To deal with this query HC11 cells have been starved of insulin at the same time as serum and growth aspect, then stimulated with either insulin or EGF within the presence of reduced levels of PI three kinase or mTOR inhibitors. The results in figure five detect differences during the p70S6 kinase phosphorylation and kinase activity toward RPS6 that was dictated by the stim ulatory agent. Insulin stimulation of Akt was PI 3 kinase dependent, and phosphorylation of p70S6 kinase was PI three kinase and mTOR dependent. Insulin stimulation resulted in PI 3 kinase and mTOR dependent RPS6 phosphorylation. In con trast, the stimulation of RPS6 phosphorylation by EGF was partially independent of PI three kinase and mTOR path techniques. This further RPS6 phosphorylation correlated with elevated p70S6 kinase phosphorylation at Thr 421 and Ser 424, the autoinhibitory website reported to contribute to its action in vivo.
Since higher ranges of PI three kinase and mTOR inhibitors entirely eliminated selelck kinase inhibitor this signal, it appears that EGF involves Akt and mTOR to activate p70S6 kinase and that residual reduced degree activity selleck of p70S6 kinase may be enhanced by EGF dependent phosphorylation at Thr 421 and Ser 424. Consequently, we conclude that both insulin and EGF stimulate the PI 3 kinase Akt mTOR p70S6 kinase pathway, but that EGF modulates p70S6 kinase activity within a manner not activated by insulin. Additionally, differences while in the phosphorylation of 4E BP1 and elF4E had been detected in response to insulin and EGF. There may be mTOR independent 4E BP1 and elF4E phosphorylation in response to insulin which is not detected with EGF, suggesting that insulin stimulation of those pathways may perhaps be distinctive from that seen with EGF i.
e. that insulin signaling may phosphorylate these sub strates by way of a pathway besides mTOR. Dexamethasone contributes to your inhibition of p70S6 kinase during lactogenic differentiation of HC11 cells The studies described above addressed quick term stimu lation of cells with EGF. Subsequent, the long lasting activation of signal transduction pathways dependent on PI 3 kinase stimulation was examined in HC11 cells. HC11 cells were taken care of with lactogenic hormone mix while in the presence or absence of EGF and LY294002 for occasions as much as 24 hrs. Cell lysates had been analyzed by western blot ting for phosphorylation of p70S6 kinase. Within the HC11 cells stimulated using the lactogenic hormone combine DIP the activation of p70S6 kinase on threonine 389 totally diminished by approximately twelve hrs, whereas while in the cells stimulated with DIP inside the presence of EGF the acti vation of p70S6 kinase persisted for 24 hrs.