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

Received for publication, April 8, 2004, and in revised form, June 28, 2004Published, JBC Papers in Press, July 19, 2004, DOI 10.1074/jbc.M403936200

Annalisa Natalicchio‡, Luigi Laviola‡, Claudia De Tullio‡, Lucia Adelaide Renna‡,Carmela Montrone‡, Sebastio Perrini‡, Giovanna Valenti§, Giuseppe Procino§, Maria Svelto§,and Francesco Giorgino‡¶

From the ‡Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology andMetabolic Diseases, and the §Department of General and Environmental Physiology, University of Bari, 70124 Bari, Italy

To investigate the role of Shc in IGF action and sig-naling in skeletal muscle cells, Shc protein levels werereduced in rat L6 myoblasts by stably overexpressing aShc cDNA fragment in antisense orientation (L6/Shcas).L6/Shcas myoblasts showed marked reduction of thep66Shc protein isoform and no change in p52Shc or p46Shc

proteins compared with control myoblasts transfectedwith the empty vector (L6/Neo). When compared withcontrol, L6/Shcas myoblasts demonstrated 3-fold in-crease in Erk-1/2 phosphorylation under basal condi-tions and blunted Erk-1/2 stimulation by insulin-likegrowth factor I (IGF-I), in the absence of changes in totalErk-1/2 protein levels. Increased basal Erk-1/2 activa-tion was paralleled by a greater proportion of phospho-rylated Erk-1/2 in the nucleus of L6/Shcas myoblasts inthe absence of IGF-I stimulation. The reduction ofp66Shc in L6/Shcas myoblasts resulted in marked pheno-typic abnormalities, such as rounded cell shape andclustering in islets or finger-like structures, and wasassociated with impaired DNA synthesis in response toIGF-I and lack of terminal differentiation into myo-tubes. In addition, L6/Shcas myoblasts were character-ized by complete disruption of actin filaments and cellcytoskeleton. Treatment of L6/Shcas myoblasts with theMEK inhibitor PD98059 reduced the abnormal increasein Erk-1/2 activation to control levels and restored theactin cytoskeleton, re-establishing the normal cell mor-phology. Thus, the p66Shc isoform exerts an inhibitoryeffect on the mitogen-activated protein kinase signalingpathway in rodent myoblasts, which is necessary formaintenance of IGF responsiveness of the MEK/Erkpathway and normal cell phenotype.

Skeletal muscle represents an important site of action for theIGFs,1 because specific high affinity IGF-I receptors are ex-

pressed in this tissue (1, 2), and both IGF-I and IGF-II stimu-late growth and differentiation of skeletal muscle cells (3).Muscle cells also synthesize and secrete IGF-I, IGF-II, andIGF-binding proteins, providing a cell model for integratedautocrine and paracrine control of mitogenic and metabolicactions by the IGFs. IGF signaling in skeletal muscle involvesactivation of specific cell surface receptors, containing a tyro-sine kinase domain within their cytoplasmic portion and un-dergoing autophosphorylation on specific tyrosine residuesupon ligand binding. Receptor autophosphorylation triggerstyrosine 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 mediatorsthat are tyrosine-phosphorylated by multiple receptor or recep-tor-associated tyrosine kinases and are capable of stimulatingthe Ras/MAP kinase pathway after binding to the Grb2 adaptor(6, 7). The Shc proteins originate by alternative use of threedistinct translation starting points on a longer transcript(p42Shc, p52Shc, and p66Shc) and of two translation startingpoints on a shorter transcript (p46Shc and p52Shc); the twomRNA transcripts are generated by alternative splicing from asingle gene and are regulated by distinct promoters (8–10). Asa consequence, p66Shc contains the entire sequence of p46Shc

and p52Shc, with one phosphotyrosine binding (PTB) domain,one src homology 2 (SH2) domain, and one collagen homology(CH1) region. In addition, p66Shc has a second CH2 domain of110 amino acids, located at the NH2-terminal portion of themolecule, that is not present in p46Shc or p52Shc. Recent reportshave established a specific role for the p66Shc isoform. p46Shc

and p52Shc are found in every cell type with similar reciprocalrelationship, whereas p66Shc expression varies from cell type tocell type and is also lacking in some cell types (8). Evidence fordivergent regulation of p66Shc versus p46/p52Shc has emergedfrom studies that demonstrated specific serine phosphorylationof p66Shc through an MEK-mediated pathway in response toinsulin (11). Knock-out experiments, in which the p66Shc genehas been selectively inactivated in mice, suggest an importantrole of this protein in the regulation of cellular responses to

* This work was supported by grants from the Ministerodell’Istruzione, Universita e Ricerca (Italy), the Cofinlab 2000, Centrodi Eccellenza Genomica Comparata: Geni Coinvolti in Processi Fisiopa-tologici in Campo Biomedico e Agrario (Italy), the Associazione ItalianaRicerca sul Cancro (Italy), and an educational grant from Pfizer Italiasrl (ARADO Program) (to F. G.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

¶ To whom correspondence should be addressed: Dept. of Emergencyand Organ Transplantation, Section on Internal Medicine, Endocrinol-ogy and Metabolic Diseases, University of Bari, Piazza Giulio Cesare,11, 70124 Bari, Italy. Tel./Fax: 39-080-547-8689; E-mail: f.giorgino@endo.uniba.it.

1 The abbreviations used are: IGF, insulin-like growth factor; Erk,

extracellular signal-regulated protein kinase; IRS, insulin receptor sub-strate; PTB, phosphotyrosine binding; SH2, Src homology 2; CH1, -2,collagen homology 1 and 2; MEK, mitogen-activated and extracellularsignal-regulated protein kinase kinase; PD98059, an inhibitor of acti-vation of MEK by Raf; MEM, minimum essential medium; BCS, bovinecalf serum; BSA, bovine serum albumin; PY99, phosphotyrosine anti-body; Akt, protein kinase B; MAP, mitogen-activated protein; FITC,fluorescein isothiocyanate; Grb2, growth factor receptor binding pro-tein-2; Sos, son of sevenless; EGF, epidermal growth factor; TBS, Tris-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 42, Issue of October 15, pp. 43900–43909, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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oxidative stress, apoptosis, and life span (12, 13). However, theintracellular signaling pathways mediating the unique actionsof p66Shc remain to be established.

