We demonstrate that the atypical MAP kinase homologue ERK3 and the cyclin-dependent kinase inhibitor p21 are ubiquitinated on their NH2
termini. With the transcriptional activator MyoD (7
), ERK3 and p21 are the second and third examples of mammalian proteins ubiquitinated in that fashion. This conclusion is based on the following evidence. First, lysineless versions of both proteins are degraded by the proteasome with kinetics similar to that of the wild-type protein in vivo. In contrast, lysineless mutants of cyclin E (12
) or Sic1 (31
) are very stable in vivo, indicating that these proteins require an internal lysine(s) for efficient ubiquitination and degradation. Second, in vivo degradation of ERK3 and p21 lysineless mutants is absolutely dependent on a functional ubiquitin conjugation pathway. Third, we clearly detected polyubiquitin conjugates of lysineless ERK3 and p21 proteins. Fourth, we showed that addition of N-terminal tags of increasing size stabilizes ERK3 and p21 by specifically interfering with their ubiquitination. Finally, and most importantly, we demonstrated the existence of a fusion peptide between the N-terminal methionine of ERK3 and p21 constructs and the C-terminal glycine of ubiquitin in intact cells.
Recent studies have shown that positioning of the ubiquitin attachment site is critical for efficient polyubiquitination and proteasome recognition. Petroski and Deshaies have shown that the context of the ubiquitin acceptor site in Sic1 affects the targeting property of the polyubiquitin chain (31
). Ubiquitination of C-terminal Sic1 lysines occurs slowly, while producing substrates poorly recognized by the 26S proteasome. In contrast, the six N-terminal lysine residues are more efficiently ubiquitinated in vitro and assemble ubiquitin chains that are more competent for degradation by the proteasome. Interestingly, these authors also showed that a single polyubiquitin chain can support efficient proteasomal degradation in vitro. The results presented here suggest that the N-terminal amine of ERK3 and p21 is both necessary and sufficient to sustain efficient degradation by the proteasome in intact cells. Accordingly, lysineless mutants of ERK3 and p21 have half-lives similar to those of their wild-type counterparts. Our findings further substantiate the idea that a single ubiquitin chain is sufficient to efficiently target a protein substrate to the proteasome in vivo.
A recent structural study has also highlighted the importance of ubiquitin acceptor site positioning. The X-ray structure of the ternary complex Skp1/β-trcp1/β-catenin has revealed that the amino group, provided by a lysine in this case, must be properly placed to be efficiently ubiquitinated by SCFβ-trcp1
). For example, displacing the lysine acceptor by 19 residues can decrease the apparent in vitro ubiquitination rate by a factor of 3. Here we show that addition of sequence tags at the N terminus greatly affects the ubiquitination status and stability of ERK3 and p21 in vivo. Most importantly, we show that the size of the tag and not its primary sequence is important for this effect. Small tags comprising less than 13 residues have a minimal effect, whereas tags larger than 5 kDa very efficiently inhibit protein ubiquitination and degradation. The observation that three Myc epitopes are sufficient to stabilize ERK3 is in good agreement with the results of Sheaff et al. (40
), who showed that addition of Myc3
tag stabilizes expression of p21 by about threefold. Thus, our results support the notion that the E3 ligase active site has a limited range of action and that displacing the NH2
acceptor site can greatly influence the rate of catalysis. Alternatively, the presence of large N-terminal tags may sterically interfere with the binding of the E3 to the substrate. More detailed in vitro studies with purified E3 ligases will allow for discrimination between these possibilities.
