NPC Valorisation Voucher - Netherlands Proteomics Centre

Teck Yew Low, Flore Kruiswijk, Roberto Magliozzi andDaniele GuardavaccaroGenotoxic stress affectsprotein synthesis throughregulation of eEF2K20 | NPC Highlights 16 | November 2012Eukaryotic elongation factor 2 kinase (eEF2K) is a crucialenzyme in repressing translation elongation. We have recentlydiscovered this protein as a substrate of the SCF βTrCP ubiquitinligase using a combined mass spectrometry-based proteininteraction screening with an in-house developed technique forphosphopeptide analysis. Focusing on the biological functionsof eEF2K, we investigated how the enzyme is controlled bygenotoxic stress and how this leads to a decrease in translationelongation and protein synthesis.Cells activate surveillance molecular networks known as DNAdamage checkpoints to protect their genome from environmentaland metabolic genotoxic stress. Depending on the type andextent of DNA lesions and the cellular context, damaged cellswith an activated checkpoint can undergo senescence, die byapoptotic cell death, or repair the damaged genome and, aftercheckpoint termination, resume their physiological functions.Genotoxic stress has a greater effect on gene expression at thelevel of mRNA translation than at the level of transcription,probably because protein synthesis requires about 40% of thetotal cellular energy, and cells need to couple stress responsesto metabolic demands. Therefore, in response to genotoxicstress, cells aim to preserve energy by reducing protein synthesisin order to be able to repair the damage.Despite the major impact of genotoxic stress on mRNA translation,no information is available on how translation elongationis affected by genotoxic stress, and there have been few studiesdirected toward understanding the regulation of protein synthesisby the DNA damage response.Translation elongation Mammalian target of rapamycin (mTOR)is a serine/threonine protein kinase that integrates growth andstress signals and promotes protein synthesis. The mTOR pathwaypromotes translation elongation by inhibiting eukaryoticelongation factor 2 kinase (eEF2K), which phosphorylates andinactivates eEF2, a factor that mediates the translocation stepof peptide-chain elongation. The activity of eEF2K is controlledunder various conditions. For example, stimuli that induceprotein synthesis trigger the inactivation of eEF2K and the subsequentdephosphorylation of eEF2. In contrast, deficiencies innutrients or energy lead to activation of eEF2K and impairmentof translation elongation.In addition to this level of regulation, eEF2K abundance iscontrolled by the ubiquitin-proteasome system. In this systemsubstrates are tagged by covalent attachment of multipleubiquitin molecules and then the polyubiquitylated proteinsare degraded by the 26S proteasome. Several enzymes areinvolved in this system, but the E3 ubiquitin ligases representthe essential regulators of ubiquitylation because they physicallyinteract with target substrates. Although it is known that