on 10-31-201405:55 AM - edited on 10-15-202111:36 AM by Closed Account
Békés M, Okamoto K, Crist SB, Jones MJ, Chapman JR, Brasher BB, Melandri FD, Ueberheide BM, Denchi EL, Huang TT. Cell Rep. 2013 Nov 14;5(3):826-38. doi: 10.1016/j.celrep.2013.10.008. Epub 2013 Nov 7. The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.