulation appears to account for the increased basal phosphorylation of Erk-1/2 in L6/Shcas myoblasts.
To investigate another signaling pathway that is largely independent of the MEK/Erk pathway, the activation state of Akt was also assessed. As shown in Fig. 5D, the level of Akt phosphorylation on Ser-473 was similarly low in control and L6/Shcas myoblasts and was increased markedly following stimulation with IGF-I. Therefore, while MEK/Erk kinases are de-regulated in the L6/Shcas myoblasts, the Akt pathway is not altered and shows normal responsiveness to IGF-I.
Cytoskeleton of L6/Shcas Myoblasts-L6 myoblasts in cul- ture typically grow as a monolayer of elongated cells (Fig. 4B). The phenotype of L6/Neo myoblasts is similar to that of untransfected L6 cells (Fig. 4B). By contrast, L6/Shcas myoblasts showed marked phenotypic abnormalities, including rounded cell shape, tendency to adhere poorly to the culture dish, and clustering in islets or finger-like structures (Fig. 4B). To investigate whether the changes in cell morphology of L6/Shcas myoblasts were associated with alterations of F-actin-containing structures and focal adhesions, the actin network and focal contacts were visualized with FITC-conjugated phalloidin and an antibody against vinculin, respectively. Control L6 myoblasts exhibited a characteristic morphology, with well developed actin stress fibers and vinculin-containing focal adhesions (Fig. 6, A and B). In marked contrast, a deep disorganization of actin cytoskeleton with strong reduction of actin fibers was found in the L6/Shcas myoblasts with reduced p66 Shc , in which actin accumulated in patches at the cell periphery (Fig. 6A). In addition, L6/Shcas myoblasts showed loss of vinculin-containing focal adhesions (Fig. 6B).
The constitutive activation of MEK/Erk could potentially result in altered turnover of G/F actin balance in L6 myoblasts with reduced p66 Shc , leading to disorganization of actin cytoskeleton and altered cell morphology (22). To explore this possibility, L6/Shcas myoblasts were treated with the MEK inhibitor PD98059 for 72 h to achieve persistent inhibition of the abnormally elevated levels of MEK-1/2 and Erk-1/2 activities, and the resulting effects on actin cytoskeleton were then examined. The levels of basal MEK-1/2 phosphorylation in control myoblasts were very low or undetectable and were not apparently affected by the PD98059 compound (Fig. 7A). On the other hand, the elevated levels of basal MEK-1/2 phosphorylation found in L6/Shcas myoblasts were markedly reduced by PD98059 (Fig. 7A). Basal Erk-1/2 phosphorylation was also efficiently inhibited by PD98059, such that after treatment with this compound Erk-1/2 phosphorylation was similarly low in L6/Shcas and control myoblasts (Fig. 7B). Inhibition of MEK and Erk phosphorylation with PD98059 had no effect on either the actin cytoskeleton or focal adhesions of control L6 myoblasts, whereas it restored the cell shape in the L6/Shcas cells, which displayed a well organized actin network and focal contacts (Fig. 6, A and B). These results indicate that the increased basal activity of Erk-1/2 in myoblasts with reduction of the p66 Shc isoform can be corrected following inhibition of MEK with PD98059 and that this results in reorganization of actin cytoskeleton and focal contacts, with consequent restoration of the cell shape.

DISCUSSION
By stably overexpressing antisense cDNA fragments specific to the Shc mRNA, L6 skeletal muscle cell lines with marked and selective reduction of p66 Shc were established. These cells show: (i) specific reduction of p66 Shc in the absence of significant changes in the other Shc isoforms, (ii) marked de-regulation of the MEK/Erk signaling pathway, with increased basal phosphorylation and blunted response of these kinases to IGF-I stimulation, and (iii) altered actin cytoskeleton and cell shape. Furthermore, correcting the overactivity of Erk results in restoration of the actin cytoskeleton, suggesting a key function of the Erk signaling pathway in the regulation of this cellular component in skeletal muscle cells.
