title: Prolonged activation of S6K1 does not suppress IRS or PI-3 kinase signaling during muscle cell differentiation.
abstract: BACKGROUND
  Myogenesis in C2C12 cells requires the activation of the PI3K/mTOR signaling pathways. Since mTOR signaling can feedback through S6K1 to inhibit the activation of PI3K, the aim of this work was to assess whether feedback from S6K1 played a role in myogenesis and determine whether siRNA mediated knockdown of S6K1 would lead to an increased rate of myotube formation.
  
  
  RESULTS
  S6K1 activity increased in a linear fashion following plating and was more than 3-fold higher after Day 3 of differentiation (subconfluent = 11.09 +/- 3.05, Day 3 = 29.34 +/- 3.58). IRS-1 levels tended to increase upon serum withdrawal but decreased approximately 2-fold (subconfluent = 0.88 +/- 0.10, Day 3 = 0.42 +/- 0.06) 3 days following differentiation whereas IRS-2 protein remained stable. IRS-1 associated p85 was significantly reduced upon serum withdrawal (subconfluent = 0.86 +/- 0.07, Day 0 = 0.31 +/- 0.05), remaining low through day 1. IRS-2 associated p85 decreased following serum withdrawal (subconfluent = 0.96 +/- 0.05, Day 1 = 0.56 +/- 0.08) and remained suppressed up to Day 3 following differentiation (0.56 +/- 0.05). Phospho-tyrosine associated p85 increased significantly from subconfluent to Day 0 and remained elevated throughout differentiation. siRNA directed against S6K1 and S6K2 did not result in changes in IRS-1 levels after either 48 or 96 hrs. Furthermore, neither 48 nor 96 hrs of S6K1 knockdown caused a change in myotube formation.
  
  
  CONCLUSIONS
  Even though S6K1 activity increases throughout muscle cell differentiation and IRS-1 levels decrease over this period, siRNA suggests that S6K1 is not mediating the decrease in IRS-1. The decrease in IRS-1/2 associated p85 together with the increase in phospho-tyrosine associated p85 suggests that PI3K associates primarily with scaffolds other than IRS-1/2 during muscle cell differentiation.
authors.affiliation: Division of Molecular Physiology, James Black Centre, University of Dundee, Dundee, UK.
authors.forename: D Lee
authors.initials: DL
authors.lastname: Hamilton
authors.forename: Andrew
authors.initials: A
authors.lastname: Philp
authors.forename: Matthew G
authors.initials: MG
authors.lastname: MacKenzie
authors.forename: Keith
authors.initials: K
authors.lastname: Baar
mesh_terms: D000818:Animals; D002460:Cell Line; D016895:Culture Media, Serum-Free; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological; D055504:Insulin Receptor Substrate Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000473:pathology; D032446:Myoblasts / Q000378:metabolism* / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D016895:Culture Media, Serum-Free; C094556:Ecm1 protein, mouse; D016326:Extracellular Matrix Proteins; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D034741:RNA, Small Interfering; D019869:Phosphatidylinositol 3-Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C512964:Rps6ka1 protein, mouse