Regulated intracellular proteolysis is now recognized as a versatile and efficient mechanism to control gene expression. The regulation of protein turnover can have a significant impact on the activity of the corresponding target gene and is associated with either a decrease or an increase in protein stability. For example, the inhibitors of NF-κB (IκB) and the cyclin-dependent kinase inhibitor p27Kip1
are expressed to high levels in resting cells but are rapidly degraded in response to cytokines or cell cycle progression, respectively (34
). In contrast, the tumor suppressor gene product p53 and the transcription factor hypoxia inducible factor 1α (HIF-1α) are both constitutively degraded in unstimulated cells. However, in response to specific stimuli, i.e., genotoxic stress for p53 (33
) or hypoxia for HIF-1 (48
), the proteins accumulate as a result of the inhibition of protein degradation, thereby allowing them to exert their biological functions. Here we report that the atypical MAP kinase homologue ERK3 is a highly unstable protein, with a half-life of ca. 30 min, that is constitutively degraded in exponentially proliferating cells. This is the first documented example of a MAP kinase family member whose activity is acutely regulated by protein turnover. Interestingly, Lu et al. (24
) recently reported that prolonged exposure to sorbitol induces polyubiquitination of the MAP kinases ERK1/2 and promotes their degradation by a mechanism dependent on the plant homeobox domain (PHD) of MEKK1. In agreement with the results presented here, no ubiquitination of ERK1/2 was observed in response to serum or other stress stimuli, such as UV radiation or anisomycin. In contrast to ERK3, which is rapidly degraded in proliferating cells, ubiquitination of ERK1/2 (and possibly other MAP kinases) may represent an alternative feedback mechanism for downregulating kinase activity upon a persistent stress stimulus.
Protein degradation by the ubiquitin-proteasome system is involved in the regulation of many important cellular processes, including signal transduction, transcription, cell cycle progression, and class I major histocompatibility complex antigen presentation (10
). Using a combination of pharmacological, biochemical, and genetic approaches, we found that the ubiquitin-proteasome pathway is responsible for the rapid degradation of ERK3. Treatment with MG-132 and lactacystin, two structurally unrelated proteasome inhibitors, markedly increased the steady-state levels of ERK3 protein without changing the levels of ERK1. Importantly, we showed that MG-132 treatment markedly increases the half-life of endogenous ERK3, clearly demonstrating that ERK3 is a direct substrate of the proteasome.
Targeting of proteins to the proteasome generally requires the attachment of a multiubiquitin chain to the substrate (17
). However, there are examples, such as ornithine decarboxylase (32
) and p21Cip1
), for which it has been demonstrated that ubiquitination is not necessary for degradation by the proteasome. Our results clearly indicate that ERK3 is ubiquitinated in vivo and that ubiquitination is a required step for efficient degradation by the proteasome. This conclusion is supported by the following observations. First, cotransfection experiments of ERK3 with HA-tagged ubiquitin revealed the accumulation of high-molecular-weight HA-ubiquitin conjugates in MG-132-treated cells. These slowly migrating HA-immunoreactive species were not observed in cells transfected with ERK1. Second, the half-life of endogenous ERK3 protein was markedly increased at the nonpermissive temperature in cells bearing a thermolabile allele of the ubiquitin-activating enzyme E1. Third, fusion of the degradation domain of ERK3 to the heterologous protein EGFP was sufficient to promote its ubiquitination (data not shown) and to target it for proteasomal degradation (Fig. ).
A role for the proteasome in the regulation of ERK3 expression was recently reported by Zimmermann et al., who showed, by using a microarray-based differential cDNA hybridization technology, that ERK3
mRNA is upregulated by various proteasome inhibitors (58
). Proteasome inhibition also resulted in the accumulation of ERK3 protein. It was concluded that proteasome inhibitors influence ERK3 expression mainly at the transcriptional level. However, these authors did not evaluate the half-life or the ubiquitination status of ERK3. Whereas our study does not exclude a role of the proteasome in the transcriptional activation of ERK3
gene, the experiments presented here clearly demonstrate the importance of this proteolytic system in directly controlling ERK3 protein stability in proliferating cells.
It is interesting that for most protein kinases shown to be unstable, destabilization of the kinase is linked to enzymatic activation. For example, activation of the serine/threonine kinases PKCα (20
) and GRK2 (41
) by their specific activators bryostatin and isoproterenol, respectively, leads to their proteasomal degradation. Similarly, the turnover of many receptor and soluble tyrosine kinases is also regulated by their phosphotransferase activity. Ligand-activated platelet-derived growth factor β receptor (31
) or constitutively activated v-Src (16
) are both ubiquitinated and targeted to the 26S proteasome. The activation-dependent destabilization of the enzyme seems to be part of a negative feedback regulatory loop aimed at decreasing overall protein kinase activity. In contrast, our findings indicate that the control of ERK3 stability is independent of its kinase activity and of activation loop phosphorylation.
From the results of mutagenesis analysis, we concluded that the destabilizing signal is contained within the first 365 amino acids of ERK3. To further define the degradation domain of ERK3, we examined the stability of a series of chimeric kinases made between the stable ERK1 kinase and ERK3. We were able to delimit two regions in the N-terminal lobe of ERK3 kinase domain that contribute to protein instability. These two regions correspond to the first 15 amino acids of ERK3 (NDR1) and parts of kinase subdomains II and III (NDR2, residues 53 to 73). As one would expect, primary sequence analysis revealed that these two regions display low identity to the corresponding ERK1 sequences. Close inspection of the sequences did not reveal any obvious resemblance with known degradation motifs. Interestingly, in a recent study aimed at defining structural determinants controlling subcellular localization and MEK recognition, Robinson et al. observed that a chimera containing the N-terminal lobe of ERK3 and the C-terminal lobe of ERK2 is expressed at a low level compared to wild-type ERK2 (44
). This observation is in agreement with the results presented here. Despite the fact that NDR1/2 are not contiguous in the primary sequence, a theoretical model of the structure of ERK33-346
revealed that the two regions lie on the same side of the molecule in close proximity. We postulate that the NDR1/2 form a docking site for assembly of an E3 ubiquitin-protein ligase. In support of this hypothesis, we showed that ERK3 N-terminal lobe is sufficient to target heterologous proteins for degradation by the proteasome. It remains to be determined whether NDR1/2 activity is regulated by posttranslational modifications or, alternatively, if the assembly of the ERK3-E3 complex is regulated by interacting proteins. The ERK3-specific E3 ligase may also be subject to direct regulation.
Our results provide strong evidence for the physiological relevance of ERK3 rapid turnover. We found that the stability of ERK3 increases with time during in vitro myogenic differentiation of C2C12 cells, resulting in a marked upregulation of the protein. The accumulation of ERK3 is concomitant with the induction of 21Cip1 protein and exit from the cell cycle, suggesting that ERK3 may contribute in some way to cell cycle withdrawal. In agreement with this idea, BrdU incorporation studies revealed that expression of stabilized forms of ERK3, but not unstable wild-type ERK3, inhibits the induction of S phase in fibroblasts. This is the first biological effect of ERK3 described to date. All of these observations suggest that unidentified cellular ERK3-specific E3 ligase(s) is able to repress the cell cycle inhibitory activity of ERK3 by promoting its ubiquitination and degradation by the proteasome. We thus propose that the biological activity of ERK3 is mainly regulated by its cellular abundance through the control of protein degradation by the ubiquitin-proteasome pathway (Fig. ). This model is reminiscent of the regulation mode of the transcription factors p53 and HIF-1α.
Model of ERK3 regulation by proteasome-mediated degradation.