Although all three Shc isoforms can be tyrosine-phosphorylated upon growth factor stimulation, p46/52 Shc are coupled to growth and survival signals, whereas p66 Shc also undergoes serine phosphorylation and mediates pro-apoptotic responses to oxidative stress (12). Specifically, the p66 Shc has been shown to regulate intracellular oxidant levels and hydrogen peroxidemediated forkhead inactivation (23), effects that are probably relevant to the reported ability of p66 Shc to control lifespan in mammals (12) and are unique to this Shc isoform. Additional evidence for different promoter regulation by methylation and histone deacetylation (10) and subcellular localization (24) strengthen the concept of the biological diversity of the Shc isoforms. Consistent with their biological diversity, p66 Shc and p46/p52 Shc have been shown to exert opposite effects on the MEK/Erk signaling pathway. Overexpression of p46/p52 Shc enhanced EGF-induced Erk activation, whereas p66 Shc overexpression had no effect (9) or inhibited (25) this response. The latter study suggested that p66 Shc might function to provide for a feedback inactivation of the Erk signaling pathway in contrast to the other Shc isoforms. Also in the present study, p66 Shc was found to exert an inhibitory effect on Erk, because reduced expression levels of p66 Shc were associated with per- FIG. 3. Immunofluorescence analysis of the subcellular localization of Erk-1/2 in L6Shcas myoblasts. Basal and IGF-I-stimulated wild-type L6 and L6/Shcas1 myoblasts were grown on glass slides, arrested at 50% confluence in serum-free medium for 24 h, and then incubated in the absence or presence of 100 nM IGF-I for 30 min. Cells were fixed and incubated with Erk-1/2 or phospho-Erk-1/2 antibodies and then with the appropriate FITC-conjugated secondary antibody, as described under "Experimental Procedures." A, total Erk-1/2 detected by immunostaining with Erk-1/2 antibodies. B, phosphorylated Erk-1/2 detected by immunostaining with phospho-Erk-1/2 antibodies. A representative of three experiments is shown. Scale bars, 10 m. sistent Erk activation. The opposite effects of p46/p52 Shc and p66 Shc on Erk activation are of pathophysiological significance, since human breast cancer tissues with high p46/p52 Shc to p66 Shc expression ratios show increased proliferative activity and are associated with poor prognosis (26).
The mechanism underlying the inhibitory effect of p66 Shc on the Erk-signaling pathway is not fully understood. In L6/Shcas myoblasts, Erk was constitutively activated and poorly responsive to IGF-I stimulation, and so was MEK, the signaling molecule upstream of Erk. By contrast, tyrosine phosphorylation of the IGF-I receptor and the p46/p52 Shc isoforms and Akt phosphorylation were normal, indicating that the increased Erk phosphorylation in myoblasts with reduced p66 Shc was not the consequence of enhanced IGF-I receptor signaling and that other signaling pathways did not exhibit constitutive activation. According to Okada et al. (25), p66 Shc is serine-phosphorylated in an MEK-dependent manner, and the phosphorylated p66 Shc binds to Grb2 but is not capable of associating with receptor tyrosine kinases. Because p66 Shc and p46/p52 Shc compete for binding to a limited pool of Grb2 molecules, increased cellular abundance of p66 Shc may result in removal of Grb2-Sos complexes from the receptor at the cell membrane and consequent inhibition of Ras activation (25). Conversely, one could envision that reduction of p66 Shc protein levels, which was achieved in this study, may allow more p46/p52 Shc binding to Grb2 in the proximity of the tyrosine kinase receptor, leading to sustained Ras and Erk activation.