The mitogenic and survival signals evoked by the IGFs havebeen extensively studied in multiple cell types, including skel-etal muscle cells. Activation of the MEK/Erk pathway in re-sponse to the IGFs appears to mediate myoblast growth anddifferentiation (14, 15). IGF-I stimulation of DNA synthesis isblocked by the MEK inhibitor PD98059 (16), and myotubes donot survive or differentiate when the MEK/Erk pathway issimilarly blocked by PD98059 (17). Furthermore, augmenta-tion of IGF-I-stimulated cell proliferation in dexamethasone-treated L6 myoblasts is associated with increased Shc anddecreased IRS-1 tyrosine phosphorylation (18, 19), suggestingthat under conditions of glucocorticoid excess the mitogenicresponse of muscle cells to the IGFs can be modulated byaltering the activity of the Shc/Erk pathway. The specific con-tribution of the p66Shc isoform to IGF-I action on skeletalmuscle cells has not been investigated.

In this study, we show that selective reduction of p66Shc inL6 skeletal muscle cells results in up-regulation of the MEK/Erk pathway, leading to increased Erk-1/2 phosphorylationand nuclear localization in the absence of IGF-I stimulation.This is associated with a dramatic perturbation of the actincytoskeleton, leading to abnormal cell shape, growth, and dif-ferentiation of the L6 myoblasts. The abnormalities in bothsignaling reactions and organization of the actin cytoskeletonare corrected by the MEK inhibitor PD98059, suggesting thatin skeletal muscle cells the p66Shc isoform may physiologicallyexert an inhibitory role on MEK/Erk, which is necessary for fullresponsiveness of this signaling pathway to the IGFs and main-tenance of normal cell morphology.

EXPERIMENTAL PROCEDURES

Cell Culture—L6 rat skeletal muscle myoblasts were cultured inEagle’s minimum essential medium (MEM) supplemented with 10%donor calf bovine serum (BCS) (both from Invitrogen), 2 mM L-gluta-mine, 100 units/ml penicillin, 100 mg/ml streptomycin, and non-essen-tial amino acids in a 5% CO2 atmosphere at 37 °C. Myoblasts weredifferentiated into myotubes with 2% horse serum (Invitrogen), 2 nM

triiodothyronine (Sigma), and 20 nM insulin. For IGF-I studies, cellswere incubated in complete medium containing 0.5% bovine serumalbumin (BSA) without calf bovine serum for 16 h, and then stimulatedwith 100 nM IGF-I (GRO PEP, Adelaide, Australia) for the indicatedtimes or left untreated. To block the MEK/Erk signaling pathway, cellswere incubated with 20 �M PD98059 (Calbiochem, Merck KGaA) for theindicated times.

Antibodies—A monoclonal phosphotyrosine antibody (PY99) andpolyclonal IGF-I R �-subunit (C-20) antibodies were purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal MEK-1/2,phospho-MEK-1/2 (Ser-217/Ser-221), Akt, phospho-Akt (Ser-473), andphospho-p42/44 MAP kinase (Erk-1/2) (Thr-202/Tyr-204) antibodieswere from Cell Signaling Technology (Beverly, MA). Polyclonal andmonoclonal Shc antibodies were from BD Transduction Laboratories(Lexington, KY). A monoclonal MAP kinase (Erk-1/2) antibody wasfrom Zymed Laboratories (San Francisco, CA). A monoclonal vinculinantibody was from Sigma-Aldrich.

Transfection Studies—To generate L6 myoblasts stably expressingreduced levels of the p66Shc isoform, a 287-bp fragment of the cDNAencoding the p52/p46Shc proteins (from nucleotide 55 to nucleotide 342),indicated as as1, was generated by amplification using the pMJ/Shcplasmid (kindly provided by Dr. J. Schlessinger, New York, NY) as atemplate. A 326-nucleotide cDNA fragment corresponding to the NH2-terminal 109 amino acids unique to p66Shc, indicated as as2, was gen-erated by PCR amplification using the rat p66Shc cDNA (kindly pro-vided by Dr. J. E. Pessin, New York, NY). To generate stabletransfectants, the cDNAs of interest were cloned in antisense orienta-tion into the mammalian expression vector pCR3.1 (Invitrogen), con-taining a G418 resistance gene, under control of the cytomegaloviruspromoter. The as1- and as2-containing plasmids were transfected intoL6 myoblasts by liposome-mediated gene transfer using LipofectAMI-NETM (Invitrogen), and stable transfectants, indicated as L6/Shcas1

and L6/Shcas2, were selected by their ability to grow in neomycin-containing medium. Multiple stable clones of L6 cells hypoexpressingShc were obtained within 4 weeks. Plasmid integration into the cellgenome was confirmed by PCR amplification of genomic DNA withforward and reverse oligonucleotide primers corresponding to thepCR3.1 (5�-TAA TAC GAC TCA CTA TAG GG-3�) and Shc (5�-CTG GCACAG TGG CCC TGT CCA TCC-3�) sequences, respectively. The amountof Shc protein expressed in transfected L6 myoblasts was determined byimmunoblotting total cell lysates with Shc antibodies, as previouslydescribed (18).

Immunoprecipitation and Immunoblotting—For immunoprecipita-tion studies, L6 skeletal muscle cells were washed twice with Ca2�/Mg2�-free PBS and then scraped in ice-cold lysis buffer containing 50mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 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 thencentrifuged at 12,000 � g for 10 min, and the resulting supernatant wascollected and assayed for protein concentration using the Bradford dyebinding assay kit with BSA as a standard. Equal amounts of cellularproteins (500 �g) were subjected to immunoprecipitation with the in-dicated antibodies overnight at 4 °C. The resulting immune complexeswere adsorbed onto 70 �l of protein A-Sepharose beads (AmershamBiosciences) for 2 h at 4 °C, washed three times with lysis buffer, andthen eluted with Laemmli buffer for 5 min at 100 °C.

