title: Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts.
abstract: 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.
authors.affiliation: Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology and Metabolic Diseases, University of Bari, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Natalicchio
authors.forename: Luigi
authors.initials: L
authors.lastname: Laviola
authors.forename: Claudia
authors.initials: C
authors.lastname: De Tullio
authors.forename: Lucia Adelaide
authors.initials: LA
authors.lastname: Renna
authors.forename: Carmela
authors.initials: C
authors.lastname: Montrone
authors.forename: Sebastio
authors.initials: S
authors.lastname: Perrini
authors.forename: Giovanna
authors.initials: G
authors.lastname: Valenti
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Procino
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.forename: Francesco
authors.initials: F
authors.lastname: Giorgino
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000502:physiology*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000276:immunology; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000276:immunology / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004791:Enzyme Inhibitors; D005419:Flavonoids; D021122:Protein Subunits; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D017526:Receptor, IGF Type 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one