Investigation into the role of DUB3/USP17 in Ras signalling

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We have uncovered a novel regulatory mechanism for the control of the Ras pathway, a major cell signalling pathway that is involved in the control of cell proliferation and survival. Specifically, we have shown that a novel enzyme (DUB-3), that we first reported to have a major impact on cell proliferation, achieves this via its action on Ras Converting Enzyme 1 (RCE1). In order for Ras to be activated properly, it undergoes a number of post-translational modifications, one of which is performed by RCE1. Previously it has been assumed that RCE1 is active upon its production by the cell. We have now shown that this simplistic view of the protease is inaccurate and that its activity is controlled by DUB-3. We have shown in a number of cell types that RCE1 is 'labelled' by the addition of ubiquitin. The addition of ubiquitin to proteins has many different effects, depending on the nature of the target protein and the specific nature of the ubiquitin linkage. These effects include labelling of the target protein for degradation, activation, inactivation or re-localisation within the cell. In the specific case of RCE1, our data has shown that removal of ubiquitin by DUB-3 inhibits the activity of RCE1 and therefore blunts its ability to process Ras. Our results also suggest that the action of DUB-3 is specific, in that other deubiquitinating enzymes cannot regulate RCE1 and that DUB-3 action upon proliferation is RCE1 dependant. Despite this insight, these exciting results highlight many questions which require further investigation. In this proposal we aim to evaluate DUB-3 and its relationship with RCE1. At this time we do not understand the type of ubiquitin linkage present on RCE1 and how this promotes or maintain its activity. Here we have presented a number of experiments that will rapidly uncover the molecular basis of this control by DUB-3. Furthermore, using microscopy analysis of cells we will visualise how RCE1 localisation is affected in the presence of DUB-3. Finally, others have shown that Ras is not the only protein that can be modified by RCE1. In fact, a number of key cell signalling protein families contain the same motif that is present in Ras and recognised and clipped by RCE1. Therefore, we will examine the effect of DUB-3 on these other pathways, aiming to gain insight into the undoubtedly complicated interplay that exists at the molecular level controlling proteins with varying roles that are all processed by RCE1.