L6 myoblasts with reduced p66 Shc levels showed rounded shape and altered growth properties when compared with control myoblasts (Fig. 4A) and were characterized by complete disruption of the actin stress fibers, focal adhesions, and cell cytoskeleton (Fig. 6). Importantly, inhibition of Erk restored the myoblast phenotype in L6/Shcas cells, suggesting a role for the MEK/Erk pathway in the regulation of actin polymerization and focal contacts. The Shc proteins have been implicated in the control of actin cytoskeleton and cell motility in multiple cell types. Fibroblasts obtained from mice with targeted disruption of the Shc gene show aberrant rounded morphology and a disorganized actin cytoskeleton (27), and down-regulation of p46/52 Shc isoforms results in reduced EGF-dependent motility in MCF-7 breast cancer cells (28). Conversely, overexpression of p46/52 Shc was found to promote some extent of organization of actin cytoskeleton and focal adhesions, associated with a random type of cell motility, in a glioblastomaderived cell line (29), and to improve motility in hepatocyte growth factor-stimulated melanoma cells (30). Even though one study demonstrated that p46/p52 Shc can translocate to the cytoskeleton and directly bind to F-actin in PC-12 cells (31), suggesting direct regulation of actin polymerization by the Shc proteins, current evidence indicates that Shc promotes cytoskeletal rearrangement in response to growth factor stimulation through the Erk pathway (27,32). In addition, the p46/ p52 Shc isoforms bind to and are tyrosine-phosphorylated by integrin-activated tyrosine kinases such as FAK and Src (33,34) and, similarly, promote Erk activation. Thus, growth fac-tor-and integrin-triggered signals converge on the Shc/Erk pathway and dynamically regulate actin fiber assembly, together with signals transduced through the FAK-p130 Cas complex. Consistent with this model, regulation of actin cytoskeleton was observed following overexpression of activated MEK, implying the Ras/Raf/MEK/Erk pathway in the p46/p52 Shc -dependent effects (29). The link between Erk and the cytoskeleton is demonstrated by the finding that activated Erk can directly phosphorylate and activate myosin light chain kinase, leading to phosphorylation of myosin light chains and subsequent promotion of the cytoskeletal contraction necessary for cell movement (35).
If MAP kinase promotes actin organization, why did constitutive activation of Erk-1 and Erk-2 cause actin fiber disassembly in the L6/Shcas myoblasts? First, persistent up-regulation of Erk activity may be inappropriate for maintenance of normal organization of the actin cytoskeleton. Consistent with this concept, up-regulation of MEK activity in rat kidney cells, obtained by expressing a temperature-sensitive v-Src mutant FIG. 5. IGF-I signaling in L6/Shcas myoblasts. Wild-type L6, L6/Neo (clones N1 and N5), and L6Shcas1 (clones C6 and D28) myoblasts were studied in the basal state or 10 min following stimulation with 100 nM IGF-I. A, tyrosine phosphorylation (top) and total protein content (bottom) of IGF-I receptors. Solubilized myoblast proteins were subjected to immunoprecipitation with IGF-I receptor antibodies, and the resulting immune complexes were analyzed by immunoblotting with phosphotyrosine (top) or IGF-I receptor (bottom) antibodies, respectively. Each immunoblot shows a representative of two experiments. B, tyrosine phosphorylation (top) and total protein content (bottom) of Shc proteins. Solubilized myoblast proteins were subjected to immunoprecipitation with Shc antibodies followed by immunoblotting with phosphotyrosine (top) or Shc (bottom) antibodies. The efficiency of immunoprecipitation of the Shc antibodies was Ͼ90% for all Shc proteins. Each immunoblot shows a representative of two experiments. C, phosphorylation and total protein content of MEK. Total cells lysates were analyzed by immunoblotting with phospho-MEK-1/2 (Ser-217/Ser-221) or MEK antibodies to assess MEK phosphorylation (top) and total protein content (bottom), respectively. A representative immunoblot (left) and the quantitation of basal (white bars) and IGF-I-stimulated (black bars) MEK phosphorylation and total MEK protein content in three independent experiments (right) are shown. *, p Ͻ 0.05 versus wild-type L6 myoblasts. D, phosphorylation and total protein content of Akt. Total cells lysates were analyzed by immunoblotting with phospho-Akt (Ser-473) or Akt antibodies to assess Akt phosphorylation (top) and total protein content (bottom), respectively. A representative immunoblot (left) and the quantitation of basal (white bars) and IGF-I-stimulated (black bars) Akt phosphorylation and total Akt protein content in three independent experiments (right) are shown.
