Cell fate is subject to translational control (2
), a principle that is underscored by the wide variety of human tumors and cancer cell lines in which the cap-dependent initiation apparatus is activated (reviewed in references 6
). In prior studies, overexpressed eIF4E was found to prevent the death of both nontransformed and transformed fibroblasts, whereas wild-type 4E-BP1 promoted apoptosis only in transformed cells, sparing nontransformed fibroblasts (28
). This raised the question of whether malignant transformation fundamentally alters translational control of apoptosis or whether it serves to increase the cellular requirement for cap-dependent translation. Here, we show that nontransformed fibroblasts undergo apoptosis when repression of cap-dependent translation is intensified. Transfer of wild-type 4E-BP1 into nontransformed fibroblasts together with pharmacological blockade of 4E-BP1 phosphorylation using the FRAP/mTOR kinase inhibitor rapamycin activated apoptosis. Mutation of 4E-BP1 at residues T37, T46, S65, or T70 eliminates phosphorylation sites, resulting in mutant proteins that are able to evade physiological mechanisms of deactivation. Transfer of these mutant forms into nontransformed fibroblasts markedly decreased their ability to form colonies and increased apoptosis in a manner dependent on the presence of specific phosphorylation sites. In general, for the wild type and each mutant 4E-BP1 protein studied, we found that proapoptotic potency paralleled its ability to repress cap-dependent translation. Surprisingly, our data did not reveal a strict monotonic relationship between cap-dependent translation and apoptosis. Instead, we found that as repression of cap-dependent translation intensified, apoptosis increased to a maximum value (observed with the A70 mutant) and that further repression (achieved with A37/A46) resulted in less apoptosis—a state associated with activation of translation through an IRES. Together with our previous observations (28
), these findings reveal that 4E-BP1 is proapoptotic in both normal and transformed fibroblasts and suggest that oncogenic transformation may confer cells with a markedly reduced capacity to block apoptosis when cap-dependent translation is suppressed by 4E-BP1. Our results also suggest that 4E-BP1-mediated control of apoptosis occurs through qualitative rather than quantitative changes in protein synthesis, mediated by a dynamic interplay between cap-dependent and cap-independent processes.
Excluding the limiting case where an insufficient quantity of structural proteins or critical enzymes is synthesized to sustain life, the role of translational control in the regulation of apoptosis is just coming into focus. In some cell types including mammalian neurons (7
) and fibroblasts (28
), pharmacological inhibition of peptide chain elongation suppresses programmed cell death. In contrast, specific inhibition of cap-dependent translation initiation by ectopic expression of 4E-BP1 stimulates apoptosis in Ras-transformed fibroblasts in a manner dependent upon its ability to sequester eIF4E (28
). Consistent with this, ectopic expression of eIF4E rescues fibroblasts from apoptotic death (29
). Here, we add to the data connecting the cap-dependent initiation apparatus to the regulation of apoptosis. Ectopic expression of 4E-BP1 in nontransformed fibroblasts (CREF) had no discernible impact on the assembly of eIF4F or on cell viability. However, inhibition of phosphorylation with rapamycin led to disassembly of eIF4F and activation of apoptosis. In accord with this result, introduction of mutant forms of 4E-BP1 lacking specific phosphorylation sites into CREF also activated apoptosis. These findings suggest that the FRAP/mTOR kinase cascade, as well as other pathways leading to 4E-BP1 phosphorylation, is an important component of the antiapoptotic signaling system activated by extracellular survival factors. In addition, these data demonstrate a close connection between cellular functions controlled by 4E-BP1 and the regulation of apoptosis, thus supporting the concept that activation of cap-dependent translation leads to the synthesis of regulatory proteins that antagonize apoptosis.
A detailed examination of the relationship between repression of cap-dependent initiation and apoptosis proved informative. The mutant forms of 4E-BP1 studied suppressed cap-dependent initiation in exactly the rank order of potency expected based on prior publications (A70 > A65 > A37/A46 > wild type) (9
). In general, activation of apoptosis followed this same pattern, with A70 > A65 > wild type. Only A37/A46 deviated from expectation, a result with several important implications. First, despite being expressed at lower levels than the other 4E-BP1 forms, A37/A46 suppressed translation most robustly. This dissociates the level of ectopic protein expression from the intensity of translational repression and strongly suggests that our findings do not result from toxicity of the mutant proteins themselves. Second, introduction of the double mutant (A37/A46) allowed us to reduce cap-dependent translation initiation by 70%, which by itself was not sufficient to achieve maximum levels of 4E-BP1-induced apoptosis. This makes it unlikely that the apoptosis observed was triggered by metabolic poisoning due to inadequate protein synthesis—pointing strongly instead to a change in the balance of factors regulating apoptosis, not those required to sustain other critical physiological functions. Third, in cells expressing the A37/A46 mutant, translation via IRES was activated concomitantly with a decline in apoptosis. Somewhat surprisingly, rather than leading to potentiation of apoptosis as might be expected from the simplest model of translational control (that cap-dependent translation rescues, and translation via IRES promotes, apoptosis), apoptosis was actually attenuated.
The data in this report together with the published literature lead us to speculate that at some threshold level, the proapoptotic effect of hypophosphorylated 4E-BP1 may be mitigated by concomitant activation of IRES-mediated translation of rescue proteins. In this connection, experimental precedent does exist for translation of known rescue moieties via IRES, including insulin-like growth factor I receptor, insulin-like growth factor II, and X-linked inhibitor of apoptosis protein (17
). Along these lines, recent findings indicate that the potent mediator of IRES-dependent translation, p97/DAP5/NAT1, is a bifunctional regulator of neuroblastoma cell fate—essential for both viability and apoptosis (37
). Alternatively, as repression of cap-dependent translation is intensified, apoptosis may be attenuated by a reduction in cap-dependent death proteins independent of any effects exerted by proteins translated using IRES.
Our prior studies (28
) together with the data reported here establish the functional import of regulating cap-dependent translation initiation in the control of programmed cell death. In addition, as the process of apoptosis unfolds, the translational machinery itself is attacked, setting up potential positive or negative feedback loops. For example, eIF4GI and eIF4GII are cleaved by factors triggering, controlling, or executing apoptosis (5
), resulting in shutdown of cap-dependent protein synthesis. In addition to the eIF4G family proteins, 4E-BP1 is also cleaved during apoptosis (2
). In accord with the general concept that cap-dependent translation machinery is suppressed during apoptosis (2
), one of the 4E-BP1 daughter fragments retains the ability to sequester eIF4E (36
). In this regard, recent work demonstrates that sequestration of eIF4E by overexpressed 4E-BP1 (28
) or by synthetic oligopeptides that associate with its eIF4G-4E-BP1 binding motif (14
) is proapoptotic. These findings indicate that the cap-dependent initiation apparatus is both a regulator and target of the apoptotic machinery and suggest that degradation of specific components of the initiation complex is an integral part of apoptotic death.
Based on current experimental evidence, it appears possible that cell fate is in part determined by a dynamic interplay between cap-dependent translation and translation via IRES. The level and activity of the 4E-BP family of proteins are major determinants of the rate of translation initiation utilizing eIF4F, the cap binding apparatus. Thus, this formulation assigns a central role to the 4E-BP family of proteins and the biochemical pathways governing their phosphorylation status in the control of apoptosis. It also highlights the need for studies in which cap-dependent and IRES-mediated translation is independently manipulated—to elucidate the rules governing which mechanism is utilized in the translation of critical mRNA species encoding proteins that regulate cell viability.