Browsing by Subject "eIF4G"

Signaling pathways converge on the translation machinery and influence protein synthesis globally or specifically on certain classes of transcripts. The experiments described in this thesis focus on regulation of translation initiation through the cap-binding protein eIF4E.

Aberrant regulation of eIF4E has important roles in several pathologies and, most notably, in tumorigenesis. Nevertheless, the understanding of the molecular con-sequences of changes in eIF4E activity remains incomplete. We employ a cell-free system to demonstrate that eIF4E function is required for efficient cap-dependent translation but inhibitory for translation of both cellular and viral RNAs relying on cap-independent mechanisms. Furthermore, we show that phosphorylation of eIF4E favors cap-independent translation in vitro.

To verify that our findings in the cell-free system are representative of an in vivo system, we also analyzed growth of an oncolytic poliovirus, relying purely on cap-independent translation, in the context of varying activity of signaling pathways. Data obtained from this virus helps to confirm that phosphorylation of eIF4E does indeed result in increased cap-independent translation. Additionally, these experiments provide important information for the clinical application of this oncolytic poliovirus, as they help to explain virus specificity and might allow for rational patient selection.

Multiple signaling pathways control protein synthesis by modulating translation initiation factors. Map Kinase Integrating Kinase 1 (Mnk1) relays signals to its major downstream target eIF4E. Activation of Mnk1 and subsequent phosphorylation of eIF4E results in changes in translation rates for subsets of mRNAs. Both the Erk1/2 and p38 MAPK pathways activate Mnk1 meaning that Mnk1 responds to growth signals through Erk1/2 and stress signals through p38 MAPK. However, it is not clear how Mnk1 mediates translational changes specific to each pathway. We investigated the activation of Mnk1 by stress and cytokines through the p38 MAPK pathway. We found that of the four different p38 MAPK isoforms, p38α alone controls acute stress and cytokine signaling to translation machinery. Furthermore, this regulatory axis is greatly diminished in neurons. We discovered that p38α expression is repressed in the brain due to two neuron-selective microRNAs, miR-124 and -128. Next, we investigated the mechanism of p38α mediated Mnk1 activation to see if it differed from Erk1/2 mediated activation. Looking at the induced binding of Mnk1 to eIF4G, we found that the dissociation rate varies depending on the activating pathways. This shows that Mnk1 is not a true convergence point of p38 and Erk1/2 MAPK pathways resulting in identical downstream effects, but that Mnk1 mediates pathway specific effects on translation factors.