For immunoblotting studies, equal amounts of cellular proteins wereresolved by electrophoresis on 7% or 10% SDS-polyacrylamide gels, asappropriate, directly or following immunoprecipitation with the specificantibodies, as indicated. The resolved proteins were electrophoreticallytransferred to nitrocellulose membranes (Hybond-ECL, Amersham Bio-sciences) using a transfer buffer containing 192 mM glycine, 20% (v/v)methanol, and 0.02% SDS. To reduce nonspecific binding, the mem-branes 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, asappropriate, and then incubated overnight at 4 °C with the indicatedantibodies. The proteins were visualized by enhanced chemilumines-cence (ECL) using horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG (Amersham Biosciences) and quantified by densitometricanalysis using Optilab® image analysis software (Graftek SA, Mir-mande, France).

Immunofluorescence Analyses—To visualize the actin cytoskeleton,L6 cells were grown on coverslips in complete medium for the indicatedtimes, then fixed with 4% paraformaldehyde and permeabilized at�20 °C with 100% methanol. Fixed cells were incubated with fluores-cein isothiocyanate-conjugated (FITC) phalloidin (purchased from Sig-ma-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 conventionalepifluorescence microscopy (Leica DMLB microscope with a Sensicam12 Bitled charge-coupled device camera, Bansheim, Germany), and allimages were captured at the same magnification.

To examine vinculin-containing focal adhesions, L6 cells grown onglass coverslips were fixed with 4% paraformaldehyde and permeabi-lized with 0.5% Triton X-100. Incubation with primary antibody againstvinculin (1:400) was conducted at room temperature for 16 h, followedby incubation with secondary Alexa488 Fluor anti-mouse goat antibody(1:500, Molecular Probes, Eugene, OR) for 1 h and washing three timeswith PBS. Cells were finally stained with TO-PRO-3 (1:10,000, Molec-ular Probes) to visualize nuclei, and coverslips were mounted using GelMount (Biomeda). Images were acquired on a Leica TCS SP2 laserscanning 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 grownon glass slides, arrested at 50% confluence in serum-free medium for24 h, and then incubated in the absence or presence of 100 nM IGF-I for30 min. To assess total Erk-1/2, control and IGF-I-treated cells werefixed with methanol/acetone (70:30, v/v) for 10 min at �20 °C. After a10-min rehydration with multiple PBS washes at 25 °C, the cells wereblocked with PBS/10% BCS for 45 min and then incubated with themonoclonal MAP kinase antibody in PBS/10% BCS (1:100) for 60 min at25 °C. Cells were then washed three times with PBS and incubatedwith 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% parafor-maldehyde for 20 min and permeabilized with 100% methanol for 5 minat �20 °C. After a 10-min rehydration with multiple PBS washes at

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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 1�,TBST) supplemented with 5.5% BCS for 45 min, and then incubatedwith the polyclonal phospho-p44/p42 MAP kinase (Thr-202/Tyr-204)antibody in Tris-buffered saline 1� (TBS) containing 50 mM Tris-HCl(pH 7.4), 150 mM NaCl/3% BSA (1:100) for 60 min at 25 °C. Cells werethen washed three times with TBST and incubated with FITC-conju-gated anti-rabbit antibodies in TBST/3% BSA (1:100) for 60 min at25 °C in the dark. Finally, cells were washed three times with PBS,mounted under glass coverslips with CITIFLUOR medium, examinedunder epifluorescent illumination with excitation-emission filters forFITC by epifluorescence microscopy (Leica DMRXA microscope), andphotographed with an Olympus inverted microscope equipped withphase-contrast and UV illumination through an FITC filter.

Thymidine Incorporation—Thymidine incorporation was carried outas previously described (18). Briefly, L6 cells were grown in 35-mmmultiwell plates to �50% confluence in MEM with 10% BCS. Followingincubation in serum-free MEM for 16 h, L6 myoblasts were incubatedwith or without 100 nM IGF-I for 16 h at 37 °C. The cells were thenincubated for 1 h at 37 °C in fresh MEM containing 0.2% BSA, 25 mM

HEPES (pH 7.6), and 1 �Ci/ml [3H]thymidine. The medium was re-moved, 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-cold10% trichloroacetic acid. The cells were then solubilized in 0.1 N NaOHfor 30 min at 37 °C, and the amount of 3H was quantitated by liquidscintillation counting. For each condition, experiments were carried outin triplicate.

Statistical Analyses—All data are expressed as mean � S.E. Data areexpressed as percentage of control or basal control values, as appropri-ate. Statistical analyses were performed by unpaired Student’s t tests.

RESULTS

Generation of L6 Skeletal Muscle Myoblasts with Reducedp66Shc Protein Levels—To determine the specific contributionof the p66Shc isoform to IGF-I action and signaling in skeletalmuscle cells, the cellular levels of this IGF-I receptor substratewere selectively reduced by expressing specific Shc antisenseRNA sequences. For this purpose, undifferentiated L6 skeletalmuscle cells were stably transfected with two independent Shcantisense sequences, designated as1 and as2. as1 represents a287-nucleotide cDNA fragment corresponding to both the p46/p52Shc and p66Shc mRNA transcripts, whereas as2 represents a326-nucleotide cDNA fragment specific to the NH2-terminal109 amino acids unique to the p66Shc mRNA transcript (Fig.1A). Independent clones of L6 cells showing decreased expres-sion of p66Shc were identified by immunoblotting with anti-Shcantibodies and selected for further analyses.

Both as1 and as2 induced a marked and selective reductionin p66Shc protein levels in multiple clones of L6 myoblasts (Fig.1, B and C). as1 decreased the p66Shc protein �90% comparedwith untransfected wild-type L6 myoblasts (p � 0.05) or L6/Neo myoblasts transfected with the empty pCR3.1 vector. Bycontrast, the protein levels of the other Shc isoforms, i.e. p52Shc

and p46Shc, were not significantly different in L6/Shcas1, L6/Neo, and untransfected L6 myoblasts (Fig. 1B). A 65% reduc-tion in p66Shc was obtained following overexpression of the as2

