Gray, Douglas A.,Blanchette, Paola.2009-03-232009-03-2320022002Source: Dissertation Abstracts International, Volume: 63-09, Section: B, page: 4046.9780612728028http://hdl.handle.net/10393/6281http://dx.doi.org/10.20381/ruor-14776The murine Unp gene encodes a ubiquitously expressed protein that fractionates with the nuclear fraction (hence its name ubiquitous nuclear protein) (Gupta, K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Gray, D. A. (1993). Oncogene 8, 2307-10). It possesses proprieties of an oncogene, such as the ability to promote tumours in a nude mouse assay (Gupta, K., Chevrette, M., and Gray, D. A. (1994). Oncogene 9, 1729--31), and its human homologue, USP4 (previously known as Unph), was shown to be over expressed in certain types of human lung tumours (Gray, D. A., Inazawa, J., Gupta, K., Wong, A., Ueda, R., and Takahashi, T. (1995). Oncogene 10, 2179--83). Although very little was known about this protein's normal function, even less on its mechanism of tumorigenicity, the predicted protein sequence gave some clues on its function. It possesses the two conserved domains present in all ubiquitin specific proteases, and the two motifs common to viral oncoproteins through which they interact with the retinoblastoma gene product pRb. In addition to these features it also possesses a region that resembles a nuclear localisation signal. A mutational approach was taken in combination with ubiquitin cleavage assays and binding assays to study Unp's function. With these, it was confirmed that Unp is a ubiquitin specific protease, its ability to cleave ubiquitin dependent on the conserved cysteine, and may be dependent on Unp phosphorylation status. Unp is a phosphoprotein, being phosphorylated on serine residue(s). It is capable of binding to pRb's hypophosphorylated as well as the hyperphosphorylated forms, a binding that is dependent on an intact conserved motif 2 (CR2). It is also capable of binding to the other pocket proteins, p107 and p130, although with different requirements of conserved regions that for pRb. With these and other results obtained, a model is proposed linking Unp's activity as a deubiquitinating enzyme and its interactions with the pocket proteins with its role as an oncogene. The data obtained also supports the newer view that ubiquitin specific proteases, have a role in the specific regulation of protein levels and not just as general ubiquitin recycling enzymes as previously believed.168 p.Biology, Molecular.Thesis