Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts.
The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110alpha, p110beta, and simultaneous knockdown of p110alpha and p110beta resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110beta-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110alpha or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Ronald W
RW
Matheny
Martin L
ML
Adamo
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D032446:Myoblasts / Q000187:drug effects / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases
D002352:Carrier Proteins; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D020033:Protein Isoforms; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C546843:mTOR protein, mouse