It has been previously reported that the cell cycle inhibitor p21 is degraded by the proteasome in a ubiquitin-independent manner (3
). This conclusion was based mainly on two sets of arguments. First is the apparent lack of involvement of the ubiquitin conjugation pathway in regulating p21 turnover based on the fact that a lysineless mutant of p21 was degraded at the same rate as wild-type p21 in intact cells. In addition, Sheaff et al. (40
) reported that overexpression of a ubiquitin mutant (UbR7) that stabilizes cyclin E expression had no apparent effect on the steady-state levels of p21. However, the half-life of p21 was not measured in UbR7-transfected cells and it is not known if this mutant ubiquitin is used by all classes of E3 ligases with similar efficiency in vivo. Here we used a robust genetic approach with an E1 temperature-sensitive cell line to evaluate the importance of the ubiquitin conjugation pathway in the in vivo degradation of p21. We unambiguously demonstrate that both endogenous and ectopically expressed wild-type or lysineless p21 is degraded in a ubiquitin-dependent manner. The second piece of evidence for ubiquitin-independent degradation of p21 was the inability to detect ubiquitin conjugates of a lysineless p21 mutant in vivo. In contrast to these studies, we clearly detected polyubiquitin conjugates of this mutant. Why was polyubiquitination of p21-0K not detected earlier? Our results reveal the presence of two apparent populations of ubiquitinated species of ERK3 and p21 with high- and low-molecular-size ubiquitin adducts (Fig. and ). However, we noticed the absence of low-molecular-size ubiquitin conjugates for both the ERK3-0K and p21-0K mutants, whereas higher-molecular-size adducts are detected. Thus, the lack of detection of low-molecular-size ubiquitination species could lead to the wrong conclusion that a lysineless mutant is not ubiquitinated in vivo (for example, see Fig. , middle panel).
The physiological significance of N-terminal ubiquitination remains to be established. In the case of ERK3, accumulating evidence suggests that N-terminal ubiquitination plays a critical role in regulating the effect of the kinase on cell proliferation. In a recent study, it was shown that expression of stable ERK3 chimeras (in which ERK3 degrons were replaced by analogous ERK1 sequences) strongly inhibits S-phase entry in fibroblasts (14
). In contrast, expression of unstable wild-type ERK3 protein had no significant effect on cell cycle progression. Notably, it was observed that ectopic expression of Myc6
-ERK3 also potently inhibits entry of cells into S phase (24
). These observations, together with the findings reported here, suggest that N-terminal ubiquitination is essential to repress the negative regulatory effect of ERK3 on cell cycle progression. However, our results do not exclude the possibility that ubiquitination of internal lysine residues may contribute in some way to the regulation of ERK3 or p21. Indeed, the absence of low-molecular-size ubiquitin adducts in lysineless mutants suggests that these ubiquitin chains are normally conjugated to internal lysine residues. Based on their apparent size, these conjugates may contain one to five ubiquitin molecules, although it is not known if these adducts occur on one or multiple lysine(s). Based on the fact that lysineless mutants of ERK3 and p21 have half-lives very similar to that of the wild-type protein, we conclude that these low-molecular-size ubiquitin conjugates play a minor role, if any, in proteasomal targeting. Modification by mono- or short ubiquitin species may serve functions other than proteolysis, such as regulation of subcellular localization (22
During the course of this work, Bloom and coworkers (4
) have independently shown that p21 is ubiquitinated on its NH2
terminus in vivo. In agreement with the findings reported here, these authors have shown that p21 degradation is dependent on a functional ubiquitin system and that addition of a Myc6
tag stabilizes the protein. However, contrary to what we observed for ERK3, they suggested that the stabilization effect of the Myc6
tag does not result from inhibition of p21 ubiquitination. This discrepancy may be intrinsic to the studied proteins or may result from differences in experimental procedures. The identification of additional proteins ubiquitinated on the NH2
terminus will help clarify this issue.
One of the key issues that needs to be addressed is the identity of the ubiquitin ligase(s) responsible for the polyubiquitination of ERK3 and p21. In the case of p21, different E3 ligases have been implicated in the regulation of its degradation. The SCFSkp2
ligase was found to catalyze the ubiquitination of p21 in vitro and to participate in the degradation of p21 specifically during S phase of the cell cycle (5
was also implicated in the rapid degradation of p21 in response to low doses of UV light (3
). Consistent with these findings, treatment of cells with antisense oligonucleotides to Cul1, Skp1, or Skp2 leads to the accumulation of p21 (46
), and physical complexes of Skp2 and p21 can be found in intact cells (3
). However, p21 half-life is unchanged in asynchronous Skp2−/−
mouse embryonic fibroblasts (3
), and the inhibitor is also unstable in postmitotic cells in the absence of Skp2 expression. These observations argue that additional ubiquitin ligases contribute to the proteasomal degradation of p21 that may be specific to the cell cycle phase or cellular context. The ring finger MDM2 is another E3 ligase that binds to p21 and has been shown to promote its degradation by the proteasome (23