Investigation into the role of DUB3/USP17 in Ras signalling

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.

Previously we have shown that the deubiquitinating protease DUB-3 can control cell proliferation. More recently we have uncovered the basic mechanism for this in that DUB-3 causes the specific deubiquitination of Ras converting enzyme 1 (RCE1). Ras undergoes a number of processing steps in its activation and subsequent re-localisation to the cell membrane. RCE1 mediates a key step in this process with the removal of a tripeptide from the C terminal CAAX motif found on Ras. The deubiquitination of RCE1 results in its loss of activity and therefore ability to properly process and activate Ras. The project detailed specifically aims to more fully understand the molecular basis for the action of DUB-3 on RCE1 and how ubiquitin contributes to the regulation of RCE1. Furthermore, at a cellular level we aim to examine changes in localisation of RCE1 upon DUB-3 expression. The CAAX motif found in Ras is also present in a range of other proteins such as members of the Rho small GTPase family. Although we can demonstrate that the action of DUB-3 is specific towards RCE1 and blunts Ras activation, it remains to be clarified if DUB-3 can exert similar effects on these related GTPases. Therefore, finally we will also examine in cell based models the effects of DUB-3 on the ability of RCE1 to process and control these other GTPases. Reports of the roles of deubiquitinating enzymes in signalling pathways are increasing, but our data represents the first suggestion of a role for the ubiquitin pathway in the regulation of Ras. Additionally, we will also gain much insight into both DUB-3 and RCE1, two proteases that at this stage are still poorly understood.