Identifizierung und Charakterisierung von Proteinphosphatasen als Hauptregulatoren zellulärer Stresssignalwege
Zusammenfassung der Projektergebnisse
PP1 is one of the major serine/threonine phosphatases in cells and responsible for dephosphorylating a huge variety of different target proteins. Thereby it is involved in the regulation of many essential signalling pathways as for example cell cycle, cell metabolism and many more. PP1 is an oligomeric enzyme which forms holophosphatase complexes with either one or more regulatory subunits. The subunits are essential for controlling the specificity and subcellular localization of the enzyme complex and thereby regulating PP1 activity. More than 200 potential PP1 regulatory subunits have been identified in high throughput screens, though their biological function remains elusive. The aim of my postdoctoral research in the Bertolotti lab was to characterize PP1 holoenzymes which are involved in stress signalling as well as identifying their targets. Attempts at identifying new stress regulated PP1 regulatory subunits were not successful most likely due to the transient nature of PP1 interacting with those regulatory subunits. However, targeted approaches revealed novel insights on the function of uncharacterised regulatory subunits. I focussed on the identification of substrates of specific PP1 holoenzymes, which have not been functionally characterized yet. In course of my studies, I established a line of high throughput interaction screens as well as global phospho-proteomic analysis to confirm the interaction with PP1 and to identify potential targets of a specific PP1 holophosphatase. Phospho-proteomic analysis of cells depleted of the regulatory subunit of interest, gave further insights into perturbed up- and downstream signalling pathways shedding light on the cellular function of the holophosphatase. However, due to the substoichiometric nature of many important phosphorylation events in signalling, the possibility to miss important substrates in those mass spectrometry analysis remains. Additionally, further in-depth validation of specific dephosphorylation events proved to be challenging due to the lack of specific phospho-antibodies. During my postodoctoral research an opportunity emerged to establish a luciferase reporter assay to measure translation of specific mRNAs in cells. The Bertolotti lab has a long-standing expertise in studying the integrated stress response (ISR), which is activated upon a huge variety of different stress cues such as amino acid deprivation, ER folding stress and many more. Depending on the stress signal, one in four ISR stress kinases (GCN2, PERK, HRI, PKR) is activated and phosphorylates the translational initiation factor 2α (eIF2α) at serine 51. This in turn activates a two-tier system: Translation is attenuated and a specific translational and transcriptional reprogramming set-up is initiated, which allows the cell to cope with stress. To investigate these stress responses, most data is generated by following global translation rates using radioactive labelling and high throughput sequencing of mRNAs associated with ribosomes. These methods give very valuable insights into translational regulation upon stress on a broad spectrum, though a specific assay to monitor the translation of single mRNAs was missing in the lab. I established a luciferase reporter assay, which allows the observation of the translation of specific mRNAs under non- and stressed conditions in living cells. I was able to recapitulate global behaviours of mRNAs under stress conditions but in a refined set-up. The translational reporter assay will be a very powerful tool for ongoing studies in the Bertolotti lab on the ISR and its impact on translational regulation.
Projektbezogene Publikationen (Auswahl)
- (2020) Protein translation buffers the cost of translation attenuation following eIF2alpha phosphorylation. Cell Rep. 32(11):108154
Schneider K, Nelson G M, Watson J L, Morf J, Dalglish M, Luh L M, Weber A, Bertolotti A
(Siehe online unter https://doi.org/10.1016/j.celrep.2020.108154)
