Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

Abstract

Background The most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation. Results We developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1’s ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E. Conclusions We show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance. Electronic supplementary material The online version of this article (10.1186/s12915-019-0658-0) contains supplementary material, which is available to authorized users.

Engineering Disulphide-Free Autonomous Antibody VH Domains to modulate intracellular pathways

“…Both miniproteins, caused dephosphorylation of eIF4E in a manner similar to PP242 through inhibition of eIF4G mediated MNK1 phosphorylation. 20 As expected, PP242 reduced 4E-BP1 phosphorylation to mediate its known effects on eIF4F complex disruption 30 . However, PP242 unlike the miniproteins also reduced phosphorylation of rS6 and AKT phosphorylation though its effects on mTORC12 (Fig.…”

“…Both constructs exhibited comparable levels of activity in immuno-precipitating eIF4E, whilst the 4E-BP1 4ALA negative control (termed 4E-BP1 YLM ) had negligible effects. VH-S4 was then shown to more e ciently disrupt the eIF4G-eIF4E interaction in cells than the weaker a nity VH domain variants (1C5, S2 and M4) using the NanoBit eIF4E:eIF4G cell-based assay 20 (Fig. 5B).…”

Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence labelled foci

“…After 4 or 24 hrs of incubation with indicated compounds, plate were processed. 25 μl of a 20-fold dilution of Nano-Glo live cell substrate (Promega) in Nano-Glo dilution buffer (PROMEGA) was added to each well and the plate shaken for 1 min at 22 °C and luminescence assessed after additional 50 minutes as described previously 43 . All luminescence assay were carried out in triplicate and luciferase activity for each well was measured by Envision Multilabel plate reader (PerkinElmer).…”

Functional Display of Bioactive Peptides on the vGFP Scaffold.

“…Transfections in 6-well plates were performed using Lipofectamine 3000 (Thermo Fisher Scienti c) with 0.5 μg of plasmid vectors per well according to the manufacturer's instructions. After 48 hrs, cells were lysed and m7GTP pull down were performed as described 50 . Brie y, 300 µg of cell lysates were incubated with 20 μl of m 7 GTP (Jena Bioscience) agarose beads for 3 hrs at 4 °C on a rotator.…”