title: Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway.
abstract: Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C₂C₁₂ skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr³⁹⁷ phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.
authors.affiliation: Medical Research Council-Arthritis Research United Kingdom Centre of Excellence for Musculoskeletal Ageing Research, School of Graduate Entry Medicine and Health, University of Nottingham, Royal Derby Hospital, Derby, United Kingdom.
authors.forename: Hannah
authors.initials: H
authors.lastname: Crossland
authors.forename: Abid A
authors.initials: AA
authors.lastname: Kazi
authors.forename: Charles H
authors.initials: CH
authors.lastname: Lang
authors.forename: James A
authors.initials: JA
authors.lastname: Timmons
authors.forename: Philippe
authors.initials: P
authors.lastname: Pierre
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wilkinson
authors.forename: Kenneth
authors.initials: K
authors.lastname: Smith
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Szewczyk
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Atherton
mesh_terms: D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors; D047468:Immunoprecipitation; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000166:cytology / Q000254:growth & development*; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D007334:Insulin-Like Growth Factor I; C546843:mTOR protein, mouse; D051416:Focal Adhesion Protein-Tyrosine Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C512964:Rps6ka1 protein, mouse; D058570:TOR Serine-Threonine Kinases
keywords: S6 kinase 1; focal adhesion kinase; hypertrophy; insulin-like growth factor-I; mammalian target of rapamycin; skeletal muscle; tuberous sclerosis complex 2