FIG. 1. Antisense-mediated inhibi-tion of p66Shc protein expression inL6 myoblasts. A, structure of p46/p52Shc

and p66Shc mRNAs. The phosphotyrosinebinding (PTB), collagen homology 1(CH1), and src homology 2 (SH2) domainscommon to all Shc isoforms, and the col-lagen homology 2 (CH2) domain specificto p66Shc are indicated. Antisense cDNAfragments corresponding to nucleotides381–668 of the p66Shc cDNA (indicated asas1) or nucleotides 1–326 of the p66Shc

cDNA (indicated as as2) were cloned inpCR3.1 and expressed in L6 myoblasts bystable transfection, as described under“Experimental Procedures.” B, proteinlevels of Shc isoforms in L6 myoblastsoverexpressing as1. Total cell lysatesfrom wild-type L6 myoblasts, L6 myo-blasts transfected with the empty vector(L6/Neo) and L6 myoblasts transfectedwith as1 (L6/Shcas1) were analyzed byimmunoblotting with Shc antibodies. Arepresentative immunoblot is shown onthe left. The quantitation of p66Shc (blackbars), p52Shc (gray bars), and p46Shc

(white bars) protein levels in wild-typeL6, L6/Neo, and L6/Shcas1 myoblasts infour independent experiments is shownon the right. *, p � 0.05 versus wild-typeL6 myoblasts. C, protein levels of Shc iso-forms in L6 myoblasts overexpressingas2. Total cell lysates from L6 myoblaststransfected with the empty vector (L6/Neo) and L6 myoblasts transfected withas2 (L6/Shcas2) were analyzed by immu-noblotting with Shc antibodies. A repre-sentative immunoblot is shown on theleft. The quantitation of p66Shc (blackbars), p52Shc (gray bars), and p46Shc

(white bars) protein levels in L6/Neo andL6/Shcas2 myoblasts in five independentexperiments is shown on the right. *, p �0.05 versus N22 myoblasts.

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cDNA construct (p � 0.05), which also did not affect p52Shc orp46Shc protein levels (Fig. 1C). Therefore, L6 skeletal musclecell lines with marked reduction of p66Shc were established bystable transfection of as1 or as2. The selective inhibition ofp66Shc expression in the absence of changes in expression levelsof the other Shc isoforms may be potentially explained bydifferent sensitivity to antisense-mediated inhibition of proteintranslation of the two mRNA transcripts encoding the Shcproteins (8–10). Therefore, both as1 and as2 interfere with themRNA transcript encoding p66Shc, whereas the mRNA tran-script encoding p46/52Shc may be resistant to interference byas1 (Fig. 1A).

Erk-1/2 Signaling in L6/Shcas Myoblasts—The Shc pro-teins regulate cellular responses through activation of theGrb2/Sos/Ras signaling cascade, leading to phosphorylationand activation of the MAP kinase family members Erk-1 andErk-2. To verify whether selective reduction of the p66Shc pro-tein levels could modify this signaling pathway, we analyzedbasal and IGF-I-induced Erk-1/2 phosphorylation in L6/Shcas1and L6/Shcas2 myoblasts with decreased p66Shc protein levels.Activation of Erk-1 and Erk-2 was evaluated by immunoblot-ting with phospho-Erk-1/2 (Thr-202/Tyr-204) antibodies in twoindependent clones of L6/Shcas1 myoblasts (C6 and D28) andtwo independent clones of L6/Shcas2 myoblasts (E15 and E21).In the L6/Shcas1 clones, basal Erk-1 and Erk-2 phosphoryla-tion was increased 411 and 360% of control, respectively (p �0.05) (Fig. 2A). By contrast, basal phosphorylation of Erk-1 andErk-2 was similar in L6 myoblasts transfected with the emptyvector (L6/Neo, clones N1 and N5) and in untransfected wild-type L6 myoblasts (respectively, p � 0.32 and p � 0.97), indi-cating that plasmid transfection and clonal selection in neomy-cin-containing medium did not affect the level of Erkphosphorylation. No change in total Erk-1 or Erk-2 proteincontent was evident in wild-type L6, L6/Neo, and L6/Shcas1myoblasts (Fig. 2A). Increased phosphorylation of Erk isoformsin the basal state was also evident in two independent clones ofL6/Shcas2 myoblasts compared with control (Fig. 2B; p � 0.05),although this change was of lower magnitude (i.e. 280 and200% increases of Erk-1 and Erk-2 phosphorylation, respec-tively, in clones E15 and E21 versus control) as compared withthat seen in L6/Shcas1 myoblasts. Total protein content ofErk-1 and Erk-2 was slightly and not significantly increased inL6/Shcas2 compared with control myoblasts (Fig. 2B). There-fore, stable overexpression of either as1 or as2 in L6 myoblastsresults in decreased p66Shc content and increased phosphoryl-ation of Erk-1 and Erk-2 not due to changes in total Erk proteincontent. L6/Shcas1 myoblasts were used in subsequent studies,because they showed greater decrease in p66Shc protein levelsand more prominent increase in Erk-1/2 phosphorylation ascompared with L6/Shcas2 myoblasts.

To assess the responsiveness of Erk-1/2 phosphorylation toIGF-I stimulation, control and L6/Shcas myoblasts weretreated with 100 nM IGF-I for various times and subjected toimmunoblotting with phospho-Erk-1/2 antibodies. In controlL6/Neo cells, IGF-I markedly increased phosphorylation ofboth Erk isoforms, which was evident after 3 min of stimula-tion, peaked at 5 min, and remained sustained up to 30 min(Fig. 2C). By contrast, Erk-1/2 phosphorylation was elevated inthe basal state in L6/Shcas myoblasts and showed no furtherincrease during IGF-I stimulation (Fig. 2C).

Activation of Erk-1/2 following phosphorylation on Thr-202/Tyr-204 results in translocation of the phosphorylated kinasesfrom the cytoplasm, where they are normally retained presum-ably via a cytoplasmic anchoring complex, to the nucleus (20).To investigate the intracellular localization of activated Erk-1/2 in myoblasts with reduced p66Shc levels, L6/Shcas cells

were studied by immunofluorescence using antibodies to totalor phosphorylated Erk-1/2 and compared with control. In con-trol L6 myoblasts, Erk-1/2 appeared uniformly distributed inthe cell cytoplasm under basal conditions. IGF-I stimulationinduced an increase in the Erk-1/2 signal in the cell nucleusand perinuclear region (Fig. 3A). Increased nuclear fluores-cence in IGF-I-treated cells was also observed using antibodiesto phosphorylated Erk-1/2 (Fig. 3B), indicating IGF-I-depend-ent nuclear translocation of the phosphorylated form of Erk-1/2. As compared with control cells, L6/Shcas myoblastsshowed a greater amount of Erk-1/2 in their nucleus already inthe basal state, which did not augment upon IGF-I stimulation(Fig. 3A). The nuclear Erk-1/2 in the unstimulated L6/Shcasmyoblasts was constitutively phosphorylated, as demonstratedby immunofluorescence with phospho-Erk-1/2 antibodies (Fig.3B). Thus, L6/Shcas myoblasts show constitutive activationand nuclear translocation of Erk-1/2 proteins in the absence ofIGF-I stimulation.

