F-Box Proteins Elongate Translation During Stress Recovery

Sci. Signal.  05 Jun 2012:
Vol. 5, Issue 227, pp. pe25
DOI: 10.1126/scisignal.2003163


Protein synthesis is energetically costly and is tightly regulated by evolutionarily conserved mechanisms. Under restrictive growth conditions and in response to various stresses, such as DNA damage, cells inhibit protein synthesis to redirect available adenosine triphosphate to more essential processes. Conversely, proliferating cells, such as cancer cells, increase protein synthetic rates to support growth-related anabolic processes. mRNA translation occurs in three separate phases, consisting of initiation, elongation, and termination. Although all three phases are highly regulated, most of the translational control occurs at the rate-limiting initiation step. New evidence has described a molecular mechanism involved in the regulation of translation elongation. DNA damage initially slowed down elongation rates by activating the eukaryotic elongation factor 2 kinase (eEF2K) through an adenosine monophosphate (AMP)–activated protein kinase (AMPK)–dependent mechanism. However, during checkpoint recovery, the SCF (Skp, Cullin, F-box–containing) βTrCP (β-transducin repeat–containing protein) E3 ubiquitin ligase promoted degradation of eEF2K, thereby allowing the restoration of peptide chain elongation. These findings establish an important link between DNA damage signaling and the regulation of translation elongation.

Protein synthesis is energetically costly (1), and accordingly, eukaryotic cells have evolved sophisticated mechanisms to tightly regulate this biological process (2). Under restrictive growth conditions or in response to various stresses, such as DNA damage, cells inhibit protein synthesis to redirect available energy to more essential processes. Although the process of mRNA translation can be divided into three stages (initiation, elongation, and termination), the majority of regulation occurs at the level of initiation (2). Consequently, most of our knowledge about translational control relates to initiation mechanisms, and much less is known about the mechanisms that regulate other stages of translation. Kruiswijk et al. now report that DNA damage specifically inhibits the elongation stage of translation (3), thus providing a regulatory mechanism linking genotoxic stress with translational regulation.

Protein synthesis is largely controlled by the mammalian target of rapamycin (mTOR) protein, which regulates translation initiation but also plays roles in peptide chain elongation (4). The best-characterized mTOR substrates are the eukaryotic initiation factor 4E (eIF4E)–binding proteins (4E-BPs) and the 70-kD ribosomal S6 kinases (S6Ks), which regulate several aspects of mRNA metabolism (5). Although both 4E-BPs and S6Ks contribute to translation initiation (4), the latter also increase elongation rates by inhibiting the elongation factor 2 kinase (eEF2K) (6). Indeed, the S6Ks phosphorylate eEF2K at Ser366, thereby preventing the inhibitory phosphorylation of eEF2 and increasing the affinity of eEF2 for the ribosome (4). Kruiswijk et al. demonstrated that, in response to genotoxic stress, the adenosine monophosphate (AMP)–activated protein kinase (AMPK) phosphorylates and activates eEF2K, resulting in a temporary slowdown in translation elongation (Fig. 1). AMPK is an important regulator of metabolic activity and inhibits mTOR signaling when intracellular adenosine triphosphate is low (7). These findings indicate that AMPK inhibits translation elongation through mechanisms that are both direct (phosphorylation of eEF2K) and indirect (inhibition of mTOR), but whether one mechanism prevails over the other under specific cellular circumstances remains to be determined.

Fig. 1

Dual regulation of eEF2K activity in response to genotoxic stress. eEF2 is an essential factor for protein synthesis because it promotes the GTP-dependent translocation of the nascent protein chain from the A site to the P site of the ribosome. eEF2 is inactivated by eEF2K through phosphorylation of Thr56. (A) According to Kruiswijk et al., DNA damage promotes the AMPK-dependent phosphorylation of eEF2K at Ser398. This phosphorylation event increases the activity of eEF2K and results in increased eEF2 phosphorylation and the inhibition of translation elongation in response to genotoxic stress. (B) During checkpoint recovery, eEF2K autophosphorylates at Ser445, and an unknown kinase (kinase X) simultaneously phosphorylates Ser441. These two residues are located within a phosphodegron that is recognized by the E3 ubiquitin ligase SCFβTrCP, which promotes eEF2K ubiquitination and degradation by the ubiquitin-proteasome system. Several ubiquitination sites have been identified in large-scale proteomics studies (K65, K162, K238, K341, K347, K485, K516, K517, K603, and K684), suggesting that SCFβTrCP promotes multisite ubiquitination of eEF2K.


