p53 vs. ISG15: Stop, you’re killing me

The stability of the p53 tumor-suppressing transcription factor is tightly regulated. In normal, unstressed cells, p53 is kept at low levels primarily by its major negative regulator MDM2 through polyubiquitylation at several N- and/or C-terminal sites, thus targeting p53 for degradation through the 26S proteasome. Under certain conditions, MDM2 also may monoubiquitylate or neddylate p53, but these primarily serve different functions. p53 also can be ubiquitylated by several other E3 ligases, but at least in the cells and tissues that have been examined, MDM2 is the major regulator of p53 stability.1 Both MDM2 and MDMX also block the activity of p53 as a transcription factor by binding the N-terminal transactivating domains. In contrast, many tumor-derived mutant p53s are much more stable and accumulate to high levels in tumor cells, although generally the ability of these mutants to act as a transcription factor is impaired. Both wild-type and mutant p53s also are covalently posttranslationally modified at many sites in a variety of different ways, including phosphorylation of serines and threonines and acetylation or methylation of lysines.2 Huang and colleagues now describe yet another posttranslational modification of p53 that regulates its stability and activity: ISGylation mediated through the interferon stimulated gene 15 (ISG15).3 ISG15 was the first ubiquitin-like modifier protein to be discovered. It is rapidly and highly induced following interferon treatment and plays important roles in regulating the innate immune response and in the defense of host cells to viral infection (reviewed in ref. 4). Like MDM2-mediated ubiquitylation of p53, ISG15 follows a similar modus operandi to ISGylate this “guardian of the genome.” ISG15 is covalently attached to lysine residues of target proteins through its C terminus via the sequential action of 3 enzymes: the E1-activating enzyme UbE1L, the E2-conjugating enzyme UbcH8, and the major E3 ligase HERC5 (Fig. 1). Unlike ubiquitin, however, there is no evidence for poly-ISGylation; also contrary to ubiquitin, the ISG15 sequence is not highly conserved and is found only in vertebrate species. Huang et al. identified 5 lysines in the N-terminal domain of p53 (K101, K120, K132, K139, and K164) and 6 (K291, K292, K320, K321, K351, and K357) in its C-terminal domain that can be conjugated with ISG15 and that partially overlap with lysines reported to be ubiquitylated (and/or neddylated) by MDM2.1,2 Interestingly, the N-terminal target lysines all reside in the structured beginning of the p53 DNA binding domain, while the 6 C-terminal targets reside in the C-terminal section of the DNA binding domain, the nuclear localization signal, or in or near the structured tetramerization domain. These locations may serve in part to prevent a misfolded p53 monomer from interacting with other p53 molecules to form dimers or tetramers. Among the 11 conjugation sites, p53 was primarily conjugated with one or two ISG15 molecules, presumably because steric interference by the larger ISG15 prevents more than one ISG15 from being attached to a p53 domain. While ISGylation has been intimately tied to protein translation, preferentially targeting newly translated proteins for ISG15 modification, its function has remained largely unknown.5 Now Huang et al. showed that p53 ISGylation provides a signal to target ribosome-linked, misfolded p53 for degradation by the 20S proteasome (Fig. 1). Interestingly ISG15 itself is a transcriptional target of p53 that, similar to Mdm2, can be upregulated by stress,6 potentially forming yet another p53 feedback loop. Figure 1. ISGylation of p53 serves to remove dominant-negative, misfolded, nascent p53 through the 20S proteasome,3 while polyubiquitylation by MDM2 and degradation by the 26S proteasome serves as the major negative regulator of p53 abundance ... The study by Huang et al. raises several important questions. Among these are: Many cellular proteins have been identified as potential ISGylation targets;4,5 are these also targeted for degradation as a consequence of misfolding? How is misfolded p53 recognized by the ISGylation system? p53 is also degraded by the 20S proteasome in a ubiquitin-independent manner (reviewed in ref. 7); is ISGylation involved in this mechanism? A significant percentage of newly synthesized proteins in mammalian cells are ubiquitylated and degraded in a quality control process. Do the ubiquitylation and ISGylation systems share in the quality control of p53 synthesis, or does each primarily serve a different quality control function(s)? The “Stop, you’re killing me!” website is well known to readers of mystery novels. We trust that master detectives Lane and Bulavin will soon provide more clues to the mysterious affair of p53 ISGylation.