IGF-I exerts a dual effect on proliferation and differentiationof skeletal muscle cells, which relies upon an intact Erk-1/2responsiveness to this growth factor (16, 17, 21). Because Erkactivation was altered in L6/Shcas compared with control L6myoblasts, IGF-I effects on DNA synthesis and cell differenti-ation were next determined. In untransfected L6 and L6/Neomyoblasts IGF-I stimulation resulted in a 3-fold increase inDNA synthesis, measured by determining the rates of [3H]thy-midine incorporation into DNA (p � 0.05 versus basal) (Fig.4A). By contrast, in L6/Shcas myoblasts the IGF-I effect onDNA synthesis was modest and statistically not significant(Fig. 4A). Differentiation into myotubes, which is largely me-diated by IGFs secreted in an autocrine manner (3), was alsoimpaired in the L6/Shcas myoblasts. Both wild-type L6 andL6/Neo myoblasts became elongated and fused to form thecharacteristic multinucleated myotubes when grown in thedifferentiation medium (Fig. 4B). By contrast, L6/Shcas myo-blasts showed elongation and some degree of alignment, butdid not develop into myotubes (Fig. 4B).

IGF-I Receptor Signaling in L6/Shcas Myoblasts—The con-stitutive activation of Erk in the absence of IGF-I stimulationin myoblasts with reduced p66Shc levels could potentially resultfrom increased activity of steps in the IGF-I signaling cascadeupstream of Erk. To explore this possibility, tyrosine phospho-rylation and total protein levels of the IGF-I receptor weremeasured 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 ofthe IGF-I receptor was very low under basal conditions and wasincreased severalfold following incubation with 100 nM IGF-Ifor 10 min in wild-type L6, L6/Neo, and L6/Shcas myoblasts(Fig. 5A). Tyrosine phosphorylation of p52Shc, the predominantShc isoform undergoing tyrosine phosphorylation in responseto IGF-I stimulation in L6 cells (18), was also low in theabsence of IGF-I in wild-type L6, L6/Neo, and L6/Shcas myo-blasts, and was increased to a similar extent in all cell linesafter stimulation with IGF-I (Fig. 5B). Tyrosine phosphoryla-tion of p66Shc was induced by IGF-I in wild-type L6 and L6/Neomyoblasts but not, as expected, in the L6 myoblasts expressinglow levels of this Shc isoform following transfection of thep66Shc antisense (Fig. 5B). These results indicate that theconstitutive activation of Erk in myoblasts with reduced p66Shc

is not the consequence of increased tyrosine phosphorylation ofthe IGF-I receptor or Shc proteins.

Erk-1/2 phosphorylation and activation in response togrowth factor stimulation is mediated by the serine kinaseMEK. To further investigate the mechanisms responsible forthe increased activity of Erk in the L6/Shcas myoblasts, the

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activation of MEK was measured by immunoblotting with spe-cific phospho-MEK (Ser-217/Ser-221) antibodies. In wild-typeL6 and L6/Neo myoblasts, MEK phosphorylation was low inthe basal state and increased 7-fold after IGF-I stimulation(Fig. 5C). In contrast, MEK phosphorylation was already aug-mented in the basal state in L6/Shcas myoblasts (5-fold versuscontrol, p � 0.05), and showed no significant changes whencells were stimulated with IGF-I. These findings parallel thechanges in Erk-1/2 phosphorylation observed in L6/Shcas myo-blasts (Fig. 2, A–C). The increased MEK phosphorylation wasnot explained by changes in MEK protein levels, which weresimilar in wild-type L6, L6/Neo, and L6/Shcas myoblasts (Fig.

5C). Thus, constitutive MEK activation in the absence of IGF-Istimulation appears to account for the increased basal phos-phorylation of Erk-1/2 in L6/Shcas myoblasts.

To investigate another signaling pathway that is largelyindependent of the MEK/Erk pathway, the activation state ofAkt was also assessed. As shown in Fig. 5D, the level of Aktphosphorylation on Ser-473 was similarly low in control andL6/Shcas myoblasts and was increased markedly followingstimulation with IGF-I. Therefore, while MEK/Erk kinases arede-regulated in the L6/Shcas myoblasts, the Akt pathway is notaltered and shows normal responsiveness to IGF-I.

Cytoskeleton of L6/Shcas Myoblasts—L6 myoblasts in cul-

FIG. 2. Erk-1/2 phosphorylation in L6Shcas myoblasts. Control L6 myoblasts (L6 and L6/Neo) and L6 myoblasts overexpressing as1 or as2were incubated in serum-free medium in the absence of IGF-I for 16 h and then harvested. Total cell lysates were analyzed by immunoblotting withphospho-p42/44 MAP kinase (Thr-202/Tyr-204) or MAP kinase antibodies to detect phosphorylated or total Erk-1/2, respectively, as indicated. A,representative immunoblot (left), and quantitation of three independent experiments (right), showing basal phosphorylation (top) and total proteincontent (bottom) of Erk-1 and Erk-2 in L6 (white bars), L6/Neo (white bars), and L6/Shcas1 (black bars) myoblasts. *, p � 0.05 versus L6 myoblasts.B, representative immunoblot (left), and quantitation of three independent experiments (right), showing basal phosphorylation (top), and totalprotein content (bottom) of Erk-1 and Erk-2 in L6/Neo (white bars) and L6/Shcas2 (black bars) myoblasts. *, p � 0.05 versus N22 and N24myoblasts. Erk-1 and Erk-2 phosphorylation and protein content were not different in N22, N24, and untransfected L6 myoblasts (data not shown).C, Erk-1/2 phosphorylation in response to stimulation with 100 nM IGF-I for various times in L6/Neo (clone N5) and L6/Shcas1 (clone C6) myoblasts(top). A MAP kinase immunoblot for control of total Erk protein loading is also shown (bottom).