for at least 24 h, or by stably expressing v-Src, has been recently shown to cause disruption of the actin cytoskeleton and loss of focal contacts (22). This effect was due to MEK-dependent inhibition of the Rho-ROCK-LIM kinase pathway (22), which promotes actin stress fiber stabilization and actomyosinbased cell contractility (36,37). A similar mechanism, i.e. ROCK inhibition by sustained MEK/Erk signaling, has been described in Ras-transformed fibroblasts (38,39). The phosphatidylinositol 3-kinase/Akt pathway was reported to be not involved in the v-Src-or Ras-induced disruption of the actin cytoskeleton (22,38), and this is consistent with the finding of normal Akt activation in L6/Shcas myoblasts in this study (Fig.  5D). The ability of constitutive Erk activation to cause changes in cell morphology and rearrangement of actin cytoskeleton has been recently demonstrated also in a macrophage cell line following overexpression of annexin 1 (40). Second, persistent MEK activation may induce altered subcellular localization of Erk kinases. To regulate the adhesion/cytoskeletal network, activated Erk has to be translocated to newly forming focal adhesions, because MEK inhibition suppresses this peripheral targeting of Erk and integrin-dependent focal adhesion assembly (41). In this study, we found that the L6 myoblasts with reduced p66 Shc had constitutive IGF-I-independent translocation of activated Erk proteins in the nucleus. It is possible, therefore, that constitutive nuclear targeting of Erk-1 and Erk-2 in the L6/Shcas myoblasts may diverge these kinases from critical sites in the cytoskeletal network, leading to impaired Erk regulation of the actin cytoskeleton.
Myoblasts with increased Erk activity and disruption of actin stress fibers showed impaired DNA synthesis in response to IGF-I stimulation and incomplete differentiation into myotubes. Changes in actin filament-associated proteins and loss of actin stress fibers can contribute to aberrant growth control, because this may alter both growth factor-and integrin-mediated entry into the S phase of the cell cycle (42)(43)(44). In addition, focal adhesion-associated proteins may play an important role in skeletal muscle differentiation, as treatment with cytochalasin D, a selective disruptor of actin filaments, reportedly inhibits differentiation of myoblasts into myotubes (45). Alternatively, impaired growth and differentiation responses of L6/ Shcas myoblasts could be due to abnormal regulation of the , and L6/Shcas1 (clones C6 and D28) myoblasts. Cell lysates were analyzed by immunoblotting with phospho-MEK-1/2 (Ser-217/Ser-221) or MEK antibodies to assess MEK phosphorylation and total protein content, respectively. Data shown are representative of three independent experiments. B, effects of PD98059 on Erk-1/2 phosphorylation in wild-type L6, L6/Neo (clone N5), and L6/Shcas1 (clones C6 and D28) myoblasts. Cell lysates were analyzed by immunoblotting with phospho-p42/44 MAP kinase (Thr-202/Tyr-204) or MAP kinase antibodies to study Erk-1/2 phosphorylation and total protein content, respectively. Data shown are representative of three independent experiments. MEK/Erk signaling pathway. It is well recognized that IGF-I stimulates mitogenesis in L6 myoblasts via Erk (14) 2 and that Erk inhibition is associated with enhanced differentiation into myotubes (14). Furthermore, Erk phosphorylation is initially increased and then decreased in response to IGF-I, this biphasic and opposite response being required for the stimulatory effects of IGF-I on myoblasts proliferation and differentiation, respectively (21). In L6/Shcas myoblasts, the phosphorylation levels of both MEK and Erk were persistently elevated and unresponsive to IGF-I (Figs. 2 and 5). Constitutively active MEK/Erk has been shown to block S-phase entry in fibroblasts and other cell types (40,46), and this may explain the lack of mitogenic response of the L6/Shcas myoblasts to IGF-I, even in the presence of normal IGF-I signaling through Akt (Fig. 5D). In addition, the activation levels of MEK and Erk were persistently higher in the L6/Shcas myoblasts compared with control myoblasts examined at various times during differentiation. 2 The inappropriateness of this high MEK/Erk signaling activity may have similarly led to impaired terminal differentiation into myotubes.
In conclusion, this study shows that the p66 Shc isoform exerts a physiologically relevant, inhibitory signaling effect on the Erk pathway in skeletal muscle myoblasts, which is necessary for coordinated actin cytoskeleton polymerization and normal IGF-I responsiveness of MEK/Erk. Loss of p66 Shc in L6 myoblasts results in an altered cell phenotype resembling that of transformed cells, with rounded shape, disruption of actin fibers, growth factor-insensitive DNA synthesis, and inability to undergo complete differentiation. In future studies, it will be important to assess whether variations in p66 Shc expression levels may contribute to phenotype changes in other cell types, including cancer cells.