Genotoxic stress inhibits global protein synthesis (8), but information regarding the mechanisms governing this response is surprisingly sparse. In nearly all cases, however, DNA damage inhibits mTOR signaling through a p53-dependent mechanism. p53 induces the transcription of the mRNAs encoding the lipid phosphatase PTEN, the mTORC1 component TSC2 (tuberous sclerosis protein 2), and the HIF-1 (hypoxia-inducible factor 1)–responsive protein REDD1 (regulated in development and DNA damage responses), which all act to inhibit mTOR activity (9-11). p53 also transactivates Sestrin1 and Sestrin2, which can repress mTOR signaling in an AMPK-dependent manner (12). Ataxia telangiectasia mutated (ATM)–dependent mechanisms also converge on mTOR inhibition in response to cellular stresses (13, 14), suggesting that proximal elements in the DNA damage response pathway also regulate protein synthesis. Although DNA damage inhibits mTOR-dependent translation initiation, Kruiswijk et al. demonstrated a direct role for AMPK at the level of translation elongation. These results suggest that different stages of mRNA translation may be affected depending on the cellular stressor, which may affect the cellular response or adaptation to stress.

What would be the advantage of specifically inhibiting the elongation phase of translation? As mentioned above, protein synthesis consumes a high proportion of cellular energy, and the majority of this is used by peptide chain elongation (1). It therefore makes sense that, under conditions of decreased energy supply, it would be advantageous for the cell to reduce the rate of elongation together with that of initiation to allow energy to be diverted to other processes. Another possibility is that the inhibition of peptide chain elongation would prevent the disassembly of polysomes during checkpoint activation, which would prevent mRNAs from being degraded or sequestered in stress granules. Although this mechanism might ensure that translation can rapidly resume upon DNA repair, it also requires that elongation can be restored during checkpoint recovery. Kruiswijk et al. demonstrated that during checkpoint silencing, eEF2K is degraded by the ubiquitin-proteasome system through the ubiquitin ligase SCFβTrCP. These mechanisms appear to be involved in restoring elongation rates and suggest that SCFβTrCP regulates the recovery from genotoxic stress.

Kruiswijk et al. demonstrated that eEF2K is a target of SCFβTrCP, suggesting that other components of the translational machinery may be targeted by SCFβTrCP or other ubiquitin ligases. Consistent with this notion, programmed cell death 4 (PDCD4), which binds to and inhibits the eukaryotic initiation factor 4A (eIF4A), is also targeted for degradation by SCFβTrCP (15). The ubiquitin ligase Atrogin-1 (also known as muscle atrophy F-box; MAFbx) promotes the degradation of the initiation factor eIF3f (16). In addition, the poly-A–binding protein (PABP)–interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA, is targeted for degradation by EDD (also known as Rat100), a HECT (homology to E6-AP carboxy terminus) domain family member (17). These findings underscore the important role played by ubiquitin ligases in the regulation of protein synthesis (Fig. 2) and suggest that other translational regulatory factors could be targeted by the ubiquitin-proteasome system.

Fig. 2

The diverse relationships between E3 ligases and mRNA translation. In addition to the regulation of eEF2K by SCFβTrCP, as described by Kruiswijk et al., this ubiquitin ligase also regulates PDCD4, an inhibitor of eIF4A and translation initiation. These results indicate that SCFβTrCP regulates both the initiation and elongation phases of translation, by promoting the degradation of PDCD4 and eEF2K, respectively. Two other E3 ligases regulate protein synthesis at the level of translation: EDD promotes the degradation of Paip2, an inhibitor of PABP and translation initiation, and Atrogin-1 (also known as MAFbx) promotes the degradation of eIF3f, an eIF3 isoform that participates in ribosome recruitment to mRNA. Together, these results indicate that several E3 ligases participate in translational control.