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ture typically grow as a monolayer of elongated cells (Fig. 4B).The phenotype of L6/Neo myoblasts is similar to that of un-transfected L6 cells (Fig. 4B). By contrast, L6/Shcas myoblastsshowed marked phenotypic abnormalities, including roundedcell shape, tendency to adhere poorly to the culture dish, andclustering in islets or finger-like structures (Fig. 4B). To inves-tigate whether the changes in cell morphology of L6/Shcasmyoblasts were associated with alterations of F-actin-contain-ing structures and focal adhesions, the actin network and focalcontacts were visualized with FITC-conjugated phalloidin andan antibody against vinculin, respectively. Control L6 myo-blasts exhibited a characteristic morphology, with well devel-oped actin stress fibers and vinculin-containing focal adhesions(Fig. 6, A and B). In marked contrast, a deep disorganization ofactin cytoskeleton with strong reduction of actin fibers was

found in the L6/Shcas myoblasts with reduced p66Shc, in whichactin accumulated in patches at the cell periphery (Fig. 6A). Inaddition, L6/Shcas myoblasts showed loss of vinculin-contain-ing focal adhesions (Fig. 6B).

The constitutive activation of MEK/Erk could potentiallyresult in altered turnover of G/F actin balance in L6 myoblastswith reduced p66Shc, leading to disorganization of actin cy-toskeleton and altered cell morphology (22). To explore thispossibility, L6/Shcas myoblasts were treated with the MEKinhibitor PD98059 for 72 h to achieve persistent inhibition ofthe abnormally elevated levels of MEK-1/2 and Erk-1/2 activi-ties, and the resulting effects on actin cytoskeleton were thenexamined. The levels of basal MEK-1/2 phosphorylation incontrol myoblasts were very low or undetectable and were notapparently affected by the PD98059 compound (Fig. 7A). Onthe other hand, the elevated levels of basal MEK-1/2 phospho-rylation found in L6/Shcas myoblasts were markedly reducedby PD98059 (Fig. 7A). Basal Erk-1/2 phosphorylation was alsoefficiently inhibited by PD98059, such that after treatmentwith this compound Erk-1/2 phosphorylation was similarly lowin L6/Shcas and control myoblasts (Fig. 7B). Inhibition of MEKand Erk phosphorylation with PD98059 had no effect on eitherthe actin cytoskeleton or focal adhesions of control L6 myo-blasts, whereas it restored the cell shape in the L6/Shcas cells,which displayed a well organized actin network and focal con-tacts (Fig. 6, A and B). These results indicate that the increasedbasal activity of Erk-1/2 in myoblasts with reduction of thep66Shc isoform can be corrected following inhibition of MEKwith PD98059 and that this results in reorganization of actincytoskeleton and focal contacts, with consequent restoration ofthe cell shape.

DISCUSSION

By stably overexpressing antisense cDNA fragments specificto the Shc mRNA, L6 skeletal muscle cell lines with markedand selective reduction of p66Shc were established. These cellsshow: (i) specific reduction of p66Shc in the absence of signifi-cant changes in the other Shc isoforms, (ii) marked de-regula-tion of the MEK/Erk signaling pathway, with increased basalphosphorylation and blunted response of these kinases to IGF-Istimulation, and (iii) altered actin cytoskeleton and cell shape.Furthermore, correcting the overactivity of Erk results in res-toration of the actin cytoskeleton, suggesting a key function ofthe Erk signaling pathway in the regulation of this cellularcomponent in skeletal muscle cells.

Although all three Shc isoforms can be tyrosine-phosphory-lated upon growth factor stimulation, p46/52Shc are coupled togrowth and survival signals, whereas p66Shc also undergoesserine phosphorylation and mediates pro-apoptotic responsesto oxidative stress (12). Specifically, the p66Shc has been shownto regulate intracellular oxidant levels and hydrogen peroxide-mediated forkhead inactivation (23), effects that are probablyrelevant to the reported ability of p66Shc to control lifespan inmammals (12) and are unique to this Shc isoform. Additionalevidence for different promoter regulation by methylation andhistone deacetylation (10) and subcellular localization (24)strengthen the concept of the biological diversity of the Shcisoforms. Consistent with their biological diversity, p66Shc andp46/p52Shc have been shown to exert opposite effects on theMEK/Erk signaling pathway. Overexpression of p46/p52Shc en-hanced EGF-induced Erk activation, whereas p66Shc overex-pression had no effect (9) or inhibited (25) this response. Thelatter study suggested that p66Shc might function to provide fora feedback inactivation of the Erk signaling pathway in con-trast to the other Shc isoforms. Also in the present study,p66Shc was found to exert an inhibitory effect on Erk, becausereduced expression levels of p66Shc were associated with per-

FIG. 3. Immunofluorescence analysis of the subcellular local-ization of Erk-1/2 in L6Shcas myoblasts. Basal and IGF-I-stimu-lated wild-type L6 and L6/Shcas1 myoblasts were grown on glass slides,arrested at 50% confluence in serum-free medium for 24 h, and thenincubated in the absence or presence of 100 nM IGF-I for 30 min. Cellswere fixed and incubated with Erk-1/2 or phospho-Erk-1/2 antibodiesand then with the appropriate FITC-conjugated secondary antibody, asdescribed under “Experimental Procedures.” A, total Erk-1/2 detectedby immunostaining with Erk-1/2 antibodies. B, phosphorylated Erk-1/2detected by immunostaining with phospho-Erk-1/2 antibodies. A repre-sentative of three experiments is shown. Scale bars, 10 �m.

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sistent Erk activation. The opposite effects of p46/p52Shc andp66Shc on Erk activation are of pathophysiological significance,since human breast cancer tissues with high p46/p52Shc top66Shc expression ratios show increased proliferative activityand are associated with poor prognosis (26).

