Translational research? Ribosome integrity and a new p53 ... - Nature

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Mar 24, 2006 - p53 tumor suppressor checkpoint. JT Opferman*,1 and GP Zambetti*,1. 1 Department of Biochemistry, St. Jude Children's Research Hospital, ...
Cell Death and Differentiation (2006) 13, 898–901

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Translational research? Ribosome integrity and a new p53 tumor suppressor checkpoint JT Opferman*,1 and GP Zambetti*,1 1

Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN 38138, USA * Corresponding authors: JT Opferman and GP Zambetti, St Jude Children’s Research Hospital, Department of Biochemistry, 332 N. Lauderdale, Memphis, TN 38105, USA. Tel: þ 1 901 495 5524; Fax: þ 1 901 525 8025; E-mail: [email protected], [email protected]

Cell Death and Differentiation (2006) 13, 898–901. doi:10.1038/sj.cdd.4401923; published online 24 March 2006 The p53 tumor suppressor senses a variety of cellular stresses, such as DNA damage and inappropriate proliferation, and responds accordingly by inducing cell cycle arrest or apoptosis. The ability of p53 to regulate cell division and survival is considered essential for blocking tumor development. A recent article by Sulic et al.18 provides convincing genetic evidence that p53 also surveils the state of protein synthesis. Here, we focus on how p53 may detect alterations in translation and the consequence of this monitoring process on maintaining normal cell homeostasis.

Introduction: the p53 Signaling Pathway DNA damage and oncogene-driven proliferation trigger the p53 tumor suppressor signaling pathway, leading to cell cycle arrest or apoptosis. It is generally accepted that these activities provide an opportunity to correct the problem (e.g., repair DNA lesions) or to eradicate the damaged cell (e.g., induce apoptosis). Without p53-dependent control mechanisms, corrupted cells continue to proliferate and survive unabated. It is therefore not surprising that p53 is essential for preventing cancer. Indeed, somatic mutations in p53 are the most common genetic alterations detected in human malignancies.1 Mutation of p53 usually disrupts DNA binding, which precludes it from regulating gene expression either as a transcriptional activator or repressor, depending on the target gene. Many p53 responsive genes have been identified that impact cell cycle checkpoints (e.g., p21Cip1 14-3-3s, PTPRV) and cell death (Bax, Noxa and Puma).2 A key downstream target of p53 is Mdm2, an E3 ubiquitin ligase that attenuates p53 function within the context of an essential negative feedback loop.3 The accumulation of functional p53 protein in response to a cellular insult results in higher levels of Mdm2, which inactivates p53 in part by targeting it for ubiquitindependent proteasomal degradation. DNA damage activates and stabilizes p53 by turning on the Atm, Atr and/or DNA-PK PI3-like kinases, which phosphorylate p53 in the N-terminus preventing Mdm2 binding.4 By contrast, oncogene activation, which promotes inappropriate cell proliferation, induces the expression of p19Arf (p14ARF in humans) encoded by the Ink4a locus.5 Arf is a nucleolar protein that binds and

inactivates Mdm2 by blocking its E3 ubiquitin ligase activity and by sequestering it in the nucleolus.5 Arf also suppresses ribosomal RNA processing through the inactivation of nucleolar protein B23 ribonuclease.6 It is now clear that the Arf-Mdm2-p53 axis is critical for maintaining normal cell growth and proliferation as the abrogation of this signaling pathway promotes tumorigenesis. Mutation of p53, loss of Arf by gene deletion or silencing, or Mdm2 overexpression through gene amplification or translational control mechanisms, results in cancer.5

