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. 2014 Oct 15;289(48):33215–33230. doi: 10.1074/jbc.M114.608927

FIGURE 7.

FIGURE 7.

ERK1/2 versus mTORC1 phosphorylation of S6, effects of MEK1/2 inhibitor PD184352/U0126 and mTOR inhibitor rapamycin. Cells were preincubated with DMSO (vehicle), 10 μm U0126, 10 μm PD184352, and/or 50 nm rapamycin for 30 min, as indicated, before incubating with 10 nm insulin for 10 min (ERK1/2 and IRS1) or 30 min (S6 and S6K1). Quantified phosphorylation data are normalized to the effect of insulin in control. Mean ± S.E. (error bars) is shown; n = number of cell preparations from different subjects. Shown is the effect of MEK1/2 inhibitor PD184352 (yellow), mTORC1 inhibitor rapamycin (cyan), or PD184352 + rapamycin (green), compared with vehicle/control (blue), on phosphorylation of ERK1/2 at Thr202/Tyr204 rapamycin (n = 5) compared with control (n = 5) (A) or S6 at Ser235/236, PD184352 (n = 7), rapamycin (n = 3), and PD184352 + rapamycin (n = 3) compared with control (n = 7) (B); or S6K1 at Thr389, PD184352 (n = 6), rapamycin (n = 4), and PD184352 + rapamycin (n = 6) compared with control (n = 6) (C); or IRS1 at Ser307, PD184352 (n = 6) compared with control (n = 6) (D). E, effect of MEK1/2 inhibitor U0126 (yellow) compared with vehicle/control (blue) on phosphorylation of S6 at Ser235/236. F, simulations of model variables ERK1/2-Thr202/Tyr204-P, S6-Ser235/236-P, and S6K-Thr389-P (blue) compared with the effect of MEK1/2 inhibitor PD184352 implemented as reduced activation of ERK1/2 by 50, 75, 83, 88, 90, 92, and 93% (left panels, yellow); mTORC1 inhibitor rapamycin implemented as reduced mTORC1 activation by 50, 75, 83, 88, 90, 92, and 93% (middle panels, cyan); or the combined effect of PD184352 + rapamycin implemented as a combination of the two effects (right panels, green). The time points for corresponding experiments in A–C are indicated with gray bars. G, insulin signaling network with the cross-talk from ERK1/2 to S6. Indicated in yellow text is the inhibition of MEK1/2-catalyzed phosphorylation of ERK1/2 by PD184352/U0126, and shown in blue text is the inhibition of mTORC1 with rapamycin. For more details, see the legend to Fig. 1. a.u., arbitrary units.