The mechanism underlying the inhibitory effect of p66Shc onthe Erk-signaling pathway is not fully understood. In L6/Shcasmyoblasts, Erk was constitutively activated and poorly respon-sive to IGF-I stimulation, and so was MEK, the signalingmolecule upstream of Erk. By contrast, tyrosine phosphoryla-tion of the IGF-I receptor and the p46/p52Shc isoforms and Aktphosphorylation were normal, indicating that the increasedErk phosphorylation in myoblasts with reduced p66Shc was notthe consequence of enhanced IGF-I receptor signaling and thatother signaling pathways did not exhibit constitutive activa-tion. According to Okada et al. (25), p66Shc is serine-phospho-rylated in an MEK-dependent manner, and the phosphorylatedp66Shc binds to Grb2 but is not capable of associating with

receptor tyrosine kinases. Because p66Shc and p46/p52Shc com-pete for binding to a limited pool of Grb2 molecules, increasedcellular abundance of p66Shc may result in removal of Grb2-Soscomplexes from the receptor at the cell membrane and conse-quent inhibition of Ras activation (25). Conversely, one couldenvision that reduction of p66Shc protein levels, which wasachieved in this study, may allow more p46/p52Shc binding toGrb2 in the proximity of the tyrosine kinase receptor, leadingto sustained Ras and Erk activation.

L6 myoblasts with reduced p66Shc levels showed roundedshape and altered growth properties when compared with con-trol myoblasts (Fig. 4A) and were characterized by completedisruption of the actin stress fibers, focal adhesions, and cellcytoskeleton (Fig. 6). Importantly, inhibition of Erk restoredthe myoblast phenotype in L6/Shcas cells, suggesting a role forthe MEK/Erk pathway in the regulation of actin polymeriza-tion and focal contacts. The Shc proteins have been implicatedin the control of actin cytoskeleton and cell motility in multiple

FIG. 4. Abnormal growth and differentiation properties of L6 myoblasts with reduced p66Shc levels. A, basal (white bars) andIGF-I-stimulated (black bars) [3H]thymidine incorporation into DNA in wild-type L6, L6/Neo (clones N1 and N5), and L6/Shcas1 (clones C6 andD28) myoblasts. Cells were grown in complete medium to �50% confluence and then incubated in serum-free medium for 16 h in the absence orpresence of 100 nM IGF-I, as described under “Experimental Procedures.” Data represent the quantitation of basal and IGF-I-stimulated DNAsynthesis from five independent experiments. *, p � 0.05 versus basal. B, phenotypic characteristics of wild-type L6, L6/Neo (clone N5), andL6/Shcas1 (clone C6) cells before (top) and after (bottom) differentiation into myotubes, carried out as described under “Experimental Procedures.”Magnification, �200.

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cell types. Fibroblasts obtained from mice with targeted dis-ruption of the Shc gene show aberrant rounded morphologyand a disorganized actin cytoskeleton (27), and down-regula-tion of p46/52Shc isoforms results in reduced EGF-dependentmotility in MCF-7 breast cancer cells (28). Conversely, overex-pression of p46/52Shc was found to promote some extent oforganization of actin cytoskeleton and focal adhesions, associ-ated with a random type of cell motility, in a glioblastoma-derived cell line (29), and to improve motility in hepatocytegrowth factor-stimulated melanoma cells (30). Even though onestudy demonstrated that p46/p52Shc can translocate to thecytoskeleton and directly bind to F-actin in PC-12 cells (31),suggesting direct regulation of actin polymerization by the Shcproteins, current evidence indicates that Shc promotes cy-toskeletal rearrangement in response to growth factor stimu-lation through the Erk pathway (27, 32). In addition, the p46/p52Shc isoforms bind to and are tyrosine-phosphorylated byintegrin-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/Erkpathway and dynamically regulate actin fiber assembly, to-gether with signals transduced through the FAK-p130Cas com-plex. Consistent with this model, regulation of actin cytoskel-eton was observed following overexpression of activated MEK,implying the Ras/Raf/MEK/Erk pathway in the p46/p52Shc-de-pendent effects (29). The link between Erk and the cytoskeletonis demonstrated by the finding that activated Erk can directlyphosphorylate and activate myosin light chain kinase, leadingto phosphorylation of myosin light chains and subsequent pro-motion of the cytoskeletal contraction necessary for cell move-ment (35).

If MAP kinase promotes actin organization, why did consti-tutive activation of Erk-1 and Erk-2 cause actin fiber disassem-bly in the L6/Shcas myoblasts? First, persistent up-regulationof Erk activity may be inappropriate for maintenance of normalorganization of the actin cytoskeleton. Consistent with thisconcept, 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/Shcasmyoblasts. Wild-type L6, L6/Neo (clonesN1 and N5), and L6Shcas1 (clones C6 andD28) myoblasts were studied in the basalstate or 10 min following stimulation with100 nM IGF-I. A, tyrosine phosphorylation(top) and total protein content (bottom) ofIGF-I receptors. Solubilized myoblast pro-teins were subjected to immunoprecipita-tion with IGF-I receptor antibodies, andthe resulting immune complexes were an-alyzed by immunoblotting with phospho-tyrosine (top) or IGF-I receptor (bottom)antibodies, respectively. Each immuno-blot shows a representative of two exper-iments. B, tyrosine phosphorylation (top)and total protein content (bottom) of Shcproteins. Solubilized myoblast proteinswere subjected to immunoprecipitationwith Shc antibodies followed by immuno-blotting with phosphotyrosine (top) or Shc(bottom) antibodies. The efficiency of im-munoprecipitation of the Shc antibodieswas 90% for all Shc proteins. Each im-munoblot shows a representative of twoexperiments. C, phosphorylation and to-tal protein content of MEK. Total cellslysates were analyzed by immunoblottingwith phospho-MEK-1/2 (Ser-217/Ser-221)or MEK antibodies to assess MEK phos-phorylation (top) and total protein content(bottom), respectively. A representative im-munoblot (left) and the quantitation of ba-sal (white bars) and IGF-I-stimulated(black bars) MEK phosphorylation and to-tal MEK protein content in three inde-pendent experiments (right) are shown. *,p � 0.05 versus wild-type L6 myoblasts.D, phosphorylation and total protein con-tent of Akt. Total cells lysates were ana-lyzed by immunoblotting with phospho-Akt (Ser-473) or Akt antibodies to assessAkt phosphorylation (top) and total pro-tein content (bottom), respectively. A rep-resentative immunoblot (left) and thequantitation of basal (white bars) andIGF-I-stimulated (black bars) Akt phos-phorylation and total Akt protein contentin three independent experiments (right)are shown.