Kruiswijk et al. reported that the degradation of eEF2K is coupled with its activation, because autophosphorylation of eEF2K within a βTrCP phosphodegron was required for its degradation (Fig. 1). Although the data presented by Kruiswijk et al. support this model, several questions remain about the role of AMPK in the degradation of eEF2K. Mutation of critical residues within the phosphodegron effectively inhibited eEF2K degradation, but Kruiswijk et al. did not determine whether inhibition of AMPK activity affected eEF2K stability. In addition, AMPK is activated in response to many cellular challenges, including hypoxia and glucose deprivation, but these events did not affect eEF2K stability. This suggests that eEF2K may be protected from being degraded in response to some types of stress or that additional effector proteins are specifically activated by genotoxic stress. Evidence for the latter comes from the finding that eEF2K autophosphorylates at only one of the two phospho-residues (Ser445, but not Ser441) present in the phosphodegron (18), suggesting the involvement of an additional protein kinase in the regulation of Ser441. This protein kinase may specifically be regulated by DNA damage, which would render the degradation of eEF2K specific to this situation. Unfortunately, Kruiswijk et al. did not determine whether the single mutation of Ser441 or Ser445 is sufficient to inhibit eEF2K degradation in response to genotoxic stress, and thus at the moment, it is unknown whether this potential heterologous kinase is essential for eEF2K degradation.

The proposed mechanism of coupled activation-degradation of eEF2K in response to genotoxic stress is analogous to the coupled phosphorylation-dephosphorylation of eIF2α in the unfolded protein response (19). In response to endoplasmic reticulum stress, the PKR-like endoplasmic reticulum kinase is activated and phosphorylates eIF2α on Ser51 to reduce the global rate of translation initiation. However, selective mRNAs bearing upstream open reading frames are preferentially translated under these conditions, including growth arrest and DNA damage–inducible protein 34 (GADD34). GADD34 forms a complex with the catalytic subunit of protein phosphatase 1 to specifically promote the dephosphorylation of eIF2α and restore global protein synthesis during stress recovery.

Available data indicate that eEF2K is an important regulatory hub targeted by various signaling pathways and protein kinases. In addition to AMPK and autophosphorylation events, phosphorylation of eEF2K is regulated by S6K, 90-kD ribosomal S6 kinase (RSK), and the stress-activated kinase p38δ (6). Based on the suggestion by Kruiswijk et al. that eEF2K stability is linked with its activation, more experimentation will be required to determine whether other activation mechanisms affect eEF2K stability. Clearly, identification of the kinase that targets Ser441 will be important to better understand the regulation of eEF2K stability. An interesting possibility is that phosphorylation of Ser445 may be a prerequisite for that of Ser441, as has been shown for many substrates of glycogen synthase kinase 3 (GSK3) that require priming phosphorylation sites. Although Ser441 does not lie within an optimal GSK3 phosphorylation motif, its possible involvement could be easily tested using pharmacological inhibitors and genetically altered cells. Another area of investigation resides in the possibility that DNA damage redirects the translational machinery during checkpoint activation. In support of this, a study aimed at identifying how the translatome is modified by DNA damage found that mRNAs coding for DNA repair enzymes are selectively recruited to polysomes after ultraviolet irradiation (20). It will be interesting to determine the specificity of this response and how it could be exploited for improving the treatment of cancer cells with DNA-damaging agents.

References and Notes

Funding: P.P.R. is supported by grants from the Canadian Cancer Society Research Institute, the Cancer Research Institute, and the Natural Sciences and Engineering Research Council of Canada. S.M. is supported by grants from the Canadian Institutes for Health Research and the Cancer Research Society. P.P.R. and S.M. hold Canada Research Chairs in Signal Transduction and Proteomics and in Cellular Signaling, respectively.
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