Molecular Cross-talk between MEK1/2 and mTOR Signaling during Recovery of 293 Cells from Hypertonic Stress

Abstract

To investigate the role for initiation factor phosphorylation in de novo translation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. As de novo translation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge upon de novo translation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuated de novo protein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promote de novo translation.

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

“…To determine whether ERK activity was responsible for 4E-BP1 phosphorylation here, we repeated experiments in the absence or presence of PD184352, a characterized inhibitor of ERK. 38 As shown in Figure S3C, inhibition of ERK did not prevent phosphorylation of 4E-BP1 in refed cells, either in the presence or absence of raptor. Ablation of rictor (lane 10 vs lane 6) led to increased p70S6K activity possibly reflecting increased expression of raptor.…”

The Cap-Dependent Translation Apparatus Integrates and Amplifies Cancer Pathways

“…3) including the Ras-regulated PI3K/Akt/mTOR 39 and Erk pathways. 40 This markedly diminishes the ability of the 4E-BPs to compete with eIF4G, thus favoring formation of intact eIF4F complexes.…”

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

“…The opposite conclusion, that eIF4E phosphorylation does not affect the rate of protein synthesis, or even decreases it, has been drawn from studies with Mnk overexpression (51), cell-free translation (52), recovery of cultured cells from hypertonic stress (53,54), and Indinavir, a human immunodeficiency virus protease inhibitor (55). Whole animal studies have also generated conflicting results.…”