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.
Natalicchio
Annalisa
A
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology and Metabolic Diseases, University of Bari, Italy.
Laviola
Luigi
L
De Tullio
Claudia
C
Renna
Lucia Adelaide
LA
Montrone
Carmela
C
Perrini
Sebastio
S
Valenti
Giovanna
G
Procino
Giuseppe
G
Svelto
Maria
M
Giorgino
Francesco
F
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
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