The role of reversible ubiquitylation in EGF signalling

Deubiquitylases (DUBs) have been implicated in the regulation of cell signaling processes. However, the role of DUBs in the regulation of the Epidermal growth factor receptor (EGFR) signaling is not completely understood. This study has aimed to identify DUBs involved in the regulation of EGFR signaling and downstream cascades. The first part of this study has characterized the role of the DUB USP15 in the regulation of the mitogen activated protein kinase (MAPK) cascade, a pathway downstream of EGFR. An interaction between USP15 and the MAPK negative regulator BRAP had been previously reported. When we tested the USP15 depletion phenotype on MAPK signaling we observed a paradoxical decrease in MAPK activation. Examination of upstream components of the MAPK cascade revealed a decrease in the levels of the CRAF kinase following USP15 depletion. Concordant depletion of CRAF also caused a reduction in MAPK activation, showing that depletion of CRAF phenocopied that of USP15. This work demonstrated that USP15 has a dual role in the regulation of the MAPK through BRAP and CRAF. The dominant signaling effect in the cell lines studied is through maintenance of CRAF levels. We employed a previously characterized GFP-DUB library to identify DUBs that exhibited EGF dependent distributions. One such DUB, USP46, exhibited MAPK dependent recruitment onto multi-vesicular bodies (MVB). To further characterize USP46 we generated a set of cell lines expressing GFPUSP46 and catalytically inactive GFP-USP46-C44S using the Flp-in system. While the Flp-in cells lines did not exhibit the same EGF dependent recruitment onto the MVB compartment, they did localize to Saponin resistant punctate structures. Furthermore, I observed differential activation of downstream EGFR signaling pathways that USP46 may play an undetermined role in EGF signaling. We combining stable isotope labeling of amino acids in culture (SILAC) with immuno-precipitation (IP) to quantitatively identify interactors of USP46 using mass spectrometry. We identified a 13 number of candidate interactors and confirmed a novel interaction between USP46 and FBXO11 using western blotting. Next we aimed to identify DUBs that regulate the retrograde trafficking pathway from the MVB to the trans Golgi network (TGN). We used the localization of the cation independent mannose 6-phosphate receptor (CIM6PR) as readout of the retrograde trafficking. CI-M6PR constitutively recycles from the TGN to the endo-lysosomal pathway, delivering newly synthesized acid hydrolases, required for degradative action of the lysosome. Depletion of USP8 trapped CI-M6PR in aberrant endosomes and caused a concomitant missorting of the acid hydrolase, Cathepsin D. Cathepsin D is activated through limited proteolysis in the acidic environment of the endolysosomal pathway. Depletion of USP8 caused a decrease in the mature cellular form of Cathepsin D. The mislocalization of CI-M6PR could be rescued by re-expression of GFP-USP8. The activated EGFR is degraded via the lysosome and depletion of USP8 has been demonstrated to cause a delay in the degradation of EGFR. The results presented here suggest that the decrease in active acid hydrolases observed in USP8 depleted cells, may contribute to the delay in EGFR degradation.