Control of Ribosome Biogenesis Ribosome biogenesis and protein synthesis are critical processes inextricably linked to cell growth and proliferation. It has been estimated that proliferating cells expend more than 80% of their cellular energy on the synthesis, transport, processing and assembly of ribosomal subunits.7 Coordination of synthesis occurs in the nucleolus, a nuclear suborganelle where rRNA is transcribed, processed, and assembled with ribosomal proteins to produce ribosomal subunits. The process of translation and ribosomal biogenesis is tightly regulated at a variety of levels and can therefore respond dynamically to cellular stimuli delivered through growth factor receptors and nutrient sensors. Many signal transduction pathways activated by growth factor stimulation (including Myc, Ras and PI3K) can induce ribosome synthesis and/or increase the rate of translation initiation. For example, the BCR/ABL oncoproteins activate MDM2 mRNA translation via the RNA binding protein La resulting in the downmodulation of p53.8 Thus, cells are hardwired to respond to environmental stimuli by inducing the synthesis of protein translational machinery to coordinate cell growth (increase in cell size) with proliferation (increase in cell number). Uncoupling protein synthesis from cell growth and proliferation can result in tumorigenesis. Indeed, several gene products involved in ribosomal biogenesis and protein translation are associated with tumor susceptibility (e.g., mutated ribosomal protein S19 in Diamond-Blackfan Anemia and mutated DKC1 in dyskeratosis congenital),9,10 suggesting that cancer cells may have defects in ribosomal control. Therefore, it has been hypothesized that ribosomal biogenesis may represent a critical pathway that must be monitored in order to preserve the fidelity of protein synthesis. The S6 ribosomal protein, one of at least 33 ribosomal proteins, forms the 40S ribosomal subunit along with 18S rRNA. The S6 protein is the principal substrate of the S6 kinase (S6K) and its phosphorylation is induced as cells enter the cell cycle in response to growth factor stimulation.11 S6K activity is positively regulated by mTOR, the target of the anticancer drug rapamycin, and phosphorylated S6 has been associated with enhanced translation of 50 TOP (50 UTR

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terminal oligopyrimidine tract) mRNAs.12,13 The 50 TOP class of mRNA includes several ribosomal proteins and elongation factors (eEF1A1 and eEFA2), many of which act in a feedforward loop to enhance protein synthesis.14 The proteins encoded by 50 TOP mRNAs have been shown to act as proto-oncogenes inducing cell growth and proliferation. For example, EEF1A2, which encodes a protein elongation factor, is amplified and overexpressed in 25% of primary ovarian tumors.15 Inhibition of mTOR by rapamycin blocks cell proliferation, decreases S6K activity and represses translation of 50 TOP mRNAs.16 Interestingly, rapamycin and derivative compounds are currently being developed as chemotherapeutic agents for treating ovarian cancer, as well as other tumor types.

The S6 Ribosomal Biogenesis Checkpoint Thomas and co-workers developed a conditional model for deleting the gene encoding the S6 ribosomal protein to genetically assess the requirement of ribosomal biogenesis for cell growth and proliferation.17 Following inducible deletion of the gene encoding S6 ribosomal protein in the liver of adult mice, they unexpectedly demonstrated that livers from fasted mice were capable of increasing cell size in response to the re-addition of food despite a deficiency in 40S ribosomes. However, upon S6 deletion, liver cells failed to proliferate following partial hepatectomy. Despite the formation of active cyclin-CDK4 complexes, S6-deficient liver cells failed to induce cyclin E expression and enter S phase. These keystone findings suggested the existence of a previously unrecognized checkpoint preventing cell proliferation, but not growth, downstream of a deficiency in ribosomal biogenesis.