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for at least 24 h, or by stably expressing v-Src, has been re-cently shown to cause disruption of the actin cytoskeleton andloss of focal contacts (22). This effect was due to MEK-depend-ent inhibition of the Rho-ROCK-LIM kinase pathway (22),which promotes actin stress fiber stabilization and actomyosin-based cell contractility (36, 37). A similar mechanism, i.e.ROCK inhibition by sustained MEK/Erk signaling, has beendescribed in Ras-transformed fibroblasts (38, 39). The phos-phatidylinositol 3-kinase/Akt pathway was reported to be notinvolved in the v-Src- or Ras-induced disruption of the actincytoskeleton (22, 38), and this is consistent with the finding ofnormal Akt activation in L6/Shcas myoblasts in this study (Fig.5D). The ability of constitutive Erk activation to cause changesin cell morphology and rearrangement of actin cytoskeleton hasbeen recently demonstrated also in a macrophage cell linefollowing overexpression of annexin 1 (40). Second, persistentMEK activation may induce altered subcellular localization ofErk kinases. To regulate the adhesion/cytoskeletal network,activated Erk has to be translocated to newly forming focal

adhesions, because MEK inhibition suppresses this peripheraltargeting of Erk and integrin-dependent focal adhesion assem-bly (41). In this study, we found that the L6 myoblasts withreduced p66Shc had constitutive IGF-I-independent transloca-tion of activated Erk proteins in the nucleus. It is possible,therefore, that constitutive nuclear targeting of Erk-1 andErk-2 in the L6/Shcas myoblasts may diverge these kinasesfrom critical sites in the cytoskeletal network, leading to im-paired Erk regulation of the actin cytoskeleton.

Myoblasts with increased Erk activity and disruption of ac-tin stress fibers showed impaired DNA synthesis in response toIGF-I stimulation and incomplete differentiation into myo-tubes. Changes in actin filament-associated proteins and loss ofactin stress fibers can contribute to aberrant growth control,because this may alter both growth factor- and integrin-medi-ated entry into the S phase of the cell cycle (42–44). In addition,focal adhesion-associated proteins may play an important rolein skeletal muscle differentiation, as treatment with cytocha-lasin D, a selective disruptor of actin filaments, reportedlyinhibits differentiation of myoblasts into myotubes (45). Alter-natively, impaired growth and differentiation responses of L6/Shcas myoblasts could be due to abnormal regulation of the

FIG. 6. Actin cytoskeleton and focal adhesions in L6/Shcasmyoblasts and the effects of PD98059 treatment. Control L6/Neoand L6/Shcas myoblasts were incubated with 20 �M PD98059 for 72 hor left untreated. A, effects of PD98059 on the actin cytoskeleton incontrol L6/Neo (clone N5) and L6/Shcas1 (clone C6) myoblasts. Cellswere stained with FITC-phalloidin, as described under “ExperimentalProcedures.” Scale bars, 10 �m. B, effects of PD98059 on focal adhesionsin control L6/Neo (clone N5) and L6/Shcas1 (clone C6) myoblasts. Cellswere stained with vinculin antibodies (green) and TO-PRO-3 (blue) tovisualize focal adhesions and nuclei, respectively, as described under“Experimental Procedures.” Scale bars, 40 �m.

FIG. 7. Effects of PD98059 on Erk and MEK phosphorylation inL6/Shcas myoblasts. Wild-type L6, L6/Neo, and L6/Shcas myoblastswere incubated with 20 �M PD98059 for 72 h or left untreated. A, effectsof PD98059 on MEK-1/2 phosphorylation in wild-type L6, L6/Neo (cloneN5), and L6/Shcas1 (clones C6 and D28) myoblasts. Cell lysates wereanalyzed by immunoblotting with phospho-MEK-1/2 (Ser-217/Ser-221)or MEK antibodies to assess MEK phosphorylation and total proteincontent, respectively. Data shown are representative of three independ-ent experiments. B, effects of PD98059 on Erk-1/2 phosphorylation inwild-type L6, L6/Neo (clone N5), and L6/Shcas1 (clones C6 and D28)myoblasts. Cell lysates were analyzed by immunoblotting with phos-pho-p42/44 MAP kinase (Thr-202/Tyr-204) or MAP kinase antibodies tostudy Erk-1/2 phosphorylation and total protein content, respectively.Data shown are representative of three independent experiments.

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MEK/Erk signaling pathway. It is well recognized that IGF-Istimulates mitogenesis in L6 myoblasts via Erk (14)2 and thatErk inhibition is associated with enhanced differentiation intomyotubes (14). Furthermore, Erk phosphorylation is initiallyincreased and then decreased in response to IGF-I, this bipha-sic and opposite response being required for the stimulatoryeffects of IGF-I on myoblasts proliferation and differentiation,respectively (21). In L6/Shcas myoblasts, the phosphorylationlevels of both MEK and Erk were persistently elevated andunresponsive to IGF-I (Figs. 2 and 5). Constitutively activeMEK/Erk has been shown to block S-phase entry in fibroblastsand other cell types (40, 46), and this may explain the lack ofmitogenic response of the L6/Shcas myoblasts to IGF-I, even inthe presence of normal IGF-I signaling through Akt (Fig. 5D).In addition, the activation levels of MEK and Erk were persis-tently higher in the L6/Shcas myoblasts compared with controlmyoblasts examined at various times during differentiation.2

The inappropriateness of this high MEK/Erk signaling activitymay have similarly led to impaired terminal differentiationinto myotubes.

In conclusion, this study shows that the p66Shc isoform ex-erts a physiologically relevant, inhibitory signaling effect onthe Erk pathway in skeletal muscle myoblasts, which is neces-sary for coordinated actin cytoskeleton polymerization and nor-mal IGF-I responsiveness of MEK/Erk. Loss of p66Shc in L6myoblasts results in an altered cell phenotype resembling thatof transformed cells, with rounded shape, disruption of actinfibers, growth factor-insensitive DNA synthesis, and inabilityto undergo complete differentiation. In future studies, it will beimportant to assess whether variations in p66Shc expressionlevels may contribute to phenotype changes in other cell types,including cancer cells.

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