Prolonged activation of S6K1 does not suppress IRS or PI-3 kinase signaling during muscle cell differentiation

Abstract

BackgroundMyogenesis 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.ResultsS6K1 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.ConclusionsEven 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.

Distinct amino acid–sensing mTOR pathways regulate skeletal myogenesis

“…Most likely, mTORC1 itself, rather than S6K1, phosphorylates IRS1 on Ser-307 ( Shah and Hunter, 2006 ) to effect the inhibition of PI3K-Akt signaling, subsequently suppressing myogenic differentiation. Indeed, it has been reported that activation of S6K1 does not affect the IRS1-PI3K pathway in myoblast differentiation ( Hamilton et al ., 2010 ). Although this mTORC1-IRS1 pathway is considered a negative feedback loop in other cellular contexts, it appears to be the major if not only function of mTORC1 in myogenesis.…”

High mobility group box 2 regulates skeletal muscle development through ribosomal protein S6 kinase 1

“…It is controversial whether S6K1 regulates myoblast differentiation. Knockdown of S6K1 did not affect the myotube formation as well as MyHC and Myogenin expression 34,35 . However, treating C2C12 cells with S6K1 inhibitor decreased the fusion index, indicating S6K1 is required for the myoblast fusion program 23 .…”

From Nutrient to MicroRNA: a Novel Insight into Cell Signaling Involved in Skeletal Muscle Development and Disease

“…Interestingly, ribosomal S6 kinase 1 (S6K1), a major target of mTORC1, is dispensable for myoblast differentiation and nascent myofibers formation in muscle regeneration despite its well-known functions in muscle growth, hypertrophy, and maintenance 50 , 52 , 57 , 60 , 61 . However, evidence also indicated that S6K1-mediated phosphorylation of Rictor negatively regulates the capacity of mTORC2 to phosphorylate Akt-S473 and persistent activation of S6K1 does not influence IRS1-PI3K signaling during myoblast 62 , 63 differentiation 62 . However, in addition, rapamycin-sensitive mTOR signaling governs two distinct stages of skeletal myogenesis: initial myotube/myofiber formation in an mTOR kinase-independent manner and myotube/myofiber maturation in an mTOR kinase-dependent mechanism 50 .…”