Ribosomal Biogenesis Checkpoint is p53 Dependent A recent report by Volarevic and co-workers offers genetic insight into the basis of how ribosomal biogenesis and protein synthesis feedback to regulate cell cycle progression.18 To investigate this apparent novel cell cycle checkpoint, the S6 ribosomal protein gene was conditionally deleted in the T-cell lineage using CD4-Cre transgenic mice. While the deletion of both alleles of the S6 gene resulted in the complete block in Tcell development (more than 50% of the mice lacked a thymus and the remainder contained 100-fold fewer T cells), deletion of only a single allele of S6 did not perturb thymic development. Therefore, a single allele of S6, which results in a 50% reduction in mRNA without altering basal S6 protein levels, is sufficient to sustain ribosomal biogenesis in thymocytes during development. Despite normal thymic development, mice lacking one copy of the S6 protein gene exhibited a reduction in the number of T lymphocytes in the peripheral lymphoid organs. In response to stimulation through the T-cell receptor, both control and S6 haploinsufficient T lymphocytes rapidly transformed from small resting lymphocytes to large blasts. However, the S6 haploinsufficient blast T cells failed to proliferate due to a cell cycle block, reminiscent of the S6-deficient hepatocytes that could undergo cell growth but not cellular proliferation.17

Breeding of the S6 haploinsufficient mice to Bcl-2 transgenics partially rescued the numbers of peripheral T cells, but merely preventing apoptosis could not rescue the proliferative defect in stimulated T cells. Bcl-2 suppresses p53-mediated cell death but not its ability to arrest the cell cycle, therefore, the contribution of p53 to the translational-defect checkpoint was assessed by co-deleting the S6 and p53 alleles. In T-cell development, loss of p53 fully restored thymic cellularity in mice lacking both alleles of S6, but not T-cell numbers in peripheral lymphoid organs. Of note and still unexplained, S6 is required at some level even in the absence of p53 to maintain the homeostatic expansion and survival of peripheral T lymphocytes. In contrast, deletion of p53 in S6 haploinsufficient T cells rescued the proliferative defect and restored near normal numbers of T lymphocytes in the spleen. Presumably, deletion of p53 in S6 heterozygous T cells inactivates both arms of the checkpoint by eliminating the cell cycle arrest and apoptotic responses, and the diminished level of S6 expression is sufficient to permit cell expansion. Therefore, these data indicate that a defect in ribosome biogenesis induced by S6-deficiency activates a p53-dependent cell cycle checkpoint to prevent the survival and proliferation of defective T lymphocytes.

Tumor Suppressors and Ribosomal Biogenesis Stress One important mechanism for regulating ribosomal biogenesis occurs at the level of transcription. In the nucleolus, RNA polymerase I (Pol I) and III (Pol III) complex activities generate ribosomal RNA (rRNA), small nuclear RNAs (snRNA) and transfer RNA (tRNA) all of which are required for protein synthesis. The tumor suppressors p53 and Rb individually repress the transcriptional activity of both Pol I and Pol III transcription of ribosomal subunit genes by preventing the assembly of the RNA polymerase complexes.19,20 As a result, p53 and Rb can directly repress the synthesis of rRNA (Pol I) and 5S rRNA, snRNAs and tRNAs (Pol III) thereby interfering with the assembly of ribosomal subunits. Thus, loss of function mutations in either p53 or Rb tumor suppressors may result in the aberrant synthesis of ribosomal subunits and increase translation. Conversely, alterations in ribosome assembly and function likely activate p53 to downregulate translational processes.

How is the Checkpoint Sensed? How might p53 ‘sense’ a deficiency in ribosome number or activity in proliferating cells? It has been suggested that p53 monitors ‘nucleolar stress’ as a result of errors in rRNA processing and therefore induces cell cycle arrest. The existence of a p53-dependent signaling mechanism between ribosomal biogenesis and cell cycle control was established when a nucleolar protein involved in rRNA processing and assembly known as BOP1 was identified.21 Expression of a dominant negative version of BOP1 causes defects in rRNA processing that induces a cell cycle arrest, which was alleviated by deletion of p53. Therefore, p53 can functionally link ribosome biogenesis to cell cycle progression. In the case Cell Death and Differentiation

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Figure 1 In unstressed cells (Normal), the integrity of the nucleolus (yellow) is maintained sequestering Arf and p53, as well as ribosomal proteins including L5, L11, and L23. In the nucleoplasm (blue) ubiquitylation of p53 by Mdm2 targets the degradation of p53 preventing cell cycle arrest or induction of proapoptotic genes. Upon S6 deletion, ribosome deficiency in the proliferating cell (Nucleolar Stressed) may make available Arf, L5, L11 and/or L23 to promote p53 stabilization by inhibition of Mdm2. The increased activity of p53 in response to alterations in translation induces the expression of target genes that enforce cell cycle arrest (e.g. p21) and elicit apoptosis (e.g. Puma), resulting in the destruction of the stressed cell

of S6 haploinsufficiency in T lymphocytes, it is unlikely that a limitation in the amount of 40S ribosomes induced problems in rRNA processing directly. Perhaps deficiencies in translational capacity can feedback to elicit an increase in the production of new ribosomal proteins and rRNA triggering a p53 response. It has been hypothesized that the nucleolus is a stress sensor responsible for maintaining low levels of p53 expression. Rubbi and Milner demonstrated using focused micropore irradiation, that p53 levels are not stabilized by high levels of nuclear irradiation unless the nucleolus is disrupted.22 By directly destabilizing the nucleolus in the absence of DNA damage, they demonstrated that p53 levels are stabilized. From these data, they proposed a model in which the integrity of the nucleolus regulates p53 activity irrespective of DNA damage. Perhaps in proliferating T lymphocytes S6haploinsufficiency and the resulting paucity of 40S ribosomes disrupts the nucleolus thereby stabilizing p53 and inducing cell cycle arrest (see Figure 1). Consistent with this possibility, Volarevic and co-workers reported that S6-haploinsufficiency in stimulated T lymphocytes did not activate key DNA damage repair proteins implying that DNA damage is not responsible for the p53-dependent checkpoint.18 The absence of a DNA damage response may point to the possible involvement of Arf, which is harbored within the nucleolus, and can activate p53 independently of the Atm/Atr signaling pathway (Figure 1). Perturbations in ribosome assembly could cause nucleolar alterations that ‘unleash’ Arf to target Mdm2, indirectly leading to functional p53 and cell cycle arrest and/or death. It will be interesting to test whether Arf deletion can also rescue T-cell development in S6-deficient mice. An alternative, but not mutually exclusive, mechanism for p53 sensing the integrity of the translation process centers on the binding of Mdm2 to the ribosomal proteins L5, L11 and L23.23–27 It is therefore tempting to speculate that upon translational stress that L5, L11 and/or L23 become accessible to negatively feedback upon Mdm2 and activate p53 (Figure 1). Likewise, p53 is covalently Cell Death and Differentiation

associated with 5.8S RNA,28 which interacts with L5 and 5S RNA, both of which bind Mdm2.23 Whether 5.8S RNA plays a direct role in the regulation of p53 activity or indirectly through complex formation with Mdm2/L5/5S RNA is not yet known. A detailed biochemical analysis of each of these interactions before and after translation-mediated stress in response to S6 deficiency should be enlightening and provide insight into the p53-sensing mechanism of altered protein synthesis.

Concluding Remarks It is well established that p53 serves as a checkpoint regulator of cell cycle progression in response to DNA damage and oncogene-induced hyperproliferation. The recent studies by Volarevic’s group have now firmly placed p53 within a ribosome assembly and protein synthesis signaling pathway. How p53 senses stress signals due to abnormal protein translation is not yet clear. It is likely that the answer lies within the realm of the nucleolus, which is the central factory for ribosome biosynthesis and assembly, and where the key regulators of p53 reside at some point (Arf, Mdm2, L5/L11/L23 and p53 itself). While the physiological relevance of p53 responding to ribosomal assembly-protein translation-related stress remains to be determined, these surprising findings add to the growing number of targets and pathways that p53 monitors to maintain normal cell integrity and eliminate damaged cells. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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