Identification of Substrates for the E3 Ubiquitin Ligase RNF13 (2012)

Undergraduates: Mohsin Ali, Jeffrey P. Bocock

Faculty Advisor: Ann Erickson
Department: Biology

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E3 ubiquitin ligases play an essential role in protein regulation by tagging their substrates with one or more ubiquitin tags, which often act as proteolytic signals. Ring finger protein 13 (RNF13) is a transmembrane E3 ubiquitin ligase upregulated in cancer tissues. In stressed cells, it localizes to the inner nuclear membrane (INM). To identify proteins which RNF13 ubiquitinates in the nucleus, we used SILAC labeling and mass spectroscopy (MS) and analyzed the results with the bioinformatic tool DAVID. We predicted that INM proteins ubiquitinated by RNF13 would decrease in quantity when RNF13 expression is increased due to increased turnover by proteolysis. MS analysis revealed that the NuRD complex, a chromatin remodeling complex, Trim28, a regulator of transcription factors, and Arp2, a regulator of the actin cytoskeleton all showed at least a 2-fold decrease in expression level when RNF13 expression was increased. To validate these results, we used western blotting to compare the concentration of these proteins in cells expressing empty vector, wild-type RNF13 and mutant RNF13 unable to ligate ubiquitin to substrates. We confirmed that expression level of the putative substrates did decrease at least 1.2 fold, but found that this degrease still occurred in the cells expressing RNF13, but without the ability to ubiquinate. These results suggest that along the INM, RNF13 may be using a mechanism other than ligation of ubiquitin to